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==== Front PLoS BiolPLoS BiolplosplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1576918310.1371/journal.pbio.0030123Research ArticleBiotechnologyOncologyIn VitroNeutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase Aptamer Inhibition of RET RTKCerchia Laura 1 Ducongé Frédéric 2 Pestourie Carine 2 Boulay Jocelyne 3 Aissouni Youssef 3 Gombert Karine 2 Tavitian Bertrand 2 * de Franciscis Vittorio 1 * Libri Domenico [email protected] 3 1 Istituto per I'Endocrinologia e Oncologia Molecolare “G. Salvatore”, CNR, Naples, Italy2 CEA/DSV/DRM Service Hospitalier Frédéric Joliot, INSERM E-103, Orsay, France3 Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique (CNRS), Gif sur Yvette, FranceJoyce Gerald Academic EditorScripps Research Institute, United States of America* To whom correspondence should be addressed. E-mail: [email protected] (BT), Email: [email protected] (VD) LC, FD, BT, VdF, and DL conceived and designed the experiments. LC, FD, CP, JB, YA, and KG performed the experiments. LC, FD, BT, VdF, and DL analyzed the data. VdF and DL contributed reagents/materials/analysis tools. BT, VdF, and DL wrote the paper. A patent application was filed covering the D4 aptamer and its use in diagnostic and therapeutics of cancer. 4 2005 22 3 2005 22 3 2005 3 4 e12327 10 2004 2 2 2005 © 2005 Cerchia et al2005Cerchia et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. A Small RNA That Neutralizes a Protein Linked to Tumor Development Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance. The strategy used to select aptamers that bind a tyrosine kinase mutated in certain cancers holds promise for targeting other members of this biomedically important class of proteins. ==== Body Introduction The identification of tumor-specific molecular markers is a powerful tool in cancer diagnostics, and the targeting of tumor-specific pathways is the best hope for developing nontoxic and efficient anticancer therapies. Targeting of cancer cells relies on the development of molecular beacons, suited for in vivo applications, that are endowed with the required affinity, specificity, and favorable pharmacokinetic properties. With the systematic evolution of ligands by exponential enrichment (SELEX) technology [1,2], specific macromolecular ligands—aptamers—can be generated by screening very large pools of oligonucleotides containing regions of random base composition with reiterated cycles of enrichment and amplification. At each cycle, the individual oligonucleotides with affinity for the desired target are kept, those with affinity for the sham target are rejected, and the population is enriched in oligonucleotides that distinguish between sham and real target. Aptamers that recognize a wide variety of targets, from small molecules to proteins and nucleic acids, and from cultured cells to whole organisms, have been described [3,4,5,6,7,8,9,10]. These oligonucleotides generally meet the requirements for in vivo diagnostic and/or therapeutic applications: Besides their good specificity and affinity, they are poorly immunogenic, and the SELEX technology can now accept chemically modified nucleotides for improved stability in biological fluids [11]. Conspicuously, less than fifteen years after the first applications of the technique, several lead compounds, including an anti-vascular endothelial growth factor aptamer [12], are currently under clinical trials [13]. Receptor tyrosine kinases (RTKs) are involved in a variety of signaling processes that regulate cell growth and proliferation and in several cancers [14]. RTKs are privileged targets for cancer therapy, which is underscored by the promising outcome of clinical trials with small molecules or antibody inhibitors [14]. In the present study, we validated a general strategy to target transmembrane receptors by SELEX. The RET (rearranged during transfection) RTK is physiologically stimulated by any member of the glial cell line-derived neurotrophic factor (GDNF) family [15,16]. Germline mutations in the RET gene are responsible for constitutive activation of the receptor and for inheritance of multiple endocrine neoplasia (MEN) type 2A and 2B syndromes and of familial medullary thyroid carcinoma [17,18,19,20]. Mutations in the extracellular domain of RET, responsible for MEN2A syndrome, lead to constitutive dimerization of two mutated RET molecules. Conversely, a single point mutation, within the RET catalytic domain, that causes the MEN2B syndrome, involves an intramolecular mechanism to convert RET into a dominant transforming gene. Therefore, RET constitutes a model system of choice [20], in that the transforming mutations located in the extracellular domain simplify the issue of intracellular accessibility for a molecule targeting the receptor mutated in the extracellular domain (in its monomeric or dimeric form) and might provide alternative models (e.g., RET with mutations of the 2B kind) for controls or to elucidate the mode of target recognition. Here we adopted a whole-cell SELEX strategy to target RET in a complex environment that is expected to expose a native protein to the selection procedure, thus best mimicking in vivo conditions. We obtained aptamers that not only recognize the extracellular domain of RET, but also block RET downstream signaling and subsequent molecular and cellular events. The fact that aptamers with antioncogenic activity were isolated in the absence of a specific selective pressure suggests that our method could be used to identify active macromolecules with potential therapeutic interest against other transmembrane receptors. Results A library of 2′-fluoropyrimidine (2′F-Py), nuclease-resistant RNAs was subjected to a differential SELEX protocol against intact cells expressing different forms of the human RET oncogene (Figure 1). For the selection step, PC12 cells were used that express the human RETC634Y mutant receptor (PC12/MEN2A). RETC634Y is mutated in the extracellular domain and forms spontaneously active homodimers on the cell surface, which induces biochemical and morphological changes that mirror the RET-dependent human pheochromocytoma phenotype of MEN2 syndromes [21]. The counterselection necessary to avoid selecting for aptamers that nonspecifically recognized the cell surface included a first step against parental PC12 cells in order to eliminate nonspecific binders of the PC12 cell surface, followed by a second counterselection step against PC12/MEN2B cells that expressed an allele of RET (RETM918T) mutated in the intracellular tyrosine kinase domain. PC12/MEN2B and PC12/MEN2A cells have a similar morphology, but the extracellular domain of the RETM918T receptor is identical to the wild type and, in the absence of the ligand and co-receptor, remains monomeric. This step was originally aimed at selecting aptamers that recognize specifically the dimeric form of the extracellular domain. 10.1371/journal.pbio.0030123.g001Figure 1 Schematic Protocol for the Selection of PC12/MEN2A Cell-Specific Aptamers A pool of 2′F-Py RNAs was incubated with suspended parental PC12 cells (Counterselection 1). Unbound sequences were recovered by centrifugation and incubated with adherent PC12/MEN2B cells (Counterselection 2). Unbound sequences in the supernatant were recovered and incubated with adherent PC12/MEN2A cells for the selection step (Selection). Unbound sequences were discarded by several washings, and bound sequences were recovered by phenol extraction. Sequences enriched by the selection step were amplified by RT-PCR and in vitro transcription before a new cycle of selection. After 15 rounds of selection, the pool of remaining sequences bound PC12/MEN 2A cells in a saturable manner with an apparent Kd approximating 100 nM. From this pool, 67 sequences were cloned and analyzed. Two individual sequences (D14 and D12) dominated the selection and constituted together more than 50% of the clones, four other sequences represented together 25% of the clones, and eight sequences were present only once. As is often the case for a selection against a complex target [7,22] (and in contrast to in vitro SELEX on purified proteins) we found almost no similarity among sequences, except for clones D24 and D4, which shared common sequence motifs and structure prediction (Figure 2A). 10.1371/journal.pbio.0030123.g002Figure 2 Predicted Structure and Association Constants of D4 and D24 (A) Comparison of a secondary structure prediction for the D4 and D24 aptamers. Structures were predicted using MFOLD software version 3.1 (available at http://www.bioinfo.rpi.edu/applications/mfold/). (B) Binding curve of the D4 aptamer on PC12/MEN2A. D4 was 32P-radiolabeled and incubated at different concentrations on cell monolayers. The background binding value for a D4 scrambled sequence is subtracted from every data point. Scatchard analysis (inset) was used for the evaluation of the binding constant. (C) Binding of the 32P-labeled D4 aptamer to several cell lines expressing (or not) human RET. Binding was performed on the cell lines indicated in the same condition at 50 nM, and the results are expressed relative to the background binding detected with the starting pool of sequences used for selection. Expression of RET could not be detected by Western blot in HeLa, NBTII, PC12wt and NIH3T3 cells, whereas PC12/MEN2A and NIH3T3/MEN2A express RETC634Y and PC12/MEN2B and NIH3T3/MEN2B express RETM918T. We assessed binding to PC12/MEN2A cells of all individual aptamers that were found more than once and also of some unique sequences (including D4 and D24). Several sequences bound PC12/MEN2A cells with apparent Kd values ranging from 30 to 70 nM (Figure 2B and unpublished data), but not parental PC12, rat-derived bladder carcinoma (NBTII), or human cervical carcinoma (HeLa) cells (Figure 2C and unpublished data). As a first attempt to deconvolute the complex pool of winning aptamers, we first produced a recombinant fragment of RET, EC-RETC634Y [23], but all attempts to identify in the winning pool aptamers binding to EC-RETC634Y were fruitless. Likewise, SELEX against this purified EC-RETC634Y protein gave rise to aptamers unable to recognize the PC12/MEN2A cells, suggesting that they did not bind to the RET protein present in its native conformation on the cell surface. Consequently, we screened the winning pool of aptamers for the ability to interfere with the biological activity of RET. To this end, we used an in vitro cell system in which we assessed the capability of each aptamer to inhibit RETC634Y autophosphorylation and receptor-dependent downstream signaling. Mutant RETC634Y, expressed in PC12/MEN2A cells, forms homodimers on the cell surface that cause constitutive activation of its tyrosine kinase activity [24] and induce several downstream signaling cascades, including the activation of extracellular signal-regulated protein kinase (ERK) [25]. As previously reported [25], levels of phosphorylated RET and ERK were constitutively high in untreated PC12/MEN2A cells due to the presence of the active RETC634Y allele. Surprisingly, some of the tested aptamers inhibited RETC634Y and ERK phosphorylation, compared to the control starting pool and to the other aptamers (Figure 3A and unpublished data). In all experiments, inhibition of phosphorylation was more rapid and quantitative for ERK than for RETC634Y. We believe that this is due to a different sensitivity to changes in RET tyrosine kinase activity of the two processes and/or to differences in the half-lives of the phosphorylated forms of the two proteins [26]. In a dose-response experiment (Figure 3B, left panel), the best inhibitor, D4, was effective at a concentration of 200 nM to inhibit RETC634Y autophosphorylation up to 70% and to drastically reduce ERK phosphorylation. Time-activity studies showed that the treatment of PC12/MEN2A cells at 200 nM for 1 h was sufficient to significantly inhibit RETC634Y autophosphorylation and to drastically reduce ERK phosphorylation (Figure 3B, right panel). 10.1371/journal.pbio.0030123.g003Figure 3 Effect of Selected Aptamers on RETC634Y Activity (A) PC12/MEN2A cells were either left untreated or treated for 16 h with 150 nM of the indicated RNA aptamer, or the starting RNA pool (pool). Cell lysates were immunoblotted with anti-(phospho)-ERK (pErk), then stripped and reprobed with anti-ERK (Erk) to confirm equal loading. Values below the blots indicate signal levels relative to untreated controls. (B) PC12/MEN2A cells were treated for 1 h with increasing amounts of D4 (left blots) or with 200 nM D4 for the indicated incubation times (right blots). Cell lysates were immunoblotted with anti-(Tyr-phosphorylated)-RET (pRet) or anti-(phospho)-ERK (pErk) antibodies, as indicated. To confirm equal loading the filters were stripped and reprobed with anti-RET (Ret) or anti-ERK (Erk) antibodies, respectively. In (A) and (B), “C” indicates mock-treated cells. Quantitations were done on the sum of the two RET- or ERK- specific bands, and values are expressed relative to the control, arbitrarily set to 1. Standard deviations are indicated (n = 4). Comparison of the predicted structures of D4 and of the related clone D24 (Figure 2A) suggests that a conserved stem-internal loop-stem is crucial for binding. Consistently, we found that replacing the apical loop with a stable tetraloop (UUGC) or deleting nucleotides not included in the conserved structure did not significantly affect binding of D4 to PC12/MEN2A cells (unpublished data). However, only the full-length D4 inhibits RETC634Y signaling, demonstrating that binding is necessary but not sufficient for inhibition. A 2′F-Py RNA oligonucleotide of identical composition but with a scrambled sequence (D4Sc) was ineffective for both binding and inhibition. The D4 aptamer bound to PC12/MEN2A with an estimated apparent Kd of 35 ± 3 nM (Figure 2B), but also to PC12/MEN2B cells (Figure 2C and unpublished data), suggesting that one of the counterselection steps employed in the SELEX procedure was ineffective in this case. The D4 aptamer bound to transfected NIH3T3 cells expressing at similar levels the two mutant forms (RETC634Y and RETM918T) of the RET receptor (NIH/MEN2A and NIH/MEN2B, respectively [Figure 2C; see also below]). Binding was dependent on expression of human RET, as D4 did not recognize parental untransfected PC12, NIH3T3 cells, or other cell lines, including rat NBTII, human HeLa cells, and mouse MN1 (Figure 2C and unpublished data). Interestingly, the latter, a mouse motor neuron-neuroblastoma fusion cell line, expresses the mouse RETwt, suggesting some species-specificity in RET recognition by D4. Finally, D4 bound a human neuroblastoma cell line (SK-N-BE) that naturally expresses endogenous RET (L. Cerchia et al., personal communication). Consistently with what was observed for the pool of winning aptamers, D4 was unable to bind the purified EC-RETC634Y protein (unpublished data), thus supporting the specificity for the membrane-bound RET. We next determined whether D4 could inhibit wild-type RET. Cells from a PC12-derived cell line expressing the human wild-type RET (PC12/wt) were stimulated with a mixture containing GDNF and soluble GDNF family receptor α1 (GFRα1), and either treated with the D4 aptamer or with the starting pool of 2′F-Py RNA as a negative control. As shown in Figure 4A, the D4 aptamer, but not the control RNA pool, strongly inhibited GDNF-induced phosphorylation of RET (left panel) and of the downstream effector ERK (middle panel). A similar inhibitory effect was observed in PC12-α1/wt cells, a PC12-derived cell line that stably expresses both human RET and GFRα1 (unpublished data). In contrast, D4 was inactive in inhibiting the signaling triggered by the unrelated nerve growth factor (NGF) receptor tyrosine kinase TrkA, thus indicating that D4-induced inhibition of ERK phosphorylation was specific for RET intracellular signaling (Figure 4A, right pane) 10.1371/journal.pbio.0030123.g004Figure 4 D4 Aptamer Inhibits RETwt but Not RETM918TActivity (A) PC12/wt cells were treated for 10 min with GDNF (50 ng/ml) and soluble GFRα1 (1.6 nM), or 5 min with NGF (100 ng/ml), together with 200 nM of either the D4 aptamer or the starting RNA pool. “C*” indicates cells treated with GDNF and GFRα1 in the absence of aptamer. (B) PC12/MEN2B cells were starved for 6 h and then treated for 1 h with 200 nM D4 or the starting RNA pool. Cell lysates were immunoblotted with anti-(Tyr-phosphorylated)-RET or anti-(phospho)-ERK antibodies, as indicated (see Figure 3 legend). In (A) and (B), “C” indicates mock-treated cells. Quantitations were done as in Figure 3, and relative abundances are expressed relative to controls, arbitrarily set to 1. Standard deviations are indicated (n = 4). Although the D4 aptamer binds PC12/MEN2B cells, treating these cells with 200 nM D4 for 1 h (Figure 4B) or longer, or at higher D4 concentrations (unpublished data), did not interfere with signaling due to the monomeric RETM918T. This further confirms that inhibition of ERK phosphorylation is not a nonspecific effect of exposing the cells to the D4 aptamer. The kinase and the biological activities of RETM918T, although constitutive, are responsive to GDNF stimulation in the presence of GFRα1 [27,28]. Similarly to the inhibition of RETwt activity, the treatment of PC12/MEN2B cells by D4 abolished the GDNF-dependent overstimulation of RET and ERK phosphorylation (unpublished data). These data strongly suggest that D4 inhibits exclusively the dimerization-dependent RET activation. We then searched for phenotypic effects of D4 on RET-dependent cell differentiation and transformation. First we measured neurite outgrowth in PC12-α1/wt cells following GDNF stimulation. As shown in Figure 5, cells extended long neurite-like processes in response to a 48-h exposure to GDNF (Figure 5B) with respect to the nonstimulated control cells (Figure 5A). Treatment of the cells with the D4 aptamer (Figure 5C), but not with the D4Sc scrambled control (Figure 5D), significantly decreased the proportion of neurite outgrowth (Figure 5E). To biochemically monitor differentiation, we determined the levels of the nerve growth factor-inducible protein (VGF) in cell extracts following 48 h of treatment. VGF is an early gene that is rapidly induced by both NGF and GDNF in PC12 cells [29]. As expected, in GDNF-treated cells, VGF expression was stimulated and, consistent with the phenotypic effects reported above, treatment with D4, but not with D4Sc, kept the VGF levels close to basal (Figure 5F). 10.1371/journal.pbio.0030123.g005Figure 5 D4 Aptamer Inhibits the GDNF-Induced Differentiation of PC12-α1/wt Cells Cells were either left unstimulated (A), stimulated with GDNF (B), or with GDNF together with D4 or D4Sc (C and D, respectively). Following 48 h of GDNF treatment, the percentage of neurite outgrowth was calculated. The data represent the average of three independent experiments and are expressed as percentage of neurite-bearing cells/total cells analyzed (E). Following 48 h of treatment, cells were lysed and proteins immunoblotted with anti-VGF antibodies. Equal loading was confirmed by immunoblotting with anti-ERK antibodies as indicated (F). Upon expression of either RETC634Y or RETM918T, NIH3T3 cells show drastic changes in their morphology [24]. We treated NIH/MEN2A and NIH/MEN2B cells stably expressing the RET mutants with D4 for 72 h, and analyzed the morphological changes induced by the aptamer. As shown in Figure 6, NIH/MEN2A and NIH/MEN2B cells have a spindle shape, long protrusions, and a highly refractive appearance (Figure 6B and 6E, respectively). As expected, D4-treated NIH/MEN2A cells (Figure 6C) reverted to a flat and polygonal morphology similar to the parental NIH3T3, whereas no morphological changes were observed in NIH/MEN2B (Figure 6F), which is consistent with the notion that constitutive signaling from RETC634Y, but not from RETM918, is inhibited by D4. On the other hand, treatment with D4Sc had no effects on any cell line (Figure 6D and unpublished data). 10.1371/journal.pbio.0030123.g006Figure 6 D4 Aptamer Reverts the Transformed Morphology of NIH/MEN2A Cells NIH3T3-derived cell lines were either left untreated (A, B, and E) or treated with D4 (C and F) or D4Sc (D), and the cells were maintained in culture for 72 h. Each experiment was repeated a minimum of three times. Discussion RTKs are involved in a variety of signaling pathways that affect cell growth and differentiation. Targeting specifically RTKs holds potential for dissecting the molecular mechanisms of receptor function, but also for diagnosis and therapeutics of cancer [14]. Here we employed a modified SELEX procedure to target the RET RTK, and we obtained nuclease-resistant RNA ligands capable of binding and inhibiting the protein on the cell surface. Aptamers against recombinant heregulin 3 (HER3) RTK have been recently isolated and shown to inhibit the heregulin-induced activation of the HER3/HER2 dimer [30]. However, finding the most efficient binders and inhibitors is likely to generally rely on the recognition of the target protein in its native state. In the case of transmembrane receptors, whole-cell SELEX offers the advantage of selecting molecules capable of recognizing the target protein in its natural glycosylation state and presented in its physiological environment. An important drawback of this strategy is the lack of knowledge of the identity and abundance of the effective targets and the possibility that unwanted aptamers may dominate the selection, preventing the emergence of the molecules of interest. However, the abundance of the target protein and an appropriate selection scheme might provide sufficient selective pressure to favor the wanted aptamers [10]. The D4 aptamer binds to different cell types, provided that human RET is expressed on the cell surface, and specifically inhibits both RET and ERK phosphorylation, strongly suggesting that RET is the bona fide target of D4. Interestingly, aptamers isolated by whole-cell SELEX were unable to bind purified EC-RETC634Y and, conversely, aptamers coming from the selection with purified EC-RETC634Y were unable to bind the membrane-bound RET. Thus, it is likely that D4 binding is dependent on the association of RET with the cellular membrane, which might reflect changes in the receptor's conformation/modification state or, alternatively, might imply unidentified molecular components interacting with RET at the cell surface. This latter possibility is supported by a recent report demonstrating that the presence of heparan sulfate glycosaminoglycan on the cell surface is required for RET-dependent GDNF intracellular signaling [31]. Our interpretation of the D4 aptamer's mode of action relies upon three observations: (1) D4 binds with similar affinities to cells expressing RET in a monomeric or dimeric form; (2) D4 inhibits dimerization-dependent RET activation, as a consequence either of GDNF stimulation of RETwt or RETM918T or of constitutive dimerization of the RETC634Y mutant; and (3) D4 does not inhibit a monomeric form of RET that is constitutively activated by a mutation in the intracellular kinase domain (RETM918T). These results taken together are compatible with the notion that D4 acts by interfering with the formation of a stable, active RET dimer, regardless of whether dimerization is caused by the formation of the RET/GDNF/GFRα1 complex or by the direct interaction of two mutated RETC634Y proteins. This might occur either by D4 binding to monomeric RET, which would impede subsequent formation of the dimer, or by binding directly to the dimer. Differential whole-cell SELEX strategies (this work; see also [5,7,8,10]) can be employed to identify new markers on the surface of a given cell type, define the specificity of a cellular state, and/or allow in vivo targeting for diagnostic and therapeutic applications. The identification of lead compounds by reiterated affinity selection on living cells appears crucial when the molecular target is a membrane-bound or large transmembrane protein for which the conformation is frequently dictated by the interaction with other molecules, including membrane constituents [31]. Given that several of these proteins, as transmembrane receptors, integrins, and adhesion molecules, are involved in cell proliferation, apoptosis, and differentiation, aptamers for these targets could be promising prognostic tools in human therapy for widespread, devastating diseases such as cancer and neurodegeneration. Materials and Methods Cell culture and immunoblot analysis Growth conditions for PC12 cells and derived cell lines were previously described [32]. NIH/MEN2A and NIH/MEN2B cells were obtained from NIH3T3 cells stably transfected with vectors expressing human RETC634Y and RETM918T. To assess the effects of aptamers on RET activity, cells (160,000 cells per 3.5-cm plate) were serum-starved for 2 h and then treated with the indicated amount of RNA aptamers or the starting RNA pool after a short denaturation-renaturation step. When indicated, 2.5S NGF (Upstate Biotechnology, Lake Placid), GDNF (Promega), or recombinant rat GFRα1-Fc chimera (R&D Systems, Minneapolis, Minnesota, United States) were added to the culture medium. Cell extracts and immunoblotting analysis were performed as described [23]. The primary antibodies used were anti-RET (C-19), anti-VGF (R-15), and anti-ERK1 (C-16) (all three, Santa Cruz Biotechnology, Santa Cruz, California, United States); and anti-(Tyr-phosphorylated) RET and anti-phospho-44/42 MAP kinase (also indicated as anti-[phospho]-ERK) monoclonal antibodies (E10) (both from Cell Signaling, Beverly, Massachusetts, United States). Four independent experiments were performed. Cell transformation and neurite outgrowth bioassay PC12-α1/wt or NIH3T3 cells were plated at equal density on 12-well culture plates. Aptamers were added at 3 μM final concentration to the growth medium. To ensure the continuous presence of a concentration of at least 200 nM, this treatment was renewed every 24 h, which takes into account the half-life of the D4 aptamer in 10% serum (approximately 6 h, unpublished data). At least 15 random fields were photographed every 24 h with a phase-contrast light microscope. To evaluate the effects of D4 on cell differentiation, cells were pretreated for 6 h with 400 nM D4 or D4Sc and then incubated with 50 ng/ml GDNF together with 3 μM of the appropriate aptamer (see above). At 24 and 48 h of GDNF stimulation, 50 cells per frame were counted and scored as having neurites or not. A neurite was operationally defined as a process outgrowth with a length more than twice the diameter of cell body. Ex vivo SELEX The SELEX cycle was performed essentially as described [33]. Transcription was performed in the presence of 1 mM 2′F-Py and a mutant form of T7 RNA polymerase (T7Y639F, kind gift of R. Souza) [11] was used to improve yields. 2′F-Py RNAs were used because of their increased resistance to degradation by seric nucleases. The complexity of the starting pool was roughly 1014. 2′F-Py RNAs (1–5 nmol) were heated at 85 °C for 5 min in 3 ml of RPMI 1640, snap-cooled on ice for 2 min, and allowed to warm up to 37 °C before incubation with the cells. Two counterselection steps were performed per cycle. To avoid selecting for aptamers nonspecifically recognizing the cell surface, the pool was first incubated for 30 min at 37 °C with 107 PC12 cells, and unbound sequences were recovered by centrifugation. These were subsequently incubated with 107 adherent PC12/MEN2B cells, expressing a human RET receptor mutated in the intracellular domain (RETM918T), and unbound sequences were recovered for the selection phase. This step was meant to select sequences recognizing specifically the human RET receptor mutated in the extracellular domain (RETC634Y) expressed on PC12/MEN2A cells. The recovered sequences were incubated with 107 adherent PC12/MEN2A cells for 30 min at 37 °C in the presence of nonspecific competitor RNA (total yeast RNA) and recovered after several washings with 5 ml of RPMI by total RNA extraction (Extract-All, Eurobio, Les Ulis, France). During the selection process, we progressively increased the selective pressure by increasing the number of washings (from one for the first cycle up to five for the last three cycles) and the amount of nonspecific RNA competitor (100 μg/ml in the last three cycles), and by decreasing the incubation time (from 30 to 15 min from round 5) and the number of cells exposed to the aptamers (5 × 106 in the last three cycles). To follow the evolution of the pool we monitored the appearance of four-base restriction sites in the population, which reveals the emergence of distinct families in the population [34]. After 15 rounds of selection, sequences were cloned with TOPO-TA cloning kit (Invitrogen, Carlsbad, California, United States) and analyzed. Binding experiments Binding of individual aptamers (or the starting pool as a control) to PC12 cells and derivatives was performed in 24-well plates in triplicate with 5′-32P-labeled RNA. 105 cells per well were incubated with various concentrations of individual aptamers in 200 μl of RPMI for 10 min at 37 °C in the presence of 100 μg/ml polyinosine as a nonspecific competitor. After extensive washings (5 × 500 μl of RPMI), bound sequences were recovered in 350 μl of SDS 0.6%, and the amount of radioactivity recovered was normalized to the number of cells by measuring the protein content of each well. Binding of individual sequences to different cell lines was performed in the same condition at 50 nM only. For the binding curve of D4 to PC12/MEN2A cells (see Figure 2B), nonspecific binding was assessed using a 5′-32P-labeled naive pool of 2′F-RNAs (i.e., the starting pool of the selection), and the background values obtained were subtracted from the values obtained with the D4 aptamer. Apparent Kd values for each aptamers were determined by Scatchard analysis according to the equation [bound aptamer]/[aptamer] = −(1/Kd) × [bound aptamer] + ([T]tot/Kd) where [T]tot represents the total target concentration. Supporting Information Accession Numbers The Swiss-Prot (http://www.ebi.ac.uk/swissprot/) accession numbers for the proteins discussed in this paper are ERK (P27361), GDNF (P39905), GFRα1 (P56159), NGF (P01138), RET RTK (P07949), TrkA (P04629), and VGF (P20156). This work was supported by the European Union contract QLG1–2000–00562 (Oligonucleotide Ligands Imaging, OLIM ), the European Molecular Imaging Laboratory (EMIL) network, the CNRS, the Association por la Recherche contre le Cancer (grant 3527) and the MIUR-FIRB (Ministero dell'Istruzione, dell'Università e della Ricerca Fondo per gli Investimenti della Ricerca di Base) grant RBNE0155LB. FD was supported by a Commissariat à l'Energie Atomique (CEA) fellowship. We wish to thank M. Buckingham, E. Brody, M. S. Carlomagno, L. Di Giamberardino, C. Ibanez, C. Mann, S. Tajbakhsh and J.J. Toulmé for critical reading of the manuscript and fruitful discussions, and R. Souza for the gift of a T7Y639F RNA polymerase-expressing plasmid. Abbreviations 2′F-Py2′-fluoropyrimidine ERKextracellular signal-regulated protein kinase GDNFglial cell line-derived neurotrophic factor GFRGDNF family receptor α1 HERheregulin MENmultiple endocrine neoplasia NGFnerve growth factor RETrearranged during transfection RTKreceptor tyrosine kinase SELEXsystematic evolution of ligands by exponential enrichment VGFnerve growth factor-inducible protein ==== Refs References 1 Tuerk C Gold L Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990 249 505 510 2200121 2 Ellington AD Szostak JW In vitro selection of RNA molecules that bind specific ligands. Nature 1990 346 818 822 1697402 3 Ulrich H Magdesian MH Alves MJ Colli W In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion. 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==== Front Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-4-131581117710.1186/1476-4598-4-13Short CommunicationInactivation of MAP kinase signalling in Myc Transformed Cells and Rescue by LiCl inhibition of GSK3 Al-Assar Osama [email protected] Dorothy H [email protected] Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK2 Institute for Cancer Studies, Division of Genomic Medicine, Medical School, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK2005 5 4 2005 4 13 13 23 10 2004 5 4 2005 Copyright © 2005 Al-Assar and Crouch; licensee BioMed Central Ltd.2005Al-Assar and Crouch; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. c-Myc oncogene is an important regulator of cell cycle and apoptosis, and its dysregulated expression is associated with many malignancies. Myc is instrumental in directly or indirectly regulating the progression through the G1 phase and G1/S transition, and transformation by Myc results in perturbed cell cycle. Also contributory to the control of G1 is the Ras effector pathway Raf/MEK/ERK MAP kinase. Together with GSK3, ERK plays an important role in the critical hierarchical phosphorylation of S62/T58 controlling Myc protein levels. Therefore, our main aim was to examine the levels of MAPK in Myc transformed cells in light of the roles of ERK in cell cycle and control of Myc protein levels. We found that active forms of ERK were barely detectable in v-Myc (MC29) transformed cells. Furthermore, we could only detect reduced levels of activated ERK in c-Myc transformed cells compared to the non-transformed primary chick embryo fibroblast cells. The addition of LiCl inhibited GSK3 and successfully restored the levels of ERK in v-Myc and c-Myc transformed cells to those found in non-transformed cells. In addition, LiCl stabilised Myc protein in the non-transformed and c-Myc transformed cells but not in v-Myc transformed cells. These results can provide an important insight into the role of MAPK in the mechanism of Myc induced transformation and carcinogenesis. ==== Body Background The c-Myc oncogene is one of the most frequently dysregulated genes in human tumours. Myc was originally identified as the cellular homolog of the transforming part of the viral isolate MC29 [1]. The c-Myc oncogene is a member of the basic-helix-loop-helix-leucine-zipper transcription (bHLH-ZIP) factors, which are essential for different cellular processes [2]. Paradoxically, c-Myc promotes both cell cycle progression and apoptosis under low serum condition [3,4]. c-Myc regulates the cellular processes by controlling a large number of target genes [5,6] through heterodimerization with its biological partner Max [7-9]. The abundance of the Myc-Max heterodimer is effectively controlled by the short lived Myc protein [10]. The Myc protein is under tight and complex control mechanisms [11]. Critical phosphorylation events determining the protein half life occur in Myc homology box I (aa45-aa65) [10]. These detrimental events involve the hierarchical phosphorylation of S62 and T58 by ERK1/2 MAPK and GSK3β, respectively [12]. It is widely accepted that these kinases are involved in the phosphorylation events at these residues although other reports question the role of MAPK [13]. These two kinases are part of two different Ras effector pathways. The presence of different Ras isoforms provides for selective activation of specific Ras effector pathway, although this can only be shown in vivo [14]. It has been reported that PI-3 kinase is most effectively activated by M-Ras and R-Ras and to a less extent by H-Ras [15,16]. On the other hand, Raf-1 is most effectively activated by K-Ras [17,18]. This selective activation of different Ras effector pathways has opposing effects on Myc controlled functions. Whereas the activation of Raf fails to suppress Myc induced apoptosis, the activation of PI-3 Kinase can effectively suppress it [19]. A key component of the PI3-kinase/Akt (PKB) pro-survival pathway is GSK3 [20], whereas the active phosphorylated form of ERK1/2 MAPK is a downstream signal in the signalling cascade Ras/Raf/MEK [21]. The ERK1/2 MAPK is one of three major MAPK signalling pathways, which also includes JNK/SAPK and p38 kinase. Constitutive activation of MEK/ERK has been reported in cancer cells [22,23], with a possible role in cell transformation and oncogenesis [24]. The constitutive activation of MAPK ERK1/2 could be linked to the mitogen independence reported for oncogenes like Ras [25], Raf [26], Jun [27] and Myc [4]. Therefore, one of the aims of this study was to examine the status of active ERK2 in Myc transformed chick embryo fibroblasts (CEF), the ideal model for Myc induced transformation. Our second aim was to examine the possibility of a cross talk between ERK2 and GSK3 in Myc transformed fibroblasts using LiCl to inhibit GSK3. Reports on signalling between GSK3 and ERK1/2 are very scarce. Nonetheless, a recent report has demonstrated that GSK3β was a natural activator of the JNK/SAPK pathway [28]. Furthermore, it has been demonstrated that GSK3β could be phosphorylated on Ser9 and therefore inactivated by ERK1/2 mediated pathways, mainly through p90rsk but also through a novel mechanism downstream of ERK1/2 [29]. These findings need to be verified in transformed phenotype. Results and Discussion We have found that v-Myc (MC29) transformed fibroblasts have almost non-detectable active ERK2 (Figure 1A). A control experiment using the SFCV vector without an insert was performed in parallel with every experiment to exclude any effect for the transfection procedure. Cells transfected with the control vector gave identical results to the non-transfected control CEF cells. The addition of 100 mM LiCl was very successful in restoring (not fully) the levels of active ERK2 in v-Myc transformed fibroblasts to those found in non-transformed fibroblasts within the time scale of the experiment. The barely detectable basal levels of phosphorylated ERK2 in v-Myc transformed fibroblasts showed an increase after the addition of LiCl at the earliest time point of 20 minutes (31% of basal levels in non-transformed control CEF). These levels were almost completely restored to the levels found in non-transformed CEF after 80 minutes (83% of basal levels in non-transformed control CEF). Figure 1 The effect of LiCl on the levels of active ERK in Myc transformed and non transformed fibroblasts. (A) A time course for the effect of LiCl on the levels of active ERK2 in v-Myc transformed fibroblasts (panel 1). Total ERK levels were not affected and were used as a loading control (panel 2). Panel 3 is a graphical representation of the ERK2 levels in Li+ and K+ treated v-Myc cells after normalisation to ERK2 levels in non-transformed control CEF (identical to CEF transfected with empty vector) growing under normal conditions. (B) A time course for the effect of LiCl on the levels of active ERK2 in c-Myc transformed fibroblasts (panel 1). Total ERK levels were not affected and were used as a loading control (panel 2). Panel 3 is a graphical representation of the ERK2 levels in Li+ and K+ treated c-Myc cells after normalisation to ERK2 levels in non-transformed control CEF growing under normal conditions. (C) A time course for the effect of LiCl on the levels of active ERK2 in non-transformed control CEF (panel 1). Total ERK levels were not affected and were used as a loading control (panel 2). Panel 3 is a graphical representation of the ERK2 levels in Li+ and K+ treated cells after normalisation to ERK2 levels in non-transformed control CEF growing under normal conditions. The same pattern of expression was seen in several independent experiments for all the panels. On the other hand, c-Myc transformed fibroblasts have shown attenuated but detectable active ERK2 levels compared to the non-transformed CEF. The addition of 100 mM LiCl fully restored the levels of active ERK2 to those found in non-transformed CEF (Figure 1B). The reduced basal levels of ERK2 in c-Myc transformed fibroblasts showed an increase at the earliest time point of 20 minutes (88% of basal levels in non-transformed control CEF) after the addition of LiCl and were comparable to the levels seen in the non-transformed CEF after 60 minutes. In the non-transformed CEF, the addition of LiCl enhanced the levels of active ERK2 considerably (Figure 1C). The increase in the levels of active ERK2 in CEF after the addition of LiCl was detectable after 20 minutes (153% of basal levels in non-transformed control CEF) and peaked after 40 minutes (350% of basal levels in non-transformed control CEF). In addition, adding 100 mM of LiCl increased the levels of inactive phosphorylated GSK3 α/β in a time dependent manner (Figure 2) in agreement with the pattern seen for the restored levels of active ERK2. We confirmed the activation of the pro-survival pathway PI3K signalling pathway after the addition of LiCl by the inhibition of apoptosis in Myc transformed fibroblasts (Figure 3). Compared to the KCl control, LiCl treatment resulted in 2.1, 3.6 and 2.4 fold reduced apoptosis in v-Myc, c-Myc and non-transformed control fibroblast cells, respectively. The different cell populations showed variable number of apoptotic cells after serum starvation. This is expected since v-Myc is a stronger inducer of proliferation and apoptosis than c-Myc [30,31]. Other researchers have also demonstrated that inhibition of GSK3 using LiCl was contributory to apoptosis inhibition[32]. Other GSK3 inhibitors can be used to further support these findings. Figure 2 The effect of LiCl on the levels of GSK3 in non-transformed fibroblasts. (A) A western blot showing the levels of inactive or phosphorylated GSK3 α/β in non-transformed fibroblasts after the addition of Li+ or K+ salt control for the time points indicated. (B) A western blot showing the levels of phosphorylated and non-phosphorylated GSK3 α/β of the same samples in (A) above after stripping and re-probing of the blot with the appropriate antibody. Figure 3 The effect of LiCl on the apoptosis levels of serum starved Myc transformed and non-transformed fibroblasts. (A) A graphical representation of the percentage of apoptotic v/c-Myc transformed and non transformed fibroblasts 17 hours after serum deprivation and addition of Li+ or K+. The error bars are the standard error of three independent experiments. The total number of cells counted for each experiment was 50 cells. (B) &(C) Representative sections of Hoeschst 33258 stained c-Myc cells after K+ and Li+ treatment, respectively. Similar to what we have observed in our Myc transformed cells, previous researchers have demonstrated that ERK1/2 activity was repressed in c-Raf-1 (Raf22W), v-Ha-Ras, and v-Src transformed cells by a single-specificity tyrosine phosphatase [33]. A more recent report has also demonstrated attenuated levels of ERK2 in v-Jun transformed CEF cells, which was attributed to inefficient signalling between Ras and Raf, and increased levels of MAPK phosphatase [34]. In light of the roles of ERK and GSK3 in Myc protein phosphorylation and stability, we investigated the effect of LiCl addition on Myc protein half life in c-/v-Myc transformed fibroblasts. Figures 4A and 4B show that the addition of LiCl results in dramatic stabilisation of Myc in the non-transformed and c-Myc transformed cells, respectively. Surprisingly, although endogenous Myc is hardly detectable in non-transformed CEF (t1/2 < 1 minute), it was 30 fold more stable after the addition of LiCl (Figure 4A). We verified this using immunoprecipitation (data not shown). In comparison, c-Myc protein was 4 fold more stable after LiCl treatment. Not surprisingly, both endogenous Myc in non-transformed control fibroblasts and exogenously expressed Myc in c-Myc transformed fibroblasts had similar half life values after the addition of LiCl (30 and 32 minutes, respectively). However, LiCl failed to further stabilise Myc in v-Myc transformed fibroblasts (Figure 4C). Myc protein half life was 40 minutes compared to 38 minutes in the control cells. Since MC29 v-Myc has T58>M (T61>M in chicken), we did not expect any effect for LiCl on Myc protein half life in these cells, although it was a necessary control. Other researchers documented a small effect for LiCl on Myc stability (2 fold) in immortalised cell lines [35].. Figure 4 Myc protein turnover after the addition of LiCl in Myc transformed and non transformed fibroblasts. (A) Endogenous Myc protein in Li+ and K+ treated non-transformed fibroblasts. This was identical to fibroblasts transfected with empty vector. Panel 1 is the scanned image of a western blot autoradiograph. Panel 2 shows the half life values of the Myc protein under the different conditions. CEF represents the non-transformed control fibroblasts (B) Turnover of the Myc protein in Li+ and K+ treated c-Myc transformed fibroblasts. Panel 1 is the scanned image of a western blot autoradiograph. Panel 2 shows the half life values of the Myc protein under the different conditions. CEF represents the non-transformed control fibroblasts (C) Turnover of the Myc protein in Li+ and K+ treated v-Myc transformed fibroblasts. Panel 1 is the scanned image of a western blot autoradiograph. Panel 2 shows the half life values of the Myc protein under the different conditions. CEF represents the non-transformed control fibroblasts. The experiments were independently repeated three times and one representative experiment is shown. We can conclude that LiCl has direct effects on the hierarchical phosphorylation of S62 and T58 (S65 and T61 in chicken) by controlling the levels of active ERK2 and GSK3, respectively. The results in this study show that this is important for the Myc half life in the non-transformed and c-Myc transformed fibroblasts but not in the v-Myc transformed cells. In this context, other researchers have found that S62 phosphorylation was necessary for Myc stabilization following Ras activation or serum stimulation[36]. Conclusion In this short communication we provided important findings about Myc induced transformation. The abrogation of active MAPK in Myc transformed cells can potentially provide an insight into the mechanism of Myc induced transformation. Clarification of the mechanism of ERK2 inactivation in Myc transformed CEF is needed. Furthermore, it is critical to examine the implications of the differences in active ERK2 levels between v-Myc and c-Myc transformed cells and the possible role this has in Myc induced transformation and protein stability. Last, we need to elucidate on the possibility of a cross-talk between GSK3 and ERK, as this could be a very important mechanism for controlling the Myc protein. Methods Cell Culture, Transfection and Inhibition Studies Cell culture and transfection of the appropriate SFCV-Myc construct (10 μg) together with RCAN(A) helper (4 μg) into secondary CEF were performed as described previously [37]. A control experiment using the SFCV vector without an insert was used with every experiment as a transfection control. After G418 Neomycin selection (BDH, UK), cultures were expanded and used for the subsequent studies. At this stage, cells transfected with vectors containing either c-Myc or v-Myc were fully transformed as determined by anchorage independent growth and visible transformation characteristics, such as metamorphosis (data not shown). LiCl was added for 30 minutes at a final concentration of 100 mM to exponentially growing CEF, c-Myc or v-Myc cells before harvesting for western blotting. KCl was used in all the experiments as a salt control. For the Myc protein turnover studies, a protein synthesis inhibitor (emetine from Sigma, UK) was added 30 minute after the addition of either LiCl or KCL at a final concentration of 0.1 mM [38] for the indicated times shown in the figure. Apoptosis Induction and Measurement Apoptosis was induced by incubating the cells in a medium containing 0.2% serum for 17 hours. Serum starvation for longer periods of time resulted in apoptosis in almost all of the v-Myc cell population. To measure the percentage of the apoptotic cells, the cell population was divided into adherent and suspended cells. The adherent cells were trypsinised, washed in 1× phosphate buffered saline (PBS) and fixated in ice cold 3:1 glacial acetic acid/methanol solution. Then, the cells were permealised at room temperature using a solution of 1× PBS/0.1% triton X-100 for 5 minutes. The cells were then stained in a solution of 2.5 μg/μl Hoeschst 33258 (Sigma, UK) in 1× PBS/0.1 % triton X-100 on ice and protected from light. After that, the cells were washed twice in 1× PBS/0.1% triton X-100, made adherent onto a slide using a cytospin, and viewed and counted under an epi-fluorescent microscope using a DAPI filter. We treated the suspended cells in exactly the same way with the exception that they did not need trypsinisation. To calculate the total number of apoptotic cells in both adherent and suspended cells, we used the following formula: SDS PAGE Western Blotting and Protein Half Life Measurement Cell lysates were prepared by lysing cultures in SDS-sample buffer containing 1% SDS without bromophenol blue or mercaptoethanol. Protein concentration was measured using Micro BCA reagent (Pierce, UK) before loading onto 7.5% SDS-PAGE gels. Transfer to nitrocellulose and western blotting was performed essentially as described previously [37], except that incubation with the primary antibody was performed in 2.5% BSA in TBS-Tween20 for ERK western blots. Active phosphorylated ERK was detected using rabbit polyclonal antibodies (catalogue number 9101, New England Biolabs, UK) and total phosphorylated and non-phosphorylated ERK levels were detected using rabbit polyclonal antibody (catalogue number 71–1800, Zymed, UK). Inactive phosphorylated GSK3 α/β (Ser21/9) protein was detected using a rabbit polyclonal antibody (catalogue number 9331S, Cell Signaling Technology, UK) and phosphorylated and non-phosphorylated GSK3 α/β levels were detected using a rabbit polyclonal antibody raised against GSK3 β but detected both GSK3 α and β (catalogue number 9332, Cell Signaling Technology, UK). Full length Myc protein was expressed in our laboratory and was used to raise rabbit polyclonal antibodies against. To re-probe a blot, it was first submerged in a solution containing 100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris HCl pH 6.7 at 50°C for 1 hour, with agitation. Then, the blot was washed in a solution containing TBS/0.1% Tween20 for 10 minutes three times at room temperature. Last, the blot was blocked and probed as above with the appropriate antibody. Equal loading of lanes was determined by staining the polyacrylamide gel after transfer onto a nitrocellulose membrane in coomassie blue solution for 2 hours (50% methanol, 10% acetic acid, 0.25% coomassie blue R-250) and de-staining in a solution containing 10% methanol and 5% acetic acid for 4 hours. The different band intensities were analysed using the Kodak 1D image analysis software and then they were plotted on a linear graph for the ERK levels. For the calculation of the Myc protein half life, the densitometric values were plotted on a semi-logarithmic graph against time and then fitted to an exponential line. 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==== Front Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institue of Environmental Health Sciences 10.1289/ehp.7783ehp0113-00070815929893ResearchArticlesSustained Exposure to the Widely Used Herbicide Atrazine: Altered Function and Loss of Neurons in Brain Monoamine Systems Rodriguez Veronica M. Thiruchelvam Mona Cory-Slechta Deborah A. Environmental and Occupational Health Sciences Institute, and Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USAAddress correspondence to D.A. Cory-Slechta, Environmental and Occupational Health Sciences Institute, 170 Frelinghuysen Rd., Piscataway, NJ 08854 USA. Telephone: (732) 445-0205. Fax: (732) 445-0131. E-mail: [email protected] express our appreciation to R. Reeves and M. Virgolini for their input. This work was supported by grant ES10791 from the National Institute of Environmental Health Sciences. The authors declare they have no competing financial interests. 6 2005 24 2 2005 113 6 708 715 22 11 2004 24 2 2005 Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. The widespread use of atrazine (ATR) and its persistence in the environment have resulted in documented human exposure. Alterations in hypothalamic catecholamines have been suggested as the mechanistic basis of the toxicity of ATR to hormonal systems in females and the reproductive tract in males. Because multiple catecholamine systems are present in the brain, however, ATR could have far broader effects than are currently understood. Catecholaminergic systems such as the two major long-length dopaminergic tracts of the central nervous system play key roles in mediating a wide array of critical behavioral functions. In this study we examined the hypothesis that ATR would adversely affect these brain dopaminergic systems. Male rats chronically exposed to 5 or 10 mg/kg ATR in the diet for 6 months exhibited persistent hyperactivity and altered behavioral responsivity to amphetamine. Moreover, when measured 2 weeks after the end of exposure, the levels of various monoamines and the numbers of tyrosine hydroxylase-positive (TH+) and -negative (TH−) cells measured using unbiased stereology were reduced in both dopaminergic tracts. Acute exposures to 100 or 200 mg/kg ATR given intraperitoneally to evaluate potential mechanisms reduced both basal and potassium-evoked striatal dopamine release. Collectively, these studies demonstrate that ATR can produce neurotoxicity in dopaminergic systems that are critical to the mediation of movement as well as cognition and executive function. Therefore, ATR may be an environmental risk factor contributing to dopaminergic system disorders, underscoring the need for further investigation of its mechanism(s) of action and corresponding assessment of its associated human health risks. atrazinedopaminehypothalamuslocomotor activitymicrodialysisprefrontal cortexstriatumsubstantia nigraunbiased stereology ==== Body Atrazine (ATR; 2-chloro-4-ethylamino-6-isopropylamino-s-triazine), a chlorinated member of the family of s-substituted triazines, is one of the most widely employed herbicides in the world, with an estimated 76.4 million pounds used annually in the United States alone. It acts to suppress photosynthesis by inhibiting electron transfer at the reducing site of chloroplast complex II (Eldridge et al. 1999). Although it has limited solubility in water, ATR is frequently detected in ground and surface waters in agricultural regions (Colborn and Short 1999). Studies also reveal that ATR can be transported into the home, presumably tracked by soil (Lioy et al. 2000). Human exposure has been confirmed (Adgate et al. 2001; Clayton et al. 2003), and, in fact, approximately 60% of the U.S. population is exposed to ATR (Birnbaum and Fenton 2003). Recent reports indicate that acute dietary exposures range from 0.234 to 0.857 μg/kg/day, and corresponding figures for chronic dietary exposure are 0.046 to 0.286 μg/kg/day, considering all commodities with U.S. Environmental Protection Agency (EPA) tolerances and drinking water (Gammon et al. 2005). Occupational exposure to ATR, as measured in mixer-loader-tender applicators, was reported to be approximately 2.8 mg ATR/day of work, with an absorbed dose of 1.8–6.1 μg/kg/day based on a 5.6% dermal absorption rate (Gammon et al. 2005). An earlier study of manufacturing workers reported a total ATR exposure of 10–700 μmol (~ 2.157–151.004 mg) per work shift (Catenacci et al. 1993). The understanding of the potential of ATR to serve as a contributing factor to human disease and dysfunction is currently extremely limited. Epidemiologic studies have linked environmental and/or occupational ATR exposure to increased mortality (Sathiakumar et al. 1996), and to non-Hodgkin’s lymphoma (MacLennan et al. 2003; Sathiakumar and Delzell 1997). In experimental models, however, a growing experimental literature documents deleterious hormonal and reproductive system effects of ATR. In rodents, reported effects include reductions in testosterone levels; increases in tri-iodothyronine (Friedmann 2002; Stoker et al. 2000, 2002); suppression of immune function (Rooney et al. 2003), of luteinizing hormone (LH), and of prolactin surges (Cooper et al. 2000); the appearance of mammary gland tumors; a disruption of regular ovarian cycles; and the induction of pseudopregnancies (Cooper et al. 1996; Laws et al. 2000). The effects of ATR on ovarian function in female rats have been ascribed to changes in function of catecholamines in the hypothalamus, specifically decreases in norepinephrine (NE) and increases in dopamine (DA) in this region (Cooper et al. 1998). In correspondence with this observation, in vitro studies in PC12 cells show concentration-dependent decreases in intracellular DA after exposure to 12.5–200 μM ATR for 6, 12, 18, and 24 hr and decreases in NE release and intracellular NE concentrations after exposures to 100 and 200 μM ATR for 12, 18, and 24 hr (Das et al. 2000, 2003). In addition, reductions in the expression of DA β-hydroxylase [but not of tyrosine hydroxylase (TH)] were observed. The inhibitory effects of ATR on intracellular NE content and NE release, but not on DA intracellular content, were reversed when PC12 cells were co-incubated with ATR and agents known to enhance transcription, phosphorylation, or activity of TH and DA β-hydroxylase, such as 8-bromo-cAMP, forskolin, or dexamethasone (Das et al. 2003). These findings suggest that ATR could disrupt catecholamine metabolism by altering its biosynthetic enzymes. The fact that ATR can adversely affect hypothalamic catecholamine systems has notable implications because such effects would be unlikely to be restricted to this particular region, but could affect brain catecholamine systems more generally and thus affect pathways critical to the control of movement (nigrostriatal dopaminergic systems) and of complex cognitive functions (mesocorticolimbic dopaminergic systems). If so, then ATR exposures may also serve as a risk factor for neurodegenerative diseases and/or dysfunctions associated with these systems, which include Parkinson’s disease, schizophrenia, and attention deficit disorder, among others (Crossman 2000; Epstein et al. 1999; Viggiano et al. 2003). Indeed, epidemiologic studies have linked pesticides to an increased odds ratio for Parkinson’s disease (Breysse et al. 2002), and various pesticides that affect catecholaminergic systems have been shown to produce characteristics of Parkinson’s disease in experimental models (Betarbet et al. 2000; Reeves et al. 2003; Thiruchelvam et al. 2000b). The potential for neurotoxic effects of ATR in vivo, however, particularly chronic effects, has received almost no experimental attention. Oral exposure of rats to 1,000 mg/kg ATR for 4–11 days decreased rearing in the open field (Ugazio et al. 1991), whereas acute exposure of rats to 100 mg/kg decreased spontaneous Purkinje cell firing rate and cerebellar potentials evoked by electrical stimulation (Podda et al. 1997). The objective of the present study was to evaluate the potential for sustained low-level ATR exposure to affect two critical catecholamine pathways of the brain: the nigrostriatal DA pathway, involved in the mediation of movement (Crossman 2000), and the mesocorticolimbic DA pathway critical to complex cognitive functions (Clark et al. 2004; Remy and Samson 2003). For this purpose, we evaluated locomotor activity across the course of exposure, whereas monoamine levels in striatum, prefrontal cortex, nucleus accumbens, and hypothalamus and stereologic cell counts of TH-positive (TH+) and TH-negative (TH−) cells in the midbrain were evaluated 2 weeks after cessation of exposure. Further, this study sought to determine mechanisms by which any changes in dopaminergic function in these pathways might be produced by examining the acute effects of ATR on striatal DA release using microdialysis. Materials and Methods Chronic ATR Exposure Subjects, exposure, and experimental design. Thirty male Long-Evans rats purchased from Taconic Farms (Germantown, NY) were housed individually in plastic cages in a temperature- and humidity-controlled vivarium room with a 12-hr dark/light cycle (lights on 0600 hr). Food intake was restricted to maintain body weights at 300 g, and water was available ad libitum during the entire experiment. In our experience, this protocol sustains health and viability to a greater degree than does ad libitum feeding. At 9 months of age, exposure to 0, 5, or 10 mg/kg ATR mixed in food was initiated with continuation of ad libitum access to distilled drinking water. These doses of ATR were chosen based on reports for the rat of an oral median lethal dose (LD50) of 1,869 mg/kg (U.S. EPA 2001), a no observed adverse effect level (NOAEL) of 3.3 mg/kg/day, and a lowest observed adverse effect level (LOAEL) of 34.5 mg/kg/day for this route of administration measured as body weight loss. A chronic dietary NOAEL of 1.8 mg/kg/day and LOAEL of 3.65 mg/kg/day were also reported (U.S. EPA 2001). We recorded body weights and food consumption periodically over the entire duration of the experiment. All procedures were carried out in accord with National Institutes of Health and University of Medicine and Dentistry of New Jersey Animal Use and Care Committee Guidelines (Institute of Laboratory Animal Resources 1996). The experimental design is summarized in Figure 1A. We recorded locomotor activity at 2, 3, and 6 months of ATR exposure and 2 weeks after cessation of exposure. At the 2-month time point, we measured locomotor activity on 3 consecutive days, with animals receiving an intraperitoneal (ip) injection of saline 5 min before the session during the first 2 days, and an injection of d-amphetamine sulfate (1 mg/kg) on day 3. Only a single locomotor activity session was carried out at the 3-and 6-month time points and at 2 weeks after the termination of ATR exposure. Locomotor activity was recorded during the light phase (from 0900 hr to 1300 hr) of the light/dark cycle using methods described below. Two weeks after cessation of ATR exposure, rats were sacrificed by decapitation, brains were removed, and hypothalamus, prefrontal cortex, nucleus accumbens, and striatum were dissected on ice and frozen for HPLC analysis. The remaining tissue was postfixed in 4% paraformaldehyde for immunohistochemistry and stereologic counts. Locomotor activity measurement. Each rat was individually placed in an automated locomotor activity chamber equipped with infrared photobeams (Opto-Varimex Minor; Columbus Instruments International Corporation, Columbus, OH). Horizontal, vertical, and ambulatory activities were simultaneously measured and data were collected over the course of a 45-min session. Measurement of monoamine levels. Tissues were sonicated in 0.1N perchloric acid and centrifuged. Supernatants were stored at −80°C until analyzed for monoamine content. Pellets were digested in 0.5 M sodium hydroxide for measurements of protein concentration using reagents from Bio-Rad (Hercules, CA). We measured monoamines and their metabolites using HPLC with electrochemical detection as described elsewhere (Thiruchelvam et al. 2000a). Briefly, a Waters pump 515 plus autosampler (Waters Corporation, Milford, MA) was joined to a chromatographic column (Alltech Associates Inc, Deerfield, IL). The amperometric potential was set at 600 mV relative to the silver/silver chloride, and the sensitivity of the detector was set at 100 ρA (microdialysates) or 1 ηA (tissue samples). The mobile phase was an isocratic 0.1 M monobasic phosphate solution containing 0.5 mM sodium octyl sulfate, 0.03 mM EDTA, and 12–14% vol/vol methanol. Results generated by these determinations were analyzed with the Empower Pro program (Empower Software, Waters Corporation) and are expressed in picograms per milliliter of microdialysate or nanograms per milligram of protein of tissue. DA turnover was expressed as the ratio of dihydroxyphenylacetic acid (DOPAC) to DA. Tyrosine hydroxylase immunohistochemistry. Five randomly selected paraformaldehyde-fixed brains from each treatment group were cut into 30-μm sections, collected in cryoprotectant, and stored at −20°C for immunolabeling studies. Sections were rinsed with 0.1 M phosphate buffer (PB), blocked with 10% normal goat serum for nonspecific binding, and incubated in TH primary antibody (Chemicon, Tamecula, CA) for 48 hr at a dilution of 1:3,500 in PB with 0.3% Triton X-100 and 10% normal goat serum. Sections were then incubated with a secondary antibody 1:200 (Vector Laboratories Inc., Burlingame, CA) overnight. Sections were washed and incubated with avidinbiotin solution from Vectastain ABC reagents (Vector Laboratories) for 1 hr and developed in 3–3′-diaminobenzidine tetrachloride and H2O2 in 0.05 M Tris buffer. Sections were counterstained with cresyl violet after TH staining. We counted total numbers of TH+ and Nissl-stained neurons (TH−) in substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA) using the optical fractionator method as described below. Stereologic analysis. After delineation of the SNpc and VTA at low magnification (4× objective), one side of every fourth section from the entire midbrain region was sampled at higher magnification (100× objective) using the stereology module of the Stereo Investigator imaging program (MicroBrightField Inc., Williston, VT) with an Olympus Provis microscope (Olympus America, Melville, NY). We used the optical fractionator method, an unbiased quantitative technique, for counting TH+ (TH+ and cresyl violet+ neurons) and TH− (cresyl violet+ only) cells. Criteria for TH+ and TH− neurons were determined as previously described (Barlow et al. 2004; Thiruchelvam et al. 2004). We determined the mean thickness by measuring two fields from five sections per sample, and the entire depth of field was sampled, ignoring the upper and lower 0.5 μm. All samples were evaluated by one experimenter without knowledge of treatment status. Chemicals. ATR at 98% purity was purchased from Chem Services Inc. (West Chester, PA). Reagents for microdialysis, HPLC analysis, methylcellulose, and cresyl violet were purchased from Sigma (St. Louis, MO). Acute ATR Exposure Subjects, exposure, and experimental design. Thirty male Long Evans rats weighing between 270 and 320 g purchased from Taconic Farms were habituated to constant standard laboratory conditions of humidity, temperature, and dark/light cycle (lights on 0600 hr) as described above. As shown in Figure 1B, we used microdialysis to evaluate changes in striatal DA release after acute intraperitoneal (ip) exposures to ATR in sessions lasting 7 hr. Surgery. After a habituation period of at least 1 week, rats were anesthetized with pentobarbital (30–40 mg/kg ip) and every 30 min thereafter received an injection of atropine sulfate (0.3 mg/kg ip) to avoid respiratory failure during the cannula implantation. Once anesthetized (assessed by absence of corneal reflex), the rat was placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA), the skull was exposed, and a hole was drilled for placement of a guide cannula (MD-2250; Bioanalytical Systems Inc., West Lafayette, IN) over the right striatum, using stereotaxic coordinates (anterior-posterior, +1.0 mm, medio-lateral, −2.0 mm with reference to bregma, dorso-ventral, −3.4 mm from flat skull) according to the atlas of Paxinos and Watson (1986). The cannula was fixed to the skull with anchor screws and acrylic cement. After surgery, rats were individually housed for a recovery period of 5–7 days with food restricted to keep body weight at 300 g and water was available ad libitum. Microdialysis. A probe of concentric design (MD-2262, tip 2 mm; Bioanalytical Systems, Inc.) was inserted into the guide cannula. The dialysis probe was continuously perfused at a flow rate of 2.5 μL/min through a liquid swivel from an automated system (Bioanalytical Systems Inc.) with a physiologic Ringer’s solution containing 147 mM NaCl, 4.0 mM KCl, 1.2 mM CaCl2, and 1 mM MgCl2, pH 6.0–6.5. Sample collection occurred every 30 min. The first hour of sampling was discarded to avoid erroneous data due to probe insertion. After three baseline samples, rats received an ip injection of vehicle (1% methyl-cellulose) or ATR (100 or 200 mg/kg), and five subsequent samples of perfusate were collected. In order to probe characteristics of DA release, a high potassium solution (91 mM NaCl, 60 mM KCl, 1.2 mM CaCl2, and 1 mM MgCl2, pH 6.0–6.5) replaced the normal Ringer’s solution, and two samples were collected under these conditions. Normal Ringer’s solution was subsequently restored, and two additional samples of perfusate were collected. Collection vials contained 3.75 μL 0.1 M HClO4 solution. Collected samples were immediately frozen at −80°C until monoamine quantification by HPLC as described above. Histology. At the completion of micro-dialysis sampling, rats were overdosed with sodium pentobarbital and transcardially perfused with an isotonic saline solution followed by 10% formaldehyde. Brains were postfixed in 10% formalin overnight and then transferred to 30% sucrose. Brains were sectioned in 50 μm coronal slices, mounted, stained with cresyl violet, and coverslipped. Cannula placement for the microdialysis study was confirmed under microscopic analyses. Statistical Analyses We analyzed total locomotor activity counts, body weight, and food consumption using repeated-measures analysis of variance (RMANOVA; treatment × time) followed by post hoc tests as appropriate. Responsivity to d-amphetamine and changes in neurotransmitter levels in various brain regions were evaluated by one-way ANOVA with post hoc assessments in the event of main effects of treatment. To provide a more conservative analysis of changes in cell counts, because counts in both regions were derived from the same animals (brains), RMANOVA was carried out based on changes in TH+ and TH− cells in both SN and VTA (but not total counts because that was the sum of the TH+ and TH− cells), followed by post hoc testing as appropriate. We evaluated the effects of ATR on microdialysis by RMANOVAs with treatment and time as factors, followed by post hoc evaluation in the case of main effects or interactions. In all cases, statistical significance was defined as p ≤ 0.05. Results Chronic ATR Exposure Gross effects of treatment. No treatment-related changes in body weight or food consumption were detected at any point during the course of the exposure (data not shown), nor did any other signs of overt toxicity manifest at any point. Locomotor activity. In contrast to the other time points of measurement, the assessment of locomotor activity after 2 months of ATR exposure actually involved three sessions, the first two of which were preceded by an ip injection of saline and the third by 1 mg/kg d-amphetamine sulfate. No treatment-related changes in locomotor activity were seen in either of the sessions preceded by saline. However, in the third session, the administration of d-amphetamine increased locomotor activity of all three groups relative to levels of activity after saline administration [session 2; F(2,26) = 3.63, p < 0.041]. Additionally, these increases were modified by ATR treatment in that the 10-mg/kg dose further enhanced locomotor activity by an additional 70% (Figure 2A) relative to the increases in the 0- and 5-mg/kg groups, as confirmed in post hoc analyses. At the remaining time points of measurement, single locomotor activity sessions were carried out in the absence of drug administration. Under these conditions, after 3 months of ATR exposure, we found pronounced increases in locomotor activity again at the 10-mg/kg dose of ATR [F(2,26) = 3.62, p = 0.041], with levels of horizontal activity that exceeded those of controls and the 5-mg/kg group by approximately 50% (Figure 2B). These treatment-related effects were also evident in the measurement of locomotor activity at the 6-month time point [F(2,24) = 3.45, p = 0.048] and again when measured 2 weeks after the termination of ATR treatment [F(2,24) = 4.42, p = 0.024], when levels remained at 40% above control. Changes in monoamine levels. Measured 2 weeks after the termination of ATR exposure, changes in DA content (Figure 3A) were significant in striatum [F(2,23) = 3.61, p = 0.044] as well as in frontal cortex [F(2,21) = 3.82, p = 0.039]. Statistical analysis confirmed decreased levels of DA (~ 20%) in relation to treatment in striatum, with post hoc assessments indicating efficacy at the 10-mg/kg ATR dose with a similar but nonsignificant trend at 5 mg/kg. In contrast, levels of DA were increased in prefrontal cortex in an inverse U-shaped fashion, with post hoc assessment confirming a significant increase at 5 mg/kg (by 30–40%), with levels declining back toward control values at 10 mg/kg. Both doses of ATR reduced serotonin (5-HT) levels in hypothalamus [Figure 3B; F(2,21) = 5.19, p = 0.015] by 10–15%. Chronic ATR exposure also decreased levels of NE in frontal cortex [Figure 3C; F(2,21) = 3.84, p = 0.038], with post hoc assessments showing the effect with the 10-mg/kg dose producing reductions of approximately 15–20%. Although a trend toward increases in NE in nucleus accumbens was suggested, it was associated with significant variability and therefore not statistically significant. We observed no changes in levels of the metabolites of either 5-HT (5-hydroxyindole acetic acid) or DA [DOPAC, homovanillic acid (HVA)] or DA turnover (DOPAC:DA) in any region. Unbiased stereologic counts of cells in the midbrain. Changes in numbers of cells in the regions of the cell bodies of the two major DA pathways are shown in Figure 4 for a sample of five randomly selected animals from each treatment group; numbers are shown for TH+, TH−, and total cells in SNpc and for corresponding data for the VTA. Because these regions were from the same brains, we performed a more conservative statistical analysis based on RMANOVA to examine the impact of treatment on numbers of cells using counts of TH+ and TH− from each region (not including total counts). That analysis confirmed a significant main effect of treatment [F(2,36) = 5.53, p = 0.02] and no interaction of treatment by region, indicating that cell loss occurred in both regions and, moreover, in both TH+ and TH− cells. These effects were primarily attributable to the 10-mg/kg dose of ATR, as confirmed in subsequent post hoc tests; the mean reductions in cell numbers ranged from 9 to 13% in the 10-mg/kg dose group, whereas those in the 5-mg/kg group ranged from 0 to 3%. Acute ATR Exposure That systemic administration of ATR can indeed directly affect brain dopaminergic systems was further confirmed in microdialysis experiments. The impact of acute ip administration of ATR (100 or 200 mg/kg) on levels of DA in striatum, as assessed via microdialysis, is presented in Figure 5A. Acutely, ATR significantly decreased basal DA release, as shown in the inset in Figure 5A [main effects: treatment, F(2,19) = 4.88, p = 0.02; sampling time: F(9,18) = 26.77, p < 0.0001; treatment by time interaction: F(18,171) = 2.77, p = 0.0003]. Post hoc tests confirmed decreases as measured at 90, 120, 150, and 180 min postadministration of ATR. By 150 min, the decrements averaged approximately 40% and were seen in both the 100-mg/kg and the 200-mg/kg treatment groups. We also observed a dose-dependent decrease in DA release when the system was challenged with 60 mM high potassium solution for 60 min [F(2,19) = 3.717, p = 0.0434]. Although the control group showed a 1,256% increase from baseline in response to potassium (210 min time point), corresponding values for the 100-mg/kg and 200-mg/kg ATR groups were 729 and 427%, respectively, from baseline. After high potassium perfusion, the system was flushed again with normal Ringer’s solution; levels of DA declined in all groups, and no treatment-related differences were evident during the remaining 60 min of sampling. The increase in DA seen in the first sample (240 min time point) after high potassium infusion was due to dead volume of the microdialysis sample collection system. Analysis of striatal DOPAC levels in the dialysates revealed only a significant effect of sampling time [F(9,18) = 13.735, p < 0.0001]. One-way ANOVA at each time point did not show any difference among groups in DOPAC concentration during the course of the experiment (Figure 5B). Similarly, analysis of HVA levels showed a significant effect of sampling time [F(9,18) = 6.074, p < 0.0001] but no effect of group or group × sampling time interaction (Figure 5C). Administration of vehicle (1% methyl-cellulose) or 100 mg/kg ATR did not result in acute observable effects in these rats, but some rats injected with 200 mg/kg ATR exhibited hypoactivity during the first 2 hr after injection, after which levels appeared normal. Histologic analysis confirmed that cannula placement was appropriately located in dorsal striatum for all rats. Discussion The present study demonstrates that sustained low-level ATR exposure in diet can adversely affect both major long-length dopaminergic tracts of the central nervous system, resulting in persistent increases in locomotor activity, alterations in responsivity to the indirect DA agonist amphetamine, changes in monoamine levels, and, ultimately, loss of neurons in the midbrain. Thus, adverse effects of ATR are not restricted to endocrine and reproductive systems or to hypothalamic regions of brain. The effects observed here cannot be ascribed to acute toxicity because the half-life of ATR in tissue ranges from 31.3 to 38.6 hr, and 95% of the ATR administered is excreted within 7 days of dosing, whereas changes in monoamines and numbers of neurons were measured 2 weeks posttreatment. Moreover, the doses used here did not produce any changes in body weight or food consumption or any signs of overt toxicity. The observations of protracted changes in neurotransmitter levels coupled with neuronal loss have particular significance, given the critical roles of the nigrostriatal and mesocorticolimbic dopaminergic systems in controlling fine motor behavior and complex cognitive function, respectively (Clark et al. 2004; Crossman 2000; Remy and Samson 2003). Dysfunctions of dopaminergic systems include Parkinson’s disease, schizophrenia, attention deficit disorder, and learning and memory impairments. Collectively, the present findings raise the possibility that ATR exposure could be a contributory risk factor for such disorders. Chronic ATR exposure caused cell loss not only to TH+ immunoreactive cells but also to TH− cells in the VTA and SNpc. The non-dopaminergic neuronal subpopulation in these regions includes GABAergic (Deniau et al. 1978), calbindin (Gerfen et al. 1985), and cholecystokinin-like immunoreactive neurons (Seroogy and Fallon 1989). The lack of selectivity of effects makes it likely that ATR will exhibit neurotoxicity, including cytotoxicity to other neuronal populations in other brain regions, although other regions were not examined in the present study. Additionally, ATR may exert neurotoxic effects on other cell types of the brain as well, such as glial cells. The specificity and mechanism(s) of ATR effects within the central nervous system remain to be determined, and such assessments are clearly warranted based on the findings presented here. Chronic ATR increased locomotor activity, an effect present after 3 months of exposure, persisted for 6 months and was still evident even 2 weeks after cessation of exposure. Moreover, rats treated for 2 months with 10 mg/kg ATR exhibited an enhanced locomotor activity response to a d-amphetamine challenge. Amphetamine is known to promote the release of DA and a decrease in its re-uptake into the presynaptic terminal (Cooper et al. 2003). Thus, the increases in locomotor activity could reflect ATR-induced up-regulation of striatal DA receptors, as might be expected to occur in response to the corresponding reduction in basal DA levels (Figure 3A) or DA release produced by ATR (Figure 5A). Placement in a novel environment such as the locomotor activity chamber could increase DA, activating up-regulated DA receptors and thereby producing hyperactivity (Badiani et al. 1998), a hypothesis in agreement with the increases in locomotor activity induced by amphetamine sulfate (Mao et al. 2001). The locomotor hyperactivity observed here differs from findings of a previous study in which 1,000 mg/kg ATR administered for 4–11 days decreased rearing in the open field (Ugazio et al. 1991). Such decreases could reflect acute toxicity of a high dose of ATR because the chemical was administered immediately before the behavioral evaluation in that study, coupled with a decline in DA release that would accompany its administration and be expected to reduce activity levels, as was observed. The reductions noted here in levels of DA, NE, and 5-HT observed, respectively, in striatum, prefrontal cortex, and hypothalamus at the 10-mg/kg ATR dose could be due to inhibitory effects on synthesis in these monoamine pathways. Precursors of DA and NE (tyrosine) and of 5-HT (tryptophan) undergo the same hydroxylation process via TH or tryptophan hydroxylase, respectively. Both enzymes are pteridin-dependent aromatic amino acid hydroxylases and are highly homologous, reflecting a common evolutionary origin from a single genetic locus (Cooper et al. 2003). In an in vitro study using PC12 cells in which NE and DA were decreased by ATR, the NE effect was reversed when cells were co-incubated with agents known to enhance transcription and phosphorylation of dopamine β-hydroxylase and TH (Das et al. 2003), consistent with the possibility that ATR may have inhibitory effects on these enzymes. Previous studies have reported changes in hypothalamic DA and/or NE levels after acute ATR administration at 100 mg/kg by gavage to male rats (Cooper et al. 1998). We did not observe such changes in the chronic exposure paradigm used here, a difference that could reflect initiation of compensatory mechanisms to maintain a constant production of these neurotransmitters under conditions of chronic exposure. Alternatively, catecholamine levels were determined in specific hypothalamic nuclei in that study, whereas here we examined the hypothalamus in its entirety, thus possibly diluting any regional changes (Cooper et al. 1998). Chronic ATR exposure did decrease hypothalamic 5-HT levels, effects consistent with its known alterations of neuroendocrine systems, including the release of LH and prolactin. Serotonergic neurons from the dorsal and medial raphe nuclei project to hypothalamus, activating the hypothalamo–pituitary–adenocortical (HPA) and the hypothalamo–pituitary–gonadal (HPG) axes in the rat (Fuller 1996; Jorgensen et al. 1998). Agents that disrupt 5-HT transmission are known to alter the HPA and HPG axes (Fuller 1996; Fuller and Snoddy 1990). Furthermore, selective degeneration of the midbrain dorsal and ventromedial region of the hypothalamus induced by 5,7-dihydroxytryptamine reduces LH levels (van de Kar et al. 1980). Taken together, it can be inferred that reductions in hypothalamic 5-HT resulting from ATR could affect both the HPA and HPG axes and thereby alter other organ systems of the body with which these systems interact. DA and NE alterations in prefrontal cortex are also notable given the critical role of this structure in mediating executive function, including working memory (Dreher et al. 2002). Dysfunction of this region is also involved in cognitive deficits, altered stress responsivity, hyperactivity disorder, and schizophrenia (Mostofsky et al. 2002; Tam and Roth 1997; Viggiano et al. 2003). An increase in cortical DA levels, such as observed at 5 mg/kg, could be due to an increase in DA synthesis, decreased degradation, or altered re-uptake. It is worth noting that autoreceptors on DA terminals in the prefrontal cortex regulate release but not synthesis of DA (Cooper et al. 2003), which may explain why augmented DA concentrations in this region are not corrected after ATR exposure. Findings from the microdialysis component of these experiments are consistent with such an assertion and show alterations in the dynamics of DA in striatum after acute ATR treatment. A dose-dependent decrease in striatal DA release as observed here would normally trigger compensatory mechanisms such as decreased re-uptake rate and increased production and release of DA. As is evident from Figure 5, none of these compensatory mechanisms appears to be operative, at least within the time frame encompassed by these experiments. The observed decline in both basal and stimulated DA release could have several explanations. First, it could reflect a generalized inhibition of DA synthesis, given the absence of group differences at the end of the experiment. DA is distributed mainly in two functional presynaptic compartments, a cytoplasmic pool and a vesicular pool. Potassium-induced release is Ca2+ dependent and occurs from the vesicular pool (Du et al. 1999), which is the newly synthesized pool (Lamensdorf et al. 1996). Another possibility is that ATR decreases the firing rate of striatal and/or SN neurons, decreasing DA release. Additionally, TH exists in two kinetic forms, with differential affinities for tetrahydrobiopterin (cofactor for TH). The proportion of TH in the high-affinity state appears to be a function of neuronal firing rate (Cooper et al. 2003). A dose of 100 mg/kg ATR decreased cerebellar cell firing rate after 60 and 90 min, with rates returning to normal by 180 min; this inhibitory effect lasted up to 180 min after exposure to 200 mg/kg ATR (Podda et al. 1997). ATR could also be acting on ionotropic GABA receptors. Binding of RO15-4513 (an inverse agonist of the GABAA receptor benzodiazepine site) was inhibited when cortical membranes were incubated with ATR (Shafer et al. 1999). This would increase the influx of chloride ions, leading to hyperpolarization of cells, preventing depolarization that would, in turn, decrease DA release. No changes in levels of the DA metabolites DOPAC and HVA were observed in the microdialysis component of this study, although DA levels were altered. DA is converted to DOPAC intraneuronally after re-uptake, whereas extraneuronal DA is converted to HVA by the enzymes catechol-O-methyltransferase and monoamine oxidase. The lack of change in DOPAC and HVA could reflect the relatively modest nature of the changes in DA, leaving a sufficiently high concentration of DA in the intracellular space to maintain constant levels of DOPAC and HVA production. Further, DA re-uptake was not impaired. The decreases in DOPAC and reductions in extracellular DOPAC and HVA after potassium stimulation across sampling time in the microdialysis component of this study agree with results of other such studies (Holson et al. 1998; Robinson and Camp 1991; Stanford et al. 2000; Westerink and Tuinte 1986) and may reflect initial damage caused by the probe insertion, which recovers after several hours. The decrease in DOPAC levels that occurs with the increase in extracellular DA after potassium perfusion is thought to reflect a decrease in intracellular DA metabolism by monoamine oxidase (Camarero et al. 2002). At the present time, the health-related impacts that ATR exposures may exert in human populations remain unknown. Assessments of occupational exposures have been limited, and systematic studies of environmental exposures have not been undertaken. Although the doses of ATR used in these studies are higher than those estimated for human exposures, additional considerations must be applied to such comparisons. First, data projecting human exposure are always limited because they are dependent upon when exposures occurred relative to the time of measurement and do not provide measurement of the tissue of interest (i.e., the brain) in such cases. Second, the doses used here are low for the experimental species (rat), being consistent with previously reported NOAEL and LOAEL levels. Furthermore, even at these levels, the doses may not have been as high as administered because probably not all the ATR ingested would have been absorbed; a portion of it could have been easily eliminated through the feces, suggesting that lower actually absorbed doses could underlie the deleterious effects observed in this study. In summary, the collective findings from this study demonstrate that ATR may have broad effects on brain monoamine systems and thereby influence a wide range of behavioral functions. Clearly, additional studies are needed to unravel the targets of ATR and the mechanism(s) of its effects, as well as the ultimate human health consequences of such exposures for behavioral and/or neurologic dysfunctions. Figure 1 Experimental designs for the chronic ATR exposure component of the study (A) and for acute ATR exposure for microdialysis studies (B). Abbreviations: MC, methylcellulose; SNpc, substantia nigra pars compacta. Figure 2 Horizontal locomotor activity (group mean as percentage of control ± SEM). (A) Effect of a 1-mg/kg dose of amphetamine sulfate administration on locomotor activity after 2 months of ATR exposure. (B) Spontaneous locomotor activity measured at 3 months and 6 months of ATR exposure, and 2 weeks after cessation of ATR exposure. RMANOVA was followed by Fisher’s post hoc test. Absolute values (total counts ± SEM) for control animals are 8094.44 ± 1822.95 (amphetamine sulfate challenge after 2 months of ATR exposure); 3860.56 ± 703.66 (3 months of ATR exposure); 3168.60 ± 550.36 (6 months of ATR exposure), and 4257.00 ± 588.45 (2 weeks after cessation of ATR). TX, treatment. Significantly different from control group, p < 0.05 (n = 8–10 rats per treatment group). Figure 3 Levels of DA (A), 5-HT (B), and NE (C; presented as the group mean as a percentage of control ± SEM) in striatum, prefrontal cortex, nucleus accumbens, and hypothalamus 2 weeks after cessation of ATR exposure (6 months). One-way ANOVAs for each region were followed by Fisher’s post hoc test. Absolute values (ng/mg protein ± SEM) for control animals are, for DA: 122.25 ± 8.54 (striatum), 2.83 ± 0.21 (prefrontal cortex), 6.09 ± 0.55 (hypothalamus), 78.70 ± 7.12 (nucleus accumbens); for 5-HT: 2.46 ± 0.27 (striatum), 15.45 ± 1.31 (prefrontal cortex), 14.44 ± 0.46 (hypothalamus), 8.22 ± 0.68 (nucleus accumbens); and for NE: 11.44 ± 0.77 (prefrontal cortex), 34.65 ± 1.67 (hypothalamus), 1.63 ± 0.14 (nucleus accumbens). Significantly different from control group, p < 0.05 (n = 7–10 rats per treatment group). Figure 4 Numbers of TH+, TH−, or total cells in SNpc (top row) or in VTA (bottom row) measured using unbiased stereology and determined 2 weeks after termination of ATR treatment. Each point represents the value for an individual animal, with n = 5 randomly selected per treatment group counted. Black bars represent group medians. Figure 5 Time course (group mean as percentage of basal release ± SEM) of striatal release of DA (A; baseline DA release shown in inset), DOPAC (B), and HVA (C) over the course of microdialysis. Microdialysates were collected every 30 min; high potassium infusion (60 mM K+) lasted 1 hr, after which normal Ringer’s solution was restored for 1 hr longer. 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==== Front BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-6-261652448010.1186/1471-2180-6-26Research ArticleTemporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling Urnowey Sonya [email protected] Toshihiro [email protected] Vira [email protected] Koji [email protected] Tadamichi [email protected] Sailen [email protected] Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama 36688-0002, USA2 Department of Preventive Dentistry, Kyushu Dental College, Kitakyushu 803-8580, Japan3 Division of Microbiology and Oral Infection, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-7-1, Nagasaki 852-8588, Japan2006 8 3 2006 6 26 26 21 11 2005 8 3 2006 Copyright © 2006 Urnowey et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Porphyromonas gingivalis is the foremost oral pathogen of adult periodontitis in humans. However, the mechanisms of bacterial invasion and the resultant destruction of the gingival tissue remain largely undefined. Results We report host-P. gingivalis interactions in primary human gingival fibroblast (HGF) cells. Quantitative immunostaining revealed the need for a high multiplicity of infection for optimal infection. Early in infection (2–12 h), P. gingivalis activated the proinflammatory transcription factor NF-kappa B, partly via the PI3 kinase/AKT pathway. This was accompanied by the induction of cellular anti-apoptotic genes, including Bfl-1, Boo, Bcl-XL, Bcl2, Mcl-1, Bcl-w and Survivin. Late in infection (24–36 h) the anti-apoptotic genes largely shut down and the pro-apoptotic genes, including Nip3, Hrk, Bak, Bik, Bok, Bax, Bad, Bim and Moap-1, were activated. Apoptosis was characterized by nuclear DNA degradation and activation of caspases-3, -6, -7 and -9 via the intrinsic mitochondrial pathway. Use of inhibitors revealed an anti-apoptotic function of NF-kappa B and PI3 kinase in P. gingivalis-infected HGF cells. Use of a triple protease mutant P. gingivalis lacking three major gingipains (rgpA rgpB kgp) suggested a role of some or all these proteases in myriad aspects of bacteria-gingival interaction. Conclusion The pathology of the gingival fibroblast in P. gingivalis infection is affected by a temporal shift from cellular survival response to apoptosis, regulated by a number of anti- and pro-apoptotic molecules. The gingipain group of proteases affects bacteria-host interactions and may directly promote apoptosis by intracellular proteolytic activation of caspase-3. ==== Body Background Porphyromonas gingivalis, a gram-negative anaerobe, is a major colonizer of gingival tissues, causing severe forms of adult periodontitis, in which the gingival fibroblast suffers extensive damage [1]. As replication inside mammalian cells is a common strategy adopted by many pathogenic bacteria, P. gingivalis infection has served as an important study model. A flurry of recent research has unraveled various pathways of interaction between oral cells and P. gingivalis [2]. Infection of various cell types by P. gingivalis activated cytokines and chemokines of potential importance in pathology, such as TNF-α, IL-1β, IL-6 and IL-8, the exact roles of which in adult periodontitis remain to be determined [3-7]. P. gingivalis encodes a number of proteases, collectively known as 'gingipains', which have received considerable attention due to their multiplicity and potent activity [8,9]. The major members of this family include two Arg gingipains (RgpA and RgpB), and a Lys gingipain (Kgp) that are trypsin-like cysteine proteinases, specific for -Arg-Xaa- and -Lys-Xaa- peptide bonds, respectively. The gingipains were shown to regulate P. gingivalis adhesion and invasion positively as well as negatively depending on the cell type [10-12]. In a murine model of periodontitis, all contributed to virulence [13]. Recently, we and others have characterized a new protease of P. gingivalis, named endopeptidase O (PepO), and provided evidence for its importance in invasion and growth in human gum epithelial (HGE) and human lung epithelial (HEp-2) cells in culture [14,15]. A number of intracellular pathogens, including bacteria, viruses and parasites, either cause or suppress apoptosis of the infected mammalian cell by regulating a battery of pro- and anti-apoptotic genes [16-18]. Interestingly, various P. gingivalis protease preparations have been demonstrated to promote apoptosis when exogenously added to cells in culture [19-25]. The exact mechanism of this 'extrinsic' apoptosis is unknown but is believed to be triggered by the degradation of cell adhesion molecules such as neural cadherins and integrins by the proteases [25-28], which also causes detachment of the target cell from the tissue. It has been postulated that in periodontitis, apoptotic signals may overwhelm the normal anti-apoptotic forces that maintain periodontal vessels [29]. In contrast, P. gingivalis infection of human gingival epithelial (HGE) cells led to an anti-apoptotic response that paralleled the induction of mitochondrial anti-apoptotic Bcl-2 protein [30]. In a recent study externalization of membrane phosphotidylserine (PS) was noted immediately after P. gingivalis infection of HGE cells, suggesting an apoptosis-like response [31]. However, this was reversible, as the PS was internalized after 1 day of infection, and activation of the protein kinase AKT resulted in an anti-apoptotic response. Clearly, it is important to determine whether different gingival cell types respond differently to P. gingivalis and whether the gingipains play multiple regulatory roles in growth and pathogenesis. Although the fibroblast layer constitutes bulk of the gingival tissue, the molecular details of its interaction with P. gingivalis remain poorly understood. We decided to use primary human gingival fibroblast (HGF) cells in our studies for their obvious physiological relevance. In this communication, we analyze the status of programmed cell death or apoptosis in P. gingivalis-infected primary HGF cells and show that apoptosis is regulated in a stage-specific manner through the activation of an array of intracellular anti- and pro-apoptotic signalling molecules. Results Optimal infection of HGF cells by P. gingivalis requires a high m.o.i Previous studies in which mammalian cells were infected with P. gingivalis in tissue culture typically used high multiplicities of infection (m.o.i.) – about 100–200 bacteria per human cell. It was not clear why such large number of bacteria were apparently needed for infection. A few studies that addressed the quantitative aspects of infection paid attention to the percentage of infecting bacterial cells but not to the percentage of infected mammalian cells. For example, in one study tests of m.o.i. 10, 100, 1000 and 10000 showed that the highest percentage of bacteria invaded HGE cells at 100 m.o.i. [32]. In another study using KB oral epidermoid carcinoma epithelial cells (likely to be HeLa cells), only about 2% bacteria attached to the cell monolayer at 50 m.o.i. of added P. gingivalis [11]. Single mutants lacking either protease RgpA or RgpB showed similar efficiency; however, when the rgpA rgpB double mutant was tested, about 60% bacteria attached to the epithelial cells. To explain this unexpected result, it was postulated that the Arg-gingipains may play a negative role in cell attachment by digesting the cellular receptors [11]. In all these experiments the percentage of infected KB cells remained undetermined. Role of KgP was also not tested. As attachment and invasion are the obligatory first steps in infection, we considered it important to determine the optimal efficiency of infection of HGF cells before proceeding to study the subsequent events. To achieve this, we first quantified the efficiency of invasion. Washed wild type and triple gingipain mutant (rgpA rgpB kgp) P. gingivalis [33] were added to HGF monolayers and the infected HGF cells were counted by fluorescence staining with P. gingivalis antibody. A representative set of results (Fig. 1) show that there was a modest increase in the percentage of HGF cells infected with increasing m.o.i. Invasion reached a plateau at about 200 m.o.i., when roughly half of the cells were infected (Fig. 2). The triple protease mutant P. gingivalis fared worse, infecting only about a third of all cells at the same m.o.i. Although it is possible that some cells were infected at levels below the threshold of visible fluorescence, and therefore, were scored as uninfected, we can conclude that infection of HGF by P. gingivalis is a relatively inefficient process and that the Arg- and Lys-gingipains are not absolutely essential for invasion but improve the efficiency. Based on these findings (Fig. 2) and to achieve equal invasion we used an m.o.i. of 200 for the triple mutant and 80 for the wild type P. gingivalis in all subsequent experiments. Next, to determine the optimal time of release of progeny P. gingivalis from the infected cells, we assayed for bacterial colony forming units (cfu) in the HGF growth media at different times post-infection. This was done by plating serial dilutions of the media on agar plates as described in Materials and Methods. Maximal release of progeny bacteria occurred at 72 h post-infection coinciding with significant lysis of the monolayer (data not shown). Thus, we carried out all of our signalling experiments at earlier time points, usually not exceeding 48 h post-infection. P. gingivalis causes late apoptosis in infected HGF cells At various times post-infection P. gingivalis-infected HGF cell monolayers were fixed and multiparametric staining including TUNEL staining for apoptosis was performed. Representative data (Fig. 3) show little apoptosis at early time point but significant apoptosis from 24 h onwards. Apoptosis was confirmed by propidium iodide-Annexin V staining (data not shown). The triple gingipain-mutant P. gingivalis also caused apoptosis but appeared to be slower and less effective. As TUNEL is largely qualitative, we extended these studies with more detailed analyses (Fig. 4). A quantitative estimate of apoptotic nucleosome release was conducted, which supported the TUNEL kinetics and revealed that apoptosis by P. gingivalis is indeed a relatively late process in infection and that the mutant is less proficient in this regard (Fig. 4A). The DNA fragmentation paralleled activation of caspase-3, a major executioner caspase, using a synthetic fluorogenic substrate (Fig. 4B). We then investigated the activation of seven strategic caspases, including caspase-3, by immunoblot detection of their cleaved, active fragments, and the results (Fig. 4C) showed activation of caspase-6, caspase-7 and caspase-9 in addition to caspase-3, but not caspase-8, -10, or -12. Activation of caspases was found to occur late in infection, starting at around 24 h, coincident to apoptotic DNA damage. These results strongly suggested a role of specific caspases in P. gingivalis-induced apoptosis of HGF cells. For a direct measure of the intracellular caspase activity, we attempted to detect the breakdown products of αII-spectrin (also known as non-erythroid α-spectrin or α-fodrin). In apoptotic cells, the 240 kDa αII-spectrin undergoes a series of caspase-mediated cleavage to generate a major 120 kDa fragment, although a 150 kDa intermediate is sometimes detected as well [34]. Immunoblot analysis of P. gingivalis-infected HGF cell lysate (Fig. 4D) revealed the 120 kDa product, but only at late stages of infection, between 36 h and 48 h. The wild type P. gingivalis and the triple protease mutant produced essentially identical breakdown products that appeared much later in mutant-infected cells (48 h instead of 36 h). These results supported a caspase-induced breakdown of αII-spectrin and also matched the late-stage apoptosis and the slower kinetics of apoptosis by the mutant. P. gingivalis protease(s) directly cleave and activate procaspase-3 in vitro Although the full impact of gingipains in host-bacterial interaction is still under investigation, their role in invasion is well-documented. As mentioned, exogenously added gingipains trigger extrinsic cell death, at least one mechanism of which involves the cleavage of external cell adhesion molecules such as cadherins and integrins [23,25]. Whether they have a role in the intrinsic pathways of apoptosis remains unknown. Although activated caspases are hallmark effectors of apoptosis, recent evidence suggests that non-caspase proteases such as cathepsins, calpains, granzymes, and the proteasome complex may have a direct caspase-like role in mediating apoptosis, but perhaps only in specific cell types or under specific signals [35,36]. Because P. gingivalis is a known producer of highly active proteases in the infected cells, it was worth testing whether these proteases might directly process and thereby activate an executioner caspase. To this end, we incubated purified recombinant procaspase-3 with P. gingivalis culture supernatant, and tested for cleavage. Results (Fig. 5A) show that the 34 kDa procaspase-3 was indeed cleaved to characteristic 17 kDa and 12 kDa fragments. The product sizes roughly matched with those produced by caspase-8-mediated cleavage of procaspase-3. Enzymatic activity of caspase-3 was confirmed by using the specific substrate peptide, DEVD (Fig. 5B). Similar amounts of culture supernatant made from the triple gingipain mutant exhibited little processing activity. Finally, the processing activity was destroyed by heat, in agreement with its protease nature. These results raise the interesting possibility that the Arg- and Lys-gingipains have the potential to directly contribute to apoptosis by activating intracellular caspase-3. P. gingivalis infection of HGF cells leads to early activation of NF-kappa B The proinflammatory transcription factor NF-kappa B (NF-κB) is generally responsible for suppression of apoptosis, although there are exceptions where it promotes apoptosis or has no apparent role in apoptosis [37]. In a number of obligatory parasites, infected cells are protected from apoptotic signals, mainly as a result of activation of NF-κB. In light of the apparent lag in apoptosis in the early stages of P. gingivalis infection it was important to determine whether NF-κB is activated in these cells. For a preliminary test, we first used an engineered HEK293 (hamster embryonic kidney fibroblasts) cell line that contained a chromosomal reporter luciferase gene under the control of NF-κB enhancers. Because of the convenience of luciferase assay, these cells are commonly used for rapid initial screening of NF-κB activating agents before detailed studies are done in more relevant cells. For example, we have shown previously that infection of these cells with respiratory syncytial virus leads to activation of NF-κB and hence, luciferase activity [38]. Conversely, inhibitors of NF-κB reduced the luciferase activity back to basal levels. As shown (Fig. 6A), a surge of luciferase activity was also detected at around 15 h following P. gingivalis infection of HEK293 cells. The levels gradually subsided from 24 h onward, suggesting loss of NF-κB activity. The gingipain-deficient strain also activated NF-κB albeit substantially weakly. SN50, a cell-permeable peptide inhibitor of NF-κB [39], strongly reduced luciferase activity, demonstrating specificity. Having obtained positive results in luciferase assay, we proceeded to the physiologically relevant HGF cells and investigated NF-κB activation by direct measurement of the nuclear levels of the p65 subunit of NF-κB. In unstimulated cells, p65 is held back in the cytoplasm in complex with the inhibitors of κB, such as IκBα. Various signals including infection by many pathogens lead to the phosphorylation of IκBα via IκB kinase (IKK), followed by degradation of phospho-IκBα, allowing the nuclear import of p65 via its nuclear localization signal, where it activates transcription of NF-κB-dependent genes [40]. Thus, the appearance of NF-κB in the cell nucleus is indicative of its activation. It is seen (Fig. 6B) that P. gingivalis infection of HGF cells led to nuclear translocation of p65 early in infection that started to decline around 24 h. Phosphorylation of IκB was also detectable as early as 3 h p.i. (Fig. 6C), suggesting a correlation. A common upstream pathway leading to activation of NF-κB in many cells involves phosphoinositol-3 kinase, PI3K, and curiously, an inhibitor of PI3K (LY294002) inhibited IκB phosphorylation in these cells (Fig. 6C). This led us to study the potential role of PI3K in P. gingivalis-mediated activation of the NF-κB signalling, described next. P. gingivalis infection activates the PI3K-AKT pathway In mammalian cells, the PI3K/AKT pathway generally acts in a pro-survival, anti-apoptotic role [31,41]. A multitude of external stimuli activate intracellular PI3K that catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate into the 3,4,5-triphosphate. The latter activates protein kinase PDK, which then phosphorylates AKT (also known as protein kinase B or PKB), thereby activating AKT. The activated AKT phosphorylates a large number of substrates such as Forkhead transcription factor (FKHR), IKK, Bad, caspase-9, and GSK-3. Together, these events inactivate the pro-apoptotic players such as Bad, and activate anti-apoptotic players such as NF-κB, leading to a net suppression of apoptosis. We, therefore, determined the phosphorylation status of these signalling molecules in HGF cells during P. gingivalis growth using phosphospecific antibodies, and the results are presented in Fig. 7. It can be seen that all the major players of the pathway, namely PDK, AKT, Forkhead and GSK-3 were phosphorylated at comparable kinetics, early in infection. Total AKT protein levels did not change, confirming that the effect was truly on the phosphorylation status and not on protein synthesis. These anti-apoptotic phosphorylations occurred relatively early (≤ 6 h) and appeared to reach maximal levels by 6 h. In fact, at later times (between 12 and 24 h) phosphorylations reverted back to near pre-induction levels. The gingipain-deficient mutant also activated the same pathways with a similar kinetics. In all cases, the PI3K inhibitor, LY294002, abolished the phosphorylation, demonstrating that PI3K is indeed the most upstream kinase in this P. gingivalis-activated pathway. The same inhibitor also strongly inhibited the activation of NF-κB by P. gingivalis (Fig. 6A, striped bar), further supporting a role of PI3K in survival. Together, these results strongly support our premise that the early cellular response in P. gingivalis-infected HGF is anti-apoptotic whereas the late response is pro-apoptotic. Temporal transcriptional induction of anti- and pro-apoptotic family genes by P. gingivalis Studies over the last two decades have enlisted a large family of cellular genes that take part in either preventing or promoting apoptosis [42]. As the decision between cellular life and death is a net resultant of these two opposing forces, we decided to obtain a comprehensive view of expression of both families in P. gingivalis-infected HGF cells. The steady-state mRNA levels of major members of the two families were determined by reverse transcription and quantitative real-time PCR, and the results are presented in Fig. 8 as fold-induction over uninfected levels. The top two rows represent anti-apoptotic genes, and the rest are pro-apoptotic. The general trend that emerged from these results is that the two families were induced differently. The anti-apoptotic genes were induced early in infection, generally reaching their maximal levels by 12 h and then diminishing to variable extents over the rest of the infection period. The induction of a number of anti-apoptotic genes was sharply inhibited by the PI3K inhibitor, LY294002, as well as by the NF-κB inhibitor, SN50, further correlating the anti-apoptotic gene profile with the survival pathway. A representative example is Bfl1, the most robustly activated anti-apoptotic gene (Fig. 8, first box); both inhibitors caused 60–70% reduction of its induction. Similar inhibitory effect was also seen for three other genes tested, namely, Boo, Bcl-2, Survivin (data not shown). In sharp contrast, the vast majority of the pro-apoptotic genes reached their maximum expression in later periods of infection, peaking between 24 and 36 h. Most were expressed poorly or not at all until about 12 h p.i. As in other pathways, the gingipain-deficient mutant activated these genes with similar overall kinetics. For the majority of the genes the mutant was weaker than the wild type although there were a few exceptions, such as the expression of Bcl-w, Bik, Bax, MOAP-1 was comparable in wild type and mutant-infected cells, and the induction of Bfl-1 and Bcl-XL expression was somewhat stronger in mutant-infected cells (Fig. 8). We carried out immunoblot (Western) analysis of selected representative proteins, namely Bcl-XL, Survivin, Bax and Bad, and the results essentially paralleled those of the transcript levels (data not shown). In conclusion, the general trend was that the expression of anti-apoptotic and pro-apoptotic genes was turned on early and late in infection, respectively, in a mutually exclusive chronological order. Discussion One major finding in our study is the inefficient invasion by P. gingivalis, at least in cell culture, explaining the high m.o.i. traditionally used by all investigators. Although the exact mechanism needs further research, bacterial fimbriae, which are proteinaceous appendages extending from the bacterial cell surface, are known to play specific and important roles in bacterial adhesion and invasion, likely through an interaction with integrin and fibronectin of the host cell [43-45]. It is possible that these interactions are relatively inefficient and slow. Our results support the previous demonstration that Arg-gingipains enhance fimbriae-fibronectin binding, leading to the suggestion that the functional epitopes of cellular receptors of P. gingivalis are cryptic and that the Arg-gingipains expose them [46]. This is also consistent with our finding that the protease-deficient mutant P. gingivalis is a generally poor invader (Fig. 2). The second major finding here is that infection of primary gingival fibroblast cells by P. gingivalis, an important gum pathogen, leads to apoptosis, which may in part underlie the extensive tissue damage seen in gingivitis. Interestingly, early in infection, cellular anti-apoptotic genes are induced and postpone apoptosis; at later times, they give way to pro-apoptotic genes, and apoptosis ensues. As apoptosis is an exceedingly complex process involving a large variety of signalling molecules, we have focused our attention to selective major players. The anti-apoptotic early phase in P. gingivalis-infected HGF cells is characterized by the activation of PI3K/AKT pathway. Our results (Fig. 7) strongly suggest that this pathway is largely responsible for the activation of pro-survival transcription factor NF-κB. The anti-apoptotic function(s) most likely facilitates P. gingivalis growth by thwarting premature dismantling of the host cell. The response of HGF and HGE cells to P. gingivalis infection, therefore, has interesting similarities and differences. Like HGF cells, infection of HGE cells resulted in suppression of apoptosis, which required phosphorylation-mediated activation of AKT and was inhibited by LY294002 [31]. However, HGF cells eventually became apoptotic whereas HGE cells appeared to remain anti-apoptotic. Thus, the fibroblasts and epithelial cells of the same tissue may differently regulate the apoptotic modulators, the mechanism of which must await further study. Caspases are a class of cysteine proteases that includes several representatives involved in apoptosis [42]. They are activated via a proteolytic cascade, cleaving and activating other caspases, eventually degrading downstream targets and promoting cell death (Fig. 9). The main "initiator" caspases at the upper end of the cascade are caspase-8 and caspase-9. Caspase-8 is activated in response to death receptors (such as Fas) whereas caspase-9 is activated by the release of cytochrome c via the intrinsic mitochondrial stress pathway. Activation of caspase-9 but not caspase-8 in P. gingivalis-infected HGF cells (Fig. 4) strongly points to the activation of the mitochondrial pathway. Clearly, caspase-9 then activates the downstream "executioner" caspases, namely caspase-3, -6 and -7. Spectrin fragments serve to distinguish between caspase-3 and calpain; while caspase-3 cleavage generates the 120 kDa fragment calpain produces 150 and 145 kDa fragments [34,47]. Our results, therefore, confirms caspase-3 activation (Fig. 4) and rules out a role of calpain in P. gingivalis-mediated apoptosis. A novel apoptotic pathway has been discovered recently, in which an endoplasmic reticulum (ER) resident caspase, namely caspase-12, is activated by ER stress that may be triggered by heavy traffic of glycoproteins through the ER. For example, we have shown that infection by respiratory syncytial virus, a cytoplasmic RNA virus expressing three glycoproteins that traffic through the ER, causes late apoptosis in lung epithelial cells by activating ER-stress and caspase-12 [16]. Together, results presented here suggest that P. gingivalis activates apoptosis through the mitochondrial pathway and not the ER or Fas pathway. Based on these findings, we present a working hypothesis of P. gingivalis-activated apoptosis in HGF cells in Fig. 9. We emphasize that it is not a comprehensive list and that we have provided experimental evidence only for the key players in each major branch. In the anti-apoptotic early phase, we have demonstrated activation of PDK, AKT / PKB, the AKT substrates (GSK-3 and FKHR) and the IKK substrate (IκB-α) (Fig. 7). The involvement of PI3K and NF-κB was also uncovered by the use of specific inhibitors. Transcriptional activation of a battery of anti-apoptotic genes is also indicated (Figs. 8, 9), which was abrogated by inhibitors of either PI3K or NF-κB. The pro-survival role of the PI3K/NF-κB pathway is explained by the recent demonstrations that transcription of these anti-apoptotic genes is NF-κB-dependent [48]. In the pro-apoptotic late phase of infection, we surmise a major role for the mitochondria. One of the best characterized mechanisms used by mitochondria to induce cell death is the release of pro-apoptotic proteins into the cytosol [49]. Cytochrome c, the first molecule shown to be released, complexes with apoptosis protease-activating factor 1 (Apaf-1) and exposes domains of Apaf-1 that activate caspase-9. A proteolytic cascade ensues that eventually activates caspases-3, -7 and -6 (Fig. 9), and our results show activation of all these caspases (Fig. 4). Nucleosomal DNA degradation (Figs. 3, 4) suggested the activation of DNA-fragmentation factor (DFF), although the involvement of AIF and endonuclease G, released from stressed mitochondria (Fig. 9) cannot be ruled out. Most importantly, the kinetics of activation of these proteins was in accord with the shift from an anti- to a pro-apoptotic response. Further evidence for the shift came from transcriptional data of anti- and pro-apoptotic genes. The mitochondrial Bcl-2 family [49] comprises of both kinds, such as the anti-apoptotic members Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and the pro-apoptotic members Bax, Bak, Bok, Bid, Bad, Puma, Bmf, Bim, Bok, Noxa and Hrk/DP5. Some of the most highly activated genes in Fig. 8 indeed belong to these two families and their functions match with the temporal shift from survival to death. However, the relative contribution of each of these proteins in regulating apoptosis is currently unknown. The direct processing of procaspase-3 by P. gingivalis protease(s) (Fig. 5) adds a novel dimension to 'intrinsic' apoptosis by bacteria. Interestingly, structural analysis of the catalytic subunit of Rgp revealed a caspase-like fold, suggesting a common ancestor [50]. Moreover, homology mapping suggested that a single protease clan, named CD [51] or the caspase-hemoglobinase fold [52], encompasses gingipains and caspases as well as bacterial clostripain and legumains (processing proteases) [53,54]. As the processing by gingipains generated enzymatically active caspase-3 (Fig. 5), it is reasonable to conjecture that the cleavage occurred at or close to the natural processing site. The processing defect of the rgpA rgpB kgp mutant suggests that the cleaved peptide bond would be next to either an Arg or a Lys. The natural processing in procasapse-3 is known to occur next to the Asp175, shown here in bold within the substrate motif IETD: CR164GTELDCGIETD175SGVDDDMACHK186. We noticed Arg164 and Lys186 nearby (as underlined in the sequence), 11 residues upstream and downstream of the Asp175, respectively. We speculate that one or more of the gingipains cleave the procasapse-3 at either or both of these sites to produce the activated caspase-3. This is currently being tested. The physiological significance of this activation also needs to be resolved. It is possible that the anti-apoptotic functions antagonize this activation and prevents it from happening too early in infection. Moreover, note that the mutant strain is not completely defective in activating apoptosis and caspase-3 in HGF cells (Fig. 4). Thus, the three gingipains, although not absolutely essential, seem to be needed for optimal apoptotic response. We note here that the gingipains may be partially sequestered by serum proteins; human [55] and bovine (our unpublished results) serum albumin, for example, are excellent substrates for gingipains. It is also possible that serum will contain unidentified inhibitors of gingipains [55]. On the other hand, the presence of serum likely approximates the physiological infection of the gum and provides an optimal nutrient level for intracellular replication of the bacteria. For the best compromise, therefore, we allowed the bacteria to invade initially for 90 min in the absence of serum, and then replaced it with serum-supplemented medium for further growth (Materials and Methods). It is tempting to speculate that the delayed apoptosis of the host cell most likely allows P. gingivalis extra time to replicate intracellularly to a higher yield and at the same time offers early protection to the infected cells against cytotoxic mediators of the host immune system. An extended period of nuclear DNA integrity may also allow the cell to transcriptionally activate genes that modulate immune or inflammatory response. Apoptosis then occurs at the late stage of infection when the replicated bacteria must destroy the host cell anyway in order to egress and infect neighboring cells for fresh nutrients and continued growth. Clearly, a detailed knowledge of how P. gingivalis regulates the balance between multiple apoptotic signalling molecules in chronological order will shed important light on the mechanism of tissue damage in gingivitis and may provide a pharmacological regimen to control the infection. Conclusion P. gingivalis infection of human gingival epithelial cells initially triggers a survival response through the activation of PI3K/AKT pathway, resulting in the activation of NF-κB and a family of anti-apoptotic proteins. This likely allows optimal growth of the bacteria. At later stages of infection the anti-apoptotic proteins subside and pro-apoptotic ones are turned on, leading to apoptosis of the infected cell. Bacterial proteases of the gingipain family play important roles in various aspects of infection, including proteolytic activation of caspases by processing, which may directly contribute to the observed apoptosis. Thus, P. gingivalis and the host gingival cell interact via a variety of pathways that are relevant in the pathology and degradation of the gingiva. Materials and methods Materials Antibodies against the following antigens were obtained from commercial sources: p65, Sp1 (Santa Cruz Biotechnology); phospho-AKT, caspases-3, -6, -7, -8, -9 (Cell Signaling Technology); caspase-10 (Oncogene); caspase-12 (Sigma); IκB-α (Biomol); nonerythroid alpha-spectrin (Chemicon International); Bcl-XL, Survivin, Bax and Bad (Sigma-Aldrich). Other phosphospecific antibodies were from New England Biolabs. The phosphatase inhibitor cocktail set II (final concentration of 1 M imidazole, 0.1 M sodium fluoride, 0.115 M sodium molybdate, 0.2 M sodium orthovanadate, and 0.4 M sodium tartrate) and the inhibitors LY294002, SN50 and staurosporine were from Calbiochem (San Diego, CA). "Complete protein inhibitor cocktail" was from Roche, one mini tablet of which was added per 10 ml of buffer (final concentration) as prescribed by the manufacturer. Bacteria and culture conditions Wild-type P. gingivalis (ATCC 33277) and the isogenic mutant strain (rgpA rgpB kgp) [33] were grown anaerobically (85% N2, 10% H2, and 5% CO2) at 37°C in brain-heart infusion broth supplemented with yeast extract (0.5%), hemin (5 μg/ml), and menadione (0.5 μg/ml), essentially as described [56]. Bacteria were grown to mid-log phase (A600 in the range of 0.6–0.8), harvested by centrifugation, washed with phosphate-buffered saline (PBS) and resuspended in DMEM supplemented with 2 mM L-glutamine. For each experiment the final concentration of the suspension was determined by measurement of A600 and appropriate dilutions were made to achieve the desired m.o.i. The bacterial number was confirmed retroactively by viable counting of colony forming units (cfu) on agar plates supplemented with hemin and menadione [32,56]. Culture of HGF cells Primary cultures of HGF cells were made from biopsies of healthy human gingival obtained from dental clinics. The tissues were washed several times in PBS and DMEM, then cut into small pieces and placed in a T-25 cm2 flask with complete medium containing DMEM supplemented with 10% FBS, 2 mM L-glutamine, 2.5 μg/ml fungizone, and 5000 U/ml penicillin/ streptomycin. When ready for passage, the fibroblast cells were cultured as monolayers in the same complete media without fungizone at 37°C in a standard 5% CO2 incubator. Infection of HGF cells with P. gingivalis HGF cells were used at 80 to 90% confluency for all experiments. Before infecting with P. gingivalis, the monolayers were washed three times with PBS. P. gingivalis, resuspended in DMEM without FBS or antibiotics, was added to the HGF monolayer at the desired m.o.i. and incubated for 90 min at 37°C in 5% CO2. The monolayer was then washed twice with PBS to remove unbound bacteria, DMEM supplemented 10% FBS and 2 mM L-glutamine was added, and growth continued at 37°C in 5% CO2. Assessment of bacterial invasion was done by an antibiotic protection assay essentially as described previously with minor changes [11,14,32]. HGF cells were first infected with washed P. gingivalis and external, nonadherent bacteria were removed as described above. The cultures were then incubated for an additional 2 h at 37°C in fresh medium containing 300 μg gentamicin and 200 μg of metronidazole per ml to kill the remaining extracellular bacteria [32]. We confirmed that the antibiotics did not affect the morphology of the HGF cells or alter their ability to exclude trypan blue. Following invasion, the HGF cells were fixed and stained as described below. Immunofluorescence studies and assays of cell death and NF-κB For multiparametric staining, HGF cells were grown on coverslips in 6-well plates to 80–90% confluency. The cells were then infected with washed P. gingivalis as above. Coverslips were washed three times with PBS and fixed in 10% trichloroacetic acid for 20 min on ice. Successive washes were then performed with 70%, 90% and 100% ethanol, and finally with PBS containing 0.2% Triton X-100. The coverslips were incubated with a rabbit antibody against P. gingivalis Fim A and then with secondary mouse antibody (TRITC-conjugated). Finally, the coverslips are mounted on a slide with DAPI-DABCO solution and observed by fluorescence microscopy in an Olympus BMAX Epifluorescence microscope [16] For routine detection of apoptosis, we used the DeadEnd Fluorometric TUNEL System (Promega) that measures apoptosis by the integrating fluorescein-12-dUTP at 3'-OH DNA ends of fragmented DNA of apoptotic cells. The fluorescein-12-dUTP-labeled DNA was visualized directly by fluorescence microscopy. Cells are grown to confluency on coverslips in 6-well plates and stained as per the manufacturer's instructions. Annexin V, conjugated with fluorescein isothiocyanate (FITC) (Sigma), was used to label phosphatidylserine on the apoptotic membrane surface, and propidium iodide (PI) to stain the nuclei (if necrotic). Quantitative assay of apoptosis was performed with a new procedure termed "Cell Death Detection ELISA" (Roche) which involves photometric enzymatic immunoassay of mono- and oligo-nucleosomes in the cytoplasmic fraction of apoptotic cell lysates. Luciferase reporter assay for NF-B was performed as described [38]. Briefly, cells in monolayer were transfected with pNFκB-Luc using Lipofectin® (Gibco Life Technologies), and infected with washed P. gingivalis cells 24 hr later. When used, inhibitors were added at the same time as the bacteria. The cells were lysed at indicated times thereafter and cleared extracts subjected to luciferase assay in a Turners Design luminometer [38]. Data acquisition and analysis In invasion assay as well as in apoptosis detection, the fluorescent HGF cells were visually counted using a subjective baseline that corresponded to uninfected controls. Changes were analyzed by one-way ANOVA and then by Student's t-test with Bonferroni correction. All numerical data were collected from at least three separate experiments. Results were expressed as mean ± SEM (error bars in graphs). Differences were considered to be significant at P < 0.05. Quantitative real-time PCR HGF cells were grown in T-25 flasks and infected with wild-type and mutant P. gingivalis as described. At different time points, mRNA from infected cells was purified using a Trizol method (Ambion). First-strand cDNA was made using the GeneAmp RNA PCR Core kit (Perkin Elmer-Applied Biosystems). Primers were designed by the Beacon Designer software v 2.13 from Premier Biosoft essentially as described previously [57], and are listed in Table 1. Real Time PCR was performed on the iCycler iQ Quantitative PCR system from BioRad Laboratories (Hercules, CA) using the iQ Sybr Green SuperMix. Gene expression measurements were calculated using the manufacturer's software; GAPDH was used as an internal control. Immunoblot (Western) analysis The infected monolayer and any control samples were washed twice in PBS containing the protease inhibitor cocktail described under Materials. When phosphoproteins were to be detected, all buffers additionally contained the phosphatase inhibitor cocktail. The cells were scraped off in PBS containing the inhibitors and centrifuged at 5,000 × g for 10 min to remove cell debris. The pellets were boiled in standard SDS sample buffer, and proteins separated by 12% SDS-PAGE and transferred to nitrocellulose [58]. Blots were probed with the appropriate antibody followed by corresponding secondary antibody coupled to horseradish peroxidase, and developed using the ECL kit (Pierce). The chemiluminescence was detected in LAS-1000 plus imaging system (Fuji Film). For NF-κB, the infected monolayer in 6-well plates was washed twice with PBS containing the protease inhibitor cocktail. Fifty microliters lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 0.1 mM EDTA, 0.1% Tween 20, 1x protease inhibitor cocktail) was then added to the cells in each well. The cells were scraped in the buffer and centrifuged at 15,000 × g for 15 min at 4°C. The resulting supernatant was used as the cytosolic extract; the pellet was washed twice with the same buffer (5,000 × g for 10 min at 4°C) and used as the nuclear fraction. Caspase cleavage and assay Overnight grown P. gingivalis cells were collected by centrifugation at 10,000 × g and washed three times with PBS. Pellets were resuspended in DMEM (without serum or antibiotic) and incubated for 4 h anaerobically (85% N2, 10% H2, and 5% CO2) at 37°C. Supernatant was collected by centrifugation (14,000 × g) and protein concentration determined by Bradford assay (Bio-Rad). To test for the cleavage activity, pre-determined amounts of the supernatant were incubated with 5 μg of purified procasapase-3 (Biomol) at 37°C in a 50 μl reaction in the following buffer: 50 mM Hepes (pH 7.5), 50 mM NaCl, 20% glycerol, 0.1% CHAPS, 1 mM DTT. The supernatant was substituted with purified caspase-8 (Biomol) in a positive control reaction and with same volume of buffer in a negative control. Portions of the reaction were analyzed by SDS-PAGE and immunoblot [57]. The rest of the reaction was assayed for caspase-3 activity using the colorimetric substrate, DEVD-pNA, which upon cleavage exhibits increased absorption at 405 nm (Calbiochem). Reactions were incubated at 30°C and followed with time in a spectrophotometer to ensure linearity. Authors' contributions SU did the major experiments; TA performed initial assays of invasion in the SB laboratory; VB carried out additional invasion studies and caspase assays; KN constructed the mutant strains; TT offered expertise and guidance to TA; SB conceived and guided the project, and wrote the paper with help from SU. All authors read and approved the final manuscript. Acknowledgements Research in the SB lab was supported in part by grants AI045803 and EY013826 from National Institute of Health (NIH), USA, and was conducted in a facility constructed with support of a Research Facilities Improvement Program Grant (C06 RR11174) from the National Center for Research Resources, NIH. Figures and Tables Figure 1 Infection of HGF cells by P. gingivalis. Wild type (WT) or triple gingipain mutant (MT) P. gingivalis was added to monolayers of HGF cells at the indicated bacteria-to-cell ratios (m.o.i). Only a few representative multiplicities are shown. Invasion was determined after killing of the external bacteria with antibiotics and staining the cells with DAPI (Blue) and P. gingivalis Fim A antibody (Pg; Red) as described under Experimental Procedures. Visually detectable red cells in the 'Pg' fields were counted in three independent observations and expressed as percentage of all the blue cells in the 'DAPI' fields. Figure 2 Multiplicity-dependent increase of HGF invasion by P. gingivalis. Invasion results obtained by staining (from Fig. 1) are plotted here. Error bars from three experiments are shown. Figure 3 Apoptosis in HGF cells by P. gingivalis infection. Infection of HGF monolayers and staining for all nuclei (DAPI, Blue) or only the apoptotic ones (TUNEL, Green) were carried out as described under Experimental Procedures. Cells were infected by wild type (WT) or the triple gingipain mutant (MT), and stained at the indicated time of infection (5 h, 24 h, 48 h). For positive control of apoptosis, uninfected HGF cells were treated with staurosporine for 5 h. Figure 4 Kinetics of apoptosis and caspase activation following P. gingivalis infection. HGF monolayers were infected with wild type (WT) or triple gingipain mutant P. gingivalis and harvested at indicated times for the following assays as described under Experimental Procedures. (A) DNA fragmentation assay using the "Cell Death Detection ELISA" (Roche); (B) Caspase-3 activity assay in cell lysates; (C) Immunoblot detection of activated (cleaved) caspases in HGF cells infected with WT, with Sp1 serving as a control for equal protein loading; note the absence of activated caspases-8, -10, -12. (D) Immunoblot detection of α-spectrin fragments in infected HGF cells; the full-length 240 kDa spectrin and the caspase-specific 120 kDa product bands are so marked. In A and B, the standard error bars are from three experiments. Figure 5 Cleavage (A) and activation (B) of procaspase-3 by P. gingivalis-excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments. Figure 6 PI3K-dependent activation of NF-κB by P. gingivalis. (A) Activation of NF-κB-dependent luciferase in HEK cells. Monolayers were infected with wild type P. gingivalis in the presence of no drug (Open bars), 20 μM SN50 (Speckled bars), 20 μM LY294002 (Striped bar), or with the triple gingipain mutant (Closed bars), and luciferase assay carried out as described in Materials and Methods. Error bars represent average of three experiments. (B) Nuclear import of NF-κB p65 subunit. HGF cells were infected with P. gingivalis (wild type = WT; gingipain mutant = MT), and nuclear extracts (40 μg protein) of infected cells (or uninfected controls = U) were analyzed for p65 by immunoblot [54]. Sp1 serves as an unchanged control. (C) Phosphorylated IκB-α, an indicator of NF-κB activation, was detected by immunoblot of the total infected HGF cell extract using a specific antibody [54]. This phosphorylation was undetectable when 20 μM LY294002 (PI3K inhibitor) was included in the culture medium (+I). Figure 7 Activation of members of the PI3K/AKT pathway. HGF monolayer was infected with wild type P. gingivalis (WT) or its triple gingipain mutant (MT), and the infected cells grown in the presence (+I) or absence of 20 μM LY294002 (PI3K inhibitor). Total cell extracts (50 μg) were probed in immunoblot for the presence of total AKT or specific phosphorylated proteins of the AKT pathway as named. The two species of phospho-FKHR are indicated by closed circle and triangle. Note the early (6–12 h) activation of phosphorylation and its inhibition by the inhibitor. Figure 8 Induction of apoptosis-related gene mRNAs in HGF cells infected with wild type P. gingivalis (Triangles) or its triple gingipain mutant (Circles). Total mRNA from infected cells at different times p.i. were subjected to quantitative Real Time RT-PCR as described under Materials and Methods, using primers described in Table 1. The relative amounts of RNA were expressed as the ratio of uninfected control value (fold induction). Each box represents a specific gene as named and its generally accepted nature as anti-apoptotic ('anti') or pro-apoptotic ('pro'). Each data point is derived from three independent infection experiments with the error bar as shown. Note the general trend of early activation of anti-apoptotic genes (upper rows) and late activation of the pro-apoptotic ones (lower rows). Treatment with LY294002 (Closed circles) and SN50 (Closed triangles) inhibited activation of anti-apoptotic genes, as exemplified by Bfl-1. Figure 9 A working model for P. gingivalis-activated apoptosis in HGF cells. Note that this is a highly reductive and simplified model showing only the major signaling events and that the intermediate steps are mostly omitted for space (details in Results and Discussion). In general, the P. gingivalis-activated molecules shown in this paper are displayed more prominently. The ER stress and Fas pathways were not activated. Whether the gingipains activate caspase-3 in vivo remains a question. The box on the left lists the major anti-apoptotic molecules activated early in infection, and the box on the right shows the pro-apoptotic molecules activated later. The two sets of forces are mutually antagonistic and thus, the balance shifts from anti- to pro-apoptosis as infection progresses. Table 1 The apoptosis family genes and their PCR primers Gene, Accession # Sense Antisense Bfl-1, NM_004049 TTACAGGCTGGCTCAGGACT CCCAGTTAATGATGCCGTCT Boo, NM_020396 GAAGAAGTGGGGCTTCCAG GAAAGGGGGTCCTGAAGAAG Bcl-XL, NM_138578 GTAAACTGGGGTCGCATTGT TGGATCCAAGGCTCTAGGTG Bcl-2, NM_000633 ATGTGTGTGGAGAGCGTCAA ACAGTTCCACAAAGGCATCC Mcl-1, NM_021960 TAAGGACAAAACGGGACTGG ACCAGCTCCTACTCCAGCAA Survivin, NM_001168 GGACCACCGCATCTCTACAT GACAGAAAGGAAAGCGCAAC Bcl-w, NM_004050 GCTGAGGCAGAAGGGTTATG CACCAGTGGTTCCATCTCCT Nip3, NM_004052 CTGGACGGAGTAGCTCCAAG TCTTCATGACGCTCGTGTTC Hrk, NM_003806 CTAGGCGACGAGCTGCAC ACAGCCAAGGCCAGTAGGT Bak, NM_001188 TTTTCCGCAGCTACGTTTTT GGTGGCAATCTTGGTGAAGT Bik, NM_001197 TCTTGATGGAGACCCTCCTG GTCCTCCATAGGGTCCAGGT Bok, NM_032515 AGATCATGGACGCCTTTGAC TCAGACTGCAGGGAGATGTG Bax, NM_004324 TCTGACGGCAACTTCAACTG CACTGTGACCTGCTCCAGAA Bad, NM_004322 CGGAGGATGAGTGACGAGTT CCACCAGGACTGGAAGACTC Bim, NM_207003 TGGCAAAGCAACCTTCTGAT TCTTGGGCGATCCATATCTC Moap-1, NM_022151 CAGTGGGTGAGTTGAGCAGA GAAACATCCAGCGTCCAAAT The common names of the anti- and pro-apoptotic genes and their RefSeq accession numbers (GenBank) are shown here. The sense and antisense sequences correspond to the RT-PCR primers used for amplification of the transcripts by real-time PCR (Fig. 8). 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==== Front PLoS PathogPLoS PathogppatplpaplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1660973010.1371/journal.ppat.002002505-PLPA-RA-0242R2plpa-02-03-06Research ArticleCancer BiologyEpidemiology - Public HealthInfectious DiseasesPathologyVirologyHomo (Human)VirusesMus (Mouse)Identification of a Novel Gammaretrovirus in Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant Virus in Prostate Tumors with R462Q RNASELUrisman Anatoly 1Molinaro Ross J 23Fischer Nicole 4Plummer Sarah J 2Casey Graham 2Klein Eric A 5Malathi Krishnamurthy 2Magi-Galluzzi Cristina 6Tubbs Raymond R 6Ganem Don 478Silverman Robert H 2DeRisi Joseph L 18 1 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America 2 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America 3 Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America 4 Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America 5 Glickman Urological Institute, Cleveland Clinic, Cleveland, Ohio, United States of America 6 Anatomic and Clinical Pathology, Cleveland Clinic, Cleveland, Ohio, United States of America 7 Department of Medicine, University of California San Francisco, San Francisco, California, United States of America 8 Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, United States of America Ross Susan EditorUniversity of Pennsylvania School of Medicine, United States of America* To whom correspondence should be addressed. E-mail: [email protected] (JLD); [email protected] (RHS)3 2006 31 3 2006 2 3 e2529 11 2005 23 2 2006 © 2006 Urisman et al.2006This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals. Synopsis Prostate cancer is the most frequent cancer and the second leading cause of cancer deaths in US men over the age of 50. Several genetic factors have been proposed as potential risk factors for the development of prostate cancer, including a viral defense gene called RNASEL. A common genetic variant in this gene, R462Q, was recently implicated in up to 13% of prostate cancer cases. Given the antiviral role of RNASEL, the authors sought to examine if a virus might be present in prostate cancers associated with the R462Q variant. Using a DNA microarray designed to detect all known viral families, the authors identified a novel virus, named XMRV, in a subset of prostate tumor samples. Polymerase chain reaction testing of 86 prostate tumors for the presence of XMRV revealed a strong association between the presence of the virus and being homozygous for the R462Q variant. Cloning and sequencing of the virus showed that XMRV is a close relative of several known xenotropic murine leukemia viruses. This report presents the first documented cases of human infection with a xenotropic retrovirus. Future work will address the potential connection between XMRV infection and the increased prostate cancer risk in patients with the R462Q RNASEL variant. Citation:Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, et al. (2006) Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog 2(3): e25. ==== Body Introduction Type I interferons (IFNs) are rapidly mobilized in response to viral infection and trigger potent antiviral responses. One such response is the induction by IFN of a family of 2′5′ oligoadenylate synthetases (OAS); upon activation by virally encoded dsRNA, these enzymes produce 5′-phosphorylated 2′-5′ linked oligoadenylates (2–5A) from ATP [1]. 2–5A, in turn, is an activator of ribonuclease L (RNase L) [2], which degrades viral (and cellular) single stranded RNAs [3]. In vivo evidence for the antiviral role of the 2–5A system was provided by studies with RNase L−/− mice, which have enhanced susceptibility to infections by the picornaviruses, encephalomyocarditis virus, and Coxsackievirus B4 [4,5]. Ultimately, sustained activation of RNase L triggers a mitochondrial pathway of apoptosis that eliminates virus-infected cells [4,6–8]. Genetic lesions in RNase L impair this apoptotic response, which has raised interest in the possibility that such mutations might also contribute to malignancy [9]. In this context, several recent studies have linked germline mutations in RNase L to prostate cancer susceptibility [10–13]. Prostate cancer has a complex etiology influenced by androgens, diet, and other environmental and genetic factors [14]. While sporadic prostate cancer displays an age-related increase in prevalence, familial prostate cancer kindreds often display early-onset disease. Such kindreds, defined by having more than three affected members per family, account for 43% of early onset cases (<55 years old) and 9% of all cases [15]. The genetics of hereditary prostate cancer (HPC) is complex, and several genes have been proposed as susceptibility factors in this syndrome. Interestingly, one of these, HPC1, is linked to RNASEL [10,11]. Several germline mutations or variants in HPC1/RNASEL have been observed in HPC [10–13] (reviewed in [16]), including a common (35% allelic frequency) missense variant of RNase L, in which a G to A transition at nucleotide (nt) position 1385 (G1385A) results in a glutamine instead of arginine at amino acid position 462 (R462Q). Remarkably, a large, controlled sib-pair study implicated the R462Q RNase L variant in up to 13% of unselected prostate cancer cases [11]. One copy of the mutated gene increased the risk of prostate cancer by about 50%, whereas individuals that were homozygous for the mutation had a 2-fold increased risk of prostate cancer. The R462Q RNase L variant had a 3-fold decrease in catalytic activity compared with the wild-type enzyme [9,11]. However, while several case-controlled genetic and epidemiologic studies support the involvement of RNASEL (and notably the R462Q variant) in prostate cancer etiology [10–13], others do not [17–19], suggesting that either population differences or environmental factors may modulate the impact of RNASEL on prostatic carcinogenesis. While the antiapoptotic phenotype of RNase L deficiency has dominated previous discussions of its possible linkage to cancer, RNase L is also a key effector of the antiviral action of interferons. This led us to consider the possibility that the putative linkage of RNase L alterations to HPC might reflect enhanced susceptibility to a viral agent. To test this hypothesis, we have examined RNA derived from wild-type and RNase L variant (R462Q) prostate tumors for evidence of viral sequences, by hybridization to a DNA microarray composed of the most conserved sequences of all known human, animal, plant, and bacterial viruses [20,21]. Here we report that 40% (eight of 20) of all tumors homozygous for the R462Q allele harbored the genome of a distinct gammaretrovirus closely related to xenotropic murine leukemia viruses (MuLVs). In contrast, retroviral sequences were present in <2% of tumors bearing at least one copy of the wild-type allele (one of 66). In addition, virus-harboring cells were detected within infected prostatic tumor tissues by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). These findings represent the first detection of xenotropic MuLV–like agents in humans, and reveal a strong association between infection with the virus and defects in RNase L activity. The relation of retroviral infection to prostate cancer will require further study, but a cofactor role is not excluded. Results Detection of XMRV by Microarray-Based Screening To search for potential viruses in prostate cancer tumors, we employed a DNA microarray-based strategy designed to screen for viruses from all known viral families [20,21]. Total or polyadenylated RNA extracted from tumor tissue was first amplified and fluorescently labeled in a sequence-nonspecific fashion. The amplified and labeled fragments, which contained host as well as potential viral sequences, were then hybridized to a DNA microarray (Virochip, University of California San Francisco, San Francisco, United States) bearing the most conserved sequences of ~950 fully sequenced NCBI reference viral genomes (~11,000 70-mer oligonucleotides). The Virochip was used to screen RNA samples isolated from prostate tumors of 19 individuals (Figure 1). A positive hybridization signal suggestive of a gammaretrovirus was detected in seven of 11 tumors from patients homozygous for the R462Q RNASEL variant (QQ). In contrast, no virus was detected in three tumors from RQ heterozygotes, and only one of five tumors from RR individuals was positive. Clustering of the microarray oligonucleotide intensities (Figure 1) revealed a similar hybridization pattern in all positive cases. Furthermore, a computational analysis using E-Predict, a recently described algorithm for viral species identification [22], suggested that the same or similar mammalian gammaretrovirus was present in all positive tumors (Table S1). Thus, the Virochip detected the presence of a probable gammaretrovirus in half of the QQ tumor samples and in only one non-QQ sample. Figure 1 XMRV Detection by DNA Microarrays and RT-PCR (A) Virochip hybridization patterns obtained for tumor samples from 19 patients. The samples (x-axis) and the 502 retroviral oligonucleotides present on the microarray (y-axis) were clustered using hierarchical clustering. The red color saturation indicates the magnitude of hybridization intensity. (B) Magnified view of a selected cluster containing oligonucleotides with the strongest positive signal. Samples from patients with QQ RNASEL genotype are shown in red, and those from RQ and RR individuals as well as controls are in black. (C) Results of nested RT-PCR specific for XMRV gag gene. Amplified gag PCR fragments along with the corresponding human GAPDH amplification controls were separated by gel electrophoresis using the same lane order as in the microarray cluster. Characterization of XMRV Genome To further characterize the virus, we recovered its entire genome from one of the tumors (VP35) (Figure 2). To obtain viral clones, we first employed a direct microarray recovery technique described previously [21]. Briefly, amplified nucleic acid from the tumor tissue, which hybridized to viral microarray oligonucleotides, was eluted from two specific spots. The eluted DNA was re-amplified, and plasmid libraries constructed from this material were screened by colony hybridization using the spots' oligonucleotides as probes. The array oligonucleotides used in this case derived from the LTR region of murine type C retrovirus (MTCR) and spleen focus-forming virus (SFFV) [23]. The largest recovered fragment was 415 nt in length, and had 96% nt identity to the LTR region of MTCR, a MuLV identified in the genome of a mouse myeloma cell line (T. Heinemeyer, unpublished data). These findings established that the virus in question was indeed a gammaretrovirus, and likely a relative of MuLVs. To clone and sequence the rest of the viral genome from sample VP35, we used tumor cDNA to PCR-amplify overlapping segments using primers derived from MTCR; gaps were closed using primers from earlier recovered clones (Figure 2B and Table S2). Using a similar strategy, we have also determined the full sequence of the virus from a second tumor, VP42. Finally, a complete viral genomic sequence from a third tumor case, VP62, was obtained by PCR amplification of two ~4 Kb–long overlapping fragments jointly spanning close to the entire length of the virus (Figure 2B). The three sequenced genomes share >98% nt identity overall and >99% amino acid (aa) identity for predicted open reading frames (ORFs), and thus represent the same virus. Figure 2 Complete Genome of XMRV (A) Schematic map of the 8185 nt XMRV genome. LTR regions (R, U5, U3) are indicated with boxes. Predicted open reading frames encoding Gag, Gag-Pro-Pol, and Env polyproteins are labeled in green. The corresponding start and stop codons (AUG, UAG, UGA, UAA) as well as the alternative Gag start codon (CUG) are shown with their nt positions. Similarly, splice donor (SD) and acceptor (SA) sites are shown and correspond to the spliced 3.2-Kb Env subgenomic RNA (wiggled line). (B) Cloning and sequencing of XMRV VP35 and VP62 genomes. Clones obtained by probe recovery from hybridizing microarray oligonucleotides (blue bar) or by PCR from tumor cDNA (black bars) were sequenced. Primers used to amplify individual clones (Table S2) were derived either from the genome of MTCR (black arrows) or from overlapping VP35 clones (blue arrows). (C) Genome sequence similarity plots comparing XMRV VP35 with XMRV VP42, XMRV VP62, MuLV DG-75, MTCR, and a set of representative non-ecotropic proviruses (mERVs) (see Materials and Methods). The alignments were made using AVID [81], and plots were generated using mVISTA [82] with the default window size of 100 nt. Y-axis scale for each plot represents percent nt identities from 50% to 100%. Sequences are labeled as xenotropic (X), polytropic (P), or modified polytropic (Pm). The full genome of the virus (Figures 2 and S1) is 8,185 nt long and is distinct from all known isolates of MuLV. The genome is most similar to the genomes of exogenous MuLVs, DG-75 cloned from a human B-lymphoblastoid cell line [24], and MTCR, with which it shares 94% and 93% overall nt sequence identity, respectively. The genome also shares up to 95% nt identity with several full-length Mus musculus endogenous proviruses (Figure 2C). Phylogenetic trees constructed using available mammalian type C retroviral genomes and representative full-length proviral sequences from the mouse genome (Figures 3 and S2) showed that the newly identified virus is more similar to xenotropic and polytropic than to ecotropic genomes. Based on these findings we propose the provisional name Xenotropic MuLV-related virus (XMRV) for this agent. Figure 3 Phylogenetic Analysis of XMRV Based on Complete Genome Sequences Complete genomes of XMRV VP35, VP42, and VP62 (red); MTCR; MuLVs DG-75, AKV, Moloney, Friend, and Rauscher; feline leukemia virus (FLV); koala retrovirus (KoRV); gibbon ape leukemia virus (GALV); and a set of representative non-ecotropic proviruses (mERVs) were aligned using ClustalX (see Materials and Methods). An unrooted neighbor-joining tree was generated based on this alignment, excluding gaps and using Kimura's correction for multiple base substitutions. Bootstrap values (n = 1000 trials) are indicated as percentages. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). Translation of the XMRV genomic sequence using ORF Finder [25] identified two overlapping ORFs coding for the full-length Gag-Pro-Pol and Env polyproteins. No exogenous coding sequences, such as viral oncogenes, could be detected in the XMRV genome. The predicted Gag polyprotein is 536 aa long and is most similar to a xenotropic provirus on M. musculus Chromosome 9, with which it shares 97% aa identity (Figure S2A). The Pro-Pol polyprotein is 1,197 aa long and has the highest aa identity with MuLV DG-75 and a xenotropic provirus on M. musculus Chromosome 4, 97% and 96%, respectively (Figure S2B). An amber (UAG) stop codon separates the Gag and Pro-Pol coding sequences, analogous to other MuLVs in which a translational read-through is required to generate the full-length Gag-Pro-Pol polyprotein (reviewed in [26]). Similar to other MuLVs [23,24,27–31], the Env polyprotein of XMRV is in a different reading frame compared with Gag-Pro-Pol. The Env protein sequence is 645 aa long, and has the highest amino acid identity with the Env protein of an infectious MuLV isolated from a human small cell lung cancer line NCI-417 [32] and MuLV New Zealand Black 9–1 xenotropic retrovirus (NZB-9–1) [28]), 95% and 94%, respectively. The XMRV Env protein also shares similarly high identity with several murine xenotropic proviruses (Figure S2C). Conserved splice donor (AGGTAAG, position 204) and acceptor (CACTTACAG, position 5,479) sites involved in the generation of env subgenomic RNAs [33] were found in the same relative locations as in other MuLV genomes. A multiple sequence alignment of XMRV Env and corresponding protein sequences of other representative MuLVs (Figure 4) showed that within three highly variable regions (VR), VRA, VRB, and VRC, known to be important for cellular tropism [34–36], XMRV has the highest aa identity with xenotropic envelopes from MuLVs NZB-9–1, NFS-Th-1 [37], and DG-75. Although unique-to-XMRV aa are present in each of the three VRs, based on the overall similarity to the known xenotropic envelopes, we predict that the cellular receptor for XMRV is XPR1 (SYG1), the recently identified receptor for xenotropic and polytropic MuLVs [38–40]. Figure 4 Multiple-Sequence Alignment of Protein Sequences from XMRV and Related MuLVs Spanning SU Glycoprotein VRA, VRB, and VRC, Known to Determine Receptor Specificity Env protein sequence from XMRV (identical in VP35, VP42, and VP62; red); MTCR; MuLVs DG-75, NZB-9–1, NFS-Th-1, MCF247, AKV, Moloney, Friend, and Rauscher; and polytropic proviruses MX27 and MX33 [77] were aligned using ClustalX. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). VRs are boxed. Dots denote residues identical to those from XMRV, and deleted residues appear as spaces. The long terminal repeat (LTR) of XMRV is 535 nt long and has the highest nt identity with the LTRs from xenotropic MuLVs NFS-Th-1 (96%) and NZB-9–1 (94%). The XMRV LTRs contain known structural and regulatory elements typical of other MuLV LTRs [33,41]. In particular, the CCAAT box, TATAAAA box, and AATAAA polyadenylation signal sequences were found in U3 at their expected locations (Figure S3A). U3 also contains a glucocorticoid response element sequence AGA ACA GAT GGT CCT. Essentially identical sequences are present in genomes of other MuLVs. These elements have been shown to activate LTR-directed transcription and viral replication in vitro in response to various steroids including androgens [42–45]. In addition, presence of an intact glucocorticoid response element is thought to be the determinant of higher susceptibility to FIS-2 MuLV infection in male compared with female NMRI mice [46,47]. Despite these similarities, single nt substitutions unique to XMRV and an insertion of an AG dinucleotide immediately downstream from the TATA box are present in U3 (Figure S3A). Consistent with these findings, a phylogenetic analysis based on U3 sequences from XMRV and from representative xenotropic MuLV provirus groups [48,49] showed that XMRV U3 sequences formed a well-separated cluster most similar to the group containing NFS-Th-1 and NZB-9–1 (Figure S3B). The 5′ gag leader of XMRV, defined as the sequence extending from the end of U5 to the ATG start codon of gag, consists of a conserved non-coding region of ~200 nt, containing a proline tRNA primer binding site as well as sequences required for viral packaging [50,51] and the initiation of translation [52,53]. The non-coding region is followed by a ~270-nt region extending from the conserved CTG alternative start codon of gag. This region represents the most divergent segment of the genome compared with other MuLVs (Figures 5 and 2C). Unlike ecotropic MuLVs, where translation from this codon adds an ~90 aa N-terminal leader peptide in frame with the rest of the Gag protein, thus generating a glycosylated form of Gag [54], XMRV has a stop codon 53 aa residues downstream from the alternative start. Interestingly, both MuLV DG-75 and MTCR gag leader sequences are also interrupted by stop codons, and therefore are not expected to produce full-length glyco-Gag. Furthermore, a characteristic 24-nt deletion was present in this region of the XMRV genome, which is not found in any known exogenous MuLV isolate. However, a shorter deletion of nine nt internal to this region is present in the sequences of several non-ecotropic MuLV proviruses found in the sequenced mouse genome (Figure 5). In cell culture, expression of intact glyco-Gag is not essential for viral replication [55,56]. However, lesions in this region have been associated with interesting variations in pathogenetic properties in vivo [57–61]. For example, an alteration in ten nt affecting five residues in the N-terminal peptide of glyco-Gag was found to be responsible for a 100-fold difference in the frequency of neuroinvasion observed between CasFrKP and CasFrKP41 MuLV strains [62]. In addition, insertion of an octanucleotide resulting in a stop codon downstream of the CUG start codon prevented severe early hemolytic anemia and prolonged latency of erythroleukemia in mice infected with Friend MuLV [58]. While we do not yet know the pathogenetic significance of the lesions in XMRV glyco-Gag, the high degree of sequence divergence suggests that this region may be under positive selective pressure and therefore may be relevant to the establishment of infection within the human host. Figure 5 Multiple-Sequence Alignment of 5′ gag Leader Nucleotide Sequences from XMRV and Related MuLVs Sequences extending from the alternative CUG start codon to the AUG start codon (underlined) of gag derived from XMRV VP35, VP42, and VP62 (blue); MTCR, MuLVs DG-75, and Friend; and a set of representative non-ecotropic proviruses (mERVs) were aligned with ClustalX (see Materials and Methods). Predicted amino acid translation corresponding to the VP35 sequence is shown above the alignment (red); asterisk indicates a stop. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). Dots denote nt identical to those from XMRV, and deleted nt appear as spaces. Association of XMRV Infection and R462Q RNASEL Genotype To further examine the association between presence of the virus and the R462Q (1385G->A) RNASEL genotype, we developed a specific nested RT-PCR assay based on the virus sequence recovered from one of the tumor samples (VP35, see above). The primers in this assay (Figure S1) amplify a 380-nt fragment from the divergent 5′ leader and the N-terminal end of gag. The RT-PCR was positive in eight (40%) of 20 examined tumors from homozygous (QQ) individuals. In addition, one tumor from a homozygous wild-type (RR) patient was positive among 52 RR and 14 RQ tumors examined (Figure 1 and Table 1). Interestingly, this case was associated with the highest tumor grade among all XMRV-positive cases (Table S3). PCR specific for the mouse GAPDH gene was negative in all samples (unpublished data), arguing strongly against the possibility that the tumor samples were contaminated with mouse nucleic acid. Collectively, these data demonstrate a strong association between the homozygous (QQ) R462Q RNASEL genotype and presence of the virus in the tumor tissue (p < 0.00002 by two-tail Fisher's exact test). Table 1 XMRV Screening by gag Nested RT-PCR XMRV Sequence Diversity in Samples from Different Patients To examine the degree of XMRV sequence diversity in different patients, we sequenced the amplified fragments from all nine samples, which were positive by the nested gag RT-PCR. The amplified gag fragments were highly similar (Figure 6A) with >98% nt and >98% aa identity to each other. In contrast, the fragments had <89% nt and <95% aa identity with the most related exogenous sequence of MuLV DG-75. Several corresponding endogenous non-ecotropic sequences were more similar to the XMRV fragments, including the xenotropic provirus from M. musculus Chromosome 9, which was <98% identical on the nt level. Nevertheless, all XMRV-derived fragments were more similar to each other than they were to any other sequence. Figure 6 Comparison of XMRV Sequences Derived from Tumor Samples of Different Patients (A) Phylogenetic tree based on the 380 nt XMRV gag RT-PCR fragment from the nine positive tumor samples (red) and the corresponding sequences from MTCR; MuLVs DG-75, MCF1233, Akv, Moloney, Rauscher and Friend; and a set of representative non-ecotropic proviruses (mERVs). The sequences were aligned using ClustalX, and the corresponding tree was generated using the neighbor-joining method (see Materials and Methods). Bootstrap values (n = 1000 trials) are indicated as percentages. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). (B) Phylogenetic tree based on a 2500-nt pol PCR fragment from the 9 XMRV-positive tumor samples. The tree was constructed as described in (A). In addition to the gag gene, we also examined the same patient samples for sequence variation in the pol gene. We sequenced PCR fragments obtained with a set of primers targeting a 2500-nt stretch in the pol gene (Figure S1). Similar to the gag fragments, the amplified pol fragments were highly similar (Figure 6B) and had >97% nt and >97% aa identity to each other. In contrast, the fragments had <94% nt and <95% aa identity with the most related sequence, that of MuLV DG-75. Interestingly, XMRV-derived pol sequences were less similar to and approximately equidistant from the examined representative xenotropic and polytropic endogenous sequences. Close clustering of the sequenced gag and pol fragments (Figure 6) indicates that all microarray and RT-PCR positive cases represent infection with the same virus. On the other hand, the degree of sequence variation in the examined fragments is higher than that expected from errors introduced during PCR amplification and sequencing. The frequency of nt misincorporation by Taq polymerase has been estimated as 10−6 − 10−4 ([63] and references therein), compared with the observed rate of up to 2% in the gag and pol fragments. These findings suggest that the observed XMRV sequence variation is a result of natural sequence diversity, consistent with the virus being independently acquired by the affected patients, and argue against laboratory contamination as a possible source of XMRV. Detection of XMRV in Tumor-Bearing Prostatic Tissues Using FISH To localize XMRV within human prostatic tissues, and to measure the frequency of the infected cells, XMRV nucleic acid was visualized using FISH on formalin-fixed prostate tissues. A SpectrumGreen fluorescently labeled FISH probe cocktail spanning all viral genes was prepared using cDNA derived from the XMRV isolate cloned from patient VP35 (Materials and Methods). Distinct FISH-positive cells were observed in the tumors positive for XMRV by RT-PCR (e.g., VP62 and VP88) (Figure 7). To identify cell types associated with the positive FISH signal, the same sections were subsequently stained with hematoxylin and eosin (H&E). Most FISH-positive cells were stromal fibroblasts (Figure 8A), including those undergoing cell division (Figure 8B). In addition, occasional infected hematopoietic cells were also seen (Figure 8C). XMRV FISH with concurrent immunostaining for cytokeratin AE1/AE3 to achieve specific labeling of epithelial cells [64] showed no XMRV-infected cells that also had the epithelium-specific staining, confirming their non-epithelial origin (Figure 8C). While the XMRV nucleic acid was usually present within nuclei (Video S1), suggesting integrated proviral DNA, some cells showed cytoplasmic staining adjacent to the nucleus, suggestive of viral mRNA and/or pre-integration complexes in non-dividing cells (Figure 8A). Figure 7 Detection of XMRV Nucleic Acid in Prostatic Tissues Using FISH Prostatic tumor tissue sections from QQ cases VP62 (A–C) and VP88 (D–F) were analyzed by FISH using DNA probes (green) derived from XMRV VP35 (top right enlargements). Nuclei were counterstained with DAPI. The same sections were then visualized by H&E staining (left panels). Scale bars are 10 μm. Arrows indicate FISH positive cells, and their enlarged images are shown in the bottom right panels. Figure 8 Characterization of XMRV-Infected Prostatic Cells by FISH and FISH/Immunofluorescence Using a tissue microarray, prostatic tumor tissue sections from QQ case VP62 were analyzed by FISH (green) using DNA probes derived from XMRV VP35 (left panels). Nuclei were counterstained with DAPI. The same sections were then visualized by H&E staining (middle panels). Arrows indicate FISH-positive cells, and their enlarged FISH and H&E images are shown in the top right and bottom right panels, respectively. Scale bars are 10 μm. (A) A stromal fibroblast. (B) A dividing stromal cell. (C) A stromal hematopoietic cell. The section was concomitantly stained for XMRV by FISH (green) and cytokeratin AE1/AE3 by immunofluorescence (red). We also used FISH to obtain a minimal estimate of the frequency of XMRV-infected prostatic cells. For this purpose we employed a tissue microarray containing duplicates of 14 different prostate cancer tissue specimens (Table 2). FISH with DNA probes derived from XMRV VP35 showed five to ten XMRV/FISH–positive cells (about 1% of prostate cells observed) in each of five homozygous RNase L 462Q (QQ) cases: VP29, 31, 42, 62, and 88. Patient sample VP79, also a QQ case, contained two positive cells (0.4% of total cells examined). All of the XMRV/FISH–positive cells observed were stromal cells. In contrast, three RR tissue samples and two RQ tissue samples showed one or no (<0.15%) FISH-positive cells. Two of the QQ cases, VP35 and VP90, positive by gag RT-PCR, showed only one FISH-positive cell each (Table 2). Conversely, one case, VP31, was FISH-positive, but gag–RT-PCR negative. As expected, Chromosome 1–specific probes used as a positive control specifically labeled nearly every cell from the examined case VP88, whereas a KSHV-specific probe used as a negative control did not label any cells in sections from cases VP88 and VP51, but did efficiently label 293T cells transfected with KSHV DNA (unpublished data). Thus, consistent with the microarray and RT-PCR data, detection of XMRV by FISH was associated primarily with QQ cases. In addition, in samples where XMRV was detected, all positive cells were stromal and did not account for more than 1% of all prostatic cells. Finally, differences in the numbers of XMRV-positive cells detected in the different samples could be due to heterogeneity in virus copy numbers between different patients and/or specific regions of the prostate sampled. Table 2 Frequency of XMRV-Infected Prostatic Cells Determined by FISH Detection of XMRV in Tumor-Bearing Prostatic Tissues Using IHC To identify cells expressing XMRV proteins, we assayed for the presence of Gag protein using a monoclonal antibody against (SFFV); this antibody is reactive against Gag proteins from a wide range of different ecotropic, polytropic, and xenotropic MuLV strains [65]. Using this antibody, positive signal by IHC was observed in prostatic tissues of XMRV-positive cases VP62 and VP88, both QQ (Figure 9). An enhanced alkaline phosphatase red detection method allowed Gag detection in the same cells with both fluorescence (Figure 9A–9D, left) and bright field (Figure 9A–9D, middle) microscopy. The Gag-expressing cells were observed in prostatic stromal cells with a distribution and frequency similar to that detected by FISH (Figure 9 and unpublished data). In contrast, no Gag-positive cells were observed in VP51 prostatic tissue, which is of RR genotype (Figure 9E). Figure 9 Detection of XMRV Protein in Prostatic Tissues Using Immunostaining Prostatic tumor tissue sections from QQ cases VP62 (A and B) and VP88 (C and D), as well as an RR case VP51 (E) were stained, then visualized by immunofluorescence (left) or bright field (middle) using a monoclonal antibody to SFFV Gag protein. Nuclei are counterstained with hematoxylin. Enlarged images corresponding to the positive cells are shown on the right. Scale bars are 5 μm in (A), (B), and (E) and 10 μm in (C) and (D). Discussion The results presented here identify XMRV infection in prostate tissue from approximately 40% of patients with prostate cancer who are homozygous for the R462Q variant (QQ) of RNase L, as judged by both hybridization to the Virochip microarray and by RT-PCR with XMRV-specific primers. Parallel RT-PCR studies of prostate tumors from wild-type (RR) and heterozygous (RQ) patients revealed evidence of XMRV in only one of 66 samples, clearly demonstrating that human XMRV infection is strongly linked to decrements in RNase L activity. This result supports the view that the R462Q RNase L variant leads to a subtle defect in innate (IFN-dependent) antiviral immunity. As its name indicates, XMRV is closely related to xenotropic murine leukemia viruses (MuLVs). Unlike ecotropic MuLVs, such as the canonical Moloney MuLV, which grow only in rodent cells in culture, xenotropic MuLVs can grow in non-rodent cells in culture but not in rodent cell lines. Xenotropic viruses have been isolated from many inbred as well as wild mouse strains. Studies of the distribution of non-ecotropic sequences in different mouse strains show that the diversity of xenotropic proviral sequences in wild mice is greater than that found in the inbred laboratory strains [49,66]. This finding led to the conclusion that these endogenous elements were independently and relatively recently acquired by different mouse species as a result of infection rather than inheritance [49]. Unlike ecotropic MuLVs, which can only recognize a receptor (CAT-1) specific to mouse and rat species [67–69], xenotropic viruses recognize a protein known as XPR1 or SYG1. XPR1 is expressed in all higher vertebrates, including mice, but polymorphisms in the murine gene render it unable to mediate xenotropic MuLV entry [38–40]. Thus, xenotropic MuLVs have a potential to infect a wide variety of mammalian species, including humans. Xenotropic MuLVs have occasionally been detected in cultured human cell lines. For example, MuLV DG-75 was cloned from a human B-lymphoblastoid cell line [24], and an infectious xenotropic MuLV was detected in a human small cell lung cancer line NCI-417 [32]. Although laboratory contamination, either in culture or during passage of cell lines in nude mice, cannot be ruled out as a possible source in these cases, such contamination cannot explain our results. The evidence for this is as follows: (i) XMRV was detected in primary human tissues; (ii) no murine sequences (e.g., GAPDH) could be detected in our materials by PCR; (iii) infection was predominantly restricted to human samples with the QQ RNASEL genotype; (iv) polymorphisms were found in the XMRV clones recovered from different patients consistent with independent acquisition of the virus by these individuals; and (v) viral nucleic acids and antigens could be detected in infected QQ prostate tissue by FISH and IHC, respectively. Taken together, the above evidence argues strongly against laboratory contamination with virus or cloned DNA material as the source of XMRV infection in the analyzed samples. To our knowledge, this report represents the first published examples of authentic infection of humans with a xenotropic MuLV-like agent. Although our efforts to clone the sites of XMRV integration into the host genome have been limited by the small amounts of prostate tissue available for this purpose, our work to clone such sites is ongoing and will provide an important additional piece of evidence for XMRV infection in humans. The XMRV sequence is not found in human genomic DNA, and none of the human endogenous retroviruses, including the only known gammaretrovirus-like human endogenous sequences (hERVs E and T) [70], bare any significant similarity to the XMRV genome. This indicates that XMRV must have been acquired exogenously by infection in positive subjects. From what reservoir and by what route such infections were acquired is unknown. It seems unlikely that direct contact with feral mice could explain the observed distribution of infection in our cohort, since there is no reason to believe that rodent exposure would vary according to RNASEL genotype. It is possible that infection is more widespread than indicated by the present studies, especially if, as seems likely, individuals with the wild-type RNase L clear infection more promptly than those with the QQ genotype. But if so, a cross-species transfer model of XMRV infection would require improbably high levels of rodent exposure for a developed society like our own. Thus, although the viral sequence suggests that the ultimate reservoir of XMRV is probably the rodent, the proximate source of the infection seems unlikely to be mice or rats. Provisionally, we favor the notion that the XMRV infections we have documented were acquired from other humans, i.e., that XMRV may have been resident in the human population for some time. This speculation will, however, require direct epidemiologic validation. It also remains to be determined if RNase L R462Q homozygotes are more sensitive to the acquisition of infection, or are simply less likely to clear infection once acquired. This is an important issue, since if the latter model is correct, it would imply that in younger humans, XMRV prevalence may be higher than what is observed in our prostate cancer cohort (mean age 58.7 y). We are currently developing serologic assays for use in population-based studies that should shed light on these matters. While presented work documents a clear link of XMRV infection to RNase L deficiency, we emphasize that the data we have accumulated does not mandate any etiological link to prostate cancer. Furthermore, our finding that XMRV infection is targeted to stromal cells and not to carcinoma cells and the fact that the XMRV genome harbors no host-derived oncogenes rule out two classical models for retroviral oncogenesis: direct introduction of a dominantly acting oncogene and insertional activation of such a gene. However, more indirect contributions of the virus to the tumor can certainly be envisioned. Recent work has shown that stromal cells have an active role in directly promoting tumorigenesis of adjacent epithelial cells by producing various cytokines and growth factors that serve as proliferative signals [71] or indirectly by modifying the tumor microenvironment by promotion of angiogenesis or recruitment of inflammatory mediators leading to oxidative stress [72]. In particular, cancer-associated fibroblasts stimulate growth of human prostatic epithelial cells and alter their histology in vivo [73]. It is conceivable that XMRV-infected prostatic stromal cells could produce and secrete growth factors, cytokines or other factors that stimulate cell proliferation or promote oxidative stress in surrounding epithelia. Such a paracrine mechanism could still function quite efficiently even with the relatively small number of XMRV-infected cells that characterize the lesion. Finally, we note that the identification of an exogenous infection such as XMRV could help explain why not all genetic studies have consistently identified RNase L as a prostate cancer susceptibility factor. If such an infection were linked, however indirectly, to prostate cancer risk, and if the prevalence of infection is not uniform in different populations, populations with low XMRV prevalence might be expected to show no association of RNASEL lesions to prostate cancer. Clearly, resolution of these issues will require much further investigation. We need to determine the prevalence of XMRV infection in the general population, understand its routes of transmission and tissue tropism, explore its associations with pre-maligant and other prostatic conditions, and define the biochemical interactions of the virus with the 2–5A/RNase L system. The availability of molecular clones, infectious virus stocks, and susceptible cell culture systems should greatly enhance our ability to probe these and other questions in the near future. Materials and Methods Genotyping of patients, and prostate tissue processing. All human samples used in this study were obtained according to protocols approved by the Cleveland Clinic's Institutional Review Board. Age, clinical parameters, and geographical locations of XMRV-positive prostate cancer cases are provided in Table S3. Men scheduled to undergo prostatectomies at the Cleveland Clinic were genotyped for the R462Q (1385G->A) RNASEL variant using a premade TAQMAN genotyping assay (Applied Biosystems, Foster City, California, United States; Assay c_935391_1) on DNA isolated from peripheral blood mononuclear cells. Five nanograms of genomic DNA were assayed according to the manufacturer's instructions, and analyzed on an Applied Biosystems 7900HT Sequence Detection System instrument. Immediately after prostatectomies, tissue cores were taken from both the transitional zone (the site of benign prostatic hyperplasia, BPH) and the peripheral zone (where cancer generally occurs), snap-frozen in liquid nitrogen, and then stored at −80 °C. Remaining prostate tissue was fixed in 10% neutral buffered formalin, processed, and embedded in paraffin for later histological analyses. Frozen tissue cores were transferred from dry ice immediately to TRIZOL reagent (Invitrogen, Carlsbad, California, United States), homogenized with a power homogenizer or manually using a scalpel followed by a syringe, and total RNA was isolated according to the manufacturer's instructions. The prostate tissue RNA was then subjected to RNase-free DNase I (Ambion, Austin, Texas, United States) digestion for 30 min at 37 °C. The sample was then extracted with phenol and the RNA was precipitated with isopropanol overnight at −20 °C followed by centrifugation at 12,000 g for 30 min at 4 °C. Poly-A RNA was isolated from the DNase digested total RNA using the Oligotex mRNA Midi Kit (Qiagen USA, Valencia, California, United States) as instructed by the manufacturer. The poly-A RNA concentration was measured using the RIBOgreen quantitation kit (Molecular Probes, Invitrogen), and the samples were stored at −80 °C. Microarray screening. Virochip microarrays used in this study were identical to those previously described [20–22]. Prostate tumor RNA samples were amplified and labeled using a modified Round A/B random PCR method and hybridized to the Virochip microarrays as reported previously (Protocol S1 in [21]). Microarrays were scanned with an Axon 4000B scanner (Axon Instruments, Union City, California, United States) and gridded using the bundled GenePix 3.0 software. Microarray data have been submitted to the NCBI GEO database (GSE3607). Hybridization patterns were interpreted using E-Predict as previously described [22] (Table S1). To make Figure 1, background-subtracted hybridization intensities of all retroviral oligonucleotides (205) were used to cluster samples and the oligonucleotides. Average linkage hierarchical clustering with Pearson correlation as the similarity metric was carried out using Cluster (version 2.0) [74]. Cluster images were generated using Java TreeView (version 1.0.8) [75]. Genome cloning and sequencing. Amplified and labeled cDNA from the VP35 tumor sample was hybridized to a hand-spotted microarray containing several retroviral oligonucleotides, which had high hybridization intensity on the Virochip during the initial microarray screening. Nucleic acid hybridizing to two of the oligonucleotides (9628654_317_rc derived from MTCR: TTC GCT TTA TCT GAG TAC CAT CTG TTC TTG GCC CTG AGC CGG GGC CCA GGT GCT CGA CCA CAG ATA TCC T; and 9626955_16_rc derived from SFFV: TCG GAT GCA ATC AGC AAG AGG CTT TAT TGG GAA CAC GGG TAC CCG GGC GAC TCA GTC TGT CGG AGG ACT G) was then individually eluted off the surface of the spots and amplified by PCR with Round B primers. Preparation of the hand-spotted array, hybridization, probe recovery, and PCR amplification of the recovered material were carried out according to Protocol S1. The recovered amplified DNA samples were then cloned into pCR2.1-TOPO TA vector (Invitrogen), and the resulting libraries were screened by colony hybridization with the corresponding above oligonucleotides as probes. Hybridizations were carried out using Rapid-Hyb buffer (Amersham, Piscataway, New Jersey, United States) according to the manufacturer's protocol at 50 °C for 4 h. Eight positive clones were sequenced, of which two (one from each library; clones K1 and K2R1 in Figure 2A) were viral and had 94–95% nt identity to MTCR. To sequence the remainder of the VP35 genome as well as the entire genome from the VP42 tumor, we amplified fragments of the genome by PCR using either amplified (Round B) or unamplified (Round A) cDNA prepared for original Virochip screening. This was accomplished first using a combination of primers derived from the sequence of MTCR and earlier recovered clones of XMRV. The two overlapping fragments from VP62 were amplified by PCR from cDNA generated by priming poly-A RNA with random hexamers. All PCR primers are listed in Table S2. The amplified fragments were cloned into pCR2.1-TOPO TA vector (Invitrogen) and sequenced using M13 sequencing primers. Genome assembly was carried out using CONSED version 13.84 for Linux [76]. PCR. Screening of tumor samples by gag nested RT-PCR was carried out according to Protocol S2. PCR fragments in all positive cases were gel-purified using QIAEX II gel extraction kit (Qiagen), cloned into pCR2.1-TOPO TA vector (Invitrogen), and sequenced using M13 sequencing primers. Pol PCR was carried out using amplified cDNA (Round B material) as the template. Sequence of the primers used for amplification (2670F, 3870R, 3810F, and 5190R) is listed in Table S2. Amplified products were gel-purified using QIAEX II gel extraction kit (Qiagen), and purified products were directly used for sequencing. Phylogenetic analyses. Xenotropic mERV Chromosome 1, xenotropic mERV Chromosome 4, and xenotropic mERV Chromosome 9 were chosen by BLAST querying the NCBI nr database with the complete XMRV genomes and selecting the most similar full-length proviral sequences, all of which happened to have xenotropic envelopes (Figure S2C). Polytropic mERV Chromosome 7 and polytropic mERV Chromosome 11 were chosen by selecting NCBI nr full-length proviral sequences with envelopes most similar to a prototype polytropic clone MX27 [77]. Similarly, modified polytropic mERV Chromosome 7 and modified polytropic mERV Chromosome 12 were selected on the basis of similarity to a prototype-modified polytropic clone MX33 [77]. U3 analysis was performed using previously described reference sequences: Mcv18, Mcv3, Mxv2, Mcv11, Mxv11, and HEMV18 [49]; CWM-T-15, CWM-T-15–4, CWM-T-25a, and CWM-T-25b [48]. To generate the neighbor-joining tree of complete genomic sequences (Figure 3), the sequences were first manually edited to make all genomes the same length, i.e., R to R. The edited sequences were then aligned with ClustalX version 1.82 for Linux [78,79] using default settings. The tree was generated based on positions without gaps only; Kimura correction for multiple base substitutions [80] and bootstrapping with n = 1000 were also used. All other trees were generated as above, except sequences were first trimmed to the same length, gaps were included, and Kimura correction was not used, as using these parameters did not have any significant effect on the trees. Antibodies. Monoclonal antibody to SFFV Gag protein was produced from R187 cells ([65]; ATCC: CRL-1912) grown in DMEM (Media Core, Cleveland Clinic Foundation, Cleveland, Ohio, United States) with 10% ultra-low IgG FBS (Invitrogen) until confluent. Conditioned media was collected every three days from confluent cultures. Five ml of conditioned media per preparation was centrifuged at 168 × g for 5 min at 4 °C. Supernatant was filtered through a 0.22-μm syringe filter unit (Millipore, Billerica, Massachusetts, United States) and concentrated 16-fold in an Amicon ultrafiltration unit with a 100-kDa molecular weight cutoff membrane (Millipore). Sodium azide was added to a final concentration of 0.02%. Concomitant XMRV FISH/cytokeratin immunofluorescence was performed using a mouse anti-cytokeratin AE1/AE3 (20:1 mixture) monoclonal antibody (Chemicon International, Temecula, California, United States) capable of recognizing normal and neoplastic cells of epithelial origin. FISH. The XMRV-35 FISH probe cocktail was generated using both 2.15-kb and 1.84-kb segments of the viral genome obtained by PCR with forward primer-2345, 5′ ACC CCT AAG TGA CAA GTC TG 3′ with reverse primer-4495, 5′ CTG GAC AGT GAA TTA TAC TA 3′ and forward primer-4915, 5′ AAA TTG GGG CAG GGG TGC GA 3′ with reverse primer-6755, 5′ TTG GAG TAA GTA CCT AGG AC 3′, both cloned into pGEM-T (Promega, Madison, Wisconsin, United States). The recombinant vectors were digested with EcoRI to release the viral cDNA fragments, which were purified after gel electrophoresis (Qiagen). The purified viral cDNA inserts were used in nick translation reactions to produce SpectrumGreen dUTP fluorescently labeled probe according to manufacturer's instructions (Vysis Inc., Des Plaines, Illinois, United States). Freshly baked slides of prostatic tissues or tissue microarray arrays with ~4-μm thick tissue sections were deparaffinized, rehydrated, and subjected to Target Retrieval (Dako, Glostrup, Denmark) for 40 min at 95 °C. Slides were cooled to room temperature and rinsed in H2O. Proteinase K (Dako) at 1:5000 in Tris-HCl (pH 7.4) was applied directly to slides for 10 min at room temperature. Adjacent tissue sections were also probed with SpectrumGreen dUTP fluorescently labeled KSHV-8 DNA (nts 85820–92789) as a negative control or, as a positive control with SpectrumGreen and SpectrumOrange labeled TelVysion DNA Probe cocktail (Vysis), specific for subtelomeric regions of the P and Q arms of human Chromosome 1 as a positive control to ensure the tissue was completely accessible to FISH. FISH slides were examined using a Leica DMR microscope (Leica Micro-Systems, Heidelberg, Germany), equipped with a Retiga EX CCD camera (Q-Imaging,Vancouver, British Columbia, Canada). FISH images were captured using a Leica TCS SP2 laser scanning confocal with a 63× oil objective numerical aperature 1.4 (Leica Micro-Systems) microscope. XMRV nucleic acids were visualized using maximum intensity projections of optical slices acquired using a 488-nm argon-laser (emission at 500–550 nm). TelVysion DNA Probes were visualized using maximum intensity projections of optical slices acquired using a 488-nm argonlaser (emission at 500–550 nm) and 568-nm krypton-argon-laser (emission at 575–680 nm). DAPI was visualized using maximum intensity projections of optical slices acquired using a 364–nm UV-laser (emission at 400–500 nm). Slides were subsequently washed in 2× SSC (0.3 M sodium chloride and 0.03 M sodium citrate, [pH 7.0]) to remove coverslips, and H&E stained for morphological evaluation. IHC. IHC on human tissues was performed on a Benchmark Ventana Autostainer (Ventana Medical Systems, Tucson, Arizona, United States). Unstained, formalin-fixed, paraffin-embedded prostate sections were placed on electrostatically charged slides and deparaffinized followed by a mild cell conditioning achieved through the use of Cell Conditioner #2 (Ventana Medical Systems). The concentrated R187 monoclonal antibody against SFFV p30 Gag was dispensed manually onto the sections at 10 μg per ml and allowed to incubate for 32 min at 37 °C. Endogenous biotin was blocked in sections using the Endogenous Biotin Blocking Kit (Ventana Medical Systems). Sections were washed, and biotinylated ImmunoPure Goat Anti-Rat IgG (Pierce Biotechnology, Rockford, Illinois, United States) was applied at a concentration of 4.8 μg per ml for 8 min. To detect Gag protein localization, the Ventana Enhanced Alkaline Phosphatase Red Detection Kit (Ventana Medical Systems) was used. Sections were briefly washed in distilled water and counterstained with Hematoxylin II (Ventana Medical Systems) for approximately 6 min. Sections were washed, dehydrated in graded alcohols, incubated in xylene for 5 min, and coverslips were added with Cytoseal (Microm International, Walldorf, Germany). Negative controls were performed as above except without the addition of the R187 monoclonal antibody. Concomitant XMRV FISH/cytokeratin IHC was performed on slides of prostate tissue from patient VP62. First, sections were immunostained for cytokeratin AE1/AE3 using the Alexa Fluor 594 Tyramide Signal Amplification Kit (Molecular Probes, Invitrogen). Briefly, unstained, formalin-fixed, paraffin-embedded sections cut at ~4 μm were placed on electrostatically charged slides, baked at 65 °C for at least 4 h, deparaffinized in xylene, and rehydrated through decreasing alcohol concentrations. Slides were incubated in Protease II (Ventana Medical Systems) for 3 min at room temperature and washed in phosphate-buffered saline (PBS) in peroxidase quenching buffer (PBS + 3% H2O2) for 60 min at room temperature, then incubated with 1% blocking reagent (10 mg/ml BSA in PBS) for 60 min at room temperature. The slides were incubated with cytokeratin AE1/AE3 antibody diluted in 1% blocking reagent for 60 min at room temperature and rinsed 3× times in PBS. Goat anti-mouse IgG-horseradish peroxidase (Molecular Probes, Invitrogen) was added and incubated for 60 min at room temperature. The slides were rinsed 3× in PBS. The tyramide solution was added to the slides for 10 min at room temperature and the slides were rinsed 3× in PBS. Slides were then placed in Target Retrieval solution (Dako) for 40 min at 95 °C. FISH for XMRV was performed as described above except in the absence of proteinase K treatment. After FISH, the slides were mounted with Vectashield Mounting Medium plus DAPI (Vector Labs, Burlingame, California, United States) and examined using fluorescence microscopy. Immunofluorescence images were captured using a Texas red filter with a Leica DMR microscope (Leica Micro-Systems), equipped with a Retiga EX CCD camera (QImaging). Supporting Information Figure S1 Complete Nucleotide Sequence of XMRV VP35 Numbers to the left indicate nt coordinates relative to the first nt. Predicted open reading frames for Gag, Gag-Pro-Pol, and Env polyproteins are shown below the corresponding nt. Characteristic 24-nt deletion in the 5′ gag leader is indicated with a triangle. Other genome features as well as primers used in the nested gag RT-PCR are shown as arrows. (558 KB PDF) Click here for additional data file. Figure S2 Phylogenetic Analysis of XMRV Based on Predicted Gag, Pro-Pol, and Env Polyproteins Predicted Gag (A), Pro-Pol (B), and Env (C) sequences of XMRV VP35, VP42, and VP62 (red) as well as the corresponding sequences from MTCR; MuLVs DG-75, MCF1233, Akv, Moloney, Friend, and Rauscher; feline leukemia virus (FLV); koala retrovirus (KoRV); gibbon ape leukemia virus (GALV), and a set of representative non-ecotropic proviruses (mERVs) were aligned using ClustalX. The resulting alignments were used to generate unrooted neighbor-joining trees (see Materials and Methods). Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). (186 KB EPS) Click here for additional data file. Figure S3 Comparison of XMRV U3 Region to Representative Non-Ecotropic Sequences (A) Multiple sequence alignment of U3 sequences from XMRV VP35, VP42, and VP62; MuLVs NZB-9–1 and NFS-Th-1; and from representative non-ecotropic proviruses [37,48,49]. The sequences were aligned using ClustalX (see Materials and Methods). Only sequences most similar to XMRV are shown. Glucocorticoid response element (GRE), and TATA and CAT boxes are indicated by lines. Direct repeat regions (boxed) are numbered according to the existing convention [37,49]. Triangle indicates a 190 nt insertion in polytropic proviruses [37]. XMRV-specific AG dinucleotide insertion is shown in red. Dots denote nt identical to those from XMRV, and deleted nt appear as spaces. (B) Phylogenetic tree based on U3 nt sequences. Multiple sequence alignment from (A) was used to generate an unrooted neighbor-joining tree (see Materials and Methods). Bootstrap values (n = 1000 trials) are shown as percentages. U3 sequences from XMRV are shown in red. (188 KB EPS) Click here for additional data file. Protocol S1 Probe Recovery from Hand-Spotted Microarrays by “Scratching” (83 KB PDF) Click here for additional data file. Protocol S2 XMRV gag Nested RT-PCR (172 KB PDF) Click here for additional data file. Table S1 Computational Viral Species Predictions Using E-Predict for the Virochip Microarrays Shown in Figure 1 (48 KB DOC) Click here for additional data file. Table S2 PCR Primers Used for Sequencing of XMRV Genomes (45 KB DOC) Click here for additional data file. Table S3 Age, Clinical Parameters, and Geographical Locations of XMRV-Positive Prostate Cancer Cases (39 KB DOC) Click here for additional data file. Video S1 Confocal Optical Image Planes of a Representative XMRV FISH Positive Cell Optical image planes (0.5 μm step-size) of the XMRV FISH positive cell from Figure 1A acquired using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica, Heidelberg, Germany) were reconstructed into a 3D volume set using Volocity 3.5 (Improvision, Lexington, Massachusetts, United States). Using Volocity's movie sequence editor, each volume was rotated along horizontal and vertical axes, adjusting nuclei stained DAPI (blue) channel brightness to visualize underlying XMRV FISH (green) nucleic acid signal. The resulting image frames were exported as a movie sequence. Underlying grid represents glass slide to which tissue was placed for FISH analysis. Each square unit within grid represents 4 μm in height and width. (237 KB WMV) Click here for additional data file. Accession Numbers Accession numbers from Gen Bank (http://www.ncbi.nlm.nih.gov/Genbank) are: AKV MuLV (J01998), feline leukemia virus (NC_001940), Friend MuLV (NC_001372), gibbon ape leukemia virus (NC_001885), koala retrovirus (AF151794), modified polytropic mERV Chromosome 7 (AC127565; nt 64,355–72,720), modified polytropic mERV Chromosome 12 (AC153658; nt 85,452–93,817), Moloney MuLV (NC_001501), MTCR (NC_001702MuLV DG-75 (AF221065); MuLV MCF 1233 (U13766), MuLV NCI-417 (AAC97875), MuLV NZB-9–1 (K02730), polytropic mERV Chromosome 7 (AC167978; nt 57,453–65,805), polytropic mERV Chromosome 11 (168–229,176,580), prototype polytropic clone MX27 (M17327), Rauscher MuLV (NC_001819), xenotropic mERV Chromosome 1 (AC083892, nt 158,240–166,448), xenotropic mERV Chromosome 4 (AL627077; nt 146,400–154,635), xenotropic mERV Chromosome 9 (AC121813; nt 37,520–45,770), XMRV VP35 (DQ241301), XMRV VP42 (DQ241302), and XMRV VP 62 (DQ399707). We thank Silvi Rouskin, Shoshannah Beck, James Pettay, and Jayashree Paranjape for expert technical assistance; Sanggu Kim and Samson A. Chow for technical advice; Earl Poptic for production and purification of monoclonal antibodies to Gag; Stephen T. Koury for advice; and Judith A. Drazba and Amit Vasanji for assistance with confocal imaging. Author contributions. AU, RJM, NF, DG, RHS, and JLD conceived and designed the experiments. AU, RJM, NF, SJP, KM, CMG, and RRT performed the experiments. AU, RJM, NF, SJP, GC, EAK, KM, CMG, RRT, DG, RHS, and JLD analyzed the data. AU, RJM, NF, SJP, GC, EAK, DG, RHS, and JLD contributed reagents/materials/analysis tools. AU, RJM, NF, DG, RHS, and JLD wrote the paper. Competing interests. The authors have declared that no competing interests exist. Funding. This investigation was supported by Genentech Graduate Fellowship and a grant from the Sandler Family Supporting Foundation (AU), grants from Doris Duke Charitable Foundation (JLD and DG) and the David and Lucille Packard Foundation (JLD), Howard Hughes Medical Institute (JLD and DG), by NIH/NCI grants (to RHS and GC), and a Molecular Medicine Fellowship from Cleveland State University and the Cleveland Clinic Foundation (RJM). Abbreviations 2–5A5′-phosphorylated 2′-5′ oligoadenylate aaamino acid FISHfluorescence in situ hybridization H&Ehematoxylin and eosin HPChereditary prostate cancer IFNinterferon IHCimmunohistochemistry LTRlong terminal repeat MCFmink cell focus-inducing murine leukemia virus MTCRmurine type C retrovirus MuLVmurine leukemia virus ntnucleotide(s) NZB-9–1New Zealand Black 9–1 xenotropic retrovirus ORFopen reading frame PBSphosphate-buffered saline PCRpolymerase chain reaction QQ RNASEL homozygous R462Q QR RNASEL heterozygous R462Q RNase LRibonuclease L RR RNASEL homozygous wild-type SFFVspleen focus-forming virus VRvariable region XMRVxenotropic MuLV-related virus XPR1xenotropic and polytropic retrovirus receptor ==== Refs References Kerr IM Brown RE 1978 pppA2'p5'A2'p5'A: An inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells Proc Natl Acad Sci U S A 75 256 260 272640 Zhou A Hassel BA Silverman RH 1993 Expression cloning of 2–5A-dependent RNAase: A uniquely regulated mediator of interferon action Cell 72 753 765 7680958 Dong B Silverman RH 1995 2–5A-dependent RNase molecules dimerize during activation by 2–5A J Biol Chem 270 4133 4137 7876164 Zhou A Paranjape J Brown TL Nie H Naik S 1997 Interferon action and apoptosis are defective in mice devoid of 2′,5′-oligoadenylate-dependent RNase L EMBO J 16 6355 6363 9351818 Flodstrom-Tullberg M Hultcrantz M Stotland A Maday A Tsai D 2005 RNase L and double-stranded RNA-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection J Immunol 174 1171 1177 15661870 Castelli JC Hassel BA Wood KA Li XL Amemiya K 1997 A study of the interferon antiviral mechanism: Apoptosis activation by the 2–5A system J Exp Med 186 967 972 9294150 Li G Xiang Y Sabapathy K Silverman RH 2004 An apoptotic signaling pathway in the interferon antiviral response mediated by RNase L and c-Jun NH2-terminal kinase J Biol Chem 279 1123 1131 14570908 Malathi K Paranjape JM Ganapathi R Silverman RH 2004 HPC1/RNASEL mediates apoptosis of prostate cancer cells treated with 2′,5′-oligoadenylates, topoisomerase I inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand Cancer Res 64 9144 9151 15604285 Xiang Y Wang Z Murakami J Plummer S Klein EA 2003 Effects of RNase L mutations associated with prostate cancer on apoptosis induced by 2′,5′-oligoadenylates Cancer Res 63 6795 6801 14583476 Carpten J Nupponen N Isaacs S Sood R Robbins C 2002 Germline mutations in the ribonuclease L gene in families showing linkage with HPC1 Nat Genet 30 181 184 11799394 Casey G Neville PJ Plummer SJ Xiang Y Krumroy LM 2002 RNASEL Arg462Gln variant is implicated in up to 13% of prostate cancer cases Nat Genet 32 581 583 12415269 Rennert H Bercovich D Hubert A Abeliovich D Rozovsky U 2002 A novel founder mutation in the RNASEL gene, 471delAAAG, is associated with prostate cancer in Ashkenazi Jews Am J Hum Genet 71 981 984 12145743 Rokman A Ikonen T Seppala EH Nupponen N Autio V 2002 Germline alterations of the RNASEL gene, a candidate HPC1 gene at 1q25, in patients and families with prostate cancer Am J Hum Genet 70 1299 1304 11941539 Nelson WG De Marzo AM Isaacs WB 2003 Prostate cancer N Engl J Med 349 366 381 12878745 Carter BS Bova GS Beaty TH Steinberg GD Childs B 1993 Hereditary prostate cancer: Epidemiologic and clinical features J Urol 150 797 802 8345587 Silverman RH 2003 Implications for RNase L in prostate cancer biology Biochemistry 42 1805 1812 12590567 Downing SR Hennessy KT Abe M Manola J George DJ 2003 Mutations in ribonuclease L gene do not occur at a greater frequency in patients with familial prostate cancer compared with patients with sporadic prostate cancer Clin Prostate Cancer 2 177 180 15040862 Wiklund F Jonsson BA Brookes AJ Stromqvist L Adolfsson J 2004 Genetic analysis of the RNASEL gene in hereditary, familial, and sporadic prostate cancer Clin Cancer Res 10 7150 7156 15534086 Maier C Haeusler J Herkommer K Vesovic Z Hoegel J 2005 Mutation screening and association study of RNASEL as a prostate cancer susceptibility gene Br J Cancer 92 1159 1164 15714208 Wang D Coscoy L Zylberberg M Avila PC Boushey HA 2002 Microarray-based detection and genotyping of viral pathogens Proc Natl Acad Sci U S A 99 15687 15692 12429852 Wang D Urisman A Liu YT Springer M Ksiazek TG 2003 Viral discovery and sequence recovery using DNA microarrays PLoS Biol 1 e2. 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==== Front Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-5-111655632010.1186/1476-4598-5-11ReviewApplication of cytology and molecular biology in diagnosing premalignant or malignant oral lesions Mehrotra Ravi [email protected] Anurag [email protected] Mamta [email protected] Rahela [email protected] Associate Professor Department of Pathology, Moti Lal Nehru Medical College, Allahabad, India2 Postgraduate Student Department of Pathology, Moti Lal Nehru Medical College, Allahabad, India3 Professor and Head Department of Pathology, Moti Lal Nehru Medical College, Allahabad, India4 Research Fellow Department of Pathology, Moti Lal Nehru Medical College, Allahabad, India2006 23 3 2006 5 11 11 15 8 2005 23 3 2006 Copyright © 2006 Mehrotra et al; licensee BioMed Central Ltd.2006Mehrotra et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Early detection of a premalignant or cancerous oral lesion promises to improve the survival and the morbidity of patients suffering from these conditions. Cytological study of oral cells is a non-aggressive technique that is well accepted by the patient, and is therefore an attractive option for the early diagnosis of oral cancer, including epithelial atypia and squamous cell carcinoma. However its usage has been limited so far due to poor sensitivity and specificity in diagnosing oral malignancies. Lately it has re-emerged due to improved methods and it's application in oral precancer and cancer as a diagnostic and predictive method as well as for monitoring patients. Newer diagnostic techniques such as "brush biopsy" and molecular studies have been developed. Recent advances in cytological techniques and novel aspects of applications of scraped or exfoliative cytology for detecting these lesions and predicting their progression or recurrence are reviewed here. ==== Body Introduction Oral cancer is the most common cancer and constitutes a major health problem in developing countries, representing the leading cause of death. Although representing 2–4% of the malignancies in the West, this carcinoma accounts for almost 40% of all cancers in the Indian subcontinent [1]. A key factor in the lack of improvement in prognosis over the years is the fact that a significant proportion of oral squamous cell carcinoma (OSCC) are not diagnosed or treated until they reach an advanced stage. This diagnostic delay may be caused by either patients (who may not report unusual oral features) or by health care workers (who may not investigate observed lesions thoroughly) and it is presumed that such delays are longer for asymptomatic lesions. The prognosis for patients with OSCC that is treated early is much better, with 5-year survival rates as high as 80%. In addition, the quality of life improves after early treatment, because cure can be achieved with less complex and less aggressive treatment than is necessary for advanced lesions. A significant proportion of oral squamous cell carcinomas (OSCC) develop from premalignant lesions such as leukoplakia and oral submucous fibrosis (Fig. 1). Adjuncts for detection of lesions and selection of biopsy sites include vital tissue staining (with Toluidine blue Fig. 2) and exfoliative cytology. Unfortunately, sensitivity of cytological diagnosis in a meta-analysis of 1306 cases from 14 studies showed an average of only 87.4% (ranging from 73.8 to 100%)[2]. Histological examination of tissue remains the gold standard for diagnosis and identification of malignant oral lesions. Biopsy is an invasive technique with surgical implications, technique limitations for professionals and psychological implications for most patients. It also presents limitations when the lesions are large and in these cases it is important to select the most appropriate site of biopsy. Furthermore, even though the biopsy study is fundamental, it is a diagnostic method with limited sensitivity where one of the most important features is the subjective interpretation of the examining pathologist. These issues underline the importance of discovering and developing new diagnostic methods, improving the existing ones and discovering new therapeutics targets for oral neoplastic diseases [3-6]. In recent decades, we have seen a dramatic switch from histopathological to molecular methods of disease diagnosis and exfoliative cytology has gained importance as a rapid and simple method for obtaining DNA samples. Changes occur at the molecular level before they are seen under the microscope and before clinical changes occur. Identification of high-risk oral premalignant lesions and intervention at premalignant stages could constitute one of the keys to reducing the mortality, morbidity and cost of treatment associated with OSCC. In addition, certain patients are known to be at high risk for head and neck cancer, specifically those who use tobacco or alcohol and those over 45 years of age. Such patients can be screened by physical examination, and early-stage disease, if detected, is curable. Just as visual inspection of the uterine cervix has been shown to be an unreliable means of identifying precancer and cancer, clinical inspection of the oral cavity has been shown to be equally unreliable in identifying precursor lesions and early cancers. [7,8]. In a recent study of 647 lesions interpreted by academicians to be innocuous on clinical inspection, 29 (4.5%) were confirmed to be dysplasia or carcinoma [9]. Figure 1 Clinical picture of a patient with oral submucous fibrosis of lower lip Figure 2 Clinical picture of a patient with dysplasia of lower lip showing positive toluidine blue staining. Cytological techniques Oral brush biopsy Oral cells can be obtained by different physical systems of scraping the surface of the mucosa, by rinsing the oral cavity or even by taking a sample of saliva from the patients. The reliability of the different instruments used in oral exfoliative cytology has been reviewed in different studies [10,11]. The ideal instrument used for making a good cytological smear should be easy to use in any location, cause minimum trauma and provide an adequate and representative number of epithelial cells [11]. It has been shown that a brush is an adequate instrument due to its ease in sampling and to the quality of the oral cytologic sample (Fig. 3). Brush biopsy is a simple, relatively inexpensive, high sensitive, risk-free method of screening for cancer and serves as an aid to the clinical examination (Fig. 6, 7, 8). The improved accuracy is attributed to the ease in obtaining full transepithelial cellular samples and the evaluation of smears with an image analysis system that has been adapted specifically to detect oral epithelial abnormalities by some workers [12]. Full-thickness sampling (indicated by pinpoint bleeding during procedure Fig. 4) is essential if histomorphological, evaluation of the collected cells is to yield representative findings. For example, many dysplastic lesions are first identified in the basal epithelial layers, and the diagnostic histomorphological findings may be lost as the cells mature and parakeratin and keratin are produced (Fig. 5). To the classical applications of the oral cytologic studies, such as oral candidiasis, others have been added, such as studying the epithelial infection due to Epstein-Barr virus in oral lesions of hairy leukoplakia, widening its possibilities [13]. The importance of brush biopsy has been recently emphasized in a multicenter study where nearly 5% of clinically benign-appearing mucosal lesions were sampled by this technique and later confirmed by typical scalpel biopsy to represent dysplastic epithelial changes or invasive cancer. [18] Other authors have also demonstrated the ability of the brush biopsy to uncover similar type lesions that were not clinically suspicious for carcinoma or preinvasive disease [14]. There are controversies related to the real value of this technique in the early detection of OSCC. The existence of false positives has been pointed out showing high sensitivity (90%) and low specificity (3%) [15]. Nevertheless, these data have been discussed previously [16]. In a recent study by Potter et al., four false negatives of a total 115 analysed cases were found. Although the number of false positive cases is small it is important to emphasize that the mean delay time in diagnosing a carcinoma in these cases was of 117.25 days [17]. However, more independent studies analysing its true validity and reliability as well as its applicability and its comparison with other techniques are necessary. Multiple studies with different results have been carried out, analysing the application of the cytology in the detection of dysplastic lesions. In a study from Sudan, oral scrape smear cytological analysis has been proposed as a useful early diagnostic method for epithelial atypia and therefore also for malignant oral lesions [18]. Despite the improvements in the methods used for collecting oral cytological material this methodology still presents problems in diagnosing oral cancer. Problems are mainly due to the existence of false negatives obtained as a result of a non representative sample as well as the subjectivity of the cytologic evaluation [19]. Figure 3 Technique of brush biopsy emphasizing pin-point bleeding of the oral mucosa. Figure 4 Picture demonstrating spreading of brush biopsy sample on a slide. Figure 5 Photomicrograph of a brush biopsy specimen from oral submucousfibrosis showing anucleated hyperkeratinized cells and a superficial squamous cell. (H & E × 400). Figure 6 Photomicrograph of a oral brush biopsy specimen from a patient of squamous cell carcinoma buccal mucosa showing a binucleated cell with evidence of intracellular and extracellular keratinization in a inflammatory background. (H & E × 400). Figure 7 Photomicrograph of a oral brush biopsy specimen from a patient of squamous cell carcinoma of buccal mucosa with high nucleo-cytoplasmic ratio marked atypia, and coarsely granular chromatin in a necrotic background .(Modified pap × 1000). Figure 8 Photomicrograph of a oral brush biopsy specimen from a patient of squamous cell carcinoma of buccal mucosa with high nucleo-cytoplasmic ratio coarsely granular chromatin and a multinucleated cell showing evidence of vascular invasion. (H&E × 1000). Liquid based cytology Since liquid-based cytology was developed in the 1990s various comparative studies have shown that it can offer significant advantages over conventional exfoliative cytology. Results obtained from uterine cervix examination, for example, have shown that the liquid-based preparations reduce the problems related to sampling error, poor transfer and fixation of the cellular sample [20-24]. In cervical uterine cancer screening, the liquid-based preparations have also demonstrated a significant reduction in false-negative rates as compared with those of conventional smears [20-23,25]. In a recent study from Brazil [26] the liquid-based preparations resulted in higher specimen resolution as well as presenting a better cytological morphology for pemphigus vulgaris, squamous cell carcinomas, HSV lesions and fungus infections. For HSV lesions, in particular, the observation of the cytopathological features indicative of viral infections (binucleation, multinucleated cells) greatly improved with the liquid-based technique [26]. Application of techniques Response to radiation therapy Radiotherapy is frequently used as a standard treatment for locally advanced carcinoma of oral cavity. Although the response of malignant tumours and surrounding normal tissue to various doses of ionizing radiation is generally predictable, variability in the host-tumour reaction in a specific individual makes the response unpredictable. The cytological evaluation of sequential oral smears during radiation therapy presents a unique opportunity to study the radiation response of oral malignant tumours. Earlier reports have described various cytoplasmic and nuclear changes in a variety of malignant cells evaluated by cytology after radiation therapy and included cellular enlargement, vacuolization, cytoplasmic granulation, nuclear enlargement, pyknosis, karyorrhexis, karyolysis, multinucleation, micronucleation, nuclear budding and binucleation (Fig. 9, 10). Later on micronucleation was accepted as a reliable indicator for monitoring the effectiveness of chemopreventive agents against cancer and for monitoring the toxicity of chemicals. In a study by the author comparing the post-radiation changes in normal and malignant oral cells it was found that various morphological abnormalities demonstrated a consistent significant increase with radiation dose [27]. Figure 9 Photomicrograph of malignant cells after radiation therapy showing multinucleation and micronucleation. (H&E × 1000). Figure 10 Photomicrograph of malignant cells after radiation therapy showing multiple nuclear budding. (H&E × 1000). Apoptotic cells In the smears of patients treated for OSCC, the percentage of apoptotic cells has been studied [28]. This detection can also be quite useful for monitoring patients' reaction to chemotherapy. Cytomorphometry Ogden et al. [29] suggested that quantitative techniques, based on the evaluation of parameters such as nuclear area (NA), cytoplasmic area (CA), and nucleus-to-cytoplasm area ratio (NA/CA), may increase the sensitivity of exfoliative cytology for early diagnosis of oral cancers, since these techniques are precise, objective and reproducible. Cowpe et al. [30] demonstrated that exfoliative cytology is capable of detecting malignant changes, through estimation of NA/CA using the planimeter method in Papanicolaou-stained smears. This study, published in 1985, concluded that 50 cells were sufficient to provide indication of malignant changes. Since then, a number of studies have been carried out using the technique described by these authors to evaluate the influence of diverse systemic and external factors on NA, CA and NA/CA. In these studies planimeters have been replaced by semiautomatic image analysis techniques, which are faster, more accurate and more reproducible [31,32]. Cowpe et al. [33] found that tissues undergoing malignant transformation typically show a reduction in CA before the reduction in NA. They also suggested that samples of healthy mucosa from the same patient provide the best control. Ramaesh et al. [34] used cytomorphometric techniques to assess nuclear diameter (ND) and cytoplasmic diameter (CD) in normal oral mucosa, in dysplastic lesions and in squamous cell carcinomas. They found that CD was highest in normal mucosa, lower in dysplastic lesions, and lowest in SCCs. By contrast, ND was lowest in normal mucosa, higher in dysplastic lesions, and highest in SCCs. These studies suggested that reduced nuclear size and increased cytoplasm size are useful early indicators of malignant transformation, and thus exfoliative cytology is of value for monitoring clinically suspect lesions and for early detection of malignancy. Nuclear DNA content and DNA-image cytometry Static cytometry permits the quantification of DNA content in cells obtained by exfoliative cytology. However, routine Haematoxylin-Eosin staining is inadequate for this purpose, and special techniques are required to ensure that staining intensity is in proportion to DNA content. The Feulgen reaction meets this criterion, since it is a stoichiometric procedure: in other words, each fixed molecule of Schiff's reagent corresponds to a constant and equivalent portion of the DNA molecule. The advantage of this procedure is that staining intensities (and thus DNA contents) can be determined automatically by spectrophotometry or densitometry as well as digital image analysis [35]. Using cytology and DNA-image cytometry, it is easy to prove that oral lesions with the diagnosis of lichen planus and other inflammatory diseases show no suspicious cells. A recent review of literature places the rate of malignant transformation of lichen planus to squamous cell carcinoma at 0.2% [36]. On the contrary, the presence of malignant cells was demonstrated in one of 21 cases with leukoplakia (4.76%), in all cases with erythroplakia and in all squamous cell carcinomas. A meta-analysis of 2236 cases of leukoplakia from five studies has revealed a range of malignant transformation of leukoplakia between 2.2 and 17.5%. Furthermore, Sciubba [37], Silverman et al. [38] and Mashberg et al. [39] emphasized the fact that erythroplakia, occurring as either an isolated lesion or as a component of leukoplakia (erythroleukoplakia) is a marker of severe epithelial dysplasia or carcinoma in situ. In fact, 90% of erythroplakia were histologically diagnosed as in situ or invasive carcinomas. In one study, it was shown that sensitivity of cytological diagnosis combined with DNA-image cytometry may reach 100%, whereas specificity was 97.4%. The authors reported a case of erythroplakia in which intraobserver variability among four pathologists led to diagnoses ranging from mild to severe dysplasia and because of the cytological and DNA cytometric diagnosis (severe dysplasia with DNA aneuploidy), this case was finally diagnosed on early cytological and DNA-cytometric diagnosis prior to the histological diagnosis [40]. Remmerbach et al have reported that sensitivity of cytological diagnosis combined with DNA-image cytometry was 98.2% and specificity 100%, when compared with the gold standard' of histology [41]. In a study, Maraki et al. analyzed 150 patients with histologically proven epithelial dysplasia of which 36 developed squamous cell carcinoma. DNA-cytometry showed DNA-diploidy in 105 patients. 20 patients had DNA-polyploidy and in 25 patients DNA-aneuploidy was found at the time of the initial diagnosis. Carcinoma developed in only three of the 105 diploid lesions when compared with 21 of the 25 aneuploid lesions. Remmerbach et al. [42] concluded in the clinical setting that DNA-aneuploidy might detect histologically obvious malignancy, 1–15 months prior to histology. Sudbo et al. analyzed archival material and reported that the nuclear DNA-content in cells of oral leukoplakia may be used to predict the risk of oral epithelial dysplasia up to 5 years before histological diagnosis [43]. Based on these observations, they proposed brush biopsies with cytological/DNA-cytometric examination for microscopic evaluation of white or red patches of the oral cavity (leukoplakia or erythroplakia). The finding of tumor cells or DNA-aneuploidy should lead to a total excision of the respective lesions and histological examination. Molecular analyses While the classic oral cytologic evaluation is labour intensive and requires a high degree of expertise for identifying and evaluating cells with suspicious morphology the analysis of molecular alterations is objective and tries to identify specific genetic anomalies [6]. The possibility of extracting RNA from cells obtained by scraping has recently been demonstrated emphasizing its usefulness in the early diagnosis of oral premalignant and cancerous lesions [44]. 1. Gene alterations Nowadays malignancy is considered as a process caused by the accumulation of multiple genetic alterations, which affect the cell cycle as well as normal cell differentiation. These alterations are mainly acquired (somatic) although some of them may be inherited and when they activate protooncogenes, inactivate tumour suppressor genes or affect enzymes, which repair DNA, they could lead to a malignant transformation. Most of the oral cavity carcinogens are chemical (tobacco), physical (radiation) and infectious (Human papilloma virus, Candida) mutagenic agents that may cause changes in gene and chromosome structure by point mutations, deletions, insertions and rearrangements. However, some of these changes may occur spontaneously. These genetic alterations, which occur during carcinogenesis, can be used as targets for detecting tumour cells in clinical samples [4,6,45]. Molecular analysis can identify a clonal population of cancerous cells. Mutations in the tumour suppressor gene p53 are the most frequent genetic alterations in human cancer and show a variable frequency in oral cancer [46]. Several authors have studied and in some cases demonstrated the potential clinical application of oral cytology for detecting point mutations in p53 as a specific neoplastic marker in OSCC [45,47-49]. However, other authors consider that the high number of point mutations, which can be found in p53, limit its potential clinical application in cost-effective early detection of oral cancer [50]. 2. Epigenetic alterations, Loss of hetrozygosity and Microsatellite instability The applicability of other molecular markers such as epigenetic alterations (hypermethylation of promoter regions) and genomic instability such as loss of hetrozygosity (LOH) and microsatellite instability (MSI) has also been studied. [50,51]. The main epigenetic modification in tumours is methylation and it seems that the changes in the methylation patterns can play an important role in tumorigenesis. These epigenetic alterations are often associated with the loss of genetic expression and their occurrence seems to be essential for the multiple necessary genetic events. So malignant progression takes place because these alterations can inactivate DNA repairing genes. Rosas et al. studied the methylation patterns of p16, MGMT and DAP-K genes in smears of patients suffering from head and neck cancer [50]. They detected abnormal hypermethylation patterns in both kinds of samples by a methylation specific Polymerase Chain Reaction (PCR). They proposed that this technique allows a sensitive and efficient detection of tumoral DNA and it is potentially useful for detecting and monitoring recurrences in these patients. Loss of heterogeneity (LOH) and other molecular changes indicative of oral carcinogenesis can be readily identified in exfoliated cells [52-54]. Huang et al. [55] used PCR techniques to amplify DNA from exfoliated cytology samples from oral carcinomas, for analysis of Restriction-Fragment Length Polymorphisms (RFLPs). They found that 66% of the tumours studied showed LOH at one position in the p53 sequence, while 55% showed LOH at some other location. PCR and RFLP analysis have also been used for the detection of microsatellite markers, i.e. short repetitive DNA sequences. Microsatellite mutations, LOH or instability (MI) are all characteristic of the squamous cell carcinomas of head and neck, and can thus be used as molecular markers of malignancy. Microsatellite regions are distributed along the genome and have been widely and satisfactorily used as molecular markers for carcinogenesis. Alterations in these regions have been used as clonal markers and for detecting tumoral cells among normal cells [56,57]. Several studies have demonstrated these by using microsatellite markers that alterations in certain regions of chromosomes 3p, 9p, 17p and 18q are associated with the development of head and neck squamous cell carcinomas [58,59]. Nunes et al.[60] performed a microsatellite analysis of cells sampled from the oral cavity of oral and oropharyngeal cancer patients by exfoliative cytology and by mouthwash, finding LOH in 84% of samples, though with differences depending on tumour stage. These authors suggested that techniques of this type might be useful for early diagnosis and for patient monitoring. In another study, Spafford et al. identified genetic alterations (LOH or MI) in all of the malignant lesions of the oral cavity included in their sample. [6] Conversely, none of their healthy patients showed such alterations, indicating the very high specificity of these methods. 3. Viral genome studies Archival cytology slides can also be used for HPV DNA detection with ISH. The diagnosis of metastatic lesions usually is determined by fine-needle aspiration. Human papillomavirus (HPV) is now being considered as a causative agent in a subset of HNSCC (FF). Presence of HPV DNA by in situ hybridization (ISH) in metastatic lesions from HNSCC using alcohol-fixed, archival, cytopathological material; was studied and the cytologic features of HPV-positive metastatic lesions of HNSCC were characterized; and HPV DNA and the origin of metastatic lesions was correlated [61] 4.Proliferation index and AgNOR analysis Ki 67 has been studied in oral cytological smears using Immunocytochemistry to evaluate the nature of lesion and response to treatment. Sharma et al, evaluated Ki-67 expression in cytologic scrapes from oral squamous cell carcinoma before and after 24 Gray radiotherapy in 43 patients. Ki-67 expression was seen in an extremely small number of cells. Only 10 tumours showed positive cells, and the labeling index in them varied from 0.1 % to 0.01 %. After 24 Gray irradiation, no case showed Ki-67 positive cells[62]. The validity of oral cytology for analyzing the number of keratinised cells and the nucleolar activity (AgNORs) in smoking patients has recently been demonstrated [63]. Remmerbach reported on AgNOR analysis in oral cytology and concluded that this may be used as a routine method for diagnosing oral cancer [64]. 5. Immunohistochemical identification of tumour markers The identification of tumoral markers, notably cytokeratins in smears from the oral cavity has attracted considerable interest. Cytokeratin expression profile provides useful information on cell differentiation status [64] but its potential for early diagnosis of oral cancer is limited [66]. However, certain cytokeratins, such as K8 and K19 are useful if not definitive indicators of malignancy, particularly if their presence is interpreted in conjunction with other information, such as DNA profile [67,68]. Conclusion Oral cytology is becoming increasingly important in the early diagnosis of oral cancers, as a procedure for obtaining cell samples that can then be analysed by sophisticated diagnostic techniques such as cytomorphometry, DNA cytometry, and molecular analyses. The advent of techniques like Toluidine blue staining, brush biopsy and application of sophisticated computer programs has changed the scenario and made the interpretation of findings far more reliable. than earlier. 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==== Front RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-3-221660308510.1186/1742-4690-3-22ResearchInhibition of constitutively active Jak-Stat pathway suppresses cell growth of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells Tomita Mariko [email protected] Hirochika [email protected] Jun-nosuke [email protected] Taeko [email protected] Masato [email protected] Takehiro [email protected] Yuetsu [email protected] Kazuiku [email protected] Naoki [email protected] Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan2 Division of Endocrinology and Metabolism, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan3 Division of Child Health and Welfare, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan4 Division of Immunology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan5 Department of Internal Medicine, Naha Prefectural Hospital, 1-3-1 Yogi, Naha, Okinawa 902-8531, Japan2006 9 4 2006 3 22 22 7 12 2005 9 4 2006 Copyright © 2006 Tomita et al; licensee BioMed Central Ltd.2006Tomita et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Human T-cell leukemia virus type 1 (HTLV-1), the etiologic agent for adult T-cell leukemia (ATL), induces cytokine-independent proliferation of T-cells, associated with the acquisition of constitutive activation of Janus kinases (Jak) and signal transducers and activators of transcription (Stat) proteins. Our purposes in this study were to determine whether activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells, and to explore mechanisms by which inhibition of Jak-Stat pathway kills ATL cells. Results Constitutive activation of Stat3 and Stat5 was observed in HTLV-1-infected T-cell lines and primary ATL cells, but not in HTLV-1-negative T-cell lines. Using AG490, a Jak-specific inhibitor, we demonstrated that the activation of Stat3 and Stat5 was mediated by the constitutive phosphorylation of Jak proteins. AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest mediated by altering the expression of cyclin D2, Cdk4, p53, p21, Pim-1 and c-Myc, and by apoptosis mediated by the reduced expression of c-IAP2, XIAP, survivin and Bcl-2. Importantly, AG490 did not inhibit the growth of normal peripheral blood mononuclear cells. Conclusion Our results indicate that activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells. Inhibition of this pathway may provide a new approach for the treatment of ATL. ==== Body Background Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder that occurs in individuals infected with human T-cell leukemia virus type 1 (HTLV-1) [1-3]. HTLV-1 causes ATL in 3–5% of infected individuals after a long latent period of 40–60 years [4]. The prognosis of ATL patients remains poor with a median survival time of 13 months in aggressive cases [5]. The poor prognosis of ATL patients is partly due to the innate resistance of HTLV-1-infected T-cells to apoptosis and thus to conventional chemotherapy regimens. Therefore, there is a critical need for new ATL therapies with improved efficacy over current treatments. High expression of the interleukin-2 receptor α chain (IL-2Rα) is a common feature of ATL cells and HTLV-1-infected T-cell lines [6]. One of the well-documented signalling pathways mediated by IL-2R is Janus kinase (Jak)-Signal transducers and activators of transcription (Stat) [7]. Jak proteins transduce signals by phosphorylating Stat proteins, which in turn dimerize and translocate to the nucleus to activate the expression of genes necessary for cell proliferation and differentiation [8]. Abnormal activation of Stat proteins is a common characteristic found in various human tumor cell lines and human tumors including leukemia and lymphoma [9-11]. Constitutive activation of the IL-2R-Jak/Stat signalling pathway correlates with IL-2 independence of HTLV-1-transformed cell lines [12]. Constitutive Jak1, Jak3, Stat1, Stat3 and Stat5 activation was observed in HTLV-1-infected T-cell lines [13]. Similarly, an in vitro study with uncultured leukemic cells from HTLV-1 seropositive patients with ATL also displayed constitutive activation of Jak3, Stat1, Stat3 and Stat5 [14]. These results suggest that activation of the IL-2R signalling pathway mediated by Jak-Stat may play a key role in transformation by HTLV-1. However, a causal relationship between carcinogenesis and activation of the Jak-Stat pathway in ATL has not been established, and it is not clear whether disruption of this pathway could reverse the phenotypic condition of HTLV-1-infected T-cells. AG490 is a recent addition to the synthetically derived tyrphostin family of tyrosine kinase inhibitors. Tyrphostins were designed on the basis of tyrosine and erbstatin and were all benzene malonitriles, many of which are substrate competitive but non-competitive inhibitors with respect to adenosine triphosphate [15]. AG490 selectively inhibits Jak family kinases but has no effect on other lymphocyte tyrosine kinases, including Lck, Lyn, Btk, Syk and Src [16,17]. Systemic administration of AG490 in SCID mice with disseminated human leukemic cells dependent on Jak2 for survival resulted in tumor cell apoptosis leading to complete tumor regression [16]. However, it has been reported that AG490 blocks the phosphorylation of Stat5 and Jak3, and DNA-binding activity of Stat5 of HTLV-1-transformed T-cell lines, but it fails to disrupt the growth of these leukemic cells [18]. In the present study, we evaluated the anti-tumor efficacy of AG490 against ATL and found that AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells, but not that of normal peripheral blood mononuclear cells (PBMCs). Furthermore, we investigated the possible mechanisms involved in such in vitro growth-inhibitory effect. Our findings suggested that activation of Jak-Stat signalling pathway is responsible for ATL cell proliferation and survival. Results Constitutive tyrosine phosphorylation of Stat3 and Stat5 in HTLV-1-infected T-cell lines We first examined HTLV-1-infected T-cell lines [MT-2, HUT-102 and ED-40515(-)] for the phosphorylation status of Stat3 and Stat5. All HTLV-1-infected T-cell lines displayed constitutive phosphorylation of Stat3 (Figure 1A, top panel). Constitutive phosphorylation of Stat5 was observed in MT-2 and HUT-102 (Figure 1A, third panel). In contrast, phosphorylation of Stat3 and Stat5 was not observed in HTLV-1-negative T-cell lines (Jurkat, MOLT-4 and CCRF-CEM) (Figure 1A, top and third panels), although the expression of Stat3 and Stat5 was detected in all cell lines (Figure 1A, second and forth panels). MT-2 and HUT-102 highly express HTLV-1 viral proteins, whereas ED-40515(-), a T-cell line of leukemic cell origin established from a patient with ATL, expresses little HTLV-1 viral proteins. For example, HTLV-1 transforming protein Tax was detected in MT-2 and HUT-102, but not in ED-40515(-) and all HTLV-1-negative T-cell lines (Figure 1A, second panel from the bottom). Because hypermethylation of 5' HTLV-1 long terminal repeat in ATL derived cell lines and ATL cells silenced the viral gene expression [19], ED-40515(-) cells did not express significant levels of Tax protein. These results suggested that constitutive phosphorylation of Stat3 and Stat5 seems to depend on HTLV-1 infection, but not on the expression of HTLV-1 Tax protein. Figure 1 Constitutive activation of Stat3 and Stat5 in HTLV-1-infected T-cell lines. (A) Western blot analysis of cellular lysates prepared from three HTLV-1-negative [HTLV-1 (-)] and three HTLV-1-infected [HTLV-1 (+)] T-cell lines. The blots were probed with anti-phospho-Stat3, anti-Stat3, anti-phospho-Stat5, anti-Stat5 and anti-Tax. Amounts of actin are shown as loading controls. (B) Stat-DNA binding activities in HTLV-1-negative and HTLV-1-infected T-cell lines were detected by EMSA using SIE or β-casein probe. Arrows indicate specific protein-DNA complexes. NS indicates non-specific bands. (C) Competition assay was performed with nuclear extracts of HTLV-1-infected cell lines using 100-fold excess of unlabeled wild type (W) or mutant (M) oligonucleotide as a competitor (upper panels: SIE, lower panels: β-casein). (D and E) Involvement of Stat3 and Stat5 in the formation of SIE- (D) and β-casein- (E) binding complexes in HTLV-1-infected T-cell lines. EMSA was performed with nuclear extracts of the indicated cell lines either in the absence (-) or presence of a specific Stat antibody (αStat: anti-Stat1, Stat2, Stat3, Stat4 and Stat5 antibodies). The supershifted complexes are indicated by arrowheads. Constitutive activation of Stat3- and Stat5-DNA binding activity in HTLV-1-infected T-cell lines Electrophoretic mobility shift assay (EMSA) was performed to analyze Stat-DNA binding activity of HTLV-1-infected T-cell lines using two different Stat-consensus sequences from the c-fos gene promoter [sis-inducible element (SIE)] and from the β-casein gene promoter (β-casein) (Figure 1B). Both SIE- and β-casein-binding activities were detected in the nuclear extracts of MT-2 and HUT-102 cells. SIE- but not β-casein-binding activity was detected in extracts of ED-40515(-) cells. In contrast, no significant DNA binding activity of SIE or β-casein was detected in extracts of HTLV-1-negative T-cell lines. Competition assays showed that the observed protein-DNA complexes were specific for SIE or β-casein (Figures 1C). The SIE-binding protein complexes from MT-2, HUT-102 and ED-40515(-) cells included Stat3, since the complex was supershifted by specific antibody for Stat3 (Figure 1D). The β-casein-binding protein complexes from MT-2 and HUT-102 cells included Stat5 (Figure 1E, upper panels). Stat1, Stat2 and Stat4 specific antibodies did not influence the formation of both SIE- and β-casein-complexes in any cell lines (Figures 1D and 1E). These results indicate that constitutive phosphorylation of Stat3 and Stat5 correlates with their DNA binding activities in HTLV-1-infected T-cell lines. Tax is not responsible for the induction of Stat3 and Stat5 phosphorylation in T-cells We next examined whether HTLV-1 Tax protein alters the phosphorylation status of Stat3 and Stat5. Tax-inducible T-cell line, JPX-9 expressed Tax 10 h after addition of CdCl2 and the expression persisted until 72 h after treatment (Figure 2, second panel from the bottom, lanes 4–7). Although Stat3 and Stat5 were consistently expressed in JPX-9 cells even after CdCl2 treatment, phosphorylated Stat3 and Stat5 were not detected in these cells (Figure 2, first and third panels). These results suggest that Tax is not involved in the induction of Stat3 and Stat5 phosphorylation in T-cells. Figure 2 HTLV-1 Tax does not involve in phosphorylation of Stat3 and Stat5. Cell lysates were prepared from CdCl2-treated JPX-9 cells at the indicated time points (lanes 1–7) and untreated MT-2 cells (lane 8: as a positive control). The expression of phospho-Stat3, Stat3, phospho-Stat5, Stat5 and Tax (arrow) was analyzed by Western blot. Actin expression served as a loading control. AG490 reduces constitutive activation of Stat3 and Stat5 through inhibition of Jak kinases in HTLV-1-infected T-cell lines The regulation of phosphorylation of Stat3 and Stat5 by Jak kinases was investigated with Jak selective inhibitor, AG490. AG490 reduced constitutive phosphorylation in Stat3 [MT-2, HUT-102 and ED-40515(-)] and Stat5 (MT-2 and HUT-102) in a dose-dependent manner (Figures 3A and 3B). AG490 also suppressed constitutive phosphorylation of Stat3 and Stat5 in freshly isolated ATL cells (Figure 3C). Constitutive phosphorylation of Jak1, Jak2 and Jak3 was observed in MT-2 and HUT-102 cells, and treatment of these cells with increasing concentrations of AG490 resulted in significant inhibition of phosphorylation of Jak1, Jak2 and Jak3 (Figure 3D). Constitutive phosphorylation of Jak2 but not Jak1 and Jak3 was detected in ED-40515(-) cells and treatment with AG490 inhibited phosphorylation of Jak2 in ED-40515(-) cells (Figure 3D). AG490 did not affect on phosphorylation status of glycogen synthase kinase-3β (GSK-3β) that is not regulated by Jak-Stat pathway (Figure 3E), suggesting that effect of AG490 is specific for Jak-Stat pathway. To determine whether AG490 inhibits DNA binding activity of Stat3 and Stat5 in HTLV-1-infected T-cell lines, we treated the cells with 50 μM AG490 for 24 h and performed EMSA (Figure 3F). AG490 decreased SIE- [MT-2, HUT-102 and ED-40515(-)] and β-casein- (MT-2 and HUT-102) DNA binding activity of HTLV-1-infected T-cell lines. These results suggest that AG490 reduces the constitutive activation of Stat3 and Stat5 by inhibiting three Jak kinases in HTLV-1-infected T-cell lines. Figure 3 AG490 inhibits constitutive activation of Jak and Stat in HTLV-1-infected T-cell lines and primary ATL cells. (A and B) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. (C) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. Phosphorylation status of Stat3 and Stat5 was assessed by Western blot analysis. (D) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Phosphorylation status of Jak1, Jak2 and Jak3 were assessed by Western blot analysis. (E) AG490 does not affect phosphorylation of other phosphor-protein that is not regulated by Jak-Stat pathway. HTLV-1-infected T-cell lines were treated with (+) or without (-) 50 μM AG490 for 24 h. Phosphorylation status of GSK-3β was assessed by Western blot analysis. (F) AG490 inhibits constitutive Stat3- and Stat5-DNA binding in HTLV-1-infected T-cell lines. Nuclear extracts were isolated from HTLV-1-infected T-cell lines treated with (+) or without (-) 50 μM AG490 for 24 h. Stat-DNA binding activity was assessed by EMSA using SIE or β-casein probe. AG490 inhibits the cell growth of HTLV-1-infected T-cell lines and primary ATL cells Next we examined the effect of AG490 on the growth of HTLV-1-infected T-cell lines and primary ATL cells. HTLV-1-infected T-cell lines were treated with different concentration of AG490 (0, 25 or 50 μM) and cell numbers were counted 24 and 48 h after treatment. AG490 suppressed the growth of HTLV-1-infected T-cell lines in a dose and time dependent manner (Figure 4A). The antiproliferative effects of AG490 against primary ATL cells and PBMCs from healthy donors were measured by WST-8 method (Cell Counting Kit-8; Wako Chemical, Osaka, Japan) based on the MTT assay as described previously [20]. Cell viability was determined as percentage of the control (without AG490). AG490 also inhibited the growth of PBMCs from ATL patients (ATL #1–7 in Figure 4B). In comparison, the cell growth inhibitory effect on PBMCs from healthy donors was weak (Normal #1–3 in Figure 4B). These findings indicate that AG490 inhibits the growth of cells infected with HTLV-1 but not that of uninfected PBMCs. Figure 4 AG490 reduces cell growth of HTLV-1-infected T-cell lines and primary ATL cells. (A) HTLV-1-infected T-cell lines (5 × 104/mL) were treated with 0, 25 or 50 μM AG490 for 24 or 48 h. Cell numbers were counted in triplicate by Trypan blue dye exclusion method. Data are expressed as the mean values of viable cell numbers. (B) Primary ATL cells from seven patients (ATL #1–7) and PBMCs from three healthy donors (Normal #1–3) were treated with 0, 25 or 50 μM AG490 for 48 h. Cell growth was assessed by the WST-8 method. Data are expressed as the percentages of control (untreated cells). (C) Cell-cycle analysis of HTLV-1-infected T-cell lines treated with AG490. Cells were treated in the absence (-) or presence (+) of 25 μM AG490 for 24 h. DNA content was analyzed by flow cytometry with propidium iodide staining. G1, S and G2/M indicate the stages of the cell-cycle. Data represent mean percentages of cells at each cell-cycle from three independent experiments. (D) Induction of apoptosis in HTLV-1-infected T-cell lines by AG490. Cells were treated in the absence (open bar) or presence (solid bar) of 50 μM AG490 for 48 h and stained with Annexin-V. Apoptosis was analyzed by flow cytometry. Data represent mean percentages of apoptotic cells from three independent experiments. AG490 induces cell-cycle arrest and apoptosis of HTLV-1-infected T-cell lines We then investigated the effect of AG490 on cell-cycle distribution in HTLV-1-infected T-cell lines (Figure 4C). Cells were treated with 25 μM AG490 for 24 h. Twenty-five μM AG490 inhibited cell-cycle progression, as demonstrated by the increased proportion of cells in G1 phase [MT-2: from 52% to 72%; HUT-102: from 51% to 83%; ED-40515(-): from 35% to 44%] and decreased percentage of cells in S phase [MT-2: from 36% to 18%; HUT-102: from 36% to 8%; ED-40515(-): from 51% to 43%], indicating G1 cell-cycle arrest. The effect of AG490 on apoptosis was examined by the Annexin-V method. Annexin-V binding reveals the phosphatidylserine molecules have been flipped out from the inner to the outer cell surface during apoptosis. Cells were treated with 50 μM AG490 for 48 h. AG490 increased the proportion of cells positive for Annexin-V in all cell lines (Figure 4D), indicating the increased apoptosis of AG490-treated cells. Thus, AG490 is both anti-proliferative and pro-apoptotic in HTLV-1-infected T-cell lines. Expression of cell-cycle associated genes in AG490-treated HTLV-1-infected T-cell lines and ATL cells We next examined whether AG490 induces G1 cell-cycle arrest by modulating the expression of G1 cyclins, cyclin D1 and cyclin D2, which are associated with cell-cycle progression from G1 to S phase. AG490 decreased cyclin D2 expression, however, the expression of cyclin D1 was almost unchanged (Figure 5A). Cell-cycle progression from G1 to S phase is also regulated by G1 cyclin-dependent kinases; Cdk4 and Cdk6, which bind and activate the cyclin D. AG490 inhibited the expression of Cdk4 in a dose-dependent manner but not that of Cdk6 protein (Figure 5A). These results suggest that AG490 induces G1 arrest by reducing the expression of cyclin D2 and Cdk4, which regulate the G1-S transition. The p53/p21 pathway also plays a critical role in regulating the G1-S transition. We examined the effects of AG490 on p53 and p21 levels in HTLV-1-infected T-cell lines. Expression of p53 protein was increased in AG490 treated MT-2 and HUT-102 cells. In contrast, p53 protein was almost undetectable in ED-40515(-) cells and remained unchanged in AG490-treated cells. p21 was induced in MT-2 and HUT-102 cells and remained undetectable in ED-40515(-) cells. These results indicate that p21 activation can also contribute to AG490-induced G1 arrest in p53-competent cells. AG490-treated ED-40515(-) cells did not induce G1 arrest as much as MT-2 and HUT-102 cells (Figure 4D). This might be due to absence of p53 and p21 proteins in AG490-treated ED-40515(-) cells. Cell-cycle progression from G1 to S phase is also regulated by Serin/Threonin kinase Pim-1 and c-Myc through Cdc25A activation [21,22]. pim-1 and c-myc genes are both direct targets of Stat [23,24]. AG490 decreased the expression of these proteins in all HTLV-1-infected T-cell lines (Figure 5A). AG490 also reduced the expression of cyclin D2 and increased the expression of p53 in freshly isolated ATL cells (Figure 5B). However, other proteins that were altered by AG490 in HTLV-1-infected T-cell lines were undetectable and AG490 did not change the expression of these genes in primary ATL cells (data not shown). Figure 5 Effects of AG490 on the expression of cell-cycle associated proteins. HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Amounts of cyclin D1, cyclin D2, Cdk4, Cdk6, p53, p21, Pim-1 and c-Myc were determined by Western blot analysis. (B) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of cyclin D2 and p53 was assessed by Western blot analysis. The amount of actin is shown as a loading control. Expression of anti-apoptotic genes in AG490-treated HTLV-1-infected T-cell lines and ATL cells We also examined the effects of AG490 on the expression of IAP and Bcl-2 family members, which determine the response to apoptotic stimuli. AG490 significantly altered the expression of XIAP and survivin, which are Stat-regulated genes [25,26], but not that of Bcl-xL protein in all tested cell lines (Figure 6A). Downregulation of Bcl-2 expression by AG490 was only noted in HUT-102 cells. The expression of c-IAP2 was downregulated in HUT-102 and ED-40515(-), but not in MT-2 cells. These results indicated that AG490-induced apoptosis of HTLV-1-infected T-cells is mediated by downregulation of c-IAP2, XIAP, survivin and Bcl-2 expression. AG490 reduced the expression of all these genes in freshly isolated ATL cells (Figure 6B). Bcl-2 protein was undetectable in primary ATL cells (data not shown). Cyclin D2 [27,28], Cdk4 [29], XIAP [30] and survivin [31] are Tax-responsive genes, therefore, we also examined the level of Tax expression in these cells. AG490 did not alter Tax protein levels in MT-2 and HUT-102 cells (Figure 6A). Tax protein remained at undetectable levels in ED-40515(-) and primary ATL cells after AG490 treatment (Figures 6A and 6B). Therefore, the altered expression of cyclin D2, Cdk4, XIAP and survivin was not attributable to Tax downregulation. We also examined whether AG490 could change the expression levels of other viral proteins. The expression levels of HTLV-1 envelope 46 kDa glycoprotein (gp46) and 19 kDa core protein (p19) were not changed by AG490 treatment in HUT-102 cells (Figure 6C), suggesting that the AG490 does not drop the virus levels in these cells and the effects of AG490 on these cells are not due to downregulation of viral proteins. Figure 6 Effects of AG490 on the expression of anti-apoptotic proteins. (A) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Amounts of c-IAP-2, XIAP, survivin, Bcl-2, Bcl-xL and Tax were determined by Western blot analysis. (B) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of c-IAP2, XIAP, survivin and Tax was assessed by Western blot analysis. (C) HUT-102 cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of HTLV-1 viral proteins, envelope glycoprotein gp46 and p19 core protein was assessed by Western blot analysis. The amount of actin is shown as a loading control. Discussion In this study, we demonstrated that Stat3 and Stat5 are constitutively activated in HTLV-1-infected T-cell lines and primary ATL cells, but not in HTLV-1-negative T-cell lines. Using AG490, a Jak-specific inhibitor, we showed that the activation of Stat3 and Stat5 is mediated by the constitutive phosphorylation of Jak proteins. Furthermore, we showed that AG490 inhibits the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest and apoptosis, but not that of normal PBMCs. Our results indicate that constitutive activation of Jak-Stat is responsible for the proliferation and survival of ATL cells. The mechanism for the constitutive activation of Jak-Stat after HTLV-1 infection is still unclear. HTLV-1 transforming protein Tax is considered to play a critical role in leukemogenesis and development of ATL. However, our data showed no correlation between Stat activation and Tax protein expression in HTLV-1-infected T-cell lines. Previous reports are consistent with our data in their lack of support for the involvement of Tax or the autocrine production of IL-2 or IL-15 in Stat-activation of HTLV-1-infected T-cell lines and primary ATL cells [12,14]. Expression of Stat5 mRNA is induced by HTLV-1 Tax using JPX-9 cells [32]. Using this cell line, we showed that Tax induced neither the expression nor the phosphorylation of Stat3 and Stat5 proteins. A T-cell line denoted Tax, in which a herpes samiri-based vector drives Tax gene expression, does not exhibit constitutive Stat binding activity [12]. We also showed that ATL-derived T-cell line, ED-40515(-) and primary ATL cells which did not express Tax protein at detectable level, expressed Stat proteins in the phosphorylated form. It should be noted that the leukemic cells in vivo generally do not express Tax by several mechanisms [33]. Thus, it is unlikely that Tax is involved in the induction or activation of Stat proteins or represents a target of anti-ATL drugs. Previously, Nicot and colleagues [34] reported that the p12I protein, encoded by the pX open reading frame I of HTLV-1, binds to the IL-2R β chain, resulting in activation of Stat5 through Jak1 and Jak3 activation. However, the mechanisms for the Jak2 activation in HTLV-1-infected T-cells are not elucidated. Our data demonstrating that inhibition of Stat activity led to apoptosis in HTLV-1-infected T-cell lines and primary ATL cells are in line with a previous study reporting induction of apoptosis by ectopic expression of a dominant-negative form of Stat5 in MT-2 cells [25]. Our data of a weaker effect of AG490 on the growth of normal PBMCs than that of ATL cells were consistent with a previous report showing that AG490 has no significant effect on the growth of normal B and T cells in vitro [16]. In contrast to our data, Kirken and colleagues [18] reported that although AG490 blocks the phosphorylation of Stat5 and Jak3, and DNA-binding activity of Stat5 of HTLV-1-transformed T-cell lines, MT-2 and HUT-102, it fails to disrupt the growth of these leukemic cells. Although we used lower concentration of AG490 (50 μM Max.) than this group (100 μM Max.), we observed a dose-dependent inhibition of cell growth in these cells by AG490. The precise reason for these differences is not clear, however, we cannot exclude the possibility that these differences could be attributable to variations in experimental conditions such as serum concentration (1% vs. 10%) in tissue culture medium. Perhaps for AG490 mediated growth inhibitory effect in HTLV-1-infected T-cell lines and ATL cells, active protein synthesis is required. Previous study suggested that AG490 is a Jak2-specific inhibitor and blocks leukemic cell growth of acute lymphoblastic leukemia [16]. Our data showed that AG490 also inhibited phosphorylation of Jak1 and Jak3 of MT-2 and HUT-102. Thus, three constitutively phosphorylated Jak proteins in HTLV-1-infected T-cell lines were inhibited by AG490. These results are consistent with recent studies reporting that AG490 inhibits Jak1 activated by IL-6 in myeloma cells or IL-2-induced Jak3 activity in an IL-2-dependent T-cell line [17,35], suggesting that the aforementioned three Jak proteins share AG490 sensitivity. Interestingly, AG490 does not affect other lymphocyte tyrosine kinases [16]. This may also account for the fact that AG490 is well-tolerated in mice [16,36]. Conclusion We have demonstrated that constitutive activation of Jak-Stat is responsible for the proliferation and survival of ATL cells. Previously we showed that NF-κB pathway is constitutively activated in HTLV-1-infected T-cell lines and primary ATL cells [37] and inhibition of this pathway suppresses the growth of these cells [38,39]. In addition to NF-κB pathway, our findings in this study indicate that inhibition of the Jak-Stat pathway offers a new approach for ATL treatment. Furthermore, AG490 kinase inhibitor is well tolerated in vivo, and thus presents a useful agent for this novel anti-ATL therapeutic approach. Methods Cell lines The HTLV-1-uninfected T-cell leukemia cell lines; Jurkat, MOLT-4, CCRF-CEM and HTLV-1-infected T-cell lines; MT-2 [40], HUT-102 [1] and ED-40515(-) [41] [HUT-102 was a generous gift from the Fujisaki Cell Center, Hayashibara Biomedical Laboratories, Okayama, Japan, ED-40515(-) was from Dr. M. Maeda, Kyoto University, Kyoto, Japan] were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin and 50 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO) at 37°C in 5% CO2. MT-2 is an HTLV-1-transformed T-cell line, established by an in vitro coculture protocol. The clonal origin of HUT-102 was not determined. ED-40515(-) is a leukemia T-cell line derived from a patient with ATL. JPX-9 (kindly provided by Dr. M. Nakamura, Tokyo Medical and Dental University, Tokyo, Japan) is a subclone of Jurkat cells expressing Tax under the control of the metallothionein promoter [42]. Expression of Tax was induced by addition of CdCl2 to a final concentration of 20 μM. Reagents AG490 was purchased from Calbiochem (La Jolla, CA). The anti-Tax (Lt-4), anti-gp46 (REY-7) and anti-p19 (GIN-7) antibodies were described previously [43-45]. The anti-Stat3, anti-phospho-Stat3 (Tyr705), anti-phospho-Stat5 (Tyr694) and anti-phospho-GSK-3β (Ser9) antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti-phospho-Jak1 (Tyr 1022/Tyr 1023), anti-phospho-Jak2 (Tyr 1007/Tyr 1008), anti-phospho-Jak3 (Try980), anti-cyclin D2, anti-Pim-1, anti-survivin and anti-c-IAP2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-cyclin D1 and anti-XIAP antibodies were purchased from Medical & Biological Laboratories (Nagoya, Japan). The anti-Cdk4, anti-Cdk6, anti-p53, anti-p21, anti-c-Myc, anti-Bcl-2 and anti-actin antibodies were from NeoMarkers (Fremont, CA). The anti-Stat5 and anti-Bcl-xL antibodies were from BD transduction Laboratories (San Jose, CA). Horseradish-peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ). Western blot analysis Western blot analysis was performed as described previously [46]. In brief, whole cell lysates were subjected to SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA), and then analyzed for immunoreactivity with the appropriate primary and secondary antibodies as indicated in the figures. Reaction products were visualized using Enhanced Chemiluminescence reagent, according to the instructions provided by the manufacturer (Amersham Pharmacia, Uppsala, Sweden). EMSA Nuclear extracts were prepared from AG490-treated and untreated cells and Stat3- or Stat5-DNA binding activity was analyzed by EMSA as described previously [47,48]. The probes or competitors used were prepared by annealing the following sense and antisense synthetic oligonucleotides: Stat3 consensus binding motif (SIE) derived from c-fos promoter 5'-gatcGACATTTCCCGTAAATCG-3', SIE mutant 5'-gatcGACATTTCCCGTCCCGCG-3', Stat5 consensus binding motif (β-casein) derived from β-casein promoter 5'-gatcAGATTTCTAGGAATTCAAATC-3' and β-casein mutant 5'-gatcAGATTTAGTTTAATTCAAATC-3'. To identify Stat proteins in the DNA-protein complex revealed by EMSA, we used specific antibodies for various Stat family proteins including Stat1, Stat2, Stat3, Stat4 and Stat5 (Santa Cruz Biotechnology), to elicit a supershift DNA-protein complex formation. Patient samples PBMCs from three healthy volunteers (Normal #1–3) or patients with the acute (ATL #1–4, 6 and 7) or chronic (ATL #5) type of ATL were analyzed. The diagnosis of ATL was based on clinical features, hematological characteristics, presence of serum antibodies to ATL-associated antigens and presence of HTLV-1 proviral genome in DNA from leukemic cells. PBMCs were isolated by Ficoll/Hypaque (Pharmacia LKB, Piscataway, NJ) using density gradient centrifugation. Each patient had more than 90% leukemic cells in the blood at the time of analysis. The study protocol was approved by the Human Ethics Review Committee of University of the Ryukyus, and a signed consent form was obtained from each subject. Assays for cellular proliferation The antiproliferative effects of AG490 against HTLV-1-infected T-cell lines were measured by the Trypan blue dye exclusion method. The 5 × 104 cells were incubated in the presence of 0, 25 or 50 μM AG490 in a final volume of 1 mL at 37°C. The cell numbers were counted by the Trypan blue dye exclusion method after 24 and 48 h treatment. The antiproliferative effects of AG490 against primary ATL cells and PBMCs from healthy donors were measured by WST-8 method (Cell Counting Kit-8; Wako Chemical, Osaka, Japan) based on the MTT assay as described previously [20]. Briefly, the 1 × 105 cells were incubated in triplicate in 96-well microculture plates in the presence of 0, 25 or 50 μM AG490 in a final volume of 0.1 ml for 48 h at 37°C. Thereafter, 5 μl Cell Counting Kit-8 solution [5 mM WST-8, 0.2 mM 1-Methoxy PMS (5-methylphenazinium methylsulfate) and 150 mM NaCl] was added, and the cells were further incubated for another 4 h. The number of surviving cells was measured by a 96-well multiscanner autoreader at optical density of 450 nm. Cell viability was determined as percentage of the control (without AG490). Cell-cycle analysis Cells were plated at a density of 1 × 105/ml in 60-mm tissue culture dish. Twelve hours after plating, cells were exposed to 25 μM AG490 for 24 h. Cell-cycle analysis was performed with the CycleTEST PLUS DNA reagent kit (Becton Dickinson, San Jose, CA). Briefly, cells were washed with a buffer solution containing sodium citrate, sucrose and dimethyl sulfoxide, suspended in a solution containing RNase A, and stained with 125 μg/ml propidium iodide for 10 min. Cell suspensions were analyzed on a FACS Calibur (Becton Dickinson) using CellQuest. The cell population at each cell-cycle phase was determined with ModiFit software. Assays for apoptosis Cells were plated at a density of 1 × 105/ml in 60-mm tissue culture dish. Twelve hours after plating, cells were exposed to 50 μM AG490 for 48 h. Apoptosis was quantified by staining with Annexin-V-Fluos (Roche Diagnostics, Mannheim, Germany) according to the instructions supplied by the manufacturer. Cells were analyzed on a FACS Calibur using CellQuest. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MT contributed to the concept and design, interpreted and analyzed the data, provided drafting of the article, provided critical revisions and important intellectual content, collected and assembled the data. HK, JU, TO and TM collected and assembled the data. MM, YT and KO provided study materials and critical revisions and important intellectual content. NM contributed to the concept and design, provided critical revisions and important intellectual content, obtained a funding source, provided administrative support. All authors read and approved the final manuscript. Acknowledgements This work was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science, by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. ==== Refs Poiesz BJ Ruscetti FW Gazdar AF Bunn PA Minna JD Gallo RC Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma Proc Natl Acad Sci U S A 1980 77 7415 7419 6261256 Hinuma Y Nagata K Hanaoka M Nakai M Matsumoto T Kinoshita KI Shirakawa S Miyoshi I Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera Proc Natl Acad Sci U S A 1981 78 6476 6480 7031654 Yoshida M Miyoshi I Hinuma Y Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease Proc Natl Acad Sci U S A 1982 79 2031 2035 6979048 Tajima K The 4th nation-wide study of adult T-cell leukemia/lymphoma (ATL) in Japan: estimates of risk of ATL and its geographical and clinical features. 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==== Front Indian Pacing Electrophysiol JIndian Pacing Electrophysiol JIndian Pacing and Electrophysiology Journal0972-6292Indian Pacing and Electrophysiology Group 16943966ipej060163-00ReviewsAtrial Fibrillation and Pacing Algorithms Terranova Paolo MD†Severgnini Barbara MD†Valli Paolo MD†Dell'Orto Simonetta MD†Greco Enrico Maria MD† U.O. Cardiologia e UTIC, Azienda Ospedaliera "S. Paolo" - Polo Universitario, Department of Medicine, Surgery and Odontoiatry, University of Milan, Italy† U. O. di Cardiologia, Presidio Ospedaliero "Causa Pia Ospedaliera Uboldo", Cernusco sul Naviglio, Azienda Ospedaliera di Melegnano, Milano, ItalyAddress for correspondence: Dott. Paolo Terranova, MD, U. O. Cardiologia, A. O. "S. Paolo", Dept. Medicine, Surgery and Odontoiatry University of Milan, Italy. E-mail: [email protected] 2006 1 7 2006 6 3 163 172 Copyright: © 2006 Terranova et al.2006This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Pacing prevention algorithms have been introduced in order to maximize the benefits of atrial pacing in atrial fibrillation prevention. It has been demonstrated that algorithms actually keep overdrive atrial pacing, reduce atrial premature contractions, and prevent short-long atrial cycle phenomenon, with good patient tolerance. However, clinical studies showed inconsistent benefits on clinical endpoints such as atrial fibrillation burden. Factors which may be responsible for neutral results include an already high atrial pacing percentage in conventional DDDR, non-optimal atrial pacing site and deleterious effects of high percentages of apical ventricular pacing. Atrial antitachycardia pacing (ATP) therapies are effective in treating spontaneous atrial tachyarrhythmias, mainly when delivered early after arrhythmia onset and/or on slower tachycardias. Effective ATP therapies may reduce atrial fibrillation burden, but conflicting evidence does exist as regards this issue, probably because current clinical studies may be underpowered to detect such an efficacy. Wide application of atrial ATP may reduce the need for hospitalizations and electrical cardioversions and favorably impact on quality of life. Consistent monitoring of atrial and ventricular rhythm as well as that of ATP effectiveness may be extremely useful for optimizing device programming and pharmacological therapy. AntitachycardiaAtrial fibrillationPacingPrevention algorithms ==== Body Introduction Atrial fibrillation is surely the most common arrhythmia in clinical practice and nowadays its incidence is increasing mainly due to the progressive ageing of population. Atrial fibrillation represents also the cardiac rhythm disorder that causes the highest number of hospitalizations [1] and is associated with higher mortality [2], major clinical complications such as heart failure, acute myocardial infarction, stroke [3], and impaired quality of life [4]. Atrial fibrillation is frequently associated with ventricular tachyarrhythmias. It has been calculated that 20% of the patients with conventional indication for cardioverter defibrillator implantation had atrial fibrillation before implant and that during the lifespan of the device more than 50% may develop atrial fibrillation [5,6]. Antiarrhythmic drugs have been widely used for cardioversion and maintenance of sinus rhythm, but they showed a limited and usually temporary efficacy [7]. As a consequence, several non-pharmacological therapies, including physiological pacing, pacing prevention algorithms, anti-tachycardia pacing therapies and low-energy internal cardioversion have been introduced to treat drug refractory patients and have been implemented in multifunction implantable devices [8]. Pacing prevention algorithms Antiarrhythmic benefits of atrial and dual-chamber pacing versus single-chamber ventricular pacing in reducing atrial fibrillation recurrences and in preventing progression to permanent atrial fibrillation have been clearly demonstrated in large prospective trials enrolling either sinus node disease patients or unselected patients candidate for pacemaker implantation [9-11]. It has also been suggested that availability of rate responsiveness may maximize the effect of physiological pacing [12]. Pacing prevention algorithms have been introduced in order to maximize the preventive benefits of atrial pacing. Specific mechanisms involved in atrial fibrillation prevention by pacing algorithms include premature atrial contraction suppression or conditioning, with fewer chances to initiate atrial fibrillation, short-long atrial cycle prevention and maintenance of a high degree of exit block from all natural subsidiary atrial pacemakers. Several algorithms have been introduced by the different manufacturers during the last years. To summarize, they may be classified as follows: 1) dynamic sinus rhythm overdrive, 2) premature atrial beat reaction (short-long cycle prevention and ectopy overdrive), 3) post-tachycardia overdrive to prevent early recurrence of atrial fibrillation, and 4) prevention of inappropriate rate fall after exercise. The atrial pacing preference algorithm was the first evaluated, before being implemented in market released devices, as a temporary software, named "consistent atrial pacing", which could be downloaded into a conventional dual-chamber pacemaker via telemetry using a custom research telemetry device. It included a diagnostic which could be interpreted by a special Microsoft Excel spreadsheet. Diagnostic data were available also when the algorithm was switched off (suspended). The reliability and effectiveness of such algorithm was evaluated in a prospective randomized pilot study in which 61 patients with brady-tachy syndrome were enrolled and implanted with a rate responsive dual-chamber pacemaker [13]. After downloading of the algorithm, patients were randomly assigned to algorithm programming "on" or "suspended", with crossover after 1 month. Consistent atrial pacing induced an increase in atrial pacing percentage from 77 to 96%, and was associated with an 80% reduction of premature atrial contractions' number. Atrial fibrillation recurrences were not reduced in the overall population, but they significantly decreased by 42% when considering the patients in whom the atrial pacing percentage was < 90% during the algorithm "suspended" period. Algorithm tolerance was good with no severe adverse events. The atrial rate stabilization algorithm has been evaluated (alone or combined with consistent atrial pacing) in 16 patients with brady-tachy syndrome [14]. With regard to the effects on atrial fibrillation burden, 11 patients (69%) were found to benefit significantly from the consistent atrial pacing or atrial rate stabilization algorithms (reduction > 50% of atrial fibrillation burden). In detail, 7 patients were responders to both algorithms, 2 to consistent atrial pacing only, and 2 to atrial rate stabilization only. The ability of the post-mode switching overdrive algorithm to stabilize the atrial rate after termination of treated atrial arrhythmias has been studied in 15 patients with structural heart disease and documented atrial and ventricular arrhythmias, receiving a Jewel AF device [15]. The algorithm was active in 41% of 600 spontaneous atrial tachyarrhythmia episodes. It was capable of driving and stabilizing the atrial rhythm in the presence of slow spontaneous atrial rhythm or premature atrial beats with normal atrioventricular conduction. In case of premature atrial beats with any degree of atrioventricular block, the algorithm stabilized the ventricular rate. The authors concluded that the algorithm is reliable and might be of benefit for atrial arrhythmia treatment. Efficacy of preventive pacing algorithms has been confirmed by the preliminary results of the AF Therapy Study [16]. In 97 patients with a history of paroxysmal atrial fibrillation of at least 1 year, who had experienced at least three episodes during the last 3 months and were refractory to drug therapy, activation of four different preventive algorithms was associated with a significant improvement in all pre-selected endpoints: atrial fibrillation burden, average sinus rhythm duration, mean time in-between atrial arrhythmia episodes and number of patients free of atrial arrhythmias lasting > 1 min. All benefits were observed either in patients with or without conventional indication for pacing. Similarly, in the ADOPT trial [17], in which 320 patients were enrolled in a parallel design with randomization to algorithms on vs. off, symptomatic atrial fibrillation burden was reduced by 25% in the treatment group. On the contrary, atrial fibrillation episode number, quality of life and hospitalizations did not differ between the two randomization arms. An important criticism of ADOPT trial was that only symptomatic AF was used as an end-point. Also, a retrospective analysis by the ADOPT investigators presented in Heart Rhythm 2004 suggested that less ventricular pacing is associated with less AF recurrence (although the results did not reach statistical significance). Concern about the actual effectiveness of prevention algorithms in improving clinical outcome has been raised by the conflicting results [18,19] of the most recently published studies in which arrhythmia burden was usually selected as the main endpoint. To explain that, the interaction with other critical factors has been claimed, first of all the atrial pacing site. It is well known that atrial pacing from the right atrial appendage is associated with a prolonged P wave duration with unfavorable effects on atrial conduction and refractoriness. Clinical studies suggest that combining prevention algorithms with interatrial septal pacing may lead to better clinical outcome [20]. Furthermore, the hemodynamic negative effects of unnecessary ventricular pacing in dual-chamber pacing systems may counterbalance the preventive role of pacing algorithms. Blanc et al. [21] demonstrated that pacing algorithms could reduce the atrial arrhythmia burden only in patients with ventricular pacing percentage < 70%. In the MOST study [22], in patients with sinus node disease, atrial fibrillation recurrences had reverse relationship with ventricular pacing percentage. Moreover, the OASES trial [23] showed that the AF Suppression algorithm was even more effective when the atrial lead was implanted in the low atrial septum. In patients with right atrial appendage leads, the algorithm reduced AF burden as measured by mode-switches by 49%, whilst in patients with low atrial septal leads, the AF burden was reduced by 70%. However, whereas the ADOPT and OASES trials showed that this AF suppression algorithm reduces AF, trials testing other algorithms have not shown a similar efficacy. The ATTEST trial [24] evaluated the efficacy of overdrive pacing, and high-rate pacing algorithms in patients with AF and an indication for pacing. There were no statistically significant differences in recurrence of symptomatic AF or in AF burden. ATP terminated 54% of atrial tachycardia episodes, but high-frequency pacing could not terminate AF. Another subsequent trial, quite similar to OASES one, the ASPECT [25], showed that pacing from the atrial septum resynchronizes the atrium and provides an antiarrhythmic effect that enhances the efficacy of the suppression algorithms. It is very important to recognize the differences between these trials. The ADOPT [17] and OASES trials [23] tested the efficacy of a single algorithm to prevent AF, whereas ATTEST [24] tested the efficacy of 3 preventive pacing algorithms and 2 pacing algorithms designed to terminate atrial tachyarrhythmias. Moreover, the follow-up in ATTEST trial was 3 and not 6 months. Another matter with the OASES and the ASPECT trials was the crossover design. Because the beneficial effect of atrial pacing may persist, this design may have limited the observed efficacy of the pacing algorithms. Thus the difference in results between these trials may reflect differences in study design or differences in the efficacy of the algorithms used. Finally, due to high variability of atrial fibrillation recurrence patterns [26], published studies may be underpowered to demonstrate the true benefits of prevention algorithms. In summary, 2 trials have shown that AF suppression algorithms enhances the antiarrhythmic effects of atrial pacing in patients with AF and an indication for pacing. Thus, minimizing ventricular pacing and placing the atrial lead in the septum may probably enhance the efficacy of these algorithms. Atrial antitachycardia pacing to treat atrial tachyarrhythmias It has been demonstrated that rapid atrial pacing delivered for treating atrial tachycardia or atrial flutter may be effective in restoring sinus rhythm in 60-90% of patients [27]. Maximal effectiveness can be usually obtained by delivering antitachycardia pacing at a rate that is slightly greater than the atrial arrhythmia rate. Delivering of some extrastimuli following a rapid pacing train may be more efficacious than overdrive atrial pacing at the same pacing cycle length in terminating atrial flutter [28]. High-frequency pacing may change atrial tachycardia in transient atrial fibrillation with later sinus rhythm restoration. Appropriate detection of atrial tachyarrhythmias and efficacy of pacing therapies in patients receiving a dual defibrillator have been evaluated in large series. Adler et al. [29] reported on 537 patients with ventricular arrhythmia implanted with the Medtronic 7250 dual defibrillator (Medtronic Inc., Minneapolis, MN, USA) who were enrolled in the Worldwide Jewel AF Study and followed on average for 1 year. Seventy-four percent had a documented history of prior atrial tachyarrhythmias. Seventy-one percent of the patients had atrial therapies enabled at some time during the follow up, allowing collection and analysis of 3500 atrial episodes from 167 patients. In the 7250 Dual Defibrillator Italian Registry [30], 105 patients were enrolled. Implant indication was represented by combination of ventricular and atrial tachyarrhythmias in 52% of patients, ventricular tachyarrhythmias only in 33%, and atrial tachyarrhythmias only in 14%. During a mean follow-up of 6 months, 863 treated atrial episodes were collected and analyzed. Gold et al. [31], on behalf of the Worldwide Jewel AF-Only Investigators, studied 146 patients with recurrent drug refractory atrial fibrillation without prior ventricular tachyarrhythmias, who were implanted with a dual defibrillator and were followed on average for 1 year. During the follow-up 4913 treated episodes were available for stored electrogram analysis and therapy efficacy evaluation. Atrial tachyarrhythmia detection was very good in all the studies with a positive predictive value of atrial detection ranging from 91 to 99%. The most common reasons of inappropriate detection were represented by far-field R-wave oversensing during sinus rhythm or cluster of premature atrial contractions. Reprogramming of atrial sensitivity allowed in some case avoidance of inappropriate detection due to far-field R-wave oversensing. Sensitivity of atrial tachycardia and fibrillation detection and validation of continuous detection of atrial tachyarrhythmias have been specifically addressed by Swerdlow et al. [42] who performed 80 Holter recordings with telemetered atrial electrograms in 58 patients, implanted because of combination of atrial and ventricular arrhythmias. Continuous detection of atrial tachyarrhythmia could be demonstrated in 96% of patients with spontaneous episodes. Atrial sensitivity for arrhythmia detection was 100%. It is worthwhile to stress that the atrial arrhythmia detected at very early onset was very commonly a well organized atrial tachycardia. Among 2380 episodes in the worldwide series, 63% were classified as atrial tachycardia (mean atrial cycle 278 ± 56 ms) and 37% as atrial fibrillation (mean cycle 204 ± 35 ms). In the Italian Registry, among 863 atrial episodes, 53% were automatically classified by the device as atrial tachycardia and 47% as atrial fibrillation. After revision of the stored data, among 843 appropriately detected episodes, 55% were clinically classified as atrial fibrillation and 45% as atrial tachycardia. In the AF-Only group, among 3116 episodes treated by antitachycardia pacing, 67% were classified as atrial tachycardia. Efficacy of antitachycardia pacing therapy in the three series is reported in Table 1 [29-31]. The efficacy was estimated in two ways: 1) crude estimate, defined as the proportion of successful terminations out of the total number of treated episodes; 2) adjusted estimate, using the generalized estimate equation method [30] which allows adjusting estimate for multiple episodes within a patient through a correlation structure between episodes and patients. In the worldwide study [29-31], a subanalysis performed to compare the efficacy of burst+ vs. ramp therapy for atrial tachycardia did not find any significant difference between the two therapies. An history of atrial flutter was the only independent predictor of pacing efficacy for atrial tachycardia, while no independent predictors could be identified for atrial fibrillation. No comparison was feasible between burst+ or ramp and high-frequency burst because the last was usually applied after unsuccessful delivery of burst+ or ramp therapy. Pooling all antitachycardia pacing therapies (burst+, ramp and 50 Hz burst), their efficacy consistently increased as far as atrial arrhythmia cycle lengthened. As a matter of fact, in the Italian Registry, while the relationship between efficacy of atrial burst+ and ramp vs. atrial cycle length was very high (r2 = 0.85, p < 0.001), the relationship between efficacy of 50 Hz burst and atrial cycle length was very poor, with a trend toward a reverse correlation (r2 = 0.31, p < 0.05). Such a difference could be explained by taking into account the mechanisms of action of different antitachycardia pacing techniques. During overdrive pacing, the wavefront of pacing stimulus enters the reentrant pathway in the antidromic and orthodromic direction. The antidromic wavefront blocks by collision the arrhythmia wavefront, while the orthodromic wavefront may either reset the tachycardia or stop it, when early enough to be blocked in a refractory area. Slower tachycardias, due to a wider excitable gap, may be more easily terminated in this way. Termination of atrial tachyarrhythmias by high-frequency pacing is typically preceded by tachycardia acceleration, which becomes unable to be sustained, hence resulting in arrhythmia termination and sinus rhythm restoration. It has been hypothesized that high-frequency pacing induces a new faster reentrant circuit which is unable to sustain itself [34]. This mechanism looks independent of the arrhythmia cycle. Looking at individual patients, random and wide distribution of median atrial cycles at onset during the follow-up was observed in the majority of them. A subgroup showed a narrow Gaussian distribution along either a fast (200 ms) or a slow band (250 ms). Antiarrhythmic drugs have been demonstrated to be capable of modifying atrial cycle profile. Antiarrhythmics may modify the electrophysiological properties of the atria and the arrhythmia organization pattern by lengthening the mean atrial arrhythmia cycle and by widening the temporal excitable gap [35-37]. Dijkman and Wellens [38] demonstrated that atrial arrhythmias in defibrillator patients with structural heart disease, receiving class III antiarrhythmic drugs, frequently had longer cycle lengths than atrial fibrillation. In fact, among 600 spontaneous episodes, atrial fibrillation was diagnosed in 19%, fast polymorphic atrial tachycardia in 20%, fast monomorphic atrial tachycardia in 57%, and slow atrial tachycardia in 4%. Drug-induced atrial cycle length changes may impact on atrial antitachycardia pacing therapy efficacy. In spite of that, in the AF-Only series there were no drugs that were independent predictors of therapy efficacy. Finally, increasing the delay from arrhythmia detection to therapy delivery was associated with a significant reduction in efficacy. Optimal delay has been identified in less than 5 min in the Worldwide Study and in less than 1 min in the Italian Registry. That was probably due to the fact that the majority of atrial tachyarrhythmias accelerated in few minutes after onset. Considering that slower arrhythmias can be more easily treated by pacing techniques, a short delay in therapy delivery should be recommended. The efficacy of 50 Hz burst on atrial fibrillation may be a matter of debate. Wide local capture of atrial fibrillation has been documented [39]. Data from canine studies suggested that high-frequency pacing could accelerate the local atrial fibrillation cycle length and destabilize the reentrant rhythm so that destabilized atrial fibrillation could be converted to sinus rhythm in some cases. Anyway, in humans termination of persistent atrial fibrillation by high-frequency pacing could never be demonstrated [40,41]. High-frequency pacing may terminate induced atrial fibrillation during electrophysiological study with a 33% efficacy rate [34] and atypical atrial flutter with a 60% efficacy rate [41]. In selected cases, local capture of new-onset atrial fibrillation and entrainment of a relatively large area of the atria [42] could have destabilized the arrhythmia by reducing the number of wavelets of the random reentry, hence preventing arrhythmia sustenance. Anyway, considering that arrhythmia classification is based on atrial activity at atrial lead site, misclassification of atrial tachycardia as atrial fibrillation cannot not be excluded. Furthermore, some episodes may have terminated spontaneously after therapy delivery [43]. The ASPEN-ICD and ASSIST trials will probably give more data about these new perspectives. They will test the algorithm in patients with ICDs. The former will determine if the first episodes of AF can be prevented by the algorithm, whereas the latter will test if this algorithm prevents AF in patients who have a history of atrial arrhythmia. Safety of antitachycardia pacing therapy was excellent since no ventricular arrhythmia induction was observed in any case after antitachycardia pacing delivery. These data suggest that, on a large scale, these algorithms would presumably be safe, and that it would be necessary to better determine how effective atrial ATP is, and whether it would reduce hispitalizations and/or electrical or pharmacological cardioversions and thereby improve quality-of-life. Conclusions As stated in the discussed trials, pacing prevention algorithms should probably be better and further evaluated, before having a definitive and comprehensive answer on their utility. Clinical studies showed inconsistent clinical benefits of the algorithms, although they reduce atrial fibrillation triggers such as premature atrial complexes, thus preventing short-long cycle phenomenon. Factors which may be responsible for neutral results include: 1) high atrial pacing percentage in conventional DDDR, 2) non-optimal atrial pacing site, 3) deleterious effects of high percentages of ventricular pacing, and 4) inappropriate study design and endpoint selection. Variability of atrial arrhythmia recurrence patterns and onset mechanisms suggest individual programming of prevention algorithms by using data stored in the device memory. Atrial antitachycardia pacing is an effective tool to treat atrial tachyarrhythmias and it may stop nearly 50% of arrhythmia episodes. In particular, all previously described data are more related to atrial tachycardia reduction than to atrial fibrillation decrease. Delivery of atrial therapies early after arrhythmia onset and on more organized arrhythmias may improve success rate. Associated use of antiarrhythmic drugs, mainly propafenone and flecainide, may further increase effectiveness by lengthening atrial arrhythmia cycle. Effective antitachycardia pacing therapies may reduce atrial fibrillation burden, but conflicting evidence does exist as regards this issue, probably because current clinical studies may be underpowered to detect such an efficacy. Consistent monitoring of atrial and ventricular rhythm as well as that of antitachycardia pacing effectiveness may be extremely useful for optimizing device programming and pharmacological therapy. At present the efficacy of pacing algorithms to prevent AF is by no means something that has been proved beyond reasonable doubt, so that more large-scale prospective trials are needed to answer this question. Our information is currently based on small trials and any attempt to extrapolate these data to the general population will probably produce inaccurate results. Clinical endpoints should be further investigated in future studies. 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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-4-371704294510.1186/1476-7120-4-37ReviewC-reactive protein in aortic valve disease Sanchez Pedro L [email protected] Anna Maria [email protected] Associated Professor of Cardiology, Instituto de Ciencias del Corazón (ICICOR), Hospital Clínico Universitario de Valladolid, C/Ramón y Cajal n° 3, 47005, Valladolid, Spain2 Department of Cardiology and Cardiac Surgery, CNR Institute of Clinical Physiology, Ospedale Pasquinucci, Massa, Italy2006 16 10 2006 4 37 37 21 12 2005 16 10 2006 Copyright © 2006 Sanchez and Mazzone; licensee BioMed Central Ltd.2006Sanchez and Mazzone; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aortic Valve Disease, includes a range of disorder severity from mild leaflet thickening without valve obstruction, "aortic sclerosis", to severe calcified aortic stenosis. It is a slowly progressive active process of valve modification similar atherosclerosis for cardiovascular risk factors, lipoprotein deposition, chronic inflammation, and calcification. Systemic signs of inflammation, as wall and serum CRP, similar to those found in atherosclerosis, are present in patients with degenerative aortic valve stenosis and may be expression of a common disease, useful in monitoring of stenosis progression. ==== Body Aortic stenosis (AS) is at present time the most frequent valvulopaty in developed countries, and as life expectancy increases, the incidence and prevalence of AS will also rise fundamentally at the expense of the degenerative form. The scientific interest of this valvar disorder was parked for many years, as the image tools necessary to quantify it are at our disposal and we have a certain agreement of the clinical moment for indicating the valve replacement. Nevertheless, in the past years different studies have demonstrated a common pathogenic mechanism between degenerative AS and atherosclerosis [1-3]. This is consistent with histopathological evidence that the lesions in AS involve active cellular processes that have classical "response to injury features", namely inflammatory infiltrates containing macrophages, T cells, and smooth muscle cells [4]. Therefore, investigators have needed only a half turn of the head to transfer the early past experience of blood markers of inflammation in the atherosclerosis setting to the AS scenario. C-reactive protein (CRP) is the marker of inflammation most widely studied in patients with coronary artery disease and hence has become the marker of reference for any other inflammatory-based disease [5]. On this basis, CRP has emerged as a leading candidate for a better understanding of AS pathogenesis, for predicting AS progression, and for driving therapies in AS. CRP is increased in patients with AS The first study showing increased CRP levels in patients with degenerative AS was published by Galante et al, in 2001 [6]. They compared serum CRP levels of 68 consecutive patients with severe degenerative trileaflet AS and absence of coronary atherosclerotic lesions admitted for elective cardiac surgery with 92 healthy controls. CRP levels were higher in patients with AS than in controls (0.85 ± 1.42 vs. 0.39 ± 0.50; p = 0.0001). They also showed an independent association of CRP with AS; the odds ratio for the disease according to CRP levels were 2.62 (95% confidence interval: 1.06 to 6.49). No association between CRP and aortic jet velocity, aortic valve area, or degree of calcification was found, notwithstanding that all patients had severe aortic stenosis and were waiting for surgery. Further studies have confirmed [1] and expanded these observations. Serum CRP is also elevated in patients with asymptomatic aortic stenosis [7,8], does not rise in accordance with increasing severity of valvar disease [7,8], and decreased from before to 6 months after aortic valve replacement [9]. One of the principal characteristics of AS is the important degree of valvar calcification that takes place along the different stages of the disease. Thus, investigating whether CRP could be related to the mechanism of calcification appears very atractive. The role of serum CRP in the tissue calcification process has been investigated in a recently elegant study by Warrier et al,. When aortic wall was exposed to an excess amount of CRP in an in vitro simulating model, the calcification rate of aortic wall increased as the concentration of CRP. The results of this work could revealed the role of CRP present in physiological fluid in aortic valvar calcification [10]. Data providing contribution of serum CRP to valve calcification in the clinical setting is available in patients with renal failure. Valvar calcification in patients with renal failure is associated with enhanced inflammation [11]. Furthermore, in chronic hemodyalisis patients in steady clinical conditions with no clinical evidence of either infectious or inflammatory diseases, a high CaxPO4 is associated with high CRP concentrations and hence associated with valvar calcification [12,13]. However, preliminary transversal evidence evaluating the association of CRP and calcification in patients with AS and no renal failure is controversial [6-8]; thus, the long-term predictive value of the serum CRP level for the development of aortic calcification should be addressed in future well design prospective trials. Recently, Skowasch et al, have observed localization of CRP in valve tissue of degenerative AS and degenerative aortic valve bioprostheses [14]. Furthermore, serum CRP showed a significant correlation with the valvar CRP expression (r = 0.54; P < 0.001). Consequently, we must integrate our previous understanding of the physiological role of CRP in inflamed tissues, thereby promoting local anti-inflammatory and proinflammatory effects [15]. It has been suggested that the effects of CRP on human aortic endothelial cells are similar to those seen in atherosclerotic models i.e. inducing the amplification of local inflammation and cellular damage [16,17]. CRP and progression of AS We have assessed whether serum CRP levels could predict rapid AS progression. We measured serum high sensitivity CRP in 43 asymptomatic subjects with AS at baseline and six months later. Plasma CRP concentration was significantly higher in patients with rapid AS progression (5.1 [2.3 to 11.3] mg/L) compared to patients with slow AS progression (2.1 [1.0 to 3.1] mg/L, p = 0.007). The rate of progression, was higher in patients who had a cutpoint level of CRP >3 mg/L than those who had levels ≤ 3 mg/L (66.7% vs 33.3%, p = 0.012 for aortic jet velocity and 62.5% vs 37.5%, p = 0.063 for aortic valve area, figure 1 and 2). Little is known of the mechanisms responsible for progression of AS albeit mechanical, clinical, and metabolic variables have been suggested to contribute to rapid progression of AS [18,19]. Our data suggest that elevated CRP levels may be a marker of AS progression and could have important clinical implications as interventions that reduce CRP levels may be beneficial in the prevention of AS and perhaps also in reducing AS progression. Figure 1 Rate of CRP > 3 mg/L in patients with slow and rapid aortic stenosis progression according to the assesment of aortic jet velocity (Cw). Slow and rapid progressors were considered those patients with an increase in aortic jet velocity < or ≥ 0.15 m/s respectively, during the six months follow-up. Figure 2 Rate of CRP > 3 mg/L in patients with slow and rapid aortic stenosis progression according to the assesment of the aortic valve area (AVA). Slow and rapid progressors were considered those patients with a decrease in AVA < or ≥ 0.05 cm2 respectively, during the six months follow-up. Finally, baseline CRP concentration, was similar in patients who developed symptoms compared to those asymptomatic during follow-up. Data in patients with aortic sclerosis suggest a positive association between the risk of adverse cardiovascular events and the presence of coronary artery disease (hazard ratio [HR] 3.23, p = 0.003) and enhanced inflammation (HR 2.2, p = 0.001), and not as a result of the effects of valvar heart disease per se [20]. Targeting CRP for the therapy of AS The presence of higher serum CRP levels and the tissue location of CRP in patients with AS, have raised the important question of whether medical therapies with agents such as statins and ACE inhibitors, which have already been shown to delay the progression of atherosclerosis may also affect the progression of AS. Preclinical studies, have shown that experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis-like process in the aortic valve that is inhibited by statins [21-23]; ie, atorvastatin inhibited calcification in the aortic valve by increasing eNOS protein and serum nitrite concentrations [22], and decreasing Lrp5 (low-density receptor-related protein) receptors involve in cellular proliferation and osteoblastogenesis via the beta-catenin signaling pathway [23]. Although recent retrospective clinical studies suggest that statins also may slow the hemodynamic progression of AS [24-28], the results of the SALTIRE study have been discouraging for all of us who believe that AS is an active disease process akin to atherosclerosis with lipoprotein deposition, chronic inflammation, and active leaflet calcification [29]. Therefore, It is important to analyze the neutral effect of high doses of atorvastatin in such an attractive hypothesis. First, the SALTIRE study differs not only because of its prospective design but also because the indications for therapy were different. In the retrospective trials, statin therapy was indicated for the treatment of hyperlipidemia, whereas in the prospective trial, patients in whom statins were indicated for the treatment of hyperlipidemia were excluded. Second, statin doses in the retrospective studies were lower. Finally, although the observation periods in the various studies were similar, patients in the retrospective studies were already receiving therapy at the time of inclusion in the study [30]. Aside from the already commented differences with retrospective studies, high proportion of patients were on drugs with anti-inflammatory effects (apirin, betablockers or angiotensin converting enzyme inhibitors) that could have mitigated the pleiotropic effect of statins. For example, in the retrospective trials that found a lower rate of progression among patients treated with statins, the percentage of patients on aspirin was always important and significantly higher in the group with positive results [25,27,28]. Importantly, in the SALTIRE study, half of the patients in each group were on aspirin, a prevalence that would have a bearing on the results of the study. In addition, markers of inflammation would have been useful as a means to predict or monitor the individual's response to atorvastatin. If the authors were not just looking for a reduction cholesterol effects it is surprising that they have not yet provided any information about inflammatory effects. As the main finding, Skowasch et al, [14] in their recent study showed that both valvar CRP expression and serum CRP levels were found to be lower in patients on statins. Moving forward, we must learn more about the pathogenic mechanisms of AS. 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==== Front J Orthop SurgJournal of Orthopaedic Surgery and Research1749-799XBioMed Central London 1749-799X-1-101715011710.1186/1749-799X-1-10Research ArticleThoracic myelopathy caused by ossification of ligamentum flavum of which fluorosis as an etiology factor Wang Wenbao [email protected] Linghua [email protected] Heyuan [email protected] Ronghua [email protected] Jing [email protected] Yun [email protected] Spine surgery department, Tianjin hospital, No. 406 Jiefangnan Road, Hexi District, Tianjin City, 300211, People's Republic of China2 106 Fort Washington Avenue, Room 3H, New York City, NY, 10032, USA3 Hand surgery department, Tianjin hospital, No. 406 Jiefangnan Road, Hexi District, Tianjin City, 300211, People's Republic of China2006 2 11 2006 1 10 10 6 1 2006 2 11 2006 Copyright © 2006 Wang et al; licensee BioMed Central Ltd.2006Wang et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose To evaluate the clinical feature, operative method and prognosis of thoracic ossification of ligamentum flavum caused by skeletal fluorosis. Methods All the patients with thoracic OLF, who underwent surgical management in the authors' hospital from 1993–2003, were retrospectively studied. The diagnosis of skeletal fluorosis was made by the epidemic history, clinical symptoms, radiographic findings, and urinalysis. En bloc laminectomy decompression of the involved thoracic levels was performed in all cases. Cervical open door decompression or lumbar laminectomy decompression was performed if relevant stenosis existed. The neurological statuses were evaluated with the Japanese Orthopaedic Association (JOA) scoring system preoperatively and at the end point of follow up. Also, the recovery rate was calculated. Results 23 cases have been enrolled in this study. Imaging study findings showed all the cases have ossification of ligamentum flavum together with ossification of many other ligaments and interosseous membranes, i.e. interosseous membranes of the forearm in 18 of 23 (78.3%), of the leg in 14 of 23 (60.1%) and of the ribs in 11 of 23 (47.8%). Urinalysis showed markedly increased urinary fluoride in 14 of 23 patients (60.9%). All the patients were followed up from 12 months to 9 years and 3 months, with an average of 4 years and 5 months. The JOA score increased significantly at the end of follow up (P = 0.0001). The recovery rate was 51.83 ± 32.36%. Multiple regression analysis revealed that the preoperative JOA score was an important predictor of surgical outcome (p = 0.0022, r = 0.60628). ANOVA analysis showed that patients with acute onset or too long duration had worse surgical result (P = 0.0003). Conclusion Fluorosis can cause ossification of thoracic ligamentum flavum, as well as other ligaments. En bloc laminectomy decompression was an effective method. Preoperative JOA score was the most important predictor of surgical outcome. Patients with acute onset or too long duration had worse surgical outcome. ==== Body Background Fluoride is an important element for bone mineralization. It causes an increase in bone mass by stimulation of the osteogenetic process [1]. However, over intake of fluoride may cause fluoride intoxication, so-called fluorosis [2-10]. Its typical clinical features include dental fluorosis, diffuse densification of bone, calcifications of bony insertions of many ligaments, discs, and interosseous membranes, i.e. interosseous of the ribs, forearm, and leg, posterior longitudinal ligament, transverse atlantal ligament, ligamentum flavum, and membrana obturatoria [8-10]. Thoracic spinal stenosis caused by ossification of ligamentum flavum (OLF) is a rare disease [11-20]. However, thoracic OLF caused by skeletal fluorosis is rather rare. Only 6 cases have been reported in the English literature [3,13]. The authors' purpose is to evaluate the clinical feature, operative method, and prognosis of thoracic OLF caused by skeletal fluorosis. Materials and methods All the patients with thoracic OLF, who underwent surgical management in the authors' hospital from 1993–2003, were retrospectively studied. The cases accorded with the following criteria were included. Diagnostic criteria for fluorosis: epidemic history including a long history living in a high fluorosis area; dental fluorosis; typical X-ray findings including diffuse densification of bone, calcifications of bony insertions of many ligaments, discs, and interosseous membranes, i.e. interosseous of ribs, forearm, and leg; urinalysis of fluoride may increase. Diagnostic criteria for thoracic ossification of ligamentum flavum: typical clinical symptoms and findings which included numbness in the lower limbs and below the relative segment of trunk, motor weakness in the lower extremities and difficulty in walking; physical examination showed increased lower limbs muscle tension, increased in deep tendon reflexes and appearance of pathological reflex, i.e. Babinski sign. X ray, CT scan, and MRI were used to confirm the diagnosis. For each the patient, A-P view and lateral view X-ray of the thoracic spine were taken. Then thoracic MRI was taken to ensure the diagnosis and identify the involved segments. CT scan was performed for the involved segments. A-P view, lateral view X-ray of forearms and legs and A-P view of the chest were also taken. Indication of surgery: symptoms and signs of thoracic myelopathy; CT scan and MRI demonstrated significant thoracic canal stenosis; the symptoms and signs correlation with the imaging findings. En bloc decompression was performed on each patient. In one patient combined with cervical ossification of posterior longitudinal ligament, cervical open door decompression was performed additionally. In one patient combined with lumbar stenosis, lumbar laminectomy decompression was performed additionally. Preoperative radiographic localization with a Kirschner wire was used to confirm the operative level on the morning of operation day. After induction of general anesthesia, the patient was placed prone with an indwelling bladder catheter. The abdomen was decompressed to avoid excessive epidural bleeding. According to the X-ray localization result, a midline incision was made at the appropriate level and extended to the fascia. Subperiosteal dissection of the paraspinal muscles was performed using electrocautery cutting. The spinous processes were shortened using rongeurs (not totally removed). The laminectomy was performed with high-speed drill. The width of the laminectomy was approximately one third the size of the inside of the facet. After the laminae were totally floated, it was taken off en bloc by holding the residues spinous processes (Fig 4). The ossified ligamentum flavum often adhered to the dura mater. So, much care should be paid to avoid rupture of the dura mater. Occasionally, the dura mater also ossified. In those cases, we did not take away the ossified dura mater totally, just floated it. When coexistent lesions were present at noncontinuous thoracic levels, clinical symptoms and neuro-imaging findings were examined. The level considered to be the likely cause of clinical symptoms was then surgically treated. When coexistent lesions were present at the cervical or lumbar region, the depression of the relevant region was performed. The neurological statuses were evaluated with the JOA scoring system of myelopathy preoperatively and at the end point of follow up (table 2). The recovery rate, described by Hirabayashi et al [21], indicating the degree of recovery of normal function postoperatively, was calculated as follows: (postoperative JOA score – preoperative JOA score)/(11- preoperative JOA score) ×100. Table 1 Summary of clinical features observed in 23 patients with OLF Symptoms number of cases numbness and sensory deficit 22 lower-limb weakness and gait disturbance 21 Low-back pain 8 "squeezing tight band" around chest or abdomen 6 neurological claudication 4 leg pain 12 fecal & urinary incontinence 18 knee and ankle hyperreflexia 18 positive patellar and ankle clonus 14 positive Babinski sign 15 Table 2 summary of the JOA scoring system for the assessment of myelopathy neurological status score lower-limb motor dysfunction unable to walk 0 Able to walk on flat floor with walking aid 1 Able to walk up/downstairs w/handrail 2 Lack of stability & smooth reciprocation of gait 3 no dysfunction 4 lower-limb sensory deficit severe sensory loss or pain 0 Mild sensory deficit 1 no deficit 2 trunk sensory deficit severe sensory loss or pain 0 Mild sensory deficit 1 no deficit 2 sphincter dysfunction unable to void 0 marked difficulty in micturition 1 minor difficulty in micturition 2 no dysfunction 3 Total score for a healthy patient is 11. X-ray and CT scan were performed 3 days after the operation to conform the decompression levels and decompression area. X-ray was performed at the end of the follow up to identify whether there was spinal instability. Statistical analysis Paired t test was used to analyze the differences between the results before operation and at end of follow up. Multiple linear regression was conducted to determine the quantitative variables best correlating to surgical outcome. ANOVA was used to analyse differences among the three groups according to the duration of preoperative symptom. When the results of ANOVA indicated < 0.05, further statistical analysis was followed to determine whether there was any significance difference between any two groups. The statistical results were analyzed using the Statistical Analysis System (SAS). Significance was accepted for P-values of < 0.05 in all of the above analyses. Results Clinical presentation 74 cases of thoracic OLF were surgically treated at the authors' institution between 1993 and 2003, 23 of which (16 male and 7 female) were caused by fluorosis. The 23 patients ranged in age from 42 to 72 years (mean 54.8 years). 6 cases had acute onset of clinical symptom, 4 of which had a traumatic history, 2 without markedly traumatic history. The other 17 cases did not have a traumatic history and presented with progressive symptoms. Numbness in the lower limbs and below the relative segmental was the most common initial symptom in 17 of the 23 patients (73.9%). Motor weakness in the lower extremities and difficulty in walking as initial symptoms were found in 6 patients. The details of the clinical findings are shown in table 1 and table 3. The mean duration of symptoms between initial onset and operation was 37 months (range 1 day–11 years). All of the 23 patients had a long term, high fluoride area living history. Fluoride over intake was from water in 21 cases (91.3%) or from coal smoke in 2 cases (8.7%). 22 of 23 patients (95.7%) had different levels of dental fluorosis. Urinalysis showed markedly increased urinary fluoride in 14 of 23 patients (60.9%). Table 3 Data on Patients With Ossification of Ligamentum Fluvam case no. sex & age DPS & group JOA score levels & segment number of OLF levels & number of decompression recovery rate % pre-operation follow up LEM TS LES SD Total LEM TS LES SD total 1 M, 42 15m(2) 2 2 0 1 5 4 2 1 2 9 T12–L1 (1) T12–L1 (2) 67 2 M,62 3d(1) 3 2 1 3 9 4 2 1 3 10 T12–L1 (1) T12–L1 (2) 50 3 F, 46 32m(2) 3 1 1 2 7 4 2 1 3 10 T12–L1 (1) T12–L1 (2) 75 4 M,54 12m(2) 4 1 1 3 9 4 2 2 3 11 C7–T1(1) C7–T1(2) 100 5 F,64 4y(2) 2 1 1 2 6 4 2 1 3 10 T1–4(3) T1–4(4) 80 6 M,51 5y(3) 1 1 0 2 4 3 2 1 3 9 T3–5(2) T3–5(3) 71 7 M,42 2d(1) 1 1 0 1 3 2 2 1 2 7 T7–12(5) T7–12(6) 50 8 M,65 7y(3) 1 1 1 1 4 3 1 1 1 6 T8–L1(5) T8–T12(5) 29 9 M,55 11y(3) 2 1 1 3 7 2 2 1 3 8 T7–L1(5) T8–L1(6) 25 10 F,56 15m(2) 2 1 1 2 6 3 2 1 2 8 T9–L1(4) T10–L1(4) 40 11 M,59 1d(1) 2 1 0 1 4 2 1 0 1 4 T9–L1(4) T9–L1(5) 0 12 M,45 6m(2) 4 2 1 3 10 4 2 2 3 11 T10–L1(3) T10–L1(4) 100 13 F,50 5y(3) 2 2 1 1 6 3 2 1 2 8 T10–L1(3) T10–L1(4) 40 14* M,62 1d(1) 0 0 0 0 0 0 0 0 0 0 T10–L1(3) T10–L1(4) 0 15 M,50 18m(2) 2 2 1 3 8 4 2 2 3 11 T10–L1(3) T10–L1(4) 100 16* F,48 5y(3) 2 1 1 2 6 2 1 1 2 6 T10–L1(3) T10–L1(4) 0 17 M,54 1d(1) 0 1 0 1 2 0 1 0 2 3 T3–7 11 T10–L1(4+3) T10–L1(4) 18 M,59 8y(3) 2 1 1 2 6 4 2 1 2 9 T1–T5 T1–T5(5) 60 T9–L1(4+4) 19 M,58 7y(3) 1 0 0 1 2 2 1 1 2 6 T1–6 44 T9–L1(5+4) T9–L1(5) 20 M,56 6d(1) 2 1 1 1 5 3 1 1 2 7 C7–T9(9) C7–T4(5) 33 21 F,52 7m(2) 2 1 1 2 6 4 2 2 3 11 T8–12(4) T8–12(5) 100 22 F,72 7y(3) 2 2 1 1 6 4 2 1 2 9 T9–11 T9–11 60 L3–S1(2+3) L3–S1(3+4) 23 M,58 4y(2) 1 1 0 2 4 3 2 1 2 8 C3–6 C3–6 57 T10–L1(4+3) T10–L1(4+4) DPS: duration of preoperative symptom. LEM: lower extremity motor; TS: trunk sensory; LES: lower extremity sensory; SD: sphincter dysfunction Paired t test showed that there is significant difference between the JOA score of pre-operation and followed up (P = 0.0001). The mean recover rate is 51.83%. ANOVA analysis of the three groups according to the DPS showed p = 0.0003. Further t test showed that there was significant different between group one and group tow (P = 0.0004). There was significant different between group two and group three (P = 0.003). However, there was no significant different between group one and group three (P = 0.197). Imaging study result The mean number of involved segments is 4.17, with a range from 1 to 9 (Fig 1). The ossified ligamentum flavum displayed obscuration the margin of the lamina on the A-P view X-ray in 8 patients (34.8%). On the lateral view X-ray, 12 of 23 patients (52.2%) showed high density projection into the spinal canal at the level of compression. All the ossified ligamentum flavum displayed the density of cortical bone on CT scans and sometimes had a thin gap between the laminae (Fig. 3c). All the ossified ligamentum flavum demonstrated triangular protrusion with a low-signal intensity resembling cortical bone on MR images (Fig 3a, 3b). Figure 1 A diagram of the OLF distribution of 23 patients. Figure 2 Anteroposterior view radiograph of both forearms showed significant calcifications of interosseous membranes of forearm. Figure 3 a, b. T1 and T2 weight MRI of thoracic spine showed continuous multi-level ossification of ligamentum flavum between T7–12. c. CT scan showed ossified ligamentum flavum, note that there was a thin gap between the ossified ligament and the lamina. Figure 4 The en bloc removed lamina, note the nodular ossified ligamentum flavum. X-ray of forearms, legs, and chest showed ossification of interosseous membranes of the forearm in 18 of 23 patients (78.3%) (Fig 2), of the leg in 14 of 23 patients (60.1%), and of the ribs in 11 of 23 patients (47.8%). Operation and the prognosis The operation time ranged from 2.5 hours to 4.3 hours, with a mean of 3.2 hours. The mean decompressed segments number is 4.35 (ranged from 2 to 8, table 3). Blood loss ranged from 400 ml to 2800 ml, with a mean of 850 ml. Dura mater rupture occurred in 4 patients. Deep infection occurred in one patient. No postoperative neurological deterioration occurred. All the patients were followed up from 12 months to 9 years and 3 months, with an average of 4 years and 5 months. Paired t test showed that the JOA score increased significantly at the end of follow up (P = 0.0001, table 3). The recovery rate was 51.83 ± 32.36%. Multiple regression analysis revealed that the preoperative JOA score was an important predictor of surgical outcome (p = 0.0022, r = 0.60628, table 4). However, the sex, age, preoperative symptom duration, and levels of OLF did not significantly influence the surgical outcome. ANOVA analysis showed that patients with acute onset (group 1) or too long duration (group 3) had worse surgical result (P = 0.0003) (table 3). Further t test showed that there was significant different between group 1 and group 2 (P = 0.0004). There was significant different between group 2 and group 3 (P = 0.003). However, there was no significant different between group 1 and group 3 (P = 0.197) (table 3). No postoperative instability occurred. Table 4 results of a multiple linear regression analysis of selected variables to predict surgical outcome recover rate at final follow up Variable coefficient p value Age -0.24755 0.2548 duration of preoperative symptom -0.10367 0.6378 Preoperative JOA score 0.60628 0.0022 levels of OLF -0.31515 0.1430 Discussion Etiology The thoracic OLF was first reported by Polgar [17] in 1920 with lateral radiographs. From then on, several clinical series and many case reports have been reported. However, the etiology of OLF was unclear. As most of the reported OLF's were located between T9 and T12, Barnett et al. [11] suggested that the hyper mobility of the lower thoracic spine might promote degeneration and canal stenosis. Liao's study [22] showed a high prevalence of coexisting anterior osteophytes and herniated intervertebral disc at the symptomatic OLF segments. So they concluded that OLF might be a degenerative response to the micro injury of the ligamentum flavum. The hypothesis was histologically supported by Okada and colleagues [15] who found that OLF formed in the hypertrophic ligamentum flavum with fibrocartilage proliferation, and this was thought to be a phenomenon of mechanical injury. Therefore, it was thought that the development of OLF might be secondary to the specific fiber reconstruction of the ligamentum flavum in response to mechanical stress. However, Muthukumar [13] reported two cases of OLF caused by fluorosis, recently. Wang et al. [8-10] reported fluorosis could cause ossification of a lot of ligaments. All these reports showed fluorosis might play a role in OLF. Fluoride is one of the necessary minor elements for humans, and the daily requirement is 0.05–0.07 mg/kg body weight/day [2,5]. The benefits of water fluoridation in controlling dental caries were well documented. Fluoride was first used in water for caries control in 1945 and 1946 in the United States [1] and Canada [4], respectively. However, over intake of fluoride will cause fluorosis [2-10]. Fluorosis caused by fluoride intoxication was first reported by Feil in 1930, and skeletal fluorosis was reported by Short in 1937 [7]. Normally, there are two sources of fluoride over intake, water and coal smoke. In the high fluoride area, the density of fluoride in the water is more than 5–8 mg/L, and the people drink the water directly from the well without any management. This will cause dental fluorosis, skeletal fluorosis, or even systemic fluorosis. It was reported that neurological complications occurred in approximately 10% of patients with skeletal fluorosis, usually in the later stages of the disease [7]. To date, the myeloradiculopathy caused by skeletal fluorosis was thought to be a result of compression of the spinal cord by osteophytes and vertebral osteosclerosis [7,23]. However, myelopathy caused by OLF in patients with skeletal fluorosis has been recognized recently [3,13]. So, we think fluorosis should be entertained as an etiology factor of OLF, especially in patients from endemic areas. The pathogenesis of ossification of the ligaments in this condition remains speculative. High expression of transforming growth factor beta-1 (TGF-β1) by fibroblasts was found in the ossified matrix within ossified ligaments and in chondrocytes within cartilaginous areas adjacent to the ossified ligaments [24]. TGF-β1 could have played a role in chondroid metaplasia and ectopic ossification in OLF. Recent experimental evidence points to the involvement of proto-oncogenes c-fos and c-jun in skeletal fluorosis. Zhang et al. [25] have demonstrated that exposure to excessive fluoride could stimulate the activation and proliferation of osteoblast-like cells with enhanced expression of messenger ribonucleic acid and proteins of c-fos and c-jun. Clinical feature of thoracic ossification of ligamentum flavum Thoracic OLF is rare and usually asymptomatic. The disease usually has an insidious onset and very slow progression. Analysis of previously published epidemiological data reveals that thoracic OLF most commonly involves the vertebrae between T-9 and T-12(as in our serious in figure 1), where greater mobility and vulnerability (due to spinal motion) may result in frequent mechanical injury. In our series, numbness in the lower limbs and below the relative segmental was the most common initial symptom in 17 of the 23 patients (73.9%). Motor weakness in the lower extremities and difficulty in walking as initial symptoms were found in 6 patients (26.1%). This finding is in agreement with the observations reported in previous studies [11-20]. When an extradural compressive lesion develops at the thoracic level, pressure to the spinothalamic tract, fasciculus gracilis and fasciculus cuneatus causes the numbness and lost of proprioceptive sensation in the lower limbs and below the relative segment. Upper neuron injury might occur and be caused by pressure to the cerebrospinal tract. This results in increasing muscle tension of the lower extremity, increasing in both patellar and Achilles reflexes. However, if at a lower thoracic level, the lesions exist at neighboring sites of the conus medullaris, the patellar or Achilles reflex will occasionally dissociate, or both decrease. Compressive coexistent lesions, such as cervical or/and lumbar stenosis, also influence the clinical features, as showed in the literature [26] and in our series. Although the neurological findings in our series are similar with other authors' findings, OLF caused by fluorosis has their own features. Firstly, all the patients had the character features of fluorosis. Secondly, the segment number of involved LF is more than others (figure 1). Surgical procedures Non operative method is not effective for symptomatic patients. So, early diagnosis and operation interference were recommended for the symptomatic patients. As the thoracic OLF compressed the spinal cord posteriorly, several posterior decompression methods were developed. These operative techniques include open-door laminectomy, en bloc laminectomy, fenestration, total decompression et al. [14,15,18-20]. In our cases, all the patients performed en bloc decompression. The segments are shown in table 3. The blood loss was much more when compared with our non-fluorosis cases (non published data). This was partly because the fluorosis made the soft tissue easily prone to bleeding and partly because the decompression segments are more than others. The results shown in table 3 stated that the decompression was effective. In 4 cases of our patients, ossification of dura mater occurred. Some authors also reported ossification of the dura mater together with the ossification of thoracic ligamentum flavum [14]. In those cases, severe adhesion between ossified ligamentum flavum and dura mater might occur. Much attention must be paid to avoid rupture of the dura mater. However, some times we did not remove the ossified ligament totally. We just floated it and abraded it as thinly as possible with a high speed drill. The results were satisfactory. Sometimes, rupture of the dura mater did occur. In those cases, the dura mater needed repair. Okada reported the en bloc method may induce postoperative spinal instability and preferred an open-door method [15]. However many authors reported en bloc method is safe and effective, with no postoperative spinal instability [16,26]. All patients in the present study underwent posterior thoracic laminectomy to remove the intruding ossified lesion. Efforts were made to preserve the lateral two thirds of the facet joints as much as possible to maintain the segmental stability. No postoperative instability was observed in our series. The key point is to preserve the lateral half of the facet. However, fluorosis makes the spine more rigid, decreases movement, and decreases the possibility of postoperative instability. Prognosis predictors Several authors reported some factors influenced the surgical outcome which included preoperative neurological status, duration of preoperative symptoms, level and progression of ossification, and degree of thoracic kyphosis et al [27-30]. The result of our investigation confirmed that the preoperative JOA score is the most important predictor of the recovery rate. However, the duration of preoperative symptoms was not significantly correlated with the outcome. It might be because there were six patients who suffered acute onset of the symptom, just like acute spinal cord injury. The outcomes of these patients were not all good. To study this more, we divided all the patients into three groups according to preoperative symptom duration. Group one is acute onset, the duration shorter than three days. In group two, the symptom duration is between three days and five years. In group three, the symptom duration is longer than five years. ANOVA analysis of these three groups showed p = 0.0003. It showed that there was significant difference between the groups. Further t test showed that there was significant different between group one and group two (P = 0.0004). There was significant different between group two and group three (P = 0.003). However, there was no significant different between group one and group three (P = 0.197) (table 3). The result showed that the group with acute onset or too long duration had the worse surgical result. Conclusion Fluorosis can cause ossification of thoracic ligamentum flavum, as well as other ligaments. En bloc laminectomy decompression was an effective method. Preoperative JOA score was the most important predictor of surgical outcome. 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==== Front Virol JVirology Journal1743-422XBioMed Central London 1743-422X-3-1001712938210.1186/1743-422X-3-100ResearchSmall interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes Prabhu Ramesh [email protected] Robert F [email protected] Srikanta [email protected] Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA-70112, USA2 Department of Microbiology and Immunology, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA-70112, USA2006 27 11 2006 3 100 100 23 10 2006 27 11 2006 Copyright © 2006 Prabhu et al; licensee BioMed Central Ltd.2006Prabhu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Results Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. Conclusion These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon. ==== Body Background Hepatitis C virus (HCV) is a major blood-borne human pathogen [1]. It is estimated that more than 170 million people worldwide have been infected with hepatitis C [2]. The majority of infected individuals develop life long chronic infections since only a fraction of people infected with HCV develop immunity and clear the virus infection [3]. Chronic hepatitis C virus infection can results in long-standing inflammation in the liver, which can lead to liver cirrhosis and hepatocellular carcinoma [4,5]. The only therapy currently available for HCV infection is the combination of interferon alpha and ribavirin. This therapy can effectively clear the virus infection in only a fraction of infected individuals. In the majority of patient infected the virus either does not respond to therapy or relapses when the therapy is discontinued [6-8]. Studies from our laboratories and others suggest that interferon inhibits hepatitis C virus replication by blocking it at the level of IRES mediated translation [9]. Therefore, the development of innovative approach to inhibit IRES may offer an alternative therapy for chronic hepatitis C patients that are non-responders to interferon. HCV is a positive-stranded RNA virus that belonging to the family Flaviviridae [10]. The HCV genome is approximately 9600 nucleotides in length and contains highly conserved 5' and 3' untranslated regions (UTR). These regions flanks a single large open reading frame (ORF) that encodes a large poly-protein processed into three different structural and seven nonstructural proteins. [11]. The highly conserved 5' UTR and 3' UTR sequences are required for both protein translation and virus replication [12,13]. The replication cycle of HCV occurs in the cytoplasm of infected cells making an excellent target for siRNA based antiviral development. Since many individual cannot eradicate the virus infection with interferon based combination therapy, there is great interest to use this siRNA based antiviral strategy to treat a chronic HCV infection. A number of laboratories including our own have shown that siRNA targeted to the protein coding areas of HCV can inhibit virus replication and expression [14-21]. However, these viral coding sequences may not be the best target since they show significant variation among different HCV genotypes as well as virus sub-types. The nucleotide sequences of genomes from HCV isolated from different parts of the world vary considerably and are quite heterogeneous. Six major genotypes and more than 50 sub-types of the HCV virus have been described around the world. There are 30–50% variation in the nucleotide sequences among viral genotypes and 15–30% among different sub-types [22,23]. Isolates of HCV from a single patient can show 1–5% differences in their nucleotide sequences. In the United States, 75% of chronic hepatitis C cases belong to genotype 1a and 1b, 13–15% genotype 2a and 2b and 6–7% genotype 3a [24]. Genotype 1a and 1b is common in Western Europe. Genotype 3 is most frequent in the Indian subcontinent. Genotype 4 is the most common genotype in Africa and the Middle East. Genotype 5 is found in South Africa. Genotype 6 is found in Hong Kong and Southeast Asia [25]. Therefore, selection of siRNA targeted to a highly conserved region may be appropriate for developing a rational antiviral strategy against different HCV strains. In this study, we designed the most effective siRNAs targets in the highly conserved 5' UTR of the HCV genome. Their antiviral effect on IRES mediated translation was evaluated using sub-genomic clones and full-length infectious clones. We showed here that siRNA targeted to a unique location in the stem loop-II of 5' UTR inhibits IRES function of different genotypes and silence expression of multiple HCV genotypes. Results Intracellular delivery of siRNA inhibits GFP expression from HCV-IRES Four different siRNAs (siRNA-74, siRNA-174, siRNA-207 and siRNA-245) targeting the 5' untranslated region (5'UTR) of hepatitis C virus genome were selected. The location and nucleotide sequence representing the RNAi target sites with respect to the predicted secondary structure of HCV IRES are shown in Fig. 1. pSuper-retro vector was used for intracellular production of siRNA in a liver derived cell line. Efficient transcription of siRNA from this vector occurs by host cell RNA polymerase using the H1-RNA gene promoter. As a negative control for our experiments, we used siRNA targeted to EBNA1 region of EBV. The effects of siRNA on the expression of green fluorescence protein from the IRES clones were examined by co-transfection experiments in Huh-7 cells. Initially, transfection experiments were performed to determine the optimum ratio of pSuper-retro siRNA and HCV IRES GFP plasmid for obtaining maximum inhibition of GFP from the IRES 1b clone. A ratio of 1:4 (one 100 nanogram of HCV-IRES-GFP and 400 nanogram of siRNA plasmid) produced the maximum inhibition. Using a similar experimental condition, the antiviral effect of four different siRNA targets on translation of individual HCV IRES GFP chimeric clone was examined. The specific inhibitory effect of siRNA on GFP expression was quantitated by flow cytometry analysis. The silencing of green fluorescence expression from different HCV IRES clones by four different siRNAs constructs is shown in Fig. 2. The siRNA-74 targeted to the stem loop II of HCV IRES was most efficient and completely silenced the expression of GFP in the case of all genotypes of HCV tested, siRNA-174 and siRNA-207 were moderately effective and siRNA-245 was the least effective. All three siRNA 74,174 and 207 effectively silenced expression of GFP from HCV 1a and HCV1b genotypes. These are the two most common genotypes of HCV in the United States that frequently develop resistance to interferon and ribavirin combination therapy. A control siRNA targeted to the Epstein Barr Virus nuclear antigen 1 (EBNA1) did not inhibit GFP expression in these experiments, indicating that the antiviral action of siRNA mediated gene silencing is highly specific. The numbers of GFP expressing Huh-7 cells after siRNA transfection was quantitatively measured by flow analysis (Fig. 3). It was determined that siRNA74 inhibited the IRES-GFP expression in approximately 80 to 90% of transfected Huh-7 cells in the case of all genotypes of HCV. Other siRNAs 174, 207 and 245 transfection inhibited GFP expression in only 40–60% of cells. The control siRNA specific to EBNA1 did not have any effect on GFP expression. Figure 1 Location of siRNA targets to the 5'UTR of HCV genome. Predicted secondary structure of the 5' UTR sequences (18–357). The sequence shown is that of the genotype 1a 5'UTR, HCV-H [40], and the structure based on previous studies [41-43]. Stem-loop structures are labeled for reference. The chimeric clones were made by fusing the GFP-encoding sequence, including a poly (A) tail, after the CCU sequence of the 5'UTR by overlapping PCR. The locations of siRNA targets in the stem-loop regions are shown by arrows. Figure 2 Effect of small interfering RNAs on the expression of green fluorescent protein from IRES clones of six different HCV genotypes. Huh-7 cells were co-transfected with HCVIRES-GFP plasmid with siRNA plasmid (pSuper-retro) by using the FuGENE 6 transfection reagent. After 48 hours transfected cells were observed under a fluorescence microscope. The experiments were repeated for each genotype IRES clones using different siRNA constructs. SiRNA-74 was the most effective and inhibited the expression of green fluorescence in all genotypes of HCV compared to other siRNA. Control siRNA targeted to EBNA1 had no effect on GFP expression from HCV IRES clones. Figure 3 Quantitative measurement of GFP positive Huh-7 cells by flow cytometry after siRNA transfection. Huh-7 cells were co-transfected with HCVIRES-GFP plasmid with siRNA plasmid (pSuper-retro) by using the FuGENE 6 transfection reagent. After 48 hours of transfection, cells were harvested and GFP positive cells were analysed by a flow cytometer (Becton Dickinson, BD Biosciences, Clontech). Percentage of GFP-positive Huh-7 cells was quantitatively determined after siRNA transfection using cell quest computer software. The results were expressed as percentage of control. siRNA-74 was found to be most effective in silencing GFP from all IRES clones. Intracellular delivery of siRNA74 inhibits expression of full-length clones of HCV 1a and 1b The majority of chronic hepatitis C patients in the US are infected with HCV 1a or HCV1b genotypes, two genotypes of HCV that frequently develop resistance to interferon and ribavirin combination therapy. We tested whether this highly effective siRNA-74 target in the 5' UTR region could silence the gene expression using full-length infectious clones of HCV1a and HCV1b. One HCV1a infectious clone (pCV-H77C) and two HCV1b infectious clones of HCV (pMO9.6-T7 and pCVJ4L6S) were used as targets [26-28]. To examine the antiviral effect of siRNA-74, each full-length clone were co-transfected with increasing concentration of siRNA plasmid using a two-step transfection procedure described earlier (37–38). The inhibition of core protein expression of full-length clones of HCV 1a and HCV1b due to siRNA transfection was determined by immunoperoxidase staining (Fig. 4). Complete silencing of core protein expression was seen in Huh-7 cells transfected with HCV full-length clone 1a (pCV-H77C) and 1b (pMO9.6-T7 and pCVJ4L6S). This effect appears to be very specific since cells transfected with control siRNA targeted to EBNA1 silencing of core protein was not observed. Protein lysates were made and Western blot analysis was performed using the same monoclonal antibody specific for the HCV core protein. These results suggest that siRNA-74 effectively silenced gene expression from infectious full-length clones of HCV1a and HCV1b genotypes(Fig. 5). We then examined whether the silencing of core protein expression in the transfected cells caused intracellular degradation of HCV genomic RNA. Total RNA was isolated from the transfected cells and digested with DNaseI to eliminate plasmid DNA carryover from the transfection. The levels of positive strand HCV RNA were measured by ribonuclease protection assay (RPA). The results shown in suggest that siRNA74 degraded HCV positive- strand HCV RNA in a dose dependent manner in all clones (Fig. 6). Specificity of this silencing mechanisms occurring due to intracellular RNA degradation was examined by measuring HCV RNA levels in the cells co-transfected with control siRNA (EBNA1). These differences are not due to the variation of HCV RNA in the nucleic extracts since GAPDH mRNA levels are comparable in all samples. Taken together the results of our analysis suggest that siRNA-74 targeted to the 5'UTR region can inhibit IRES mediated translation of HCV and also are highly effective in silencing the gene expression of HCV 1a and HCV 1b strain. Figure 4 Immunocytochemical staining showing the silencing of core protein from full-length clones of HCV by siRNA-74. Huh-7 cells were co-transfected with (10 μg) pSuper-retro-siRNA74 and (10 μg) full-length clones of HCV. After 48 hours, transfected cells were harvested by the treatment with trypsin-EDTA. Cells were washed with PBS and immobilized onto a glass slide by cytospin method. Then slides were blocked and stained with core antibody against HCV using a mouse monoclonal antibody. Immunostaining for HCV core protein was performed using a standard protocol. The expression of core protein of full-length HCV 1a (pCVH77C), Ib (pCVJ4L6S), and 1b (pMO9.6--T7) is observed in the presence of siRNA74 and siRNAEBNA1 (unrelated siRNA). Figure 5 Western blot analysis showing the silencing of core protein from full-length clones of HCV by siRNA transfection. Huh-7 cells were co-transfected with pSuper-retro-siRNA74 and full-length clones of HCV using the FuGENE 6 reagent. After 48 hours, transfected cells were isolated by the treatment with trypsin-EDTA. Cells were washed once with PBS and protein lysates were prepared and electrophoresed on 10% SDS-PAGE gels. The proteins were transferred to nitrocellulose membranes, blocked and immunoreacted with a primary antibody. The membrane was washed and incubated with peroxidase labeled secondary antibody and developed by ECL-chemiluminescence method. siRNA74 inhibited the expression of core protein in the case of all three full-length clones of HCV 1a and 1b. Beta actin levels were used as a control to make sure that equal amount of protein was present in the extracts. Figure 6 Ribonuclease protection assay showing siRNA expression specifically degraded intracellular HCV positive strand RNA in the transfected Huh-7 cells. Huh-7 cells were co-transfected with different concentration of siRNA74 with different full-length clones of HCV. After 48 hours, transfected cells were isolated by the treatment with trypsin-EDTA. Total RNA was isolated and subjected to RPA for positive strand HCV using a minus strand RNA probe targeted to the 5'UTR region. The degradation of HCV positive strand by siRNA74 is concentration dependent. No HCV RNA degradation was observed in the cells transfected with unrelated siRNA. Discussion Chronic HCV infection usually treated with a combination of pegylated interferon-alpha and ribavirin. However, the majority of chronic hepatitis C patients in the United States develop cellular resistance to interferon therapy. There is a need to develop new antiviral approaches to inhibit HCV replication. At present, there are several antiviral strategies that have been employed to inhibit HCV virus replication [29]. Among these, RNA interference appears to be one of the most powerful antiviral approaches to inhibit HCV gene expression in mammalian cells. RNA interference (RNAi) is a sequence specific RNA degradation process in the cytoplasm of eukaryotic cells induced by double-stranded RNA [30,31]. This process can be initiated via so called small interfering RNAs (siRNA) of approximately 19–23 base pairs. These are cleaved by double-stranded precursor RNAs by the RNase III-like enzyme dicer. These siRNAs associate with various proteins to form the RNA-inducing silencing complex (RISC), harboring nuclease and helicase activity. The antisense strand of the siRNA guides the RISC to the complementary target RNA and the nuclease component cleaves the target RNA in a sequence specific manner. This approach has been a widely used as a technique for gene knockouts for gene expression studies and as an antiviral against a number of viruses [32-34]. The RNAi approach is very specific and offers a great potential to be used as antiviral against hepatitis C virus infection. Reports from the previous studies including our own experience suggest that this siRNA-based approach is very effective by yielding up to 100-fold inhibition of virus replication [14-20]. We have used siRNA targets in the E2, NS3 and NS5B region and showed that these siRNA targets can silence HCV 1a infectious clone effectively. However, the same siRNA does not work effectively against other viral strains, because of sequence variation in the siRNA target. To develop siRNA targets that can be used for both HCV 1a and HCV1b virus and other genotypes we selected the highly conserved region of HCV. The 5' untranslated region (UTR) of HCV consisting of 341 nucleotides, is highly conserved among different viral genotypes and in clinical strains of HCV [35]. We selected four siRNA targets in the second and third stem-loop regions of secondary structure of 5'UTR. To direct the synthesis of fully processed siRNA-like transcripts in transfected Huh-7 cells a mammalian expression plasmid vector (pSuper-retro) was used. The use of the vector-based delivery is more efficient because it allows continuous transcription of siRNA in the transfected cell. We showed that intracellular expression of siRNA silences GFP expression from IRES clones. Some of the siRNA targets appear to be more efficient than others. For example, siRNA-74 is found to be most effective against six different viral IRES sequences as compared to the other three. The siRNA-174, siRNA-207 is moderately effective against HCV 1a and HCV 1b IRES. The siRNA-254 was the least effective against HCV1a and HCV1b IRES, but it was highly effective against 2b, 3a, 4a IRES. The results could be due to the fact that there are some nucleotide variations in the IRES sequences among different virus genotypes. There are also published reports suggesting that many cellular proteins binds to the 5' UTR sequence of HCV for translation of polyprotein. It is possible that the some siRNAs could not have efficiently hybridize to some sequences in the transfected cells than the others because of complex secondary structure of the 5'UTR. We extended this study and examined whether the siRNA-74 could also effectively silence gene expression of HCV 1a and 1b strain. We used full-length chimpanzee infectious clones as viral targets. The full-length HCV genomic clone was expressed in Huh-7 cells by the use of adenovirus T7 RNA polymerase. We have shown that this inducible model allows high-level expression of HCV structural and non-structural proteins that can be measured by Western blot analysis (37,38). Replication of HCV full-length genome 1a and 1b was observed in the transfected hepatic cell lines by detecting viral negative strand RNA by strand specific ribonuclease protection assay. Using co-transfection studies, it was determined that complete silencing of HCV core protein expression was observed by siRNA-74 for HCV 1a and HCV 1b infectious clones. The inhibition of viral protein expression by siRNA-74 was confirmed by an immunocytochemical method as well as by Western blot analysis. No inhibition was seen in the cells co-transfected with unrelated siRNA, suggesting that the antiviral effect of siRNA-74 is specific. These results were confirmed by looking at the stability of full-length HCV genomic RNA in the transfected cells by RPA. Silencing of the viral protein expression was due to the specific degradation of HCV genomic mRNA. Our results clearly support the hypothesis that the siRNA-74 can cause gene silencing of HCV1a and HCV1b strains. In summary, these results clearly show that the siRNA mediated viral gene silencing is a very effective antiviral strategy that has a very strong potential for curing chronic hepatitis C virus infection. The siRNA-74 is an important therapeutic target for the treatment of infection of multiple genotypes of HCV. Conclusion In the present study we identified a siRNA targeted to the stem loop II (siRNA-74) of 5'UTR of HCV that inhibited the expression of GFP in six different chimeric clones of HCV as well as inhibited the expression of the core protein and degraded the positive strand RNA in full-length clones of HCV 1a and 1b. Therefore, our results support that use of the siRNA74 as an important target for inhibiting IRES mediated translation of multiple genotypes of HCV. Methods Cell line and Transcription plasmid Huh-7 cell line was maintained in Dulbecco's Modified Media (D-MEM) containing non-essential amino acids, sodium pyruvate and 10% fetal bovine serum (In vitrogen Life Technologies, Carlsbad, CA). Chimeric clones between IRES sequences of six different HCV genotypes and green fluorescence protein used here were constructed previously [36]. A chimpanzee infectious clones pCV-H77C (HCV1a) was obtained from Jens Bukh, National Institute of Health [26,27]. Full-length HCV transcription plasmid (pNIH1a-Rz) was prepared using chimpanzee infectious clone (pCV-H77C), which contains a T7 promoter, full-length cDNA of HCV genome, followed by a cDNA copy of autolytic ribozyme from antigenomic strand of hepatitis delta virus and T7 transcriptional terminator sequences. Detailed description of transcription plasmid and method has been described previously [37,38]. A chimpanzee infectious clone (pCVJ4L6) was obtained from Jens Bukh, National Institute of Health. Transcription plasmid (pTRE-NIH1b) was prepared by addition of hepatitis delta virus ribozyme sequences and T7 transcriptional terminator at the very end of 3'UTR using the methods described in our publications. A chimpanzee infectious clone pMO9.6-T7 (HCV1b) containing an autolytic ribozyme sequence from antigenomic strand of hepatitis delta virus and T7 transcriptional terminator sequences at the 3' end was used here as described previously [37,38]. Construction of pSuper-retro vector encoding siRNAs Four different siRNAs were selected and targeted to the 5' UTR region of HCV genome (1b) using web-based Oligo-Engine software. As a control, siRNA targeted to Epstein barr virus (EBV) nuclear antigen was used [39]. A commercially available plasmid vector called pSuper-retro (Oligo- Engine) for intracellular delivery of siRNA was used. The siRNA constructs were prepared at two steps. In the first step, we synthesized a pair of (sense and anti-sense orientation) 64-nts oligos containing 19 nucleotides of HCV in sense and antisense orientations, separated by a 9-nt spacer sequence. Restriction enzymes Xho1 and Bgl II were introduced at the 5' end of sense and antisense 64 nucleotide oligos for cloning. In the second step, the sense and antisense primers were annealed by incubation at 90°C for 4 min then 72°C for 10 minutes. The annealed oligos were then slowly cooled to 10°C and ligated to the pSuper-retro vector using Xho1 and Bgl II restriction sites. The nucleotide sequences of the sense and antisense primer used to design the siRNA vectors are shown in Table 1. The recombinant clones containing the iRNA insert were selected by restriction enzyme digestion. Large-scale plasmid DNA isolation was performed using a maxi kit (Qiagen Inc). The presence of siRNA sequences was confirmed by DNA sequence analysis. Table 1 Sequences of small interfering RNAs used to target the 5' UTR of hepatitis C virus RNA Name of the siRNA Nucleotide sequence siRNA 74 S 5'-AGCGTCTAGCCATGGCGTT-3' siRNA74 AS 3'-TCGCAGATCGGTACCGCAA-5' siRNA174 S 5'-TTGCCAGGATGACCGGGTC-3' siRNA174 AS 3'-AACGGTCCTACTGGCCCAG-5' siRNA207 S 5'-CCCGCTCAATGCCTGGAGA-3' siRNA207 AS 3'GGGCGAGTTACGGACCTCT-5' siRNA245 S 5'-GACTGCTAGCCGAGTAGCG-3' siRNA245 AS 3'-CTGACCATCGGCTCATCGC-5' Effect of siRNA on expression of GFP from different IRES clones Huh-7 cells were grown in a 12- well tissue culture dish the day before transfection. The next day the cells were infected with a replicative defective adenovirus that expressed T7 RNA polymerase. After two hours, cells were co-transfected with 100 ng of HCV-IRES-GFP plasmid and different concentrations siRNAs plasmid using FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). Expression of green fluorescence was recorded at 24, 48 and 72 hours using a fluorescence microscope. The ratio of IRES plasmid to siRNA-74 plasmid required for maximum inhibition of GFP expression from the IRES clone was recorded. Using the identical condition effect of four different siRNAs on GFP expression from IRES clone was examined. The inhibitory effects of each siRNA plasmid on GFP expression from six different HCV IRES sequences were quantitatively by flow analysis. Transfected cells were harvested by treatment with trypsin-EDTA, and then resuspended in PBS (Invitrogen Life Technology, Carlsbad, CA) and subjected to flow-cytometric analysis (Becton Dickinson, BD Biosciences Clontech). The percentage of GFP-positive Huh-7 cells was quantitatively compared with control siRNA for different siRNA with different HCV genotypes. Effect of siRNA on expression of full-length HCV genome To examine the effect of siRNA on expression of full-length HCV 1a and 1b strain was examined by co-transfection experiments. We have developed a T7- based model in which expression of full-length HCV RNA genome can be reliably studied in Huh-7 cells. Detailed methodology has been described previously [37,38]. Huh-7 cells were co-transfected with 10 micrograms of HCV full-length plasmid and different concentration siRNAs plasmid using FuGENE 6 reagent. The success of intracellular delivery of each siRNA targets against full-length HCV 1a and HCV1b strain was examined by measuring core protein and positive strand HCV RNA. Immunoperoxidase Staining The extent of core protein inhibition due to siRNA74 on full-length clones of HCV1a and 1b was examined by immunostaining of transfected Huh-7 cells using a monoclonal antibody (Affinity Bioreagents, Denver. CO). Transfected Huh-7 cells were immobilized onto glass slides by cytospin method. Cells were washed with phosphate-buffered saline (PBS) pH 7.4 twice, air-dried and fixed with chilled acetone for five minutes. The cells were permeabilized by treatment with 0.05% saponin for 10 minutes at room temperature. Blocking was performed with 5% normal goat serum (Sigma Chemical Company, St. Louis, MO) diluted in minimum essential medium for 30 minutes at room temperature. Blocking for endogenous biotin-avidin was performed using blocking reagents from the kit (Avidin/Biotin Blocking Kit, Vector Laboratories Inc., Burlingame, CA) and blocking for endogenous peroxidase was done with 0.9% H2O2 for 30 minutes at room temperature. The cells were incubated with monoclonal anti-core antibody (1:100 dilution) overnight at 4°C. The next day they were washed three times and incubated with anti-mouse biotin conjugated antibody (1:1000) for one hour at room temperature. The slides were washed and incubated for 30 minutes with Elite avidin-biotin peroxidase complex (VECTOR Labs, CA). The slides were then reacted with diaminobenzidine for 10 minutes and then counterstained with hematoxylin for one minute. After dehydration, the slides were mounted with permount and observed under light microscopy. Western Blot Analysis Western blot analysis for core protein was performed on protein lysate from transfected cells using a standard protocol in our laboratory [37,38]. Briefly, transfected cells were treated with 500 μl of lysis buffer containing 150 mM sodium chloride, 50 mM Tris-HCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS and protease inhibitors (Protease Inhibitor Cocktail, Roche Biochemicals, Indianapolis, IN). Fifty micrograms of the total cell lysate was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham, Arlington Heights, and IL). The membranes were blocked with PBS containing 5% non-fat dried milk and 0.1% Tween-20 for 1 hour at room temperature. Then, the membrane was incubated with monoclonal antibody against core (Affinity Bioreagents, Denver, CO) at 1:100 dilutions for one hour. The membrane was washed three times with 0.1% Tween-20 in PBS. Following this step, the membranes were incubated with peroxidase-labeled secondary antibody (ECL Western blotting analysis system, Amersham Pharmacia Biotech UK, Amersham PLC, Buckinghamshire, England) at a dilution of 1:1000 for one hour. After this step, membranes were washed three times with PBS and developed using ECL Chemiluminescence Detection Kit (Amersham Pharmacia Biotech UK, Amersham PLC, and Buckinghamshire, England). To verify that equal amounts of protein were loaded onto each lane of the SDS-PAGE, the membrane was incubated with monoclonal antibody to beta-actin. Ribonuclease Protection Assay (RPA) Levels of HCV genomic RNA (positive strand) in the siRNA-transfected cells were examined by RPA. Total RNA was isolated from the transfected cells by the GITC method. RNA extracts were treated with DNase I (Roche Molecular Biochemicals, Indianapolis, IN) 5U/mg of RNA for one hour at 37°C to remove any residual plasmid DNA templates. RPA was performed to detect the presence of HCV-positive in transfected Huh-7 cells (Ambion, Austin, TX). The RNA probe targets the highly conserved 5' UTR of HCV genome. The plasmid pCR II-296 was linearized with Xba I and used to prepare an anti-sense RNA probe using the SP6 RNA polymerase in the presence of 32P-UTP. For RPA assays, approximately 1 × 106 cpm of the labeled anti-sense probe was added to 25 μg of RNA sample and vacuum dried. Hybridization was performed in 10 μl of the hybridization buffer after denaturing for 3 minutes at 95°C and followed by overnight incubation at 45°C. RNase digestion was performed in 200 μl of RNase cocktail (1: 100) (Ambion Inc. Austin, Texas) in a buffer consisting of 10 mM Tris, pH 7.5, 5 mM EDTA and 0.3 M NaCl for 1 hour at 37°C. Reactions were stopped by the addition of 2.5 μl of 25% SDS and 10 μl of proteinase K (10 mg/ml) at 37°C for 15 minutes. Samples were extracted with phenol/chloroform and precipitated with ethanol. The pellet was air dried and resuspended in 15 μl of gel loading buffer. The samples were then boiled for 3 minutes and separated on an 8% acrylamide/8 M urea gel. The gel was dried and exposed to X-ray film (Kodak, X-OMAT-AR). List of abbreviations HCV, hepatitis C virus; RNAi, RNA interference; siRNA, small interfering RNA, dsRNA, double stranded RNA; IRES, internal ribosome entry site; GFP, Green fluorescence protein. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors have contributed equally to the work presented in this paper. Acknowledgements This work was supported by NIH grant CA89121 (SD) and partial support from the Tulane Cancer Center. 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==== Front PLoS BiolPLoS BiolpbioplbiplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.005000506-PLBI-RA-1327R2plbi-05-01-03Research ArticleDevelopmental BiologyMolecular BiologyEukaryotesAnimalsVertebratesMammalsMus (Mouse)The RNA-Binding Protein KSRP Promotes Decay of β-Catenin mRNA and Is Inactivated by PI3K-AKT Signaling KSRP Required for β-Catenin mRNA DecayGherzi Roberto 1Trabucchi Michele 1Ponassi Marco 1Ruggiero Tina 1Corte Giorgio 12Moroni Christoph 3Chen Ching-Yi 4Khabar Khalid S 5Andersen Jens S 6Briata Paola 11 Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 2 Department of Biology, Oncology, and Genetics, University of Genova, Genova, Italy 3 Institute for Medical Microbiology, University of Basel, Basel, Switzerland 4 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America 5 King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 6 Center for Experimental Bioinformatics, University of Southern Denmark, Odense, Denmark Wickens Marv Academic EditorUniversity of Wisconsin, United States of America To whom correspondence should be addressed. E-mail: [email protected] 2007 19 12 2006 19 12 2006 5 1 e525 7 2006 3 11 2006 © 2007 Gherzi et al.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.β-catenin plays an essential role in several biological events including cell fate determination, cell proliferation, and transformation. Here we report that β-catenin is encoded by a labile transcript whose half-life is prolonged by Wnt and phosphatidylinositol 3-kinase–AKT signaling. AKT phosphorylates the mRNA decay-promoting factor KSRP at a unique serine residue, induces its association with the multifunctional protein 14-3-3, and prevents KSRP interaction with the exoribonucleolytic complex exosome. This impairs KSRP's ability to promote rapid mRNA decay. Our results uncover an unanticipated level of control of β-catenin expression pointing to KSRP as a required factor to ensure rapid degradation of β-catenin in unstimulated cells. We propose KSRP phosphorylation as a link between phosphatidylinositol 3-kinase–AKT signaling and β-catenin accumulation. Author Summary During mammalian development and adulthood, β-catenin regulates the transcription of a family of genes with multiple essential roles in cell proliferation and differentiation. β-catenin also plays a role in cancer when it carries mutations that result in uncontrolled β-catenin function. Here, we report that the lifetime of the β-catenin–encoding transcript is under regulatory control. We show that specific cellular signals relevant to proper mammalian development and implicated in tumor formation can prolong β-catenin transcript half-life, leading to the accumulation of β-catenin protein. We identify a molecular mechanism for this prolongation by showing that a protein factor responsible for β-catenin transcript instability (and thus degradation) is impaired by phosphorylation, a chemical modification. When this factor is impaired, β-catenin mRNA and protein accumulate. Our results point to an unanticipated control of β-catenin levels through regulation of its transcript half-life in response to signals related to proliferation and differentiation. The authors show that the half-life of β-catenin mRNA is prolonged by PI3K-AKT signaling, revealing a new level of control on β-catenin. citationGherzi R, Trabucchi M, Ponassi M, Ruggiero T, Corte G, et al. (2007) The RNA-binding protein KSRP promotes decay of β-catenin mRNA and is inactivated by PI3K-AKT signaling. PLoS Biol 5(1): e5. doi:10.1371/journal.pbio.0050005 ==== Body Introduction The half-life (t½) of mRNAs is regulated in a complex fashion in response to external stimuli. Whereas transcripts containing the AU-rich element (ARE) are labile, activation of signal transduction pathways induces their stabilization [1]. It is now clear that mRNA decay regulation by different signals makes a huge contribution to the global control of gene expression [1]. AREs, located in the 3′ untranslated region (3′ UTR) of many short-lived transcripts, promote mRNA deadenylation and decapping followed by degradation of the mRNA body [1,2]. Mammalian ARE-containing transcripts are thought to be deadenylated by at least one of the seven known deadenylases and degraded mainly by the exosome, a multiprotein complex containing 3′-5′ exonucleases [1,2]. A relevant role in mRNA decay has been recently demonstrated for the 5′-3′ exonuclease Xrn1 [3]. ARE-binding proteins (ARE-BPs) are trans-acting factors responsible for the decay control of labile mRNAs [1]. Some ARE-BPs are decay-promoting factors (TTP, BRF1, KSRP), and others, such as HuR, are stabilizing factors, whereas AUF1 promotes either decay or stabilization depending on the cellular context or its isoform expression profile [1]. According to the recently proposed recruitment model, destabilizing ARE-BPs interact with AREs and recruit the degradation machinery to the mRNA [4–6]. The ARE-BP KSRP, containing four contiguous K homology (KH) motifs that recognize the AREs and interact with the mRNA degradation machinery, promotes rapid decay of several ARE-containing mRNAs both in vitro and in vivo [4,6]. Activation of the stress-responsive c-Jun N-terminal kinase [7], p38 MAP kinase [8,9], MAPKAPK2 [10,11], phosphatidylinositol 3-kinase (PI3K)-AKT [12,13], and Wnt/β-catenin signaling [14] was shown to trigger stabilization of various transcripts, thereby causing large alterations in their abundance. We have previously shown that activation of the Wnt signaling pathway in pituitary αT3-1 cells induces a coordinate transcriptional and post-transcriptional regulation of select target genes [14,15]. We proposed that the integrated regulation of transcription and mRNA turnover is required to ensure rapid and relevant changes in the expression of regulatory genes [9,14]. Recently, we observed that a common set of transcripts is stabilized by either treatment with lithium chloride (LiCl), a mimicker of Wnt signaling [16], or AKT activation in pituitary αT3-1 cells (unpublished data). Among these transcripts, we found β-catenin. β-catenin plays a key role in embryonic development and tumorigenesis by controlling the expression of Wnt-responsive genes [17–19]. In response to Wnt signals, Dishevelled is recruited to the Axin complex to inhibit glycogen synthase kinase-3β, resulting in cytosolic accumulation and subsequent translocation of β-catenin to the nucleus, where it binds to transcription factors of the T-cell factor/lymphoid enhancer factor family and transactivates Wnt target genes [17,18]. Serine/threonine kinases belonging to the AKT family are major effectors of the PI3K pathway and are activated by insulin and other polypeptide growth factors [20,21]. The recruitment of AKT to the plasma membrane by the lipid products of PI3K leads to AKT phosphorylation and activation [20,21]. Once activated, AKT exerts a central role in metabolism, cell survival, motility, transcription, and cell-cycle progression [21–23]. Cross-talks between Wnt and PI3K-AKT signaling pathways have been described [24–27]. In this report, we describe that β-catenin mRNA is labile due to destabilizing elements located in its 3′ UTR and that KSRP is a major molecular determinant of β-catenin mRNA instability. β-catenin transcript can be stabilized by either Wnt or PI3K-AKT signaling activation. AKT phosphorylates KSRP at a unique serine residue favoring its interaction with the adaptor protein 14-3-3. As a consequence, KSRP fails to interact with the exosome and, in turn, β-catenin mRNA is stabilized. Results β-Catenin Is Encoded by a Labile mRNA Whose Half-Life Is Prolonged by Wnt Signaling We previously reported that the treatment of mouse pituitary αT3-1 cells with LiCl (a compound widely used to mimic Wnt signaling [16]) stabilizes select ARE-containing labile transcripts [14]. To identify additional ARE-containing transcripts whose mRNA t½ is prolonged in αT3-1 cells in response to LiCl treatment, a large-scale analysis using the ARE-cDNA microarray system was performed [28]. The microarray screening revealed that LiCl treatment significantly upregulated, among others, β-catenin mRNA (unpublished data). By both semiquantitative and quantitative RT-PCR, we confirmed that β-catenin mRNA increased by approximately fourfold in cells treated with LiCl (Figure 1A and unpublished data). The inspection of mouse β-catenin 3′ UTR revealed the presence of several U-rich regions spread over the entire sequence (classified as class III AREs [28 and references cited therein]). As shown in Figure 1B, quantitative PCR experiments demonstrated that β-catenin mRNA was unstable in intact αT3-1 cells displaying a t½ of approximately 45 min, whereas the control β2-microglobulin (β2-MG) was stable. In order to verify whether β-catenin AREs were responsible for the rapid decay of the endogenous transcript, we transfected αT3-1 cells with a reporter plasmid containing the entire β-catenin 3′ UTR placed at the 3′ end of chloramphenicol acetyltransferase (CAT) sequence. As shown in Figure 1C, CAT-β-catenin chimeric transcript displayed a short t½ while control CAT mRNA was stable. Figure 1 mRNA Encoding β-Catenin Is Labile and Is Stabilized by LiCl and Wnt-3A (A) Expression of β-catenin and β2-MG (control transcript), monitored by RT-PCR, in control and in LiCl-treated (20 mM for 6 h) αT3-1 cells. (B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (C) Quantitative RT-PCR analysis of both CAT and CAT–β-catenin transcripts in either CAT or CAT–β-catenin transiently transfected αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (D) Semiquantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either control or LiCl-treated αT3-1 cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either control-treated αT3-1, or Wnt-3A–treated (10 ng/ml, 6 h) αT3-1 cells. (F) Semiquantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either control or Wnt-3A–treated αT3-1 cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The amount of each transcript was quantitated and represented as described in (D). Next, we investigated whether Wnt signaling activation regulates β-catenin mRNA turnover in intact cells. As shown in Figure 1D, LiCl treatment significantly prolonged the t½ of β-catenin mRNA while the t½ of β2-MG was unaffected. Furthermore, treatment of αT3-1 cells with recombinant mouse Wnt-3A increased β-catenin mRNA steady-state levels (Figure 1E). Importantly, Wnt-3A treatment strongly prolonged β-catenin t½ in intact cells (Figure 1F). These results suggest that β-catenin mRNA is labile in unstimulated cells due to the presence of AREs in its 3′ UTR. Wnt signaling activation stabilizes β-catenin mRNA and induces its accumulation. PI3K-AKT Activation Stabilizes β-Catenin mRNA and Increases Both mRNA and Protein Steady-State Levels As previously reported, besides mimicking the activation of the canonical Wnt pathway, LiCl also targets PI3K-AKT signaling [29,30]. Furthermore, the results of our ARE-cDNA microarray screening revealed that LiCl treatment and AKT activation, obtained expressing a constitutively active myristylated form of AKT1 (myrAKT1 [31]), induced stabilization of a common set of mRNAs including β-catenin (unpublished data). These observations prompted us to investigate the effect of PI3K-AKT activation on β-catenin mRNA turnover. First, we assessed the contribution given by PI3K-AKT pathway to LiCl-induced β-catenin mRNA stabilization. We used both pharmacological inhibitors and an AKT dominant negative mutant [AKT1(K179M)] to block PI3K-AKT signaling. We found that treatment with either LY294002 (LY, a PI3K inhibitor [32]) or triciribine (a specific AKT inhibitor [33]) strongly reduced LiCl-induced stabilization of β-catenin mRNA in intact αT3-1 cells (Figure 2A and 2B). Similarly, AKT1(K179M) expression impaired LiCl-induced stabilization of β-catenin mRNA in vitro (Figure 2C). Figure 2 PI3K-AKT Signaling Stabilizes β-Catenin mRNA and Increases Its Expression (A) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of LY, control), DMSO plus LiCl, or LY (25 μM for 16 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of triciribine, control), DMSO plus LiCl (20 mM for 6 h), or triciribine (1 μM for 1 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (C) In vitro RNA degradation assays using S100s from either LiCl-treated mock αT3-1 or LiCl-treated αT3-1 cells expressing the AKT dominant negative mutant AKT1(K179M) (αT3-1–AKTDN). Internally 32P-labeled and capped RNA substrates were incubated with S100s for the indicated times and their decay analyzed as described in Materials and Methods. (D) Either mock αT3-1 or αT3-1–myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ-32P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either mock αT3-1 or αT3-1–myrAKT1 cells. (F) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either mock αT3-1 or αT3-1–myrAKT1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (G) Immunoblot analysis of either S100 or nuclear extracts from the indicated cell lines with anti- β-catenin, α-tubulin, and HDAC2 antibodies. Next, we expressed myrAKT1 in αT3-1 cells. As shown in Figure 2D, the kinase activity immunoprecipitated with anti-AKT antibody was fivefold higher in αT3-1–myrAKT1 than in mock αT3-1 cells, thus demonstrating that active AKT kinase was present in extracts from cells expressing myrAKT1. The steady-state levels of β-catenin mRNA were increased and β-catenin mRNA t½ was prolonged in αT3-1–myrAKT1 cells (Figure 2E and 2F). myrAKT1 expression produced a similar stabilization of β-catenin mRNA in murine C2C12 myoblasts (C2C12-myrAKT1, Figure S1). Importantly, we observed a strong increase of β-catenin protein levels in both nuclear and cytosolic compartments of αT3-1–myrAKT1 and C2C12-myrAKT1 cells compared with control cells (Figure 2G and unpublished data). These results suggest that PI3K-AKT activation stabilizes β-catenin mRNA, leading to mRNA and protein accumulation. Insulin-Induced PI3K-AKT Activation Stabilizes β-Catenin mRNA and Increases Both mRNA and Protein Steady-State Levels In order to verify whether insulin-induced AKT activation affects β-catenin mRNA stabilization, we used insulin receptor–overexpressing HIRc-B rat cells, which display strong responses to insulin [13,34]. Indeed, the kinase activity immunoprecipitated with anti-AKT antibody was approximately sixfold higher in insulin-treated than in control HIRc-B cells, and LY treatment strongly reduced insulin-dependent AKT activation (Figure 3A). Figure 3B shows that insulin increased β-catenin mRNA steady-state levels and that LY treatment almost completely blocked insulin effect. β-catenin mRNA was significantly stabilized by insulin in intact HIRc-B cells, and LY treatment strongly decreased insulin-induced β-catenin mRNA stabilization (Figure 3C). Accordingly, insulin induced β-catenin protein accumulation in both cytosolic and nuclear fractions from HIRc-B cells (Figure 3D). To investigate whether insulin affects β-catenin protein degradation rate, we treated HIRc-B cells with cycloheximide, to inhibit translational elongation, and monitored β-catenin levels in total extracts after different intervals of time. As shown in Figure 3E, insulin did not affect the rate of β-catenin protein decay. Figure 3 Insulin Stabilizes β-Catenin mRNA and Increases Its Expression (A) HIRc-B cells were treated for 1 h with DMSO (control), insulin (10−6 M) plus DMSO, or LY (25 μM for 16 h) plus insulin. Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ-32P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of β-catenin and β2-MG, monitored by RT-PCR, in control HIRc-B cells and in HIRc-B treated with either insulin or LY plus insulin. (C) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in control HIRc-B cells, in HIRc-B treated with either insulin or LY plus insulin. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (D) Immunoblot analysis of either S100 or nuclear extracts from either control- or insulin-treated HIRc-B cells with anti–β-catenin, β-actin, and HDAC2 antibodies. The amount of each band was quantitated by densitometry and insulin was found to increase β-catenin expression by 3.2- and 2.1-fold over the control in S100 and nuclear extracts, respectively. (E) HIRc-B cells were maintained for 16 h in DMEM containing 0.1% FCS; then either PBS (control) or insulin (10−6 M) was added for 1 h. Cultures were then treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared, and the levels of β-catenin quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. β-Actin immunoblotting was used to verify the equal protein loading. In conclusion, a physiological activation of PI3K-AKT signaling causes stabilization of β-catenin transcript and increases steady-state levels of both β-catenin mRNA and protein. The mRNA Destabilizing Factor KSRP Is Phosphorylated by AKT We hypothesized that AKT activation stabilizes β-catenin transcript by targeting the mRNA decay machinery. Among ARE-BPs known to affect mRNA turnover, only KSRP [4] and HuR [35] were able to specifically immunoprecipitate β-catenin mRNA in RNA-immunoprecipitation experiments (Figure 4A). We first investigated whether AKT was able to phosphorylate either KSRP or HuR. KSRP was phosphorylated by recombinant purified AKT2 in vitro, whereas HuR was not phosphorylated (Figure 4B). In silico analysis (Motif Scan, http://scansite.mit.edu/motifscan_seq.phtml) of the human KSRP primary sequence performed at medium stringency indicated serine 193 (bold in the following peptide: GLPERSVSLTGAPES) as a potential AKT phosphorylation site (asterisk in Figure 4C, left). We evaluated the ability of AKT2 to phosphorylate KSRP deletion mutants expressed as GST-fusion proteins (Figure 4C). Only the KSRP fragments including KH1, where S193 is located, were phosphorylated by AKT2 in a concentration-dependent manner (Figure 4C, lanes 1, 3, 4, and 6, and unpublished data). Importantly, myrAKT1 expression enhanced the phosphorylation of coexpressed FLAG-KSRP while not affecting FLAG-KSRP(S193A) mutant, as shown by anti-FLAG immunoprecipitation following [32P]orthophosphate metabolic labeling of intact HeLa cells (Figure 4D). In order to unambiguously identify the KSRP residue(s) phosphorylated by AKT, recombinant human KSRP was phosphorylated by AKT2 in vitro, the gel band was digested with trypsin and enriched for phosphopeptides, and the peptides were analyzed by nanoflow liquid chromatography–tandem mass spectrometry (LC-MS/MS). This led to the identification of the unique peptide SV[pS]LTGAPESVQK with phosphorylation at the second serine residue (S193) (Figure 4E). The entire sequence of the phosphorylated peptide is perfectly conserved in several mammalian species (Mus musculus, Rattus norvegicus, Canis familiaris, and Bos taurus), and the phosphorylated serine is conserved, in a corresponding position, also in nonmammalian species (Gallus gallus, Xenopus laevis, and Danio rerio) (unpublished data). S193 was mutated to alanine in KH1-4 and KH1-2, and the mutant proteins were expressed in bacteria. As shown in Figure 4F, the S193A mutation abolished AKT-dependent phosphorylation in vitro. Figure 4 KSRP Is Phosphorylated by AKT in Serine 193 (A) Immunoprecipitation of β-catenin RNA-containing ribonucleoprotein complexes. The proteins were immunoprecipitated from αT3-1 total cell extracts using the indicated antibodies. RNA was extracted from the immune complexes and analyzed by RT-PCR as indicated. (B) Either highly purified recombinant human KSRP (lanes 1 and 2) or affinity-purified GST-HuR (lanes 3 and 4) was incubated with either active recombinant AKT2 in kinase buffer or buffer alone in the presence of [γ-32P]ATP. Labeled proteins were separated by SDS-PAGE, blotted to nylon membranes, and detected by autoradiography (top panel). Immunoblot analysis of the above kinase reactions with either anti-KSRP (middle panel) or anti-GST (bottom panel) antibodies. (C) Schematic representation of KSRP and GST-fused deletion mutants. Asterisks indicate the position of a putative AKT phosphorylation site (left). AKT2 kinase assays were performed using the indicated recombinant protein substrates (middle and right). Arrows point to the position of each phosphorylated protein; the asterisk marks the position of phosphorylated AKT2. (D) In vivo [32P]orthophosphate metabolic labeling of HeLa cells transiently cotransfected with either FLAG-KSRP plus empty pcDNA3, or FLAG-KSRP plus pCMV–myr-AKT1, or FLAG-KSRP(S193A) plus pCMV–myr-AKT1. Aliquots of the lysates were immunoprecipitated with protein A–protein G–Sepharose–bound mouse control IgG (cIgG) or anti-FLAG antibody, separated by SDS-PAGE, and autoradiographed (top panel). Immunoblot analysis of the above in vivo phosphorylation reactions with anti-FLAG antibody (bottom panel). Arrows point to the position of transfected KSRP. (E) MS-MS fragmentation spectrum of the tryptic peptide SV[pS]LTGAPESVQK of KSRP showing phosphorylation of S193. (F) AKT2 kinase assays using the indicated recombinant protein substrates. Arrows point to the position of each phosphorylated protein; the asterisk marks the position of phosphorylated AKT2. Overall, these data suggest that AKT phosphorylates human KSRP at the unique site S193. KSRP Controls β-Catenin mRNA Turnover in αT3-1 Cells The results presented above indicated that KSRP was phosphorylated by AKT and led us to hypothesize that KSRP could be involved in AKT-induced stabilization of β-catenin mRNA. We and others demonstrated that KSRP regulates the stability of select mRNAs in response to different stimuli [9,36]. Thus, we investigated whether KSRP controls the decay rate of β-catenin mRNA. Stable knock-down of KSRP obtained using shRNA in αT3-1 cells (αT3-1–shKSRP, Figure 5A) led to a more than fourfold increase of the steady-state levels of β-catenin mRNA when compared to mock-transfected cells (Figure 5B). Furthermore, β-catenin mRNA was stable in αT3-1–shKSRP in vivo (Figure 5C) and in vitro (Figure S2A). Conversely, KSRP overexpression in αT3-1 cells blocked the LiCl-induced stabilization of β-catenin mRNA (Figure S2B). Importantly, β-catenin protein levels were approximately fourfold higher in αT3-1–shKSRP than in control cells, although β-catenin protein decay rates were unchanged (Figure 5D and 5E). The increase in β-catenin expression was mirrored by an increase in luciferase activity driven by two β-catenin–responsive reporters, TOP-FLASH and mouse c-myc promoter region (Figure 5F). Figure 5 KSRP Is Required for β-Catenin mRNA Degradation (A) Immunoblot analysis of total extracts from either mock αT3-1 (empty pSUPER-Puro vector-transfected) or αT3-1-shKSRP (pSUPER-Puro-shKSRP–transfected) cells using affinity-purified anti-KSRP and anti–α-tubulin antibodies. (B) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either mock αT3-1 or αT3-1–shKSRP cells. (C) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either mock αT3-1 or αT3-1–shKSRP cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (D) Immunoblot analysis of total extracts from the indicated cell lines with anti–β-catenin and α-tubulin antibodies. (E) Either mock αT3-1 or αT3-1–shKSRP cells were treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared and the levels of β-catenin quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. α-Tubulin immunoblotting was used to verify the equal protein loading. (F) Either mock αT3-1 or αT3-1–shKSRP cells were transiently transfected with either TOP-FLASH or c-myc-LUC reporter vectors, cultured for 2 d, and collected, and luciferase activity was measured. The values shown are averages (±SEM) of four independent experiments performed in duplicate. We ruled out the possibility that AKT activation could change KSRP expression levels affecting its protein stability. As shown in Figure S3, expression of myrAKT1 in αT3-1 cells did not affect either KSRP steady-state levels (Figure S3A) or protein stability (Figure S3B) Altogether, these results indicate that KSRP is crucial in controlling β-catenin mRNA decay and, in turn, β-catenin expression. 14-3-3 Interacts with Phosphorylated KSRP and Affects Its Decay-Promoting Activity To investigate the functional consequences of KSRP phosphorylation by AKT, we performed in vitro reconstitution experiments. Either nonphosphorylated or AKT2-phosphorylated KSRP was added to S100 extracts from αT3-1–shKSRP in typical in vitro degradation assays. As presented in Figure 6, KSRP promoted rapid decay of β-catenin mRNA, whereas AKT2-phosphorylated KSRP lacked its decay-promoting ability (Figure 6A). Similar results were obtained using KH1-4 as a GST fusion instead of the Baculovirus-expressed KSRP (Figure 6E). As predictable on the basis of the results shown in Figure 4F, the incubation of the mutant KH1-4(S193A) with AKT2 did not affect its β-catenin RNA decay-promoting activity (Figure 6A, lanes 17–28). Figure 6 KSRP Phosphorylation by AKT Promotes Its Interaction with 14-3-3 and Affects Its mRNA-Destabilizing Function (A) In vitro RNA degradation assays using S100s from either mock αT3-1 (lanes 1–4) or αT3-1–shKSRP cells (lanes 5–28) preincubated with BSA (lanes 1–8), GST (lanes 17–20), KSRP (30 nM, lanes 9–12), AKT2-phosphorylated KSRP (30 nM, lanes 13–16), KH1-4 (S193A) (30 nM, lanes 21–24), or AKT2-phosphorylated KH1-4(S193A) (30 nM, lanes 25–28), respectively. Internally 32P-labeled and capped RNA substrates were added, and their decay was monitored as described above. (B) Coimmunoprecipitation of FLAG-KSRP and endogenous 14-3-3 in 293T cells transiently transfected with FLAG-KSRP and either pCMV empty vector (mock 293T) or pCMV-myrAKT1 (293T-myrAKT1). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using anti–14-3-3 antibody. (C) GST pull-down of endogenous 14-3-3 from total extracts of either mock αT3-1 (lanes 1–3) or αT3-1–myrAKT1 (lanes 4–11) cells using either control GST, GST–KH1-4, or the additional KSRP deletion mutants fused with GST (as indicated, see Figure 4B for a schematic representation of KSRP deletion mutants). Proteins were analyzed by immunoblotting using anti–14-3-3 antibody. (D) Coimmunoprecipitation of either FLAG-KSRP or FLAG-KSRP(S193A) and endogenous 14-3-3 in 293T cells transiently transfected with pCMV-myrAKT1 (293T-myrAKT1) and either FLAG-KSRP or FLAG-KSRP(S193A). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using anti–14-3-3 antibody. (E) In vitro RNA degradation assays using S100s from αT3-1–shKSRP cells pre-incubated with either GST (lanes 1–4), KH1-4 (30 nM, lanes 5–8), AKT2-phosphorylated KH1-4 (30 nM, lanes 9–12), or AKT2-phosphorylated KH1-4 (30 nM) preincubated with difopein (50 nM) (lanes 13–16), respectively. Internally 32P-labeled and capped RNA substrates were added, and their decay monitored was as described above. (F) GST pull-down of endogenous 14-3-3 from total extracts of αT3-1-myrAKT1 cells using either control GST, GST–KH1-4, or GST–KH1-4 preincubated with 50 nM difopein (as indicated). Proteins were analyzed by immunoblotting using anti–14-3-3 antibody. The sequence of the peptide including S193 closely resembles the phosphoserine-containing motif that is recognized by members of the 14-3-3 protein family [37]. Indeed, in a yeast two-hybrid screening performed in order to identify molecular partners of KSRP, we found, among others, the cDNA encoding 14-3-3ζ (unpublished data). To investigate whether phosphorylation by AKT favors the interaction of KSRP with 14-3-3, we cotransfected either myrAKT1 or the empty vector (mock) together with FLAG-KSRP [9] in 293T cells. The kinase activity immunoprecipitated by anti-AKT antibody was higher in myrAKT-293T than in mock 293T cells (unpublished data). Endogenous 14-3-3 coimmunoprecipitated with FLAG-KSRP only in cells transfected with myrAKT1 (Figure 6B). Accordingly, GST–KH1-4 pulled down endogenous 14-3-3 present in extracts from αT3-1 cells stably expressing myrAKT1 but not from mock αT3-1 cells (Figure 6C). As predictable on the basis of the position of the unique AKT phosphorylation site (S193), only GST-KSRP fusion proteins containing the KH1 interacted with 14-3-3 (Figure 6C, lanes 6, 9, and 10). We examined whether phosphorylated S193 is required for KSRP interaction with 14-3-3. Coimmunoprecipitation experiments showed that wild-type KSRP interacted with 14-3-3 when coexpressed with myrAKT1 in 293T cells, whereas KSRP(S193A) did not (Figure 6D). Accordingly, the S193A mutant KH1-4 [GST–KH1-4(S193A)] failed to interact with 14-3-3 in pull-down experiments (unpublished data). To inhibit the interaction between AKT-phosphorylated KSRP and 14-3-3, we used the synthetic peptide difopein, which binds to 14-3-3 proteins with high affinity and competitively inhibits 14-3-3–ligand interaction [38]. AKT2-phosphorylated KSRP was preincubated with either a scrambled peptide (control) or difopein, as indicated, before incubation with S100 extracts from αT3-1–shKSRP cells and used in in vitro RNA degradation experiments. Data presented in Figure 6E indicate that preincubation with difopein restored the decay-promoting activity of KH1-4, preventing the interaction of phosphorylated KH1-4 protein with 14-3-3 (Figure 6F). Altogether, our results indicate that AKT-phosphorylated KSRP interacts with 14-3-3 and that this interaction impairs the decay-promoting activity of KSRP in vitro. AKT Activation Impairs the Interaction of KSRP with the Exosome We investigated whether the impairment of the mRNA decay-promoting activity of KSRP upon phosphorylation by AKT2 (see Figure 6A) was due to either reduced RNA binding or reduced interaction with the decay machinery. In vitro phosphorylation by AKT2 did not affect the β-catenin mRNA binding activity of KSRP either in the absence or in the presence of recombinant 14-3-3ζ (Figure 7A and 7B). Figure 7 AKT Activation Affects KSRP-Exosome Interaction (A) The interaction between 32P-labeled ARE β-catenin RNA and recombinant purified KSRP (increasing amounts from 30 to 300 nM) subjected to kinase reactions in the absence or in the presence of AKT2 (as indicated) was evaluated by UV-crosslinking. (B) The interaction between 32P-labeled ARE β-catenin RNA and recombinant purified KSRP (50 nM) subjected to AKT2 phosphorylation in the presence of either GST (50 nM) or GST–14-3-3 (50 nM, as indicated) was evaluated by UV-crosslinking. (C) Coimmunoprecipitation of FLAG-KSRP and either endogenous exosome components (hRrp4p and hMtr4p, as indicated) or endogenous deadenylase PARN in 293T cells transiently transfected with FLAG-KSRP and either pCMV empty vector (mock-293T) or pCMV-myrAKT1 (293T-myrAKT1). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using the indicated antibodies. (D) GST pull-down of endogenous hRrp4p and hMtr4p from total extracts of αT3-1 cells using GST–KH1-4 subjected to kinase reaction either in the absence or in the presence of AKT2. Proteins were analyzed by immunoblotting using the indicated antibodies. Asterisks indicate antibody cross-reactivity with GST-fusion proteins. (E) GST pull-down of endogenous hRrp4p and hMtr4p from total extracts of αT3-1–myrAKT1 cells using either control GST, GST–KH1-4, or GST–KH1-4 preincubated with 50 nM difopein (as indicated). Proteins were analyzed by immunoblotting using either anti-hRrp4p or anti-hMtr4p antibodies. (F) In vitro RNA degradation assays using S100s from either mock αT3-1 or αT3-1–myrAKT1 cells. Internally 32P-labeled, capped, and polyadenylated β-catenin and GAPDH RNA substrates were incubated with S100s for the indicated times, and their decay was analyzed as described in Materials and Methods. Arrows point to either poly(A)+ or poly(A)− β-catenin RNA species. Coimmunoprecipitation experiments performed in 293T cells showed that expression of myrAKT1 impaired the interaction of KSRP with core exosome components including hRrp4p and hRrp41 and with the exosome-associated factor hMtr4p [39] (Figure 7C and unpublished data), whereas it did not affect the interaction with the deadenylase PARN. Similarly, in GST pull-down experiments, GST–KH1-4 interacted with hRrp4p and hMtr4p present in αT3-1 total extracts, whereas GST–KH1-4 phosphorylated in vitro by AKT2 failed to interact (Figure 7D). Difopein, competing the interaction between GST–KH1-4 and 14-3-3 (see Figure 6F), restored the ability of GST–KH1-4 to pull-down hRrp4p and hMtr4p (Figure 7E). The evidence that myrAKT1 expression does not affect the interaction of KSRP with PARN prompted us to investigate whether β-catenin mRNA deadenylation was affected by myrAKT1 expression in αT3-1 cells. As shown in in vitro degradation experiments presented in Figure 7F, deadenylated β-catenin mRNA accumulated in αT3-1–myrAKT1 cells. Our results suggest that phosphorylation by AKT and interaction with 14-3-3 affect the ability of KSRP to interact with the exosome. Discussion Here we report that β-catenin is encoded by a labile transcript whose t½ is prolonged by Wnt and PI3K-AKT signaling. The mRNA decay-promoting factor KSRP is required to ensure rapid degradation of β-catenin transcript in unstimulated cells. AKT phosphorylates KSRP at a unique serine residue, creating a functional binding site for the molecular chaperone 14-3-3. As a consequence, AKT activation impairs KSRP ability to interact with the exoribonucleolytic complex exosome and, in turn, to promote rapid mRNA decay. β-Catenin mRNA Is Labile and Its Degradation Rate Is Controlled by the ARE-BP KSRP With its involvement in the Wnt signal transduction pathway and tumorigenesis, β-catenin is an extensively studied protein. However, the vast majority of studies focused on the control of β-catenin protein degradation upon its signal-induced phosphorylation [17–19]. Lopez de Silanes et al. [40] identified β-catenin mRNA as a target of the ARE-BP HuR in colon cancer cells, leading to the hypothesis that β-catenin is encoded by a labile mRNA. Our results demonstrate that β-catenin mRNA is labile in unstimulated cells and point to an unanticipated mechanism by which β-catenin expression can be regulated at the level of its mRNA turnover by PI3K-AKT activation. Knock-down experiments demonstrate that KSRP controls the t½ and the steady-state levels of β-catenin mRNA, thus increasing β-catenin protein levels and enhancing the β-catenin–dependent TOP-FLASH– and c-myc promoter–dependent reporter transcription. Notably, KSRP knock-down did not affect β-catenin protein degradation rate. While this manuscript was in preparation, a report from Thiele et al. [41] described the presence of alternative splicing in the 3′ UTR of human β-catenin mRNA that could influence its stability. However, we exclude that alternatively spliced isoforms exist in the 3′ UTR of mouse β-catenin mRNA in αT3-1 and C2C12 cells (Figure S4 and unpublished data). Surprisingly, Thiele et al. [41] reported that AREs in the 3′ UTR of human β-catenin mRNA are stabilizing elements. Our results indicate that β-catenin transcript is unstable in four different cell lines (αT3-1, HIRc-B, C2C12 as presented in this report, and 293T, unpublished data) and that mouse β-catenin 3′ UTR confers instability to a reporter mRNA. These data are in agreement with the general view that AREs are destabilizing elements in unstimulated cells (reviewed in [1–3]). PI3K-AKT Activation Prolongs β-Catenin mRNA Half-Life by Targeting KSRP Our data suggest that PI3K-AKT signaling stabilizes β-catenin transcript targeting the mRNA decay machinery. In the past, conflicting results on PI3K-AKT–induced control of β-catenin protein degradation have been provided [42–45]. The discrepancies might depend on the divergent regulation of protein decay in different cell types. Indeed, we have observed that insulin-induced PI3K-AKT activation does not affect β-catenin protein stability in HIRc-B cells (Figure 3E), while myrAKT1 expression slightly prolongs β-catenin protein t½ in αT3-1 cells (unpublished data). However, our results indicate that PI3K-AKT consistently induces β-catenin mRNA stabilization and protein accumulation in both cell lines. Even though our data indicate that PI3K-AKT activation, regulating the mRNA decay machinery, can lead to β-catenin protein accumulation in the absence of changes in its protein degradation rate, it is conceivable that an integrated control of both mRNA and protein stability could be required to ensure rapid and sustained changes of β-catenin expression in response to certain proliferative and differentiative cues. Notably, AKT-induced interaction of β-catenin protein with 14-3-3 has been shown to increase both β-catenin levels and its transcriptional activity [26]. Therefore, AKT-dependent interaction of 14-3-3 either with a factor involved in β-catenin mRNA turnover, such as KSRP, or with β-catenin itself can lead to accumulation of β-catenin protein. In a sense, 14-3-3 might be considered a biological switch controlling β-catenin expression at different levels. KSRP, a major regulator of β-catenin mRNA decay, is phosphorylated by AKT at a unique residue (S193). It is a general concept that destabilizing ARE-BPs, including BRF1, KSRP, and TTP, are responsible for rapid decay of labile mRNAs in unstimulated cells recruiting the mRNA decay machinery [4–6]. We and others have previously shown that activation of the PI3K-AKT pathway controls the turnover of select mRNAs targeting either BRF1 or TTP and turning off their mRNA destabilizing function [12,13,46]. It is noteworthy that, in our experimental model, KSRP and HuR are the major ARE-BPs interacting with β-catenin mRNA. PI3K-AKT activation does not affect the expression level and the phosphorylation status of HuR while inducing KSRP phosphorylation. It is possible to hypothesize that a certain signaling pathway can affect the turnover of select mRNAs regulating the function of distinct ARE-BPs, depending on the cellular context. On the other hand, the activation of different pathways can affect the turnover of different sets of transcripts targeting the same ARE-BP. In C2C12 myoblasts, MAPK p38 activation prolongs the t½ of select myogenic transcripts inhibiting KSRP function [9], whereas PI3K-AKT activation does not affect the stability of the same mRNAs (unpublished data). Similarly, p38 activation in αT3-1 cells does not affect the t½ of β-catenin mRNA (unpublished data). How the decay of distinct sets of transcripts can be specifically regulated by different signaling pathways that target the same ARE-BP is still an unsolved issue. KSRP Interacts with 14-3-3 in a Serine 193–Dependent Way Our data indicate that KSRP phosphorylation by PI3K-AKT creates a functional 14-3-3 binding site. Our current and previous findings, together with existing literature [13,47,48], suggest that 14-3-3 family members play a regulatory role on the function of some destabilizing ARE-BPs. Phosphorylation by AKT, followed by interaction with 14-3-3, impairs the ability of KSRP to promote β-catenin mRNA decay, reducing KSRP interaction with the 3′-5′ exoribonucleolytic complex exosome while leaving unaffected the ability of KSRP to interact with the mRNA. The results obtained using the high-affinity 14-3-3 competitor peptide difopein suggest that KSRP–14-3-3 interaction is implicated in this process. Unexpectedly, we found that AKT activation does not affect KSRP interaction with the deadenylase PARN and that deadenylated β-catenin mRNA accumulates when incubated with S100 from αT3-1-myrAKT1 cells in in vitro degradation assays. We previously demonstrated that KSRP associates with mRNA decay enzymes, including PARN and the exosome components [4,39]. However, our recent data indicate that, although PARN is involved in the decay of a reporter mRNA by tethered KSRP, it does not appear to play a major role in the process while tethered KSRP primarily relies on exosome function [6]. Thus, our present results are in keeping with the idea that the exosome complex is the main enzymatic machine recruited by KSRP to the RNA [6]. It is noteworthy that a large-scale proteomic analysis identified three exosome components (hRrp4p, hRrp41p, and hRrp45) and the exosome-associated helicase hMtr4p, as well as KSRP itself, as molecular partners of 14-3-3 [49]. The four KH domains that constitute the central core of the KSRP are all necessary to ensure its interaction with the entire decay-promoting machinery [6]. S193 lies in the first KH domain of KSRP. Therefore, it is not surprising that the structural changes likely produced by phosphorylation, and the consequent interaction with 14-3-3, severely impair the β-catenin mRNA decay-promoting function of KSRP. In conclusion, the expression levels of β-catenin have to be tightly regulated. As the amount of β-catenin rises, it accumulates in the nucleus, where it interacts with specific transcription factors, leading to regulation of target genes. Inappropriate activation of the β-catenin pathway is linked to a wide range of cancers, including colorectal cancer and melanoma. On the other hand, AKT has emerged as a crucial regulator of widely divergent cellular processes including apoptosis, differentiation, and metabolism. Disruption of normal AKT signaling has now been documented as a frequent occurrence in several human cancers, and it appears to play an important role in their progression. The results we obtained point to KSRP phosphorylation as a link between PI3K-AKT signaling and the control of β-catenin mRNA t½ and, consequently, of its expression. PI3K-AKT signaling activation, with consequent KSRP phosphorylation and functional deactivation, might contribute to sustain β-catenin accumulation and, as a result, activation of target genes potentially able to accelerate tumor development. Materials and Methods Reagents. LY and insulin were obtained from Sigma (http://www.sigmaaldrich.com); triciribine, from Calbiochem/EMD Biosciences (http://www.emdbiosciences.com); Wnt-3A, from R&D Systems (http://www.rndsystems.com); and histone 2B, from Roche (http://www.roche.com). Plasmids. cDNA fragments containing mouse β-catenin cDNA fragments (as shown in Figure S1) and GAPDH 3′ UTR (nucleotides 580–810) were subcloned into PstI-XbaI–restricted pCY2 [39]. A cDNA fragment containing the coding region of human KSRP (nucleotides 202-2136) was cloned into the EcoRI-XhoI sites of pCMV-Tag2B vector (Stratagene, http://www.stratagene.com) in frame with the FLAG sequence to obtain a FLAG-KSRP plasmid. Mutagenesis was performed using FLAG-KSRP and the QuickChange Site-Directed Mutagenesis kit (Stratagene) to obtain FLAG-KSRP(S193A). HA-myrAKT1 cassette was excised from the plasmid previously described by Andjelkovic et al. [31] and subcloned into pcDNA3 (Invitrogen, http://www.invitrogen.com) to obtain pCMV-myr-AKT1, AKT1(K179M) [50] in pUSEamp plasmid, and TOP-FLASH expression vectors were from Upstate Biotechnology (http://www.upstate.com). Murine c-myc promoter (−1,100 ± 580) fused with Luciferase was previously described [51]. To generate pCAT–β-catenin 3′ UTR, we cloned the entire mouse β-catenin 3′ UTR into the NotI-XbaI–restricted pcDNA3-CAT plasmid [4]. Recombinant proteins and antibodies. Affinity-purified human KSRP, expressed using the Baculovirus system, was described in Briata et al. [9]. GST-KSRP deletion mutants were previously described [4]. Amino acid 193 was mutagenized using the QuickChange Site-Directed Mutagenesis kit (Stratagene) to obtain GST–KH1-2(S193A) and GST–KH1-4(S193A), respectively. GST–14-3-3 plasmid was constructed cloning the entire coding sequence of mouse 14-3-3ζ into a pGEX vector. Mouse monoclonal anti–14-3-3β (K-19, which recognizes all the 14-3-3 family members), anti–14-3-3zeta (C-16, ζ–specific), anti-HuR monoclonal antibody 3A2, and anti-HDAC2 were obtained from Santa Cruz Biotechnology (http://www.scbt.com); mouse anti-GST was from Chemicon (http://www.chemicon.com); rabbit anti-AKT was from Cell Signaling (http://www.cellsignal.com); mouse anti–β-catenin was from BD Transduction Laboratories (http://www.bdbiosciences.com); anti-KSRP antibody [4] was affinity purified and interacted with both human and rodent KSRP [4,14, and R. Gherzi, unpublished data]; and anti-hMtr4p [38] was generated against a hMtr4p(1–369)–GST fusion protein and affinity purified. Anti-AUF1 and anti-hnRNP-A1 were a gift from Dr. Gideon Dreyfuss (Howard Hughes Medical Institute), and anti-PARN, anti-hRrp41, anti-hRrp4p, and anti-TTP polyclonal antibodies were gifts from Drs. Elmar Wahle (University of Halle), Ger Pruijn (Nijmegen Center for Molecular Life Sciences), David Tollervey (Wellcome Trust Centre for Cell Biology), and William Rigby (Dartmouth Medical School), respectively. Anti-FLAG (M2), anti–α-tubulin, and anti–β-actin monoclonal antibodies were from Sigma. Cells and transfections. Murine αT3-1 pituitary cells, rat HIRc-B fibroblasts, human HEK-293T cells (293T), and human HeLa cells were cultured in DMEM plus 10% FBS, and murine C2C12 myoblasts were cultured in DMEM plus 20% FBS. Cell transfections were performed using LipofectAMINE Plus (Invitrogen), and G418 (Invitrogen) was used at 500 to 800 μg/ml (depending on the cell line) for selection. Cell pools of transfectants were used for experiments. αT3-1 cells were starved in DMEM plus 0.5% FBS for 16 h prior to LiCl treatment, and αT3-1–myrAKT1 cells were starved in DMEM plus 0.5% FBS for 16 h prior to experiments. HIRc-B cells were starved in DMEM plus 0.1% FBS for 16 h prior to experiments or further treatments. Transient transfections of αT3-1 with either pcDNA3-CAT or pcDNA3-CAT–β-catenin plasmids were performed as described in [4] with the exception that LipofectAMINE Plus was used. Semiquantitative and quantitative RT-PCR. Cells under different culture conditions were treated with 5 μg/ml actinomycin D and harvested at the indicated times, and total RNA was isolated using RNeasy Mini Kit (Qiagen, http://www.qiagen.com) and treated with DNase I (Promega, http://www.promega.com) according to the manufacturer's instructions. First-strand cDNA was obtained with Transcriptor Reverse Transcriptase (Roche). For semiquantitative RT-PCR, 250 ng of total RNA was retrotranscribed using oligo-dT primer. β2-MG was used as an internal control for normalizing transcripts levels measured by RT-PCR. To optimize RT-PCR, preliminary dose-response experiments were performed to determine the range of first-strand cDNA concentrations at which PCR amplification was linear for each target molecule essentially as reported in Briata et al. [9]. For each species of RNA analyzed, the amount of RT-PCR product (measured as densitometric units) was plotted against the input of first-strand cDNA. For quantitative RT-PCR, 150 ng of DNase I–treated total RNA was retrotranscribed using random examers and PCRs were performed using the IQ Sybr Green Mix Super (Bio-Rad, http://www.bio-rad.com) and the MiniOpticon Real-Time PCR Detection System (Bio-Rad). The sequence-specific primers used for PCRs are listed in Table S1. RNA in vitro degradation and UV-crosslinking. 32P-labeled RNAs were synthesized and used as substrates for in vitro degradation assays as reported [7]. UV-crosslinking experiments were performed as described [7]. Immunoprecipitation of ribonucleoprotein complexes. Ribonucleoprotein complexes were immunoprecipitated from αT3-1 cell lysates as previously described [7]. Total RNA, extracted from either immunocomplexes or total cell lysates (input), was subjected to RT-PCRs. Primers are listed in Table S1. In vitro kinase assays and 32P orthophosphate metabolic labeling. AKT (1 and 2) kinase assays were performed using preactivated enzymes purchased from Upstate Biotechnology (50 ng of the active enzyme/reaction) as recommended by manufacturer. [γ-32P]ATP (3,000 Ci/mmol) was from Amersham Biosciences (http://www.amersham.com). In vivo 32P orthophosphate metabolic labeling of transiently transfected HeLa cells was performed as previously described [52], incubating cells with orthophosphate for 16 h. Isolation of phosphopeptides and LC-MS/MS and MS3 analysis. Purified recombinant KSRP was phosphorylated by AKT2 in standard kinase assays. The reactions were analyzed by gel electrophoresis; bands were excised, digested with trypsin, and enriched for phosphopeptides using titanium dioxide microcolumns; and the peptides were analyzed by automated nanoflow LC-MS/MS with a method where the neutral loss of the phosphate group activate the acquisition of a second fragment ion spectrum (an MS3 spectrum) as previously described in detail [53]. All MS/MS spectra files from each LC run were centroided and merged to a single file, which was searched using the MASCOT search engine (Matrix Science, http://www.matrixscience.com) against the publicly available human database. shRNA-mediated KSRP knock-down. pSUPER-Puro-shKSRP was previously described [9]. αT3-1 cells were transfected using LipofectAMINE Plus (Invitrogen). Transfectant pools were selected with 0.3 μg/ml puromycin (Sigma). Luciferase assays. Transient transfections of either mock αT3-1 or αT3-1–shKSRP were carried out with LipofectAMINE Plus (Invitrogen). Luciferase activity was determined after 48 h with the Dual Luciferase System (Promega) following the indications of the manufacturer and using pRL-SV40 as a normalizing vector. Supporting Information Figure S1 β-Catenin mRNA Is Stabilized by myrAKT1 Expression in Myoblasts C2C12 (A) In vitro RNA degradation assays using S100s from either mock C2C12 or C2C12 cells expressing myrAKT1. Internally 32P-labeled and capped RNA substrates were incubated with S100s for the indicated times, and their decay was analyzed as described in Materials and Methods. (B) Semiquantitative RT-PCR analysis of β-catenin mRNA in either mock C2C12 cells or C2C12 cells expressing myrAKT1. Total RNA was isolated as indicated after the addition of actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (442 KB TIF) Click here for additional data file. Figure S2 KSRP Controls β-Catenin mRNA Decay In Vitro (A) In vitro RNA degradation assays using S100s from either mock-transfected (empty pSUPER-Puro vector) αT3-1 (mock αT3-1) or pSUPER-Puro-shKSRP–transfected αT3-1 cells (αT3-1–shKSRP). (B) In vitro RNA degradation assays using S100s from either LiCl-treated mock-transfected αT3-1 cells or LiCl-treated FLAG-KSR–transfected αT3-1 cells. Internally 32P-labeled and capped RNA substrates were incubated with S100s for the indicated times, and their decay was analyzed as described in Materials and Methods. (792 KB TIF) Click here for additional data file. Figure S3 Neither KSRP Steady-State Levels nor Protein Stability Is Affected by AKT1 Activation (A) Immunoblot analysis of total extracts from the indicated cell lines with anti-KSRP and α-tubulin antibodies. (B) Either mock αT3-1 or αT3-1–myrAKT1 cells were treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared and the levels of KSRP were quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. α-Tubulin immunoblotting was used to verify the equal protein loading. (341 KB TIF) Click here for additional data file. Figure S4 A Single mRNA Form Contains the Entire 3′ UTR of Mouse β-Catenin RT-PCR analysis of total RNA from either mock αT3-1 cells (untreated or treated as indicated) or αT3-1–myrAKT1. The primers used are listed in Table S1 and schematically represented on the left. (467 KB TIF) Click here for additional data file. Table S1 Primers Used for RT-PCRs The primers used for RT-PCRs were m-β-catenin (mouse β-catenin), m-AKT1 (mouse AKT1), r-β-catenin (rat β-catenin), b-C (the entire 3′ UTR of mouse β-catenin), m-β2-MG (mouse β2-MG), r-β2-MG (rat β2-MG), CAT; CAT-β-catenin (the entire 3′ UTR of mouse β-catenin placed at the 3′ of CAT), m-β-cat Q-PCR (mouse β-catenin for quantitative PCRs, and m-β2-MG q-PCR (mouse β2-MG for quantitative PCRs). (35 KB DOC) Click here for additional data file. We are indebted to Drs. D. Tollervey, E. Wahle, G. Pruijn, G. Dreyfuss, W. Rigby, and A. Nicolin for reagents and antibodies. Author contributions. RG and PB conceived and designed the experiments. RG, MT, MP, TR, KSK, JSA, and PB performed the experiments. RG, MT, KSK, JSA, and PB analyzed the data. GC, CM, and CYC contributed reagents/materials/analysis tools. RG, MT, and PB wrote the paper. Funding. This work has been partly supported by grants from Associazione Italiana Ricerca sul Cancro, AIRC (to RG), Fondazione Telethon (GGP04012), and Istituto Superiore di Sanitá (526/A30) (to PB). Competing interests. The authors have declared that no competing interests exist. 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==== Front Indian Pacing Electrophysiol JIndian Pacing Electrophysiol JIndian Pacing and Electrophysiology Journal0972-6292Indian Pacing and Electrophysiology Group 17235373ipej070050-00Review ArticleAtrial Fibrillation and Revascularization Procedures: Clinical and Prognostic Significance. Incidence, Predictors, Treatment, and Long-Term Outcome Terranova Paolo 123Carletti Francesca 1Valli Paolo 2Dell'Orto Simonetta 2Enrico Maria Greco 2Terranova Peppino 31 Divisione e Cattedra di Cardiologia, Dipartimento di Medicina, Chirurgia e Odontoiatria, Azienda Ospedaliera "S. Paolo", University of Milan, Italy2 Unita Operativa di Cardiologia, Presidio Ospedaliero "Causa Pia Uboldo", Cernusco Sul Naviglio, Azienda Ospedaliera di Melegnano, Milano3 Divisione di Cardiologia, Azienda Ospedaliera "Luigi Sacco" - Polo Universitario, Istituto di Scienze Cliniche LITA, University of Milan, ItalyAddress for correspondence: Paolo Terranova, MD, Via Sangro 13, 20132 Milan, Italy. E-mail: [email protected] and conflict of interest disclosureFor the present study no fund has been received by any of the Authors of this paper and all the Authors state that they have no conflict of interest in this manuscript. Jan-Mar 2007 1 1 2007 7 1 50 60 Copyright: © 2007 Terranova et al.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Atrial fibrillation is the most common disorder of cardiac rhythm. In spite of simplicity of diagnosis, patients with atrial fibrillation are difficult to treat. In the recent years with the description of the phenomenon called remodelling, it has been possible to better define the principal mechanisms responsible for initiation, maintenance and, in some instances, termination of atrial fibrillation. Electrical, mechanical and anatomical remodelling indicate those alterations that, once established, may baffle any attempt to restore sinus rhythm. Atrial fibrosis is probably the most critical component of the remodelling process and appears to be mediated by several factors. Several kinds of arrhythmias, especially ventricular ones and conduction disturbances, can occur during percutaneous coronary interventions (PCI), resulting from excess catheter manipulation, intracoronary dye injection, new ischemic events, or reperfusion injury. Supraventricular arrhythmias, including atrial flutter and atrial fibrillation (AF), may also occur during or after PCI, as a complication or a sequel of the revascularisation procedure. Also post operative AF is a common complication of coronary artery bypass surgery (CABG), occurring in 5-40% of patients during the first postoperative week, depending on definitions and methods of detection. Experimental and clinical data will be discussed here. atrial fibrillationpercutaneous coronary interventioncoronary artery bypass surgery ==== Body Atrial Fibrillation and Percutaneous Coronary Interventions Atrial fibrillation (AF) is the tachyarrhythmia that has the highest prevalence among all populations. It is clinically important because affected patients have a higher risk of mortality; a deterioration in haemodynamics due to increased heart rate, loss of atrioventricular synchrony, and progressive dysfunction of the left atrium and left ventricle; and stroke and other embolic events resulting from atrial thrombi. [1,2] In addition, AF may cause significant symptoms and impair both functional status and quality of life. Percutaneous coronary interventions (PCI) have been the fastest growing major invasive procedure in the past decade. Accompanying the obvious benefit, there are certain risks, including cardiac arrhythmias. Several kinds of arrhythmias, especially ventricular arrhythmias and conduction disturbances, can occur during PCI. These arrhythmias may result from excess catheter manipulation, intracoronary dye injection, new ischemic events, or reperfusion injury, as also described in the PAMI Trials [3]. Supraventricular arrhythmias, including atrial flutter and AF, may also occur during or after PCI, as a complication or a sequel of the revascularisation procedure. However, these are not as frequent as ventricular arrhythmias. In general, AF has prognostic significance in patients treated with PCI, as it can be induced by cardiac catheterisation, especially in response to catheter placement into or out of the right atrium [4]. Atrial dysfunction (due to atrial ischemia or atrial stretching in heart failure), sino-atrial and AV nodal ischemia, congestive heart failure, sympathetic stimulation, and iatrogenic factors are the possible causes of AF in patients undergoing primary PCI for acute myocardial infarction (MI). Importance of Clinical Features The clinical characteristics of patients play an important role in the occurrence of AF during and after a PCI procedure. Kinjo et al. [4] assessed the prognostic significance of AF and atrial flutter in patients with acute MI that had been treated with PCI. In their study, patients with AF were older and in higher Killip classes, had higher rates of previous MI and previous cerebrovascular accident, had systolic blood pressures of less than 100 mmHg and heart rates' value higher than 100 beats/min, were less likely to smoke, and had higher prevalence of multivessel disease and poorer reperfusion of infarct-related artery than those without AF. AF was a common complication in patients with MI who were treated with PCI and independently influenced 1-year mortality. Cardiogenic shock, congestive heart failure, cardiac rupture, ventricular tachycardia and/or ventricular fibrillation, and stroke occurred more often in patients with AF than in those with-out AF. No significant difference was observed in the rates of recurrent infarction or recurrent ischemia. The unadjusted in-hospital mortality rate was significantly higher in patients with AF than in those without AF. But, after adjustment for demographic characteristics and clinical factors, AF was not associated with in-hospital mortality. Furthermore, when stratified by the timing of AF, both AF at admission and AF that developed during hospitalisation were not independent predictors for in-hospital mortality. Therefore, AF was not significantly associated with in-hospital mortality in patients with MI who underwent PCI. One-year mortality was higher in patients with AF than in those without AF. Most deaths were due to cardiovascular causes. Patients with AF had a greater incidence of death due to pump failure than those without AF. After adjustment for demographic characteristics and clinical factors, patients with AF remained at significantly greater risk for mortality at 1 year. Furthermore, when stratified by the timing of AF, AF during hospitalisation was independently associated with one year mortality, but AF at admission was not. The one year mortality of patients discharged alive was higher in those with AF than in those without AF. After adjustment for demographic characteristics, clinical factors, and antiarrhythmic drug use at discharge, patients with AF remained at significantly greater risk for mortality at one year [4]. Importance of Electrophysiological Predictors Inhomogeneous prolongation of sinus impulses may predict the recurrence of AF. In two recent trials from Dilaveris et al. and Gorenek et al. [5,6], P wave dispersion (P dispersion) was evaluated; it is commonly defined as the difference between the maximum and the minimum P wave duration, and maximum P wave duration (P maximum). In these trials, age, history of organic heart disease, P maximum, minimum P wave duration, and P dispersion were found to be significant univariate predictors of recurrent AF, whereas only P maximum and age remained significant independent predictors of frequent AF paroxysms in the multivariate analysis. Authors concluded that advanced age and prolonged P wave duration may be used as predictors of frequently relapsing AF. The presence of heterogeneous structural and electrophysiological properties in different portions of an atrium prone to fibrillate, may result in an inhomogeneous and discontinuous prolongation of sinus impulses [7]. Electrophysiological studies have demonstrated that individuals with a clinical history of AF have a significantly longer intra-atrial and inter-atrial conduction time of sinus impulses[8-10]. This has been confirmed by the finding of P-wave prolongation on 12-lead surface ECG and signal-averaged ECG recordings[11-13]. The importance of P-wave dispersion in predicting recurrence of AF in patients with paroxysmal AF has also been elucidated [5,14]. Thus, P-wave signal-averaged ECG could be useful to identify patients at risk for recurrence of AF after internal cardioversion [15]. Ozmen et al. [16] investigated the effects of angioplasty induced-ischemia on atrial conduction as estimated by P-wave maximum and P-wave dispersion. The study consisted of 67 consecutive patients (41 men, mean age 58±11 years) with single vessel coronary artery disease who underwent elective single vessel coronary angioplasty (left anterior descending (LAD) coronary artery in 28 patients, the right coronary artery (RCA) in 22 patients and the left circumflex coronary artery (LCx) in 17 patients). All patients underwent 12-lead surface ECG before the first inflation (baseline) and then 60s after intra-coronary balloon inflation. The maximum P wave duration, the minimum P wave duration, and P wave dispersion (Pd = Pmax - Pmin) were calculated from 12-lead surface ECG. Both values of P maximum and P dispersion were significantly higher during balloon occlusion compared with the baseline condition in coronary dilatation procedures. However, the P-wave minimum was not found to differ between baseline and during balloon occlusion. These data demonstrate that prolongation of P-wave dispersion might be a simple and useful additional marker of myocardial ischemia during PCI. Budeus et al. [17] examined the incidence of atrial late potentials in patients with a proximal stenosis of the right coronary artery and new onset of AF. They also investigated the anti-ischemic effect of a successful percutaneous transluminal coronary angioplasty (PTCA) of the right coronary artery. After successful PTCA, only three of their 15 patients were affected after one day, as well as after one month. None of the patients with a history of AF suffered from an arrhythmic recurrence within the following 6 months after successful PTCA. In that study, stenosis of the right coronary artery was associated with positive atrial late potentials. The authors concluded that a successful PTCA of the right coronary artery eliminates pre-existent atrial late potentials and may reduce the risk of AF. Atrial Fibrillation in Acute MI. Thrombolytic vs. Primary PCI Several studies in the thrombolytic era, showed that the prognostic significance of AF on mortality was attenuated by improved treatment [18-20]. Randomised studies performed in the past few years have shown that PCI is a more effective reperfusion strategy than intravenous thrombolysis[21,22]. Therefore, the incidence of AF may have decreased and the prognostic significance of AF may have been attenuated in patients with AMI who underwent PCI [4]. However, little is known concerning the incidence of AF and its effects on the prognosis of patients with AMI who are treated with PCI. A study from Akdemir and coll. [23] was conducted to compare the effects of reperfusion either by thrombolytic therapy or primary PTCA on P-wave duration and dispersion in patients with acute anterior wall MI. The Authors evaluated 72 consecutive patients, retrospectively, who had experienced an acute anterior wall MI for the first time. Patients were grouped according to the reperfusion therapy, PTCA versus thrombolytic therapy. There were no significant differences between the groups regarding age, gender, left ventricular ejection fraction (LVEF), left atrial diameter and volume, cardiovascular risk factors, and duration from symptom onset to treatment. P-wave dispersions and P-wave durations were significantly decreased after PTCA. In that study, primary PTCA reduced the incidence of AF by decreasing the P-wave maximum and P-wave dispersion. Gorenek and coll. [24] examined the coronary angiographic findings of patients who developed AF during acute MI, and the effects of primary PCI and thrombolytic therapy for restoration of sinus rhythm. This study consisted of 52 patients with acute MI who underwent primary PCI or had thrombolytic therapy and developed AF during the first 12 h of hospitalisation. On admission, and 1 month later, coronary angiography was performed in all patients. In 26 of the 52 patients primary PCI was performed and in the other 26 patients thrombolytic therapy was applied (streptokinase or r-TPA) following angiography. Right coronary artery occlusions were the most frequent causes of AF (73%). In repeated coronary angiography, the coronary artery affected by the infarct was still totally occluded in five patients in the primary PCI group and eight patients in the thrombolytic therapy group (P < 0.01). At least TIMI-3 flow was observed in rest of the patients. Twenty-one patients in PCI group, and 16 patients in thrombolytic therapy group were in sinus rhythm (SR) at the time of second coronary angiography, although there was no difference between the LVEFs of the groups, as determined by echocardiography at the time of the first coronary angiography. However, the LVEF of patients in the PCI group was higher at the time of second angiography. The authors concluded that the data showed that, because the patency of the infarct-related artery was better with primary PCI, this mode of treatment was superior to thrombolytic therapy in restoring sinus rhythm in acute MI patients. Recommendations for Management Post-PCI AF has a propensity to revert spontaneously over a period of minutes to hours, so that, usually, it does not require immediate treatment unless it produces ischemia or hemodynamic instability. Specific recommendations based on trials in international literature for therapy in these patients, are nowadays preliminary and based on consensus, since no adequate trials will have tested alternate strategies. Electrical cardioversion is very rarely required. But, when haemodynamic decompensation is prominent, electrical cardioversion is indicated, beginning with 50-100 joules with gradual increase if the initial shock is not successful. When necessary, a beta-blocker can be used for rate control, because of the combined effects of ischemia and sympathetic tone, which is usually increased in patients with AF. If an intravenous beta-blocker is preferred but it is uncertain whether such therapy will be tolerated by the patient, esmolol may be cautiously administrated since its very short half-life permits a therapeutic trial to be performed at reduced risk. If esmolol is tolerated, then a moderate or long-acting beta-blocker can be given. These drugs can be also administered in combination. Intravenous doses of verapamil or diltiazem are attractive alternatives because of their ability to slow the ventricular rate promptly, but they should be used with caution in patients with pulmonary congestion. Due to the increased risk of embolism, intravenous anticoagulation with heparin should be instituted in the absence of any contraindications, moreover if AF is still present while the patient is in the coronary care unit or in his or her room. Amiodarone and dofetelide are also effective for acute control of the ventricular response, but generally are not recommended as the drug of choice for rate control. Digoxin is one of the drugs of choice and can be used especially in patients with congestive heart failure. Atrial flutter is generally well tolerated and also tends to revert spontaneously; when necessary, it can be treated with either burst atrial pacing or electrical or pharmacologic cardioversion. Conclusions Atrial flutter may occur as a complication of PCI, but most of the time the patients' characteristics play important roles in the occurrence of this type of arrhythmia. For instance, ongoing acute MI can be the real reason for AF. Generally, AF tends to revert spontaneously, but treatment should be given promptly when necessary. If the patient is compromised by ventricular rate or by the loss of atrial contribution to cardiac output, synchronised DC cardioversion should be performed without delay. Intravenous beta-blockade can be effective for acute rate control. Calcium-channel blockers can be administered to promptly control ventricular rate. Digoxin, amiodarone and dofetilide are the drugs of choice for treating patients with acute MI with heart failure. Atrial Fibrillation after Coronary Artery Bypass Grafting Post operative atrial fibrillation (AF) is a common complication of coronary artery bypass surgery (CABG), occurring in 5-40% of patients during the first postoperative week, depending on definitions and methods of detection. A meta-analysis of controlled randomised trials confirmed that in trials using 24-h Holter ECG monitoring, the incidence of supraventricular arrhythmias was higher with Holter recordings (41.3%) than in trials with-out (19.9%) [25]. Despite recent improvements in surgical techniques and postoperative patient care, the incidence of postoperative AF seems to increase, most likely related to the existence of an increasing number of elderly patients with co-morbidities. In the majority of cases postoperative AF is transient and not life-threatening, although it may cause marked subjective symptoms, congestive heart failure, hypotension, and ischaemia, requiring pharmacological treatment or electrical cardioversion, resulting in prolonged hospital stay and additional costs in medical care [26]. Stroke is a major adverse event, complicating the immediate outcome of CABG in about 2% of cases. AF was reported to be a major determinant of stroke after CABG, preceding the occurrence of neurological complications in approximately 37% of the patients [27]. Apart from a higher risk of stroke (odds ratio, OR 2.02), postoperative AF was associated with greater in-hospital mortality (OR 1.7) and worse survival (74% versus 87%) at long-term follow-up (4-5 years) [26]. In a multivariate analysis it was an independent predictor of long-term mortality [26]. The complexity of distinguishing the intrinsic hazards due to postoperative AF from the risks related to its aetiological factors and treatment should, however, be recognised in this retrospective cohort study. The mechanisms responsible for postoperative AF are still unclear and are probably multifactorial. Since risk stratification strategies for patients undergoing CABG could lead to more targeted preventive or therapeutic interventions, large number of trials have aimed at identifying risk factors for the development of postoperative AF. Risk factors associated with AF include advanced age (OR for 10-year increase, 1.75); history of AF (OR 2.11) or chronic obstructive lung disease (OR 1.43); valve surgery (OR 1.74); peripheral vascular disease (OR 1.54); and postoperative withdrawal of (3-blockers (OR 1.91) or angiotensin-converting enzyme (ACE) inhibitors (OR 1.69) [26,28]. Among preoperative risk factors, advancing age has a significant association with the incidence of AF, a relationship that is particularly important as the number of elderly patients considered for CABG steadily increases. Advanced age was associated with increased levels of circulating norepinephrine, which could be related to imbalance in the autonomic nervous sys-tem, previously reported in some but not other studies as an independent risk factor for postoperative AF. Thoracic epidural anaesthesia, aimed at blocking excessive sympathetic activity, was, however, not effective in pre-venting postoperative AF [29]. There is still no consensus as to whether operative clinical and/or electrocardiographic characteristics further distinguish patients who would develop postoperative AF. Prolonged P-wave duration consistent with intra-atrial conduction delay, the presence of preoperative supraventricular arrhythmias, and fluctuations in autonomic balance as measured by heart rate variability were identified in some but not other studies as independent risk factors for postoperative AF [30]. Postoperative AF is probably the most important potentially reversible health care expenditure related to CABG. The recognition of the potential benefits of preventing AF after CABG is reflected by the large number of prophylactic strategies reported in the literature. In a meta-analysis including 42 randomised controlled trials, beta-blocking agents, sotalol, and amiodarone significantly reduced the incidence of postoperative AF compared with placebo, and with no marked difference between them [31]. The three drugs each prevented AF with the following odds ratios: beta-blockers 0.39, sotalol 0.35, and amiodarone 0.48) [31]. From the analysis of 10 pacing trials, atrial pacing was shown to be effective, with an odds ratio of 0.46 for biatrial pacing [31]. Biatrial pacing significantly reduced length of hospital stay by 1.5 days, but there was no evidence that the stroke rate was lowered. In another meta-analysis [32] evaluating the use of prophylactic anti-arrhythmic therapy (amiodarone, sotalol, procainamide, pacing) for the prevention of postoperative AF, the incidence of AF varied from 8% to 37% in the treatment groups and from 29% to 53% in the control groups, with a combined overall significant decrease of 0.52 (OR) in the treatment groups. When these studies were combined, there was 1,0±0,2 day overall decrease in length of hospital stay, but no significant effect on the incidence of stroke or mortality. Data on costs, available for five of the six studies that used amiodarone and one of the studies that used pacing, showed a combined insignificant decrease in cost [32]. In another randomised study from Raddy and coll. [33], amiodarone plus pacing significantly decreased the frequency of AF after open heart surgery, compared with amiodarone alone, pacing alone, and placebo. In the cost-effectiveness analysis, when compared with placebo, the probability of lower cost but higher effect (superiority) was 67% for amiodarone, 15% for pacing, and 97% for amiodarone plus pacing [33]. In the multivariate analysis, preoperative beta-blockers and amiodarone were negatively associated with hospital costs (P < 0.05). Data suggest that both amiodarone alone and the combination of amiodarone plus pacing are cost-effective compared with placebo. A meta-analysis of 8 prophylactic pacing trials [34], with 776 patients enrolled, demonstrated a significant anti-arrhythmic effect of biatrial over-drive and fixed high-rate pacing and overdrive right atrial pacing, with a relative risk reduction of approximately 2.5-fold for new-onset AF at open heart surgery. Another larger meta-analysis of 58 studies [35], conducted on 8565 patients, showed that prophylactic therapies (amiodarone, beta-blockers, sotalol, and pacing) favoured treatment for postoperative AF with an odds ratio of 0.43. A positive result for cost of hospitalisation in favour of treatment was achieved, but the statistic was not significant due to low power and large standard deviations. Beta-blockers had the greatest magnitude of effect across 28 trials (4074 patients), with an odds ratio of 0.35. The data for stroke favoured treatment by a non-significant effect size of 0.81. Similarly, a positive indication for length of stay was derived, but it too was not significant, with a weighted mean difference of -0.66. Hypomagnesaemia is frequently observed after cardiac surgery and is related to the extracorporeal circulation and the use of diuretics. In a meta-analysis of 17 trials with 2069 patients [36], magnesium supplementation reduced the risk of supraventricular arrhythmias (relative risk 0.77) but had no effect on the length of the hospital stay or mortality. Administration of prophylactic magnesium reduced the risk of postoperative AF by 29%, although the inhomogeneity among trials may limit the formulation of definitive conclusions. The effect of cardiopulmonary bypass on the incidence of AF after CABG has been addressed in several trials. A meta-analysis [37] of all observational studies comparing cardiac pulmonary bypass (2253 patients) and off-pump techniques (764 patients) in elderly patients demonstrated a significantly lower incidence of postoperative AF (odds ratio 0.70) after off-pump surgery. The results were confirmed in another meta-analysis of 37 randomised trials (3369 patients) [38], in which off-pump CABG significantly decreased AF (OR 0.58) and hospital stay (weighted mean difference, -1.0 days) but without affecting 30-day mortality or stroke rate (OR, 0.68). It should be emphasised, though, that the lower risk profile of patients undergoing off-pump CABG could contribute to a lower AF risk. Radiofrequency ablation of pulmonary vein triggers has had a remarkable high success rate for non-postoperative AF. Our own data showed that onset of AF after CABG was triggered by premature beats in 72% of patients with postoperative AF, which implies that atrial triggers may be important in the postoperative setting. It is, however, as yet unclear whether a surgical epicardial approach would be effective and safe if implemented during routine CABG procedures. The importance of the parasympathetic nervous system for the initiation of AF is still incompletely understood, although it is thought to play a role in the subsets of patients with paroxysmal non-postoperative AF. Vagal post-ganglionic neurons are located in well defined anatomic fat pads situated in two posterior epicardial regions around the heart and adjacent structures. Transvenous radiofrequency (RF) ablation at such sites has resulted in vagal denervation and improved outcome in patients with non-postoperative AF subjected to circumferential pulmonary vein ablation [39]. In animal studies the destruction of an anterior fat pad shown to contain vagal neurons resulted in decreased susceptibility to AF. The anterior fat pad, located in the aortopulmonary window, was therefore studied in humans with respect to its role in postoperative AF [40]. The authors' question was whether its removal, as routinely done during the process of placing the aortic cross-clamp, would decrease subsequent AF [40]. By stimulating the anterior fat pad in patients undergoing CABG, the sinus rate was slowed with no change in PR interval, consistent with innervation of the sinus node but not the atrioventricular node [40]. Since enhanced vagal tone is pro-fibrillatory in the atria by shortening refractoriness, the underlying theory was that removal of tissue responsible for vagal atrial influences would improve AF outcome. In this randomised study, paradoxically, 37% of patients in whom the interior fat pad was dissected developed postoperative AF compared with 7% in whom the fat pad was preserved [40]. Supportive of these findings is the reported lower incidence of AF after off-pump CABG, during which the anterior fat pad is often preserved [37]. Animal experiments support vagal denervation as an effective anti-arrhythmic strategy, which is consistent with the finding of a lower incidence of AF following vagal denervation during catheter ablation procedures. The importance of vagal influences and the role of the cardiac fat pads for developing postoperative AF demand further clinical research to determine the optimal surgical technique. The class I recommendations for prevention and management of postoperative AF are: (1) an oral beta-blocker for preventing AF, unless contraindicated (Class of Evidence: A) and (2) administration of AV node blocking agents to achieve rate control in patients who develop postoperative AF (Level of Evidence: B) [41]. Class Ila recommendations are: (1) prophylactic amiodarone for patients at increased risk of developing postoperative AF (Level of Evidence: A); (2) electrical or pharmacological cardioversion with ibutilide, when reasonable, to restore sinus rhythm, as recommended for non-surgical patients (Level of Evidence: B); (3) attempt at maintenance of sinus rhythm by administration of anti-arrhythmic medications if there is recurrent or refractory postoperative AF, as recommended for patients with coronary artery disease who develop AF (Level of Evidence: B); and (4) anti-thrombotic medication in patients who develop post-operative AF, as recommended for non-surgical patients (Level of Evidence: B). ==== Refs Benjamin EJ Wolf PA D'Agostino RB Impact of atrial fibrillation on the risk of death: the Framingham Heart Study Circulation 1998 98 946 952 9737513 Chugh SS Blackshear JL Shen WK Epidemiology and natural history of atrial fibrillation: clinical implications J Am Coll Cardiol 2001 37 371 378 11216949 Menta RH Harjai KJ Grines L Sustained ventricular tachycardia or fibrillation in the cardiac catheterization laboratory among patients receiving primary percutaneous coronary intervention: incidence, predictors, and outcomes J Am Coll Cardiol 2004 43 1765 1772 15145097 Kinjo K Sato H Prognostic significance of atrial fibrillation/atrial flutter in patients with acute myocardial infarction treated with percutaneous coronary intervention Am J Cardiol 2003 92 1150 1154 14609587 Dilaveris PE Gialafos EJ Sideris SK Simple electrocardiographic markers for the prediction of paroxysmal idiopathic atrial fibrillation Am Heart J 1998 135 733 738 9588401 Gorenek B Kudaiberdieva G Cavusoglu Y Immediate recurrence of atrial fibrillation after internal cardioversion: importance of right atrial conduction variations J Electrocardiol 2002 35 313 320 12395358 Gialafos JE P-wave dispersion Eur Heart J 1999 20 317 10099927 Tanigawa M Fukatani M Konoe A Prolonged and fractionated right atrial electrograms during sinus rhythm in patients with paroxysmal atrial fibrilla-tion and sinus sick node syndrome J Am Coll Cardiol 1991 17 403 408 1991897 Centurion OA Isomoto S Fukatani M Relationship between atrial con-duction defects and fractioned atrial endocardial electrocardiograms in patients with sick sinus syndrome PACE 2002 16 2022 2023 7694249 Papageoriou P Monahan K Boyle NG Site dependent intra-atrial conduction delay: Relationship to initiation of atrial fibrillation Circulation 1996 94 348 349 Steinberg JS Zelenkofske S Wong SC Value of P-wave signal-averaged ECG for predicting atrial fibrillation after cardiac surgery Circulation 1993 88 2618 2622 8252672 Vilani GQ Piepoli M Cripps T Atrial late potentials in patients with paroxysmal atrial fibrillation detected using a high gain, signal-averaged esophageal lead PACE 1994 17 1118 1123 7521037 Klein M Evans SJL Cataldo L Use of P-wave-triggered, P-wave signal-averaged electrocardiogram to predict atrial fibrillation after coronary artery bypass surgery Am Heart J 1995 129 895 901 7732978 Yamada T Fukunami M Shimonagata T Dispersion of signal-averaged P wave duration on precordial body surface in patients with paroxysmal atrial fibrillation Eur Heart J 1999 20 211 220 10082154 Aytemir K Aksoyek S Yildirir A Prediction of atrial fibrillation recur-rence after cardioversion by P wave signal-averaged electrocardiography Int J Cardiol 1999 70 15 21 10402041 Ozmen F Atalar E Aytemir K Effect of balloon-induced acute ischemia on P wave dispersion during percutaneous transluminal coronary angioplasty Europace 2001 3 299 303 11678388 Budeus M Hennersdorf M Dierkes S Effects of right coronary artery PTCA on variables of P-wave signal averaged electrocardiogram Ann Noninvasive Electrocardiol 2003 8 150 156 12848797 Antman EM Anbe DT Armstrong PW ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction-executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 1999 Guidelines for the Management of Patients With Acute Myocardial Infarction) Circulation 2004 110 588 15289388 Eldar M Canetti M Rotstein Z Significance of paroxysma] atrial fibrillation complicating acute myocardial infarction in the thrombolytic era. 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==== Front BMC Med ImagingBMC Medical Imaging1471-2342BioMed Central London 1471-2342-7-11726674610.1186/1471-2342-7-1Research ArticleEvaluation of 3D surface scanners for skin documentation in forensic medicine: comparison of benchmark surfaces Schweitzer Wolf [email protected]äusler Martin [email protected]är Walter [email protected] Michael [email protected] Institut für Rechtsmedizin, Universität Zürich, Zürich, Switzerland2 Centre for Geo-Information, Department of Environmental Sciences, Wageningen University, The Netherlands2007 31 1 2007 7 1 1 20 11 2006 31 1 2007 Copyright © 2007 Schweitzer et al; licensee BioMed Central Ltd.2007Schweitzer et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Two 3D surface scanners using collimated light patterns were evaluated in a new application domain: to document details of surfaces similar to the ones encountered in forensic skin pathology. Since these scanners have not been specifically designed for forensic skin pathology, we tested their performance under practical constraints in an application domain that is to be considered new. Methods Two solid benchmark objects containing relevant features were used to compare two 3D surface scanners: the ATOS-II (GOM, Germany) and the QTSculptor (Polygon Technology, Germany). Both scanners were used to capture and process data within a limited amount of time, whereas point-and-click editing was not allowed. We conducted (a) a qualitative appreciation of setup, handling and resulting 3D data, (b) an experimental subjective evaluation of matching 3D data versus photos of benchmark object regions by a number of 12 judges who were forced to state their preference for either of the two scanners, and (c) a quantitative characterization of both 3D data sets comparing 220 single surface areas with the real benchmark objects in order to determine the recognition rate's possible dependency on feature size and geometry. Results The QTSculptor generated significantly better 3D data in both qualitative tests (a, b) that we had conducted, possibly because of a higher lateral point resolution; statistical evaluation (c) showed that the QTSculptor-generated data allowed the discrimination of features as little as 0.3 mm, whereas ATOS-II-generated data allowed for discrimination of features sized not smaller than 1.2 mm. Conclusion It is particularly important to conduct specific benchmark tests if devices are brought into new application domains they were not specifically designed for; using a realistic test featuring forensic skin pathology features, QT Sculptor-generated data quantitatively exceeded manufacturer's specifications, whereas ATOS-II-generated data was within the limits of the manufacturer's specifications. When designing practically constrained specific tests, benchmark objects should be designed to contain features relevant for the application domain. As costs for 3D scanner hardware, software and data analysis can be hundred times as high compared to high-resolution digital photography equipment, independent user driven evaluation of such systems is paramount. Index terms Forensic pathology, Rough surfaces, Surface Scanning, Technology Assessment ==== Body 1. Background Courts typically rely on forensic pathologists to document injuries or skin conditions for the purpose of negotiating interpretation and legal significance. During these deliberations, newly raised hypotheses may require details not documented initially. Usually by the time a court trial opens, concerned deceased are buried or cremated and injuries of living people have healed – so the originals are never present any more. Skin surfaces featuring injuries can be fully digitized to be able to conduct a detailed re-analysis, but success relies on precise representation of injured skin. Other applications of precisely digitized surfaces in forensic pathology include the attempt to reconstructive juxtapositioning [1,2], facial [3] or dental [4] landmark projection for the purpose of quantifying shape match or non-match. However, most 3D surface scanners are not optimized for injured skin but for industrial or technical application domains such as industrial design, reverse engineering and rapid prototyping. Surfaces typically contain smooth and mostly straight or curved surfaces joined by edges or bends, holes, slots, pockets or grooves [5], which is reflected in the range of test objects used for industrial scanner evaluation [6]. For the purpose of surface scanning, spray paint may be used to reduce artifacts originating from specular reflectance, directional effects, or even discoloration. In contrast, forensic pathology of injured skin deals with complex, differently colored, locally highly reflective and small sized surface features that may still contain forensic relevance when smaller than 1 mm. We evaluated current 3D surface scanner technology for routine 3D skin surface documentation in forensic pathology, an application domain the scanners that we evaluated were not specifically designed for. The benchmark problem we posed was whether a 3D surface scanner could capture a whole body including skin findings as small as a needle mark (typically sized 0.5 – 1.0 mm) in a device setting typical for digitizing whole body surfaces within a short amount of time. We base this requirement on our observation that many forensic case re-evaluations focus on small (rather than large) findings, and are conducted at a time when a body has been cremated or buried or when wounds have been changed by surgical treatment or healing. We devised two benchmark objects to match these practical requirements. This paper reports handling, usage and 3D scan data comparison of our benchmark objects under practical constraints. 2. Methods A. Choice of scanners Choice of scanners was reduced by eliminating models clearly not suitable for the task first: upon visiting an international industry exhibition (Euromold, Frankfurt), an exhaustive internet search and visits to representatives oft two companies for preliminary field tests, all but two surface scanners were either declared to yield or effectively yielded a strikingly insufficient performance for the benchmark problem. These two devices were subsequently tested: The ATOS-II by GOM (Braunschweig, Germany), and the QTSculptor by Polygon Technology (Darmstadt, Germany). B. Benchmark objects Stability is a hard requirement for benchmark testing, so we used two [7] solid benchmark objects not subject to decay: the surface of the nasofrontal bones of a sheep skull and a washed sandstone conglomerate with quartz inclusions (Fig. 1). Realistic skin samples had been considered for scanner evaluation, but were not used because they were subject to considerable intra-object variation in their appearance and did not provide any stable 3D geometry (see Fig. 2). Figure 1 Benchmark objects used: Left: Photo of a sheep's skull containing small intrinsic bone surface features and tool marks, including add-ons such as boreholes, countersink drillings, red and black felt pen marks. Right: Photo of a sandstone conglomerate featuring different inclusions, some of dark light absorbing quality, some highly reflective. Figure 2 Skin sample (pig skin from an animal killed for nutritional purposes) illustrating the non-suitability of realistic skin samples for benchmarking 3D scanner resolution for rough surfaces. Photographs showing decay 3 days (a) and 6 days (b) post mortem. QTSculptor derived 3D surface scans at 3 days (c) and 6 days (d) (bar 1 cm) post mortem provide surfaces of similar appearance, but even at good overlap positioning a distance map (e) between the two digitized surfaces shows they are not congruent with patches of divergence exceeding 2 mm. Both objects were selected and additionally modified with countersink drillings, boreholes, scratches, and felt pen marks so they would contain a range of challenging shape features typically encountered in forensic skin pathology such as fractal granularity or roughness, holes, scratches, highly reflective patches, shape convexity matching ears, hand or feet as well as discoloration. Typical features in forensic skin pathology include superficial or deep abrasions that may contain highly reflective regions such as body fluids or attached material such as gravel (Fig. 3a/3b), injuries such as gunshot wounds (Fig. 3d/3e) as well as stab wounds caused by knife blades with by serrated (Fig. 3g) or straight (Fig. 3h) edges. Figure 3 Side-by-side comparison of real forensic skin pathology (a, b, d, e, g, h) and benchmark object features (c, f, i): Deep facial abrasion after sliding over a rough surface (a, b) containing various highly reflective surface regions, patchy dark discoloration as well as bumpy appearance. These surface shape elements are represented on a similar scale on the rock surface that we used as benchmark object (c). Superficial abrasions as found in gunshot entry wounds (d, e: arrow) are also present on skull surface (f: arrow) used as benchmark object. Curved (g: arrow) and straight (h: arrow) wound edges as found in stabs from a serrated (g) or straight (h) knife blade are represented by a bony suture of the skull (i: arrow) used as benchmark object (bar 1 cm). C. 3D scanners and data acquisition Both scanners projected collimated white light patterns for optical triangulation and manufacturers' declared specifications did not differ significantly (Table 1). Calibrations were performed using patterned calibration targets. Scans were obtained in a stationary setting without particular vibration isolation, but no vibration was incurred during the scans. Table 1 Device and setup parameters of the two surface scanners Specification/parameter [: according to manufacturers' specification] ATOSII QT-Sculptor PT-M1280 Measuring distance/Stand off used for benchmark scans about 75 cm about 80 cm Measuring volume 100 × 80 × 80 mm3 120 × 100 × 100 mm3 Camera resolution * 1280 × 1024 pixel 1280 × 960 pixel Lateral point spacing * 0.08 mm 0.08 mm Noise (depth) * 0.002 mm 0.015 mm Both scanners require objects to be captured from different directions and for this test, objects were placed on a turning table and 8 – 12 single scans were acquired. Automatic image merging for 3D surface model generation, which was done subsequentially, required reference point stickers to be placed on objects only for the ATOS-II scanner prior to the scan but not for QTSculptor. Software control of scanner was integrated with computer hardware and subsequent scan image registration was based on particular file formats for both devices. This is why we evaluated each hardware-software package sold as "3D scanner" in conjunction. The ATOS-II used for this evaluation was owned by a local government department and operated by a professional who had completed a considerable number of scans. The QTSculptor PT-M1280 was tested by a novice on the manufacturer's premises. The nature of the benchmark test had been declared to the investigators prior to the scans. We proceeded to compare resulting 3D data without further manual point-click editing of the data which – as opposed to mathematical operations applied to whole images – could be viewed as tampering with visual evidence in forensic sciences. Relevant differences pertaining to duration of worksteps are contained in Table 2. Table 2 Scanner handling Work step ATOSII QT-Sculptor Special setup of object surface Requires reference point stickers to be placed on object ~ 5 min not required Approximate user attended scan time for skull benchmark object ~ 15 min ~ 8 min Approximate user attended scan time for rock benchmark object (difficulty: convex object). ~ 30 min ~ 12 min Post processing of data ~ 5 – 15 min Rock: Number of polygons 5,005,559 3,556,544 Skull: Number of polygons 4,430,998 2,447,596 D. 3D data rendering and photographs 3D data was surface-rendered using a non-texturized gray surface structure, oblique virtual illumination and orthogonal projection (see Figures 4, 5, 6 and 7). Resulting 2D projections (best rendering resolution 10–20 μm) were complemented with similarly illuminated digital microscopy photographs of matching object surface areas (best resolution 10–20 μm). Only surfaces and no textures were processed. Figure 4 Sheep skull benchmark object. a): Photo. b) Matching the photo, this shows the 3D model obtained using ATOS-II 3D scanner. c) Matching the photo, this shows the 3D model obtained using QTSculptor scanner. Details of the scratch contained on the surface were reproduced photographically (d), on the ATOS-II scanner (e) and QTSculptor (f). See text for feature description. Bar is 5 mm. Figure 5 Rock benchmark object. a): Photo. b) Matching the photo, this shows the 3D model obtained using ATOS-II 3D scanner. c) Matching the photo, this shows the 3D model obtained using QTSculptor scanner. A detailed region of this surface was reproduced photographically (d), on the ATOS-II scanner (e) and QTSculptor (f). See text for feature description. Bar is 5 mm. Figure 6 Experimental subjective evaluation was conducted using a set of 20 comparisons just as the three visual objects (a, b, c) displayed here, evaluated by 12 participants. For each of the rows, scanner 'x' and scanner 'y' were presented in random sequence to participants who had to select the preferred match ('x' or 'y') as a forced choice. In this illustration, 'x' is QTSculptor, 'y' is ATOS-II for all three visual objects (a,b,c). a: Bone surface structure featuring finely granular roughness, and sharp edges (bottom of foramen), that blend into the surface (lateral margins of the foramen). b: Bone surface structure featuring finely granular roughness, a black felt pen dot, several small surface indents, and a suture. c: Bone surface containing a more extensive suture containing countersink drillings. Bar is 5 mm. Figure 7 Quantitative characterization of scanner performance was done using a set of 220 single surface areas, 3 of which are presented for illustration with photos of real object (a,d,g), ATOS-II-derived data (b,e,h) and QTSculptor-derived data (c,f,i). Red outlines mark homologue areas. Bar is 5 mm. E. Experimental subjective evaluation In a comparison experiment, we presented a test set containing 20 different visual objects to 12 participants: one photo of a portion of a benchmark object, and two matching images with rendered 3D data randomly placed to the left (X) or right (Y) (see Fig. 6 for illustration of 3 instances of the 20 objects). Of the 12 participants, 10 were professionally occupied with shapes (9 in forensic pathology, 1 in industrial tooling), 2 were not working with shapes (clinical researchers). None was directly involved with this study. For each of the 20 objects, participants had to indicate which scanner-derived image better matched the photo in a forced choice. A total of 240 (12 × 20) answers resulted. F. Quantitative characterization of 3D data In optical triangulation of rough surfaces using light patterns, uncertainties at each image pixel significantly depend on illumination and measurement angles at that precise location. On rough surfaces, this angle varies from one point in the original single image to the next, and so does the accuracy and sensitivity of the surface scanning system; at an extreme, the system may have no sensitivity at all for some image patches, whereas neighboring image patches may exhibit higher local accuracy and contribute to a correct overall appearance of the resulting digital surface [8]. This means, that in order to appreciate the quality of digitized rough surfaces, the inherent nature of the error requires single small regions or patches to be checked individually. We conceptually decomposed the scanned surface data (y) into a match of the ideal real object (μ) with additional deviating features (e) [9]: yscanner = μobject + edeviating_features [Eq.1] We expected the deviation of the surface data from the benchmark object to be possibly dependent on the minimal extent (s) and appearance (a) of shape elements [10]. edeviating_features = f(s,a) [Eq.2] Currently, macroscopic and microscopic inspection, including subsequent verbalization and categorization of observations, is the only technique yielding sufficient accuracy that is recognized as de-facto standard in forensic pathology. Any automatic method eligible as reference would have to be suitably accurate with a ratio of 10:1 over the method tested [11] (any lower ratios may be regarded as concession to manufacturers), and so far, no automated 3D surface digitizer was established either as reference method or accepted as standard method in forensic skin pathology by any authority. Across both benchmark objects, 220 single surface patches in the lower range of the visual scale containing distinct features were selected arbitrarily, labeled, categorized, and their smallest spatial extent was measured directly on the object using a micrometer. As a minimum of five different surface materials are required [9], surface patches were classified on the macerated skull as (a) native surface, (b) black discoloration and (c) red discoloration, and on the sandstone as (d) rough surface and (e) quartz inclusion. Shape elements were categorized as (i) granular 3D texture versus directional or streak 3d texture and (ii) repetitively monotonous versus non-repetitively sparse structures [12]. Projected 3D-appearances of homologue single surface areas of each of the two scanners were awarded binary scores as to whether they constituted a sufficient representation or not upon direct comparison with the benchmark object (illustrated in Fig. 7). Based on this, data of each 3D scanner was independently awarded a "1" for sufficiently and a "0" for insufficiently representing a particular surface area. Recognition rates were obtained by totaling these counts. Conceptual problems with confidence intervals of binary scores [13] were avoided by analysing a completely stratified dataset. Bootstrap [14] was used to determine confidence intervals. Recognition rates were correlated with size and surface categories (Tables 3 and 4) using the Chi-Square test. Logistic regression can determine continuously varying recognition rates from categorized data and was used to determine how recognition rates would continuously degrade with diminishing feature size (Fig. 8; significance levels see Table 4). Table 3 Quantitative characterization of 3D data with respect to recognition rate Items compared ATOSII QTSculptor Statistics Total surface area count recognized/not recognized on scanner data 114/106 212/8 p < 0.001 χ2 Recognition rate Cat. A > 0.7 mm 66/19 85/0 p < 0.001 χ2 Recognition rate Cat. B ≤ 0.7 mm 48/87 127/8 p < 0.001 χ2 Recognition rate (bootstrapped estimate) 73 ± 5% @ ≥ 1.2 mm 98 ± 1% @ ≥ 0.3 mm Surface categories Recognized/not recognized: Recognized/not recognized: Bone native surface 43/36 77/2 p < 0.001 χ2 Black dots on bone surface 4/1 5/0 Red dots on bone surface 1/1 2/0 Sandstone surface 66/61 121/6 Quartz inclusion (highly reflective) 0/7 7/0 Table 4 Size dependency of recognition rate Scanner model Smallest feature extent [mm] – recognized Smallest feature extent [mm] – not recognized Statistical significance (Chi Square) for fit of logistic regression model (Fig. 6) ATOSII 0.96 ± 0.71 mm logarithmic transform: -0.3 ± 0.8 mm 0.47 ± 0.75 mm logarithmic transform: -1.4 ± 1.0 mm p < 0.0001 QTSculptor 0.74 ± 0.77[] mm logarithmic transform: -0.8 ± 1.0[#] mm 0.28 ± 0.24[] mm logarithmic transform: -1.7 ± 1.0[#] mm p < 0.0183 Figure 8 Logistic regression model fit for smallest feature size (x-axis) against recognition rate for ATOS-II scanner (left) and QTSculptor scanner (right). Heteroscedastic data – i.e., data exhibiting unequal variances between groups – is assumed to yield reduced significance for statistical tests if variances are truly different; however, what appeared to be initially unequal variances was a result of differently scaled data and thus rendered homoscedastic by using a logarithmic transform [15]. G. Statistics, visualization and computer hardware Benchmark objects were photographed using a digital consumer camera (Finepix F610, Fuji Photo Film Co. Ltd., Tokyo, Japan) and a microscope-mounted (Wild M3Z, Leica-Microsystems, Glattbrugg, Switzerland) scanner camera (Progres, Jenoptik, Germany). 3D surface data was processed and visualized using IDL (Interactive Data Language, Research Systems Inc., Boulder, CO, USA) on a workstation (Intellistation 275, International Business Machines IBM, White Plains, NY, USA). Statistical computations were performed using the software packages JMP (SAS Institute Inc., Cary, NC, USA) and SYSTAT (Systat Software, Inc., Point Richmond, CA, USA). 3. Results A. Setup and handling Both scanners provided a straightforward overall approach to object setup and handling. Some reference point stickers required by the ATOS-II fell off without apparent reason, and were replaced before the scan, but not during the scan procedure. Total time requiring user attendance was less than half the time on the QTSculptor scanner than on the ATOS-II scanner. ATOS-II generated considerably larger 3D models than the QTSculptor (Table 2). B. Qualitative appreciation and individual details Qualitatively (Figure references in brackets), similarities and differences of scanner generated data can be appreciated by comparing photographs (4a, 4d, 5a, 5d, 6/photo, 7a, 7d, 7g) with matching scan data of ATOS-II (4b, 4e, 5b, 5e, 6/Y, 7b, 7e, 7h) and QTSculptor (4c, 4f, 5c, 5f, 6/X, 7c, 7f, 7i). While the overall appearance of both scanner's data seems to match the original object at first glance, individual surface patches exhibit discernible differences: A bone surface scratch lacks details on the ATOS-II generated surface (4b/1, 4e/1) but is well discernible on QTSculptor derived data (4c/1, 4f/1). The roughness of the finely granular bone surface (4a/3, 6a/photo, 7d) appears to be adequately represented on QTSculptor generated data (4c/3, 6a/X, 7f) but not on the surface documented with ATOS-II (4b/3, 6a/Y, 7e), where a markedly less granular structure is exhibited. Slightly more coarsely granular surface (6c/photo, 7g) exhibits defects on ATOS-II (6c/Y, 7h) but not on QTSculptor (6c/X, 7i). Small grooves present on the sandstone rock (5a/1, 5d/3: between arrows; 7a) are discernible on QTSculptor derived data (5c/1, 5f/3: between arrows; 7c) but not on the ATOS-II generated surface (5b/1, 5e/3; 7b); two distinct humps present on the rock (5a/2: arrows) can be differentiated on QTSculptor data (5c/2) but not on the data off the ATOS-II scanner (5b/2) where that distinction is blurred. Borehole rims (4a/2) are available for inspection in QTSculptor data (4c/2) but not on ATOS-II derived data (4c/2). Black patches (6b/photo) seem to cause the ATOS-II to represent a hole (6b/Y), whereas the QTSculptor contains a surface patch (6b/X). Shiny quartz inclusions (5a/4, 5d/5) lead to defective surface data under the ATOS-II scanner (5b/4, 5e/5) but not when scanned with QTSculptor (5c/4, 5f/5). Furthermore, ATOS-II generated surface contains some shape information not present on the object; correlates for these are locations of reference point stickers (4b/4, 5b/7, 7b). Overall the ATOS-II generated surfaces contain more details, better representation of highly reflective and finely granular rough surfaces, better representation of dark surface regions, and more surface attached to holes. C. Experimental subjective evaluation For each of the 12 individual test sets, a total of 20 votes per test set yielded an average of 17.3 ± 2.2 (min: 13/20, max: 20/20) in favor of QTSculptor generated data. In all 240 answers obtained by forced choice, 208 (87.0 ± 3.4%) votes were issued for QTSculptor generated data which is significantly more than the 32 (13.0 ± 3.4%) votes yielded by ATOS-II generated visual objects (bootstrapped standard deviation using 2000 re-samples of the size n = 100 (out of 240); p < 0.0001, Wilcoxon nonparametric test). D. Quantitative characterization of 3D data Smallest spatial extent of the 220 features used for this characterization contained in a median of 0.5 mm (25th percentile at 0.2 mm, 75th percentile at 1.0 mm). Overall recognition rate as well as recognition rate for each of the two groups – category A > 0.7 mm and category B ≤ 0.7 mm – was significantly better for QTSculptor (χ2: p < 0.001) (Table 3). Logistic regression (see Fig. 8 and Table 4) showed that recognition rate was over 90% for features at least 0.3 mm in smallest extent in the QTSculptor (Table 4: * original data with unequal variances; #: homoscedastic data after logarithmic transform). Bootstrapped recognition rate estimate for QTSculptor was 98 ± 1% for features minimally sized ≥ 0.3 mm (2000 resamples with resample size = 100). On ATOS-II generated data, logistic regression revealed a recognition rate of over 70% for surface areas at least 1.2 mm in size and bootstrapped recognition rate estimate was 73 ± 5% for features minimally sized ≥ 1.2 mm (2000 resamples with resample size = 100). Classification of 220 single surface areas yielded 79 bone surface areas, 5 bone surface areas with black discoloration, 2 bone surface areas with red discoloration, 127 sandstone and 7 quartz inclusion areas. The shape elements contained were 176 granular, 27 directional, 17 complex; patterns observed were 182 non-repetitive and 38 repetitive. No geometric characteristic (repetitive, non-repetitive; granular, directional, complex) caused significant differences in recognition rates between both scanners tested. In particular, directional shapes (such as scratches) and granular shapes (such as little indents, protrusions or rough patches) did not yield significantly different recognition rates. The ATOS-II scanner showed difficulty in digitizing highly reflective (sandstone quartz inclusions) or discolored (red, black) surface regions. Three examples representative of the total of the 220 instances that we analyzed are shown in Fig. 7: A groove on the rock (Fig. 7a) is visualized on QTSculptor data (Fig. 7c), but not on ATOS-II data (Fig. 7b). Fine granularity of rough surface (Fig. 7d) was adequately matched in data by QTSculptor (Fig. 7f) but not ATOS-II (Fig. 7e). Presence of a little ditch (Fig. 7g) could be visually discerned on data by QTSculptor (Fig. 7i) but not by ATOS-II (Fig. 7h). 4. Discussion Realistic tissue samples showed to be unreliable for benchmarking 3D scanners as decay and plasticity cause skin to exhibit varying 3D shapes. Instead, we used two solid benchmark objects that contained representative geometric aspects of relevant forensic skin pathology (Fig. 3). The targeted purpose of digitized documentation determines the means employed [16]: Is a scanner merely used to 'add a flavor of 3D'? Is 3D data obtained to examine new hypotheses for further investigation? Is data collected to confidently certify the absence of relevant injuries at a later point in time? Controversial or discriminating morphology may appear small to the naked eye and can be easily overlooked. Such injuries may include hard to detect needle-marks that raise suspicion of poisoning [17], tentative cuts which may indicate self infliction [18] or soot patterns that are important for drawing conclusions about weapon, ammunition and shooting range [19-21]. Those injuries define the size range that should be captured by a 3D scanner in order to allow for a later re-analysis of a case. Conversely, technical constraints of surface scanners intrinsically link higher resolution with a smaller field of view, and therefore a longer total scan time. That is why we tested 3D scanner performance under practical constraints, focusing on 'smallest possibly important feature' combined with 'usefully short amount of time'. Our tests show conclusively that the surface scanner QTSculptor performs faster and obtains significantly better results in the context of relevant medico-legal skin surface documentation despite similar manufacturers' specifications. User attended time to operate the scanners was about double for the ATOS-II, and generated ATOS-II data was considerably larger. We acknowledge that results of constrained tests may differ considerably from a theoretical optimum: given unlimited time and user attendance, the ATOS-II scanner may also achieve acceptable results on rough surfaces. Any digitized data requires a minimal resolution of 16 to 24 data elements in each dimension (pixel, voxel, 3D coordinate points) for an adequate representation of a real feature [22] while a resolution of 50 to 60 elements per feature would be a good resolution. Based on a quantitative analysis, smallest feature sizes that are documented by the QTSculptor ranged down to 0.3 mm for around 98.1%, pointing to an effective lateral 3D point resolution in the range of 15 to 20 μ (noise 2 μ according to manufacturer). Conversely, the ATOS-II managed to capture features sized as small as 1.2 mm in around 70%, pointing to an effective lateral 3D point resolution around 60 μ (noise 15 μ according to manufacturer). Both manufacturers declared a lateral point spacing of 80μ. The ATOS-II matched its manufacturer's specification as to resolution in this test while the QTSculptor obviously exceeds it. This would be hard to establish using automated methods: simple computable data classifiers such as mean square error only provide poor correlation with visually perceivable image quality [23]. Higher effective resolution of the QTSculptor is also indicated by the better representation of fine granularity, observed on digitized rough surfaces; a coarser grain contained in matching ATOS-II generated data indicates aliasing as a result of sub-Nyquist sampling frequency, i.e. undersampling or insufficient resolution in relation to the structure under study [24,25]. The better representation of rough surfaces, rims of boreholes and bottom regions of countersink drillings, and the faster and better acquisition of the convex and rough rock surface also indicate a better depth-of-field of the QTSculptor. Experimental subjective evaluation of the surface samples by 12 judges conclusively showed that 3D surface generation of the QTSculptor is significantly superior compared to the ATOS-II. It is known that subjective evaluation is fast and highly effective [26] and reliability increases with the number of judges; a reliability rating of 0.90 can be obtained with 10 – 50 judges [27]. This subjective evaluation matched the result of other modes of comparison that we had employed, and using projected 2D imagery seemed to be important. In evaluating 3D methods, one may have to exert specific caution not to expose oneself too much to interactive displays: 3D appearance may cause a person to perceive the quality of a 3D model as better when interactively manipulating data on a fast computer compared to its static 2D appearance [28]. Technical flaws in the surfaces obtained by the ATOS-II scanner included reference point stickers that not only created round bumps on the digitized 3D surface, but also, covered up object surface underneath. One could justify using reference stickers if they would cause the result to be of greater accuracy or if the scan process would progress significantly faster due to these stickers. Systems using point stickers might perform better in slight moving or not full stable target conditions; yet the ATOS-II scanner neither produced results of greater accuracy nor did it exceed the speed of the data capture process of the QTSculptor. An important reason for the ATOS-II scanner being outperformed by the significantly cheaper QTSculptor may be the application domain it is specifically designed for – industrial surface scanning. There, reference point sticker based methods may outperform any other 3D method in terms of accurately and precisely placing single 3D coordinates in data space while safely interpolating points in between – yet that was not the output required in this benchmark test. In fact, manufacturer's specification of what is or is not accurate may not coincide with any particular application's requirement for accuracy. Theoretical advantages of high-quality digital 3D-documentation include the option of examining complex 3D shapes from close-up at any later point in time without the limitation of depth-of-field, always producing focused 2D-imagery. This limitation of real photography cannot be overcome even by employing latest technology such as a plenoptic camera [29]. 5. Conclusion We have shown that despite similar manufacturers' specifications, one 3D scanner (QTSculptor) significantly outperformed another model (ATOS-II) both quantitatively and qualitatively under practical constraints in a specific benchmark test that was devised for an application domain neither of the two scanners had been specifically designed for – forensic skin pathology. As costs for 3D scanner hardware, software and data analysis can be hundred times as high compared to high-resolution digital photography equipment, independent user driven evaluation of such systems is paramount. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MH and WS both practically organized and supervised the benchmark testing and discussed ideas to write up the manuscript. WS planned the study, carried out the design of the test and the benchmark objects, studied relevant literature, analysed the resulting data, documented the figures, conducted the statistics and wrote the manuscript. WB and MS contributed to the content of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Rashunda Tramble, BA (University of Memphis TN), copy editor at the International Security Network of the Center of Security Studies at the ETH Zürich, Switzerland, provided professional language support. ==== Refs Brüschweiler W Braun M Thali MJ Analysis of Patterned Injuries and Injury-Causing Instruments with Forensic 3D/CAD supported Photogrammetry (FPHG): An Instruction Manual for the Documentation Process Forensic Sci Int 2003 132 130 138 12711193 Subke J Wehner HD Wehner F Szczepaniak S Streifenlichttopometrie (SLT): a new method for the three-dimensional photorealistic forensic documentation in colour Forensic Sci Int 2000 113 289 295 10978639 Goos MIM Alberink IB Ruifrok ACC 2D/3D image (facial) comparison using camera matching Forensic Science International 2006 1–2 10 17 Häusler M Schweitzer W Bär W Methodic improvements of the videosuperimposition for the identification of skulls based on photos XXth Congres of the International Academy of Legal Medicine 2006 Thompson WB Owen JC St. Germain Jd Stark SR Henderson TC Feature-based reverse engineering of mechanical parts IEEE Transactions on Robotics and Automation 1999 15 57 66 Boehler W Bordas Vincent M Marbs A Investigating laser scanner accuracy XIXth CIPA Symposium, Working Group 6 2003 ISO ISO 5725-5 Accuracy (trueness and precision) of measurement methods and results – Alternative methods for the determination of the precision of a standard measurement method 1994 Geneva: International Organization for Standardization Harding K Calibration methods for 3D measurement systems GE Research & Development Center, Technical Information Series 2001 ISO ISO 5725-1 Accuracy (trueness and precision) of measurement methods and results – General principles and definitions 1994 Geneva: International Organization for Standardization ISO ISO 5725-2 Accuracy (trueness and precision) of measurement methods and results – Basic method for the determination of repeatability and reproducibility of a standard measurement method 1994 Geneva: International Organization for Standardization Deaver DK Guardbanding and the World of ISO Guide 25 – Is There Only One Way? 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1737519206-PONE-RA-00349R110.1371/journal.pone.0000306Research ArticleHematologyImmunologyTranscriptional Repressor Gfi1 Integrates Cytokine-Receptor Signals Controlling B-Cell Differentiation Gfi1 Controls IL7 SignallingRathinam Chozhavendan Klein Christoph * Department of Pediatric Hematology/Oncology, Hannover Medical School, Hannover, Germany Papavasiliou Nina Academic EditorRockefeller University, United States of America* To whom correspondence should be addressed. E-mail: [email protected] and designed the experiments: CR. Performed the experiments: CR. Analyzed the data: CR. Wrote the paper: CK. Other: Directed the investigations: CK. 2007 21 3 2007 2 3 e30613 11 2006 20 2 2007 Rathinam, Klein.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Hematopoietic stem cell differentiation is specified by cytokines and transcription factors, but the mechanisms controlling instructive and permissive signalling networks are poorly understood. We provide evidence that CLP1-dependent IL7-receptor mediated B cell differentiation is critically controlled by the transcriptional repressor Gfi1. Gfi1-deficient progenitor B cells show global defects in IL7Rα-dependent signal cascades. Consequently, IL7-dependent trophic, proliferative and differentiation-inducing responses of progenitor B cells are perturbed. Gfi1 directly regulates expression levels of IL7Rα and indirectly controls STAT5 signalling via expression of SOCS3. Thus, Gfi1 selectively specifies IL7-dependent development of B cells from CLP1 progenitors, providing clues to the transcriptional networks integrating cytokine signals and lymphoid differentiation. ==== Body Introduction The specification of hematopoietic stem cell fate depends on the coordinated actions of cytokines and transcription factors, yet the integrative signals orchestrating transcriptional networks remain poorly understood. Distinct developmental stages of hematopoietic stem cell (HSC) differentiation pathways have been defined. HSC differentiate into common lymphoid (CLP) and common myeloid progenitor (CMP) cells that constitute early branching points of lymphoid and myeloid lineage commitment, respectively [1]. Recently, a second common lymphoid progenitor cell population (CLP2) with restricted lymphoid differentiation capacity has been characterized in TCR-α transgenic mice and proposed to represent early thymic immigrants [2]. There is evidence that early thymic progenitor cells in the thymus may derive in a CLP1- and IL7- independent pathway [3], [6]. Bone marrow B cell development originating from CLP follows a highly regulated process characterized by well defined maturation stages [4]. Bone marrow B cells in the adult mouse are supposed to originate from CLP1 cells in an IL7R-dependent pathway. IL7 mediates survival, expansion, and differentiation of progenitor B cells [5]–[10]. Gfi1 is a SNAG-domain-containing zinc-finger transcriptional repressor that was originally described to confer cytokine-independent growth in a T cell lymphoma line [11]. Gfi1 belongs to a family of proteins (named Gfi/Pag-3/Senseless) involved in cell fate determination and differentiation [12]. In the hematopoietic system, Gfi1 plays a key role in restricting HSC proliferation and preserving their functional integrity [13], [14], in controlling T cell differentiation [15], [16], and in determining the differentiation of neutrophils [17], [18] as well as dendritic cells [19]. We hypothesized that Gfi1 controls cytokine-dependent B cell differentiation. Here, we analyze the role of Gfi1 in B cell development and provide insights into the mechanisms governing the developmental dichotomy of early lymphoid progenitor cells and IL7-mediated signals. Results Characterisation of early lymphoid development in Gfi1−/− mice To address the role of Gfi1 in early B cell development, we first analyzed the numbers of lymphoid progenitor cells in Gfi1−/− and Gfi1+/+ mice. As previously reported [14], the number of CLP1 cells (Lin−Sca1+IL7Rα+ckitlow) was drastically reduced in Gfi1−/− mice (Fig. 1A upper and lower panels). In contrast, both presumptive CLP2 (Lin−Sca1+IL7Rα+ckit−B220+, Fig. 1A middle and lower panels) and ETP (Early Thymic Progenitors, Lin−CD44+CD25−CD4low/−ckit+) (Fig. 1B) were normal in relative and absolute numbers, suggesting that Gfi1 plays a role in the differentiation of CLP1 but not of CLP2 or ETP. To further assess a potential role of Gfi1 in early lymphoid development, we made use of a transgenic Gfi1GFP/Gfi1 reporter mouse that allows monitoring of the transcriptional activity of the Gfi1 locus. As shown in Fig. 1C, Gfi1 is expressed in HSC and CLP1, but not in presumptive CLP2 or ETP, indicating that the latter populations may develop in a Gfi1-independent pathway. Transcriptional activity of the Gfi1 locus was observed in bone marrow B cells at all stages of differentiation, particularly in progenitor B cells at Hardy fractions C–E (Fig. 1A). Furthermore, a dynamic expression pattern of Gfi1 was seen in vitro, when HSC were differentiated into B cells in the presence of IL7 and SCF (Fig. S1B). 10.1371/journal.pone.0000306.g001Figure 1 Developmental dichotomy of CLP1 and presumptive CLP2 progenitor cells evidenced by analysis of Gfi1−/− and Gfi1GFP/+ mice. A) FACS plots indicating relative decrease of CLP1 (upper panels) and normal numbers of presumptive CLP2 (middle panels) in Gfi1−/− bone marrow cells. Cells were pregated on Lin−IL7Rα+ cells (upper panels) and Lin−IL7Rα+Sca1+CD19− cells (middle panels), respectively. Bar diagrams indicating absolute numbers of CLP1 and CLP2 cells of Gfi1−/− and Gfi1+/+ mice (lower panel, n = 3 mice). Lineage markers included CD4, CD8, CD11c, CD11b and B220. Flow-cytometric analysis was performed on pooled BM cells from 5 mice. Shown is a representative experiment of 3. B) FACS plots indicating relative increase of ETP in Gfi1−/− thymus. Lin−CD25−CD44+ cells (upper panels) were gated (G1) and analysed for expression of c-kit and IL7Rα (lower panels). Experiments were performed on pooled thymi (n = 5 mice). Bar diagrams indicating absolute numbers of ETP of Gfi1−/− and Gfi1+/+ mice (lower panel, n = 3 mice). The total number of thymocytes was decreased by ∼6 fold in Gfi1−/− mice. Shown is a representative experiment of 3. C) Transcriptional activity of Gfi1 locus in hematopoietic progenitor cells and thymic B cells. Pooled bone marrow cells or thymocytes (n = 5) were analyzed for GFP expression in the following populations: LSK (HSC), Lin−IL7Rα+Sca1lowc-kitlow (CLP1), Lin−IL7Rα+Sca1+c-kit−B220+CD19− (CLP2) and Lin−CD25−IL7Rα−CD44+c-kit+ (ETP). Shaded histograms represent GFP fluorescence in Gfi1+/GFP cells, open histograms represent autofluorescence of Gfi1+/+ cells. The GMFI (Geo Mean Fluorescence Intensity) of Gfi1+/+ (above) and Gfi1+/GFP (below) cells is indicated. Data are representative of 3 independent experiments. As previously shown [17], total numbers of B220+ cells in the bone marrow of Gfi1−/− mice were significantly reduced, when compared to wildtype control mice (Fig. 2A). Furthermore, Hardy fraction B, proposed to represent an IL7-sensitive stage in B cell development [20], appeared proportionally reduced (Fig. 2B, Table S1). Despite distinct differences (e.g. with respect to Hardy fractions D, E, F) the bone marrow B cell compartment of Gfi1−/− mice is reminiscent of IL7−/− mice showing an incomplete block in maturation [6], [9], [10], [21]. 10.1371/journal.pone.0000306.g002Figure 2 Analysis of bone marrow B cell development in Gfi1−/− mice. A) Absolute numbers of B220+ bone marrow B lineage cells determined from bone marrow of both hind limbs (n = 4 mice). B) FACS plots indicating relative decrease of bone marrow progenitor B cells in Gfi1−/− mice according to the Hardy classification. B220+CD43+ cells were gated (G1) and further analyzed for expression of CD24 and BP-1 (middle panel). B220+CD43− cells were gated (G2) and analysed for expression of B220 and IgM (bottom panel). Flow cytometry was done in pooled cell suspensions (n = 5 mice). Shown is a representative experiment of 5. Defective in vitro differentiation of B cells Gfi1−/− mice are prone to inflammatory reactions characterized by increased systemic cytokine levels [17], [18] that might constitute extrinsic factors influencing B cell differentiation. In a subsequent series of experiments we therefore directly analyzed IL7-dependent early B cell development in vitro. We isolated Lin−Sca1+c-kit+ (LSK) cells from Gfi1+/+ and Gfi1−/− mice and determined their potential to differentiate into B cells in the presence of the cytokines SCF and IL7. In this in-vitro differentiation system, based upon an initial expansion step using a stem cell cytokine cocktail (IL3, IL6, SCF, Flt3L and TPO), complete maturation of B220+IgM+ can be seen using Gfi1+/+ HSC. In contrast to Gfi1+/+ progenitor cells, few Gfi1−/− LSK cells differentiated into B220+CD19+ (5% versus 32%) and B220+IgM+ (0.9% versus 21%) cells, respectively (Fig. 3A), suggesting that cytokine-induced B cell differentiation is impaired in the absence of Gfi1. To prove a cell autonomous and specific role for Gfi1, we transduced Gfi1−/− LSK cells with retroviruses encoding Gfi1-GFP and GFP, respectively, and cultured these genetically modified cells in SCF and IL7 containing medium. As shown in Fig. 3B, only Gfi1-transduced progenitor cells, but not GFP-transduced control cells, differentiated into B220+IgM+ B cells. In complementary experiments, we also determined whether a specific downregulation of Gfi1 in wildtype B cells would affect in vitro B cell differentiation. LSK cells were transduced with lentiviral vectors encoding a specific shRNA directed against Gfi1 or “empty” vectors, respectively. As expected, a knockdown of Gfi1 significantly impaired in vitro B cell differentiation (Fig 3C & Fig. S2). Taken together, these experiments suggest that Gfi1 is a specific, intrinsic, and cell-autonomous factor necessary for B cell development in-vitro. 10.1371/journal.pone.0000306.g003Figure 3 Defective in-vitro differentiation of Gfi1−/− B cells. A) FACS plot showing block in in vitro B cell development in the absence of Gfi1. HSC were differentiated using IL7 and SCF and analyzed for expression of B220 and CD19 (top panel) and for expression of B220 and IgM (bottom panel). Data are representative of 3 independent experiments. B) FACS plot indicating rescue of B cell differentiation upon retroviral gene transfer. LSK cells from Gfi1−/− mice were isolated and transduced with retroviral constructs encoding GFP or Gfi1-GFP. Transduced cells were cultured in the presence of SCF and IL7 for 14 days and GFP-positive cells were analyzed for expression of B220 and IgM. Data are representative of 2 independent experiments. C) FACS plot showing decreased in vitro B cell development upon knockdown of Gfi1 in HSC. HSC were transduced with lentiviral constructs encoding Gfi1 specific shRNA. Transduced cells were cultured in the presence of SCF and IL7 for 14 days and analyzed for expression of CD19 and IgM. Cells transduced with lentiviral backbone served as controls. Data are representative of 2 independent experiments. IL7-mediated signals in Gfi1−/− progenitor B cells To further assess the functional relevance of Gfi1 in orchestrating IL7Rα-dependent signal cascades, we next analyzed trophic and proliferative effects of IL7 signalling. LSK HSCs were incubated in the presence of various cytokine combinations acting on hematopoietic progenitor cells and their viability was measured by propidium iodide exclusion 48 hours later. In these experiments, we tested both LSK progenitor cells and CD48−CD150+ hematopoietic stem cells (HSCs) (45). Both progenitor cell populations expressed IL7Rα upon 48 hours of cytokine stimulation, while no IL7Rα expression could be detected at hour 0 (Fig. S3). Whereas a cytokine cocktail consisting of IL3, SCF, Flt3L, and IL6 maintained the viability of Gfi1−/− progenitor cells, Gfi1−/− cells cultured in the presence of IL7 died (Fig. 4A and Fig. S4), indicating that Gfi1 plays a specific role in IL7-mediated trophic effects. This was confirmed by RT-PCR analysis documenting deficient IL7-mediated upregulation of the mRNA encoding the antiapoptotic protein Bcl2 (Fig. 4B). To assess proliferative responses of Gfi1−/− hematopoietic stem cells, LSK cells were labelled with CFSE and cultured in the presence of IL7 or SCF & IL7. As shown in Fig. 4C, both Gfi1−/− and Gfi1+/+ HSC divided in the presence of SCF & IL7, documented by a decrease in fluorescence intensity. In contrast, IL7 did not induce proliferation in Gfi1−/− HSC. Similar results were seen when CD48−CD150+ cells were analyzed (Fig. S5). In addition to antiapoptotic and proliferative effects, IL7 also induces a differentiation program in progenitor B cells. To quantify B cell differentiation in Gfi1−/− and Gfi1+/+ cells on a clonal level, HSC were cultured on semisolid medium in the presence of various cytokine combinations. 14 days later, Colony-Forming-Units (CFU) were counted. Compared to Gfi1+/+ HSC, Gfi1−/− HSC gave rise to slightly increased numbers of CFU (8750 versus 6650) when incubated in the presence of IL3, SCF, Flt3L, IL6, potentially reflecting increased cell cycle progression in Gfi1−/− HSC (13). In contrast, IL7-induced growth of colonies was severely impaired in Gfi1−/− HSC (175 versus 2200) (Fig. 4D & Fig. S6), suggesting that IL7Rα-mediated signals are globally ineffective in the absence of Gfi1. The generation of the earliest B cell progenitors critically depends on the transcription factors E2A and EBF. Both factors co-ordinately orchestrate the expression of B cell specific genes and immunoglobulin heavy-chain gene rearrangement [22]–[24]. Pax5 is the decisive B lineage commitment factor that restricts the developmental options of early progenitors to the B cell pathway [25]. As an example of instructive effects of cytokines on transcription factors, IL7 directs commitment of CLP by upregulating expression of EBF [6], [7]. We reasoned that in the absence of Gfi1 the coordinated expression of key transcription factors might be disturbed and measured their expression levels in defined progenitor B cell populations by RT-PCR. E2A and EBF were transiently upregulated at the transition from Hardy fraction B to D in wildtype B progenitor cells, but not in Gfi1−/− progenitor cells (Fig. 4E upper and middle panels). These data were confirmed in independent experiments using B cell progenitor cells defined by the expression of B220, CD19, and IgM (Fig. S7B). As a consequence of E2A and EBF expression in progenitor B cells, Pax-5 is upregulated at the transition from Hardy fraction C to D. In line with decreased upregulation of E2A and EBF, Pax-5 transcription was not induced in Gfi1−/− B cell progenitors (Fig. 4E lower panel and Fig S7B). The number of CD19 positive B-lineage cells was also reduced in Gfi1−/− mice (Fig. 4F), either as a consequence of decreased Pax5 expression, or due to defects in fraction B cells. Thus, in the hierarchy of the signal cascade, Gfi1 appears to function upstream of IL7-mediated transcriptional activity of E2A, EBF and Pax5. Taken together, we document that the response to IL7 is severely reduced in Gfi1−/− B progenitor cells resulting in defective B cell differentiation. STAT-signalling and negative circuits In view of global IL7-unresponsiveness of Gfi1−/− cells affecting trophic, proliferative, and differentiation-inducing cellular responses, we reasoned that Gfi1-deficiency affected early IL7Rα signalling events such as activation of the JAK/STAT pathway. We isolated B220+lin− cells from Gfi1−/− and Gfi1+/+ mice and determined the levels of STAT5 phosphorylation at various time points after IL7 stimulation by Western blot. Whereas total STAT5 protein was comparable between Gfi1−/− and Gfi1+/+ control cells, IL7-induced STAT5 phosphorylation was completely absent in Gfi1−/− cells (Fig. 5A). Since Gfi1 represents a zinc finger molecule mediating transcriptional repression in the nucleus, direct effects on IL7Rα activation appeared unlikely. We therefore hypothesized that the balance of activating and inhibitory effects on JAK-STAT-signalling might be affected in the absence of Gfi1. We quantified expression levels of SOCS3, an inhibitor of STAT5 carrying a putative Gfi1/Gfi1B-binding site in its promoter [26]. Interestingly, baseline levels and IL7-induced expression levels of SOCS3 mRNA (Fig. 5B) and protein (Fig. 5C) appeared increased in Gfi1−/− progenitor B cells when compared to Gfi1+/+ cells. This finding indicates that decreased IL7-mediated signalling may at least partially be caused by preponderance of negative feedback circuits. To directly assess the role of unleashed SOCS3 expression in early B cell development, we transduced HSC from wildtype mice with retroviral constructs encoding SOCS3, expanded sorted cells in the presence of IL3, IL6, SCF, and Flt3L and evaluated IL7R-dependent signals. As shown in Fig 5D, transduced cells showed evidence of increased SOCS3 levels (upper panel). When SOCS3-overexpressing cells were stimulated by IL7, SOCS3-transduced progenitor cells revealed decreased phosphorylation of STAT5, as shown by intracytoplasmic FACS analysis (lower panel). In line with this observation, SOCS3-transduced cells also lost viability in response to IL7, whereas most control-transduced cells maintained their viability (Fig. 5E). These experiments show that elevated levels of SOCS3 are associated with decreased cellular responses to IL7 and thus provide mechanistic insights into the role of Gfi1 in negative feedback circuits controlling B cell differentiation. 10.1371/journal.pone.0000306.g004Figure 4 Defective IL7Rα signalling in Gfi1−/− progenitor cells. A) Viability of HSC in response to hematopoietic growth factors. LSK cells from Gfi1+/+ and Gfi1−/− mice were initially cultured in the presence of SCF, IL6, IL3, TPO and Flt3L for 48 hours and subsequently cultured in the presence of indicated cytokines. Cells were analyzed by PI staining and FACS 48 hours later. Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. B) Realtime PCR showing Bcl2 RNA levels. Sorted Lin−B220+ bone marrow cells were stimulated with IL7. RNA was extracted at indicated time points and reverse transcribed into cDNA. Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. C) Cell proliferation indicated by CSFE dilution. LSK cells from Gfi1+/+ and Gfi1−/− mice were initially cultured in the presence of SCF, IL6, IL3, TPO and Flt3L for 48 hours. Cells were washed, labeled with CSFE and incubated in the presence of indicated cytokines for 72 hours. Histograms representing CFSE fluorescence and division history of Gfi1+/+ (top panel) and Gfi1−/− (bottom panel) cells are shown. Data are representative of 2 independent experiments. D) CFU assay indicating decreased colony-forming-activity in Gfi1−/− HSC. LSK cells were cultured on semisolid medium in the presence of indicated cytokines. 14 days later, the absolute numbers of colony forming units of Gfi1+/+ (white bars) and Gfi1−/− (black bars) cells were enumerated and plotted. Data are representative of 2 independent experiments. E) Expression of B cell specific transcription factors in defined precursor B cell populations according to Hardy classification. mRNA expression levels of E2A (top panel) EBF (middle panel) Pax5 (bottom panel) were determined by RT-PCR. Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. F) Contour plots indicating reduced expression of CD19 in Gfi1−/− bone marrow B lineage cells (upper panels). Bar diagrams indicating absolute numbers of CD19+ cells in BM of Gfi1−/− and Gfi1+/+ mice (lower panel, n = 3 mice). Data are representative of 6 independent experiments. 10.1371/journal.pone.0000306.g005Figure 5 Defective STAT5 signalling in Gfi1−/− progenitor B cells. A) Western blot analysis showing decreased STAT5 phosphorylation in B220+ bone marrow cells upon IL7-signalling. Data are representative of 2 independent experiments. B) Realtime PCR showing increased RNA levels of SOCS3 in Gfi1−/− B cells. Sorted B220+ bone marrow cells were stimulated with IL7, RNA was extracted at indicated time points and reverse transcribed into cDNA. Expression of SOCS3 mRNA was quantified by realtime PCR. Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. C) Western blot showing protein levels of SOCS3. Sorted B220+ bone marrow cells were stimulated with IL7 and assessed for SOCS3 protein using specific polyclonal antibodies. GAPDH was detected by specific monoclonal antibodies to confirm equal protein loading. Data are representative of 2 independent experiments. D) Intra-cytoplasmic detection of SOCS3 and p-STAT5 by FACS. LSK cells from Gfi1+/+ mice were transduced with either GFP or SOCS3-GFP retroviruses, respectively. SOCS3 expression was measured by cytoplasmic staining with an anti-SOCS3 monoclonal antibody (upper panel). Sorted GFP and SOCS3-GFP transduced progenitor cells were stimulated in the presence of IL7 for 10 minutes and intra cytoplasmic staining was performed using an anti-pSTAT5 monoclonal antibody (lower panel). Shaded histograms represent cells stained with isotype control antibodies. Data are representative of 2 independent experiments. E) Viability of SOCS3 over-expressing progenitor cells in response to IL7. Sorted GFP and SOCS3-GFP transduced progenitor cells were cultured in vitro in the presence of IL7. At indicated timepoints, cells were analyzed by PI staining and FACS. Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. Modulation of IL7Rα-expression Coordinated lymphocyte development is not only critically dependent on IL7-mediated signals but also on dynamic and developmental-stage-specific regulation of the IL7Rα chain [27]–[31]. Since the IL7Rα gene contains putative binding sites for Gfi1 [32], we analyzed IL7Rα expression levels in distinct progenitor B cells in vivo. As shown in Fig. 6A, Gfi1−/− progenitor B cells at Hardy fractions D and E showed a higher mean fluorescence index of IL7Rα compared to Gfi1+/+ control cells. This finding was confirmed in independent experiments using CD19 as an unambiguous marker of the B cell lineage (Fig. S8). Similar differences were seen in an in-vitro differentiation system allowing the simultaneous detection of IL7Rα mRNA and cell surface expression (Fig. 6 B,C). These results suggest that Gfi1 directly represses IL7Rα transcription and thus allows coordinated B cell development to proceed. To directly prove binding of Gfi1 to a cognate binding site in the IL7Rα gene, we performed chromatin-immunoprecipitation studies in progenitor B cell extracts from Gfi1+/+ mice. As shown in Fig. 6 D and E, direct binding of Gfi1 could be documented to a Gfi1-binding site in intron 2 of IL7Rα, but not to putative Gfi1 binding sites present in intron 3, intron 4, or the promoter. This indicates that the IL7Rα gene is a direct target of the transcriptional repressor Gfi1. Thus, we define a complex mechanistic role for Gfi1 in IL7-mediated signalling as well as in direct downregulation of IL7Rα. 10.1371/journal.pone.0000306.g006Figure 6 Modulation of IL7Rα expression by Gfi-1. A) Histograms plots indicating IL7Rα surface expression in distinct populations of bone marrow B lineage precursor cells. BM cells from 5 mice were pooled, and IL7Rα expression was analyzed in the following subpopulations: B220+CD43+BP1−CD24− (Fraction A), B220+CD43+CD24+ (Fraction B–C), B220+CD43−IgM− (Fraction D), B220+CD43−IgM+ (Fraction E), and B220highCD43−IgMhigh (Fraction F). Shaded histograms represent IL7Rα fluorescence and open histograms represent isotype fluorescence. The GMFIi is indicated in each plot. Geometric mean fluorescence intensity index was calculated as follows: GMFIi = GMFI(antibody stained cells)−GMFI(Isotype control treated cells). Data are representative of 5 independent experiments. B–C) Realtime PCR showing IL7Rα RNA and surface expression levels during in vitro B cell development. LSK bone marrow cells isolated from Gfi1+/+ and Gfi1−/− mice were first expanded for 48 hours in a cytokine cocktail containing IL3, IL6, SCF, Flt3L, TPO and then cultured in the presence of SCF and IL7. RNA was extracted at indicated time points, reverse transcribed into cDNA and Realtime PCR was performed (B). Cell surface expression of IL7Rα during in vitro B cell differentiation. LSK bone marrow cells of Gfi1+/+ and Gfi1−/− mice were cultured in the presence of SCF and IL7. Aliquots of cells were taken at indicated time points and analyzed for expression of IL7Rα by FACS (C). Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. D) Chromatin-immunoprecipitation analysis of Gfi1 binding to the IL7Rα gene. Crosslinked chromatin from Gfi1+/+ bone marrow B220+ cells was immunoprecipitated using control goat IgG or anti-Gfi1 antibodies. PCR was performed on input DNA and immunoprecipitated DNA using primer pairs spanning Gfi1 binding sites that are present in intron 2, intron 3, intron 4, promoter and exon 8 of the IL7Rα gene, respectively. Genomic DNA and no template lanes represent controls. Data are representative of 2 independent experiments. E) Semi-quantitative real time PCR analysis. Crosslinked chromatin from Gfi1+/+ and Gfi1−/− bone marrow B220+ cells was immunoprecipitated using control goat IgG or anti-Gfi1 antibodies. Realtime PCR was performed on input and immunoprecipitated DNA using primer pairs spanning Gfi1 binding sites in intron 2 of IL7Rα gene. Data are representative of 2 independent experiments. Discussion We have shown that Gfi1 is a critical modulator of IL7-dependent B cell differentiation. Gfi1 is selectively expressed in CLP1 cells but not in presumptive CLP2 cells and controls IL7-dependent signals in B progenitor cells in bone marrow. IL7-mediated signals require integration via Gfi1 since transcriptional repression of inhibitory circuit factors and IL7Rα are critically dependent on Gfi1. Thus, the analysis of Gfi1−/− mice has shed light on a number of important questions regarding controversial issues in early lymphoid and B cell development. The identification of CLP2 cells in pre-TCR-α transgenic reporter mice [2] has led to a recent refinement in the understanding of early lymphoid development. Since the nature of CLP2 cells remains controversial, we have defined Lin−Sca1+IL7Ra+ckit−B220+ cells as “presumptive” CLP2 cells and attempted to elucidate transcriptional networks controlling the specification of CLP1 versus CLP2 cells. A detailed analysis of Ikaros−/− mice that show evidence of ongoing thymopoiesis without bone marrow B cell development revealed normal frequencies of ETPs and absence of classical bone marrow CLPs [3]. Even though this phenotype shares certain similarities to Gfi1−/− mice, it is not known whether Ikaros specifies CLP1 versus CLP2. In this respect, the observation that Gfi1 is differentially expressed in CLP1 versus presumptive CLP2 cells is interesting in light of the recently documented existence of independent B cell lineages [33], [34]. IL7 is a critical factor for survival, expansion, and differentiation of progenitor B cells in adult mice [5]–[10]. Both IL7−/− and IL7Rα−/− mice demonstrate lymphopenia associated with an incomplete block in B cell development [9], [10]. In IL7−/− mice, bone marrow production of B cells ceases after 8 weeks of age, while a pool of mature B cells is sustained that has been proposed to derive from IL7-independent fetal or perinatal precursor B cells [21]–[35]. Our analysis of Gfi1−/− B cell compartments revealed certain similarities between Gfi1−/− and IL7−/− progenitor B cells, suggesting that both IL7 and Gfi1 may act via a common pathway. However, Gfi1−/− mice and IL7−/− are not identical with respect to their B cell phenotype. For example, CLP1 cells are present in IL7−/− mice [7] and severely reduced in Gfi1−/− mice. We found that multiple IL7-mediated effects in progenitor B cells are severely reduced in the absence of Gfi1. Perhaps most significantly, IL7-induced upregulation of E2A, EBF, and Pax5, critical transcription factors controlling B cell differentiation, is severely perturbed. It should be noted however, that these effects may not be completely dependent on defective IL7-mediated signals but may result from as yet undefined other roles of Gfi1. In this respect, IL7R−/− mice show normal levels of E2A [7], suggesting that E2A is controlled by mechanisms other than IL7. Nevertheless, the net effect of decreased IL7Rα-dependent signals may at least partially account for a block in B cell development implying that Gfi1 is critically important in IL7-dependent B cell differentiation. Our findings are also relevant for an ongoing controversy bearing on the dichotomy of B1 versus B2 cells. Whereas conventional B2 cell differentiation is affected in Gfi1-deficient mice, our data suggest that B1 cell differentiation might be preserved (Rathinam and Klein, submitted). Thus, similar to PU.1 [36], [37], Gfi1 may be involved in the specification of distinct developmental fates in B cell differentiation. Recently, a B1 B cell specific progenitor has been identified in fetal and adult bone marrow [33], further supporting the concept that B1 and B2 cell differentiation represent distinct lineages. Gfi1 is a transcriptional repressor binding to cognate binding sequences in regulatory elements [38], [39]. The precise mechanism of action is under active investigation. Gfi1 and its homologue Gfi1B share a DNA binding and a SNAG (Snail and Gfi1 family of proteins) domain mediating transcriptional repression of multiple target genes, including Gfi1 itself [38], [40]. We have provided evidence for two independent and mutually non-exclusive mechanisms accounting for decreased B cell development in Gfi1−/− mice, i.e. i) regulation of negative feedback loops and ii) deficient downregulation of IL7Rα. Indirect effects of Gfi1 on cytokine receptor signalling have been shown previously in myeloid development, where impaired STAT3 signalling in Gfi1−/− precursor dendritic cells has been associated with increased transcription of PIAS3 and SOCS3 [19]. Here, we provide evidence that similar feedback mechanisms control early B cell development by integrating cytokine signals and negative circuit loops. Our data indicate that IL7-dependent STAT5 signalling is decreased in the absence of Gfi1. We cannot definitively exclude the concern that our data of decreased STAT5 phosphorylation and increased SOCS mRNA and protein levels may be confounded by the fact that the Gfi1+/+ and Gfi1−/− cell populations differ in various respects. However, SOCS3 gene transfer experiments into wildtype cells recapitulate the scenario of decreased IL7-mediated survival signals, and dysbalanced expression levels of negative feedback regulators has also been observed in Gfi1−/− myeloid cells [19]. Furthermore, the coordinated development of lymphoid cells is dependent on tightly regulated expression levels of IL7Rα. High levels of IL7Rα lead to a block in B cell development whereas low levels are permissive for B cell development [31]. In addition, at the proB to preB transition, diminishing IL7-mediated signals may permit selective expansion of B cells [28]. However, the mechanisms regulating IL7Rα expression remain largely unknown. Gfi1−/− progenitor B cells showed increased levels of IL7Rα expression in vivo and in vitro, suggesting that defects in quantitative calibration of IL7Rα signal transduction may at least partially contribute to perturbed B cell differentiation. In progenitor B cells, PU.1 has been shown to bind to IL7Rα promoter sequences, thus controlling early lymphoid development via IL7Rα expression [41]. In CD8 T cells, IL7Rα transcription is suppressed by IL7 in a Gfi1-dependent manner [32], suggesting that Gfi1 may also modulate IL7Rα expression. Furthermore, transgenic Gfi1B expression leads to defects in IL7Rα expression [15]. The murine IL7Rα a gene contains several putative Gfi1 consensus binding sites. We now could demonstrate directly by chromatin-immunoprecipitation experiments that Gfi1 binds to a cognate site in intron 2. Taken together, our findings suggest that Gfi1 directly represses IL7Rα expression in progenitor B cells. Since IL7Rα expression is a phenotypic hallmark of early lymphoid progenitor cells, this finding is of relevance with respect to the interpretation of the phenotypic characterisation of progenitor B cells in Gfi1-deficient mice. However, at the hematopoietic progenitor cell level, IL7Rα expression is not changed in Gfi1−/− mice (Fig. 6A and Fig. S3), suggesting that additional factors may contribute to differences in IL7Rα levels in lymphoid progenitor cells. In summary, we provide evidence that Gfi1 provides an important link between cytokine signalling and transcriptional regulation by modulating cytokine-receptor signalling. These insights open a new perspective for understanding lymphoid development in health and disease, B-cell mediated pathology, and potential therapeutic manipulations of cytokine-controlled transcription factor networks. Materials and Methods Mice All mice were bred and maintained under specific pathogen free conditions in the central animal facility at Hannover Medical School. Gfi1−/− mice [18] and Gfi1GFP/+ mice [42] were kindly provided by Tarik Möröy, Essen. Both mouse strains were backcrossed on C57/BL6 background for >8 generations. In Gfi1GFP/+ reporter mice, exons 1–7 of Gfi1 have been replaced by the GFP cDNA, their phenotype is similar to wildtype mice. In all experiments, age and sex matched mice were used at four to eight weeks of age. All experiments were approved by the institutional review board. Cells and cell culture In vitro cultures were performed using purified either LSK (Lin−Sca-1+c-kit+) or CD150+CD48− cells that were described recently (45). For sorting of CD150+CD48− cells, RBC depleted total BM cells were stained with 1 ug (per 1×106 cells) of biotin conjugated anti CD48 and PE conjugated anti CD150 antibodies for 15 minutes on ice. For secondary staining, cells were washed twice with PBS 2% FCS and stained with 0.25ug (per 1×106 cells) APC conjugated streptavidin antibodies, further incubated for 15 minutes and washed twice with PBS 2% FCS. Cells were resuspended in PBS 0.5% BSA and passed through nylon mesh prior to sorting. Cell sorting was performed with FACS Aria cell sorter and FACS Diva software. After every sorting, cells were reanalysed for checking its purity, and the purity was always >95%. and the following recombinant cytokines: 10 ng/ml rm-IL3, 10 ng/ml rm-IL6, 50 ng/ml rm-SCF, 50 ng/ml rh-Flt3L and 10 ng/ml rm-IL7 (all from Peprotech, Rocky Hill, NJ). To assess viability, cells were harvested after 48 hours, stained with 1 µg/mL of propidium iodide (Sigma Aldrich, Munich, Germany) and analysed by flow-cytometry. For in vitro proliferation experiments, LSK cells were sorted and labelled with 2 µM of CFDA-SE dye (Molecular probes, Karlsruhe, Germany) in 10 mL of PBS for 10 min at 37°C. Cells were washed and incubated in IMDM medium supplemented with 10% FCS, 2 mM L-Glutamine, 1% Penicillin-Streptomycin, 1 mM non-essential amino acids, 10 ng/ml rm-IL3, 10 ng/ml rm-IL6, 50 ng/ml rm-SCF, 50 ng/ml rh-Flt3L and 10 ng/ml rm-IL7 (all from Peprotech, Rocky Hill, NJ). After 72 hours of culture cells were harvested and analysed by flow-cytometry. For CFU assays, 1×105 either LSK or CD150+CD48− cells were mixed thoroughly with methocult (03234, CellSystems, St. Katharinen, Germany) and plated in the presence of the following cytokines: rm-IL3 (10 ng/ml), rm-IL6 (10 ng/ml), rm-SCF (50 ng/ml), rh-Flt3L (50 ng/ml) and rm-IL7 (10 ng/ml) (all from Peprotech, Rocky Hill, NJ). After 14 days of culture, colonies were counted using an inverted microscope (Axiovert-II Zeiss) and gridded scoring dishes. For in vivo B cell analysis, lymphoid organs were cut into small pieces, treated with collagenase D (Boehringer, Mannheim, Germany) for 30 min at 37°C, gently meshed and washed with PBS containing 50 µg/mL Dnase I (Roche, Mannheim, Germany) and 2 mM EDTA. In some experiments, BM B220+ cells were purified by labelling mononuclear cell suspensions with anti-CD45R microbeads (B220) and subsequent enrichment by immunomagnetic columns (Miltenyi Biotech, Bergisch Gladbach, Germany). For retroviral gene transfer, Sca-1+lin− cells were cultured in IMDM containing 10% FCS, 2 mM L-Glutamine, 1% Penicillin-Streptomycin, 1 mM Non-essential amino acids, 10 ng/ml rm-IL3, 10 ng/ml rm-IL6, 50 ng/ml rm-SCF, 50 ng/ml rh-Flt3L and 25 ng/ml h-TPO. BrdU assays Purified HSCs were cultured as mentioned above. 60 uM of BrdU (Sigma) was added to cells and cultured for an additional 12 hours. Cells were washed and permeabilized with FACS permeabilizing solution (BDIS, San Jose, CA). Cells were stained with anti BrdU antibodies in the presence of DNase at RT for 60 minutes. Cells were washed and analyzed by flow cytometry. Flow cytometry Single cell suspensions were analysed by flow cytometry using FACS SCAN or FACS Canto and CELLQuest software, FACS Diva software (BD Biosciences, San Jose, CA) or Flow Jo software (Tree Star, Inc., Ashland, OR), respectively. Cell sorting of defined subpopulations was performed using Moflo cell sorter (DAKO Cytomation, Glostrup, Denmark) or FACSAria cell sorter (BD Biosciences, San Jose, CA), respectively. The following monoclonal antibodies (all from BD Pharmingen, San Diego, CA except noted otherwise) were used: CD3e-FITC, -biotin &-PEcyc7, CD4-FITC, -PE, -APC, CD8 -PerCP & -PE, CD11b-FITC & -biotin, CD19-PE, CD24-FITC, CD25-FITC & -PerCP, CD34-FITC, CD43-biotin, CD44-FITC & -PE, CD48-biotin, CD117-PE & -APC, CD150-PE, B220-FITC, -PE, -biotin & -APCcyc7, BP-1-PE, IgM-FITC, -APC & -PEcyc7, Gr-1-FITC & -biotin, IL-7Rα-biotin, Sca-1-PE, Flt3-PE, BrdU- FITC, TER119-biotin, anti-mouse-STAT5, anti-mouse-STAT5-p (Cell signalling technology, Frankfurt, Germany), anti-mouse-SOCS3 (Zymed Laboratories, South San Francisco, CA), Gfi1 (N20; Santa Cruz Biotechnology), goat-anti-mouse-IgG-HRP, goat-anti-rabbit-IgG-HRP (Cell signalling technology, Frankfurt, Germany). In all experiments, cells were also stained with corresponding isotype-matched monoclonal antibodies. Cells reacted with biotinylated monoclonal antibodies were incubated with fluorochrome-conjugated streptavidin-PerCP or streptavidin-APC (BD Pharmingen). All fluorescence intensity plots are shown in log scales. Intracytoplasmic staining To detect STAT5 phosphorylation and SOCS3 expression by flow cytometry, LSK cells transduced GFP or SOCS3 GFP were stimulated with rmIL-7 for 10 min. Cells were first fixed, permeabilized using a commercially available Fix and Perm kit (Caltag Laboratories, Burlingame, CA) and stained with anti-SOCS3 and p-STAT5 antibodies respectively. Cells were washed and stained with anti-mouse-IgG-FITC secondary antibodies and detected by flow cytometry. Protein assays Extraction of proteins was carried out using standard protocols. Briefly, cells were harvested and lysed in hypotonic buffer containing 20 mM Hepes (pH 7.6), 10 mM KCl, 1 mM MgCl2, 2% glycerin, 0.1% Triton-X100, 0.5 mM DTT (Roche), 1 mM Pefabloc (Roche), 1 mM sodium-orthovandate (Sigma), 5 µg/mL protease inhibitor cocktail (Sigma). Nuclear and cytoplasmic protein fractions were separated by centrifugation (×1800g). The pellet containing nuclear proteins was re-suspended in hypertonic buffer containing 20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 20% Glycerin, 0.1% Triton-X, 0.5 mM DTT (Roche), 1 mM Pefabloc, 1 mM sodium-orthovandate, 5 µg/mL protease inhibitor cocktail and incubated for 15 min at 4°C. The lysate was subjected to centrifugation (×16,000g) and the supernatant containing the nuclear proteins was collected. Protein quantification was performed using Bradford reagent (Bio-rad, Munich, Germany). For Western blot analysis, 20 µg of protein was loaded on an 8% SDS gel, separated by electrophoresis and blotted onto nylon membranes. The membranes were exposed to anti-STAT-5, anti-STAT-5p and anti-SOCS3 respectively, followed by staining with secondary antibodies conjugated to horse radish peroxidase. The enzymatic reaction was visualized using ECL reagents (ECL kit, Amersham Biosciences, Freiburg, Germany). RNA isolation and Real Time PCR Total RNA was isolated using commercially available kit systems (“Absolutely RNA mini prep kit” - Stratagene, La Jolla, CA). cDNA was synthesised using oligo dT primer and expand reverse transcriptase (Roche). E2A, EBF and PAX5 expression was determined by Real Time PCR using the E2A specific forward primer 5′-TGACAGCTACAGCAGGGATG and reverse primer 5′-AGCGAGCCATTAACCTCAGA, EBF specific forward primer 5′ –CATGTCCTGGCAGTCTCTGA and reverse primer 5′-CAACTCACTCCAGACCAGCA, and Pax5 specific formard primer 5′- GAACTTGCCCATCAAGGTGT and reverse primer 5′-TGTCCGAATGATCCTGTTGA. SOCS3 expression was determined by Real Time PCR using a SOCS3 specific forward primer 5′-ATGGTCACCCACAGCAAGTT and reverse primer 5′-TGACGCTCAACGTGAAGAAG. Gfi1 mRNA expression in knock down studies was measured by RT-PCR using the specific forward primer 5′-CAGCTTACCGAGGCTCCCGACAGG and reverse primer 5′-CAAGACCGCTCCATGCATAGGGCTT. GAPDH specific primers were used as internal controls (forward primer 5′ GTCAGTGGTGGACCTGACC; reverse primer 5′-TGAGCTTGACAAAGTGGTCG). The PCR reaction was performed in duplicates using either a LightCycler–FastStart DNA Master SYBR Green I kit (Roche) or QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Retroviral gene transfer The murine Gfi1 cDNA and SOCS3 cDNA were cloned into the retroviral vector SFβ-91-IRES-EGFP, kindly provided by C. Baum, Hannover. Recombinant VSV-G pseudotyped retroviruses were generated using transient transfection into the packaging cell line 293GPG (43). For retroviral gene transfer, LSK progenitor cells were stimulated for 48 hours in the presence of a stem cell cytokine cocktail (see above) and transduced at a multiplicity of infection (MOI) of 10 in the presence of 8 µg/ml polybrene (Sigma). In brief, cells were exposed to recombinant retrovirus for 1 hour at 37°C, followed by spinoculation for 2 hours at ×700 g and further incubation at 37°C in 5% C02. Subsequently, cells were washed, cultured for additional 48 hours in the presence of the stem cell cytokine cocktail and used for in vitro experiments. The average transduction efficiency was 50–60%. For shRNA studies, Gfi1 specific shRNAs were designed through VectorNTI (Invitrogen) software. Two shRNAs recognising Gfi1 specific mRNA at different regions (shRNA1 and shRNA2) were cloned into pSM2 (open biosystems) lentiviral backbone. Lentiviruses were generated using 293T cell lines and viral titer was determined by counting the puromycin resistant colonies. For knocking down Gfi1 mRNA, prestimulated LSK cells were transduced as mentioned above and transduced cells were cultured in the presence of SCF+ IL7 for 14 days either in the presence or absence of puromycin (1 µg/mL). Chromatin-immunoprecipitation assay Chromatin immunoprecipitation assays were performed as described (44) with few modifications. Cells (5–10×107) were crosslinked for 10 min at room temperature by adding paraformaldehyde (1% final concentration). The crosslinking reaction was stopped by the addition of glycine to a final concentration of 0.125 M. Cells were washed in ice cold PBS and nuclei were isolated with cell lysis buffer (5 mM Pipes (pH 8.0), 85 mM Kcl, and 0.5% NP40). Nuclei were resuspended in nuclear lysis buffer (50 mM Tris (pH 8.0), 10 mM EDTA, and 1% SDS) and sonicated using a Branson 250 sonifier. Settings were optimized to yield a mean genomic DNA size of 0.2 to 0.5 kB. Chromatin was diluted 1∶3 in dilution buffer (0.01% SDS, 1% Triton-X100, 2 mM EDTA, 150 mM Nacl, and 20 mM Tris (pH 8.0), precleared with Staph A cells (BD) for 15 min at 4°C. Chromatin preparations obtained from 5×106 cells were incubated in the presence of 1 µg of control goat IgG or anti-Gfi1 (N20; Santa Cruz Biotechnology) for 3 hours and precipitated using Staph-A cells. Upon washing of the immunoprecipitates, DNA was purified by phenol/chloroform extraction and proteinase K digestion. Input and immunoprecipitated DNA samples were analysed by semiquantitative PCR (35 cycles) with primers amplifying either the IL-7Rα intron 2 (forward primer 5′ GCCCACTGTAACAAACTTCCC, reverse primer 5′ GCCTATAAAGTTTGCAAGTCC) or IL-7Rα exon 8 specific primers (forward primer 5′ CTGGACTGCCAATTCATGAGGTG, reverse primer 5′ TCTCTGTAGTCAGGGGACCTAGAG) used as control. PCR products were separated by 1.7% agarose gel and visualised after staining with ethidium bromide. Statistical analysis Data are presented as mean±SEM. Statistical significance was assessed using a 2-sided Student t test. P values >0.05 were considered to be non significant (NS) and P values <0.05 and >0.01 were represented as . P values <0.01 and >0.001 were represented as * and P values <0.001 were represented as ***. Supporting Information Figure S1 Transcriptional activity of Gfi1 locus in developing B cells. A) Gfi1 expression in defined Hardy fractions. Pooled bone marrow cells from 5 mice were analysed for GFP expression in B220+CD43+BP1− (Fraction A–B), B220+CD43+BP1+ (Fraction C–C'), B220+CD43−IgM− (Fraction D), B220+CD43−IgM+ (Fraction E) and B220highCD43−IgMhigh (Fraction F) cells. Shaded histograms represent GFP fluorescence in Gfi1+/GFP cells, open histograms represent autofluorescence in Gfi1+/+ cells. The GMFI. of Gfi1+/+ (top) and Gfi1+/GFP (bottom) cells is indicated. Data are representative of 3 independent experiments. B) Gfi-1 expression during in-vitro B cell development. LSK bone marrow cells from Gfi1+/GFP mice were cultured in the presence of SCF and IL7. Cells were harvested at indicated time points and their GFP fluorescence was determined by flowcytometric analysis. Shown is the specific geometric mean fluorescence intensity index calculated as follows: GMFIi = GMFI(Gfi1 +/GFP)−GMFI(Gfi1 +/+). Results represent the average values of duplicate samples. Data are representative of 2 independent experiments. (0.18 MB PDF) Click here for additional data file. Figure S2 shRNA-mediated knockdown of Gfi1 in hematopoietic progenitor cells. HSC from Gfi1+/+ and Gfi1−/− mice were sorted, cytokine-stimulated and transduced with lentiviral vectors (control backbone, sh-RNA1, or sh-RNA2, respectively). After 3 days in culture, cells were harvested and subjected to RT-PCR using Gfi1-specific and GAPDH-specific primer pairs. (0.09 MB PDF) Click here for additional data file. Figure S3 IL7Rα expression levels in primary and in in vitro cultured hematopoietic progenitor cells. A & B. Hematopoietic progenitor cells from Gfi1+/+ and Gfi1−/− mice were sorted and cultured in the presence of mIL3, mSCF, mIL6, mFlt3L and hTPO for 48 hours. Cells were stained with anti-IL7Rα antibodies and analyzed by flow cytometry. Note that IL7Rα expression was undetectable in non-stimulated HSCs (0.27 MB PDF) Click here for additional data file. Figure S4 Proliferation assay of hematopoietic progenitor cells in response to cytokines (CFSE-assay). LSK cells from Gfi1+/+ and Gfi1−/− mice were CSFE-labelled and incubated in the presence of cytokines for 72 hours. Histograms representing CFSE fluorescence and division history of Gfi1+/+ (top panel) and Gfi1−/− (bottom panel) cells are shown. Data are representative of 2 independent experiments. Note that Gfi1−/− cells cultured in the presence of a) IL-3+ SCF+ Flt3L+ IL-6+ TPO, b) SCF+ IL7 and c) SCF show similar proliferative responses. (0.25 MB PDF) Click here for additional data file. Figure S5 Proliferation assay of HSC in response to cytokines (BrdU-assay). Sorted CD150+CD48− cells from Gfi1+/+ and Gfi1−/− mice were cultured in the presence of either SCF + IL7 or SCF and IL7 only for 60 hours. BrdU was added and the cells were cultured for additional 12 hours. Proliferation of cells was assessed by FACS upon anti-BrdU staining. (0.23 MB PDF) Click here for additional data file. Figure S6 CFU assay determining the proliferative capacity of CD48−CD150+ HSC. CFU assay indicating decreased colony-forming-activity in Gfi1−/− HSC. CD48−CD150+ cells were cultured on semisolid medium in the presence of indicated cytokines. 14 days later, the absolute numbers of colony forming units of Gfi1+/+ (white bars) and Gfi1−/− (black bars) cells were enumerated and plotted. (0.09 MB PDF) Click here for additional data file. Figure S7 Gating strategy and analysis of B cell specific transcription factor expression in Gfi1+/+ and Gfi1−/− B cells. A) Total bone marrow cells of Gfi1+/+ and Gfi1−/−mice were stained with B220-specific antibodies and further analysed for expression of CD19 and IgM. Three fractions discriminated and designated as G1 (B220+CD19−IgM−), G2 (B220+CD19+IgM−) and G3 (B220+CD19+IgM+). Cells were sorted yielding purities of >95%. The plots were generated to indicate purity of cell populations for preparative purposes and do not reflect absolute numbers of B lineage cells in mice. The percentages of gated cell populations are as follows: In Gfi1+/+ G1 = 16%, G2 = 50%, G3 = 28%. In Gfi1−/− G1 = 53%, G2 = 20% andG3 = 21%. B) Expression of B cell specific transcription factors in G1 (B220+CD19−IgM−), G2 (B220+CD19+IgM−) and G3 (B220+CD19+IgM+) B cell fractions. mRNA expression levels of E2A (top panel) EBF (middle panel) Pax5 (bottom panel) were determined by RT-PCR. Shown are the mean values of duplicate samples. Data are representative of 2 independent experiments. (0.28 MB PDF) Click here for additional data file. Figure S8 IL7Rα expression in bone marrow B lineage cells. A) Nucleated bone marrow cells of Gfi1+/+ and Gfi1−/− mice were stained with anti-B220, anti-CD19, anti-B220, and anti-IL7Rα specific antibodies and analysed by flowcytometry. B220+ cells were gated (G1; top panel) and further analysed for expression of CD19 and IgM, yielding three distinct fractions: G1 (B220+CD19−IgM−), G2 (B220+CD19+IgM−) and G3 (B220+CD19+IgM+). B) FACS plots indicating IL7Rα surface expression in B cells. Cells of G2 (top panel), G3 (middle panel) and G4 (bottom panel) gates of Gfi1+/+ and Gfi1−/− mice were analysed for IL7Rα expression. The GMFI measuring IL7Rα expression is indicated in each plot. (0.36 MB PDF) Click here for additional data file. Table S1 Average frequency of Hardy Fraction's in the BM of Gfi1+/+ and Gfi1−/− mice. (0.02 MB XLS) Click here for additional data file. We are grateful to Tarik Möröy for providing Gfi1−/− and Gfi1+/GFP mice. We thank Matthias Ballmaier and Christina Reimer for expert help with cell sorting, Inga Sandrock for technical assistance, and Karl Welte for continuous support. Competing Interests: The authors have declared that no competing interests exist. Funding: The authors have no support or funding to report. ==== Refs References 1 Weissman IL Anderson DJ Gage F 2001 Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. 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==== Front PLoS Comput BiolPLoS Comput. BiolpcbiplcbploscompPLoS Computational Biology1553-734X1553-7358Public Library of Science San Francisco, USA 1736720410.1371/journal.pcbi.003005106-PLCB-RA-0463R3plcb-03-03-10Research ArticleComputational BiologyEvolutionary BiologyNoneMeasures of Clade Confidence Do Not Correlate with Accuracy of Phylogenetic Trees Clade Support Unrelated to AccuracyHall Barry G 1Salipante Stephen J 21 Bellingham Research Institute, Bellingham, Washington, United States of America 2 Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America Li Wen-Hsiung EditorUniversity of Chicago, United States of America To whom correspondence should be addressed. E-mail: [email protected] 2007 16 3 2007 3 3 e516 11 2006 31 1 2007 © 2007 Hall and Salipante.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Metrics of phylogenetic tree reliability, such as parametric bootstrap percentages or Bayesian posterior probabilities, represent internal measures of the topological reproducibility of a phylogenetic tree, while the recently introduced aLRT (approximate likelihood ratio test) assesses the likelihood that a branch exists on a maximum-likelihood tree. Although those values are often equated with phylogenetic tree accuracy, they do not necessarily estimate how well a reconstructed phylogeny represents cladistic relationships that actually exist in nature. The authors have therefore attempted to quantify how well bootstrap percentages, posterior probabilities, and aLRT measures reflect the probability that a deduced phylogenetic clade is present in a known phylogeny. The authors simulated the evolution of bacterial genes of varying lengths under biologically realistic conditions, and reconstructed those known phylogenies using both maximum likelihood and Bayesian methods. Then, they measured how frequently clades in the reconstructed trees exhibiting particular bootstrap percentages, aLRT values, or posterior probabilities were found in the true trees. The authors have observed that none of these values correlate with the probability that a given clade is present in the known phylogeny. The major conclusion is that none of the measures provide any information about the likelihood that an individual clade actually exists. It is also found that the mean of all clade support values on a tree closely reflects the average proportion of all clades that have been assigned correctly, and is thus a good representation of the overall accuracy of a phylogenetic tree. Author Summary The construction of phylogenetic trees, which depict past relationships between groups of DNA or protein sequences, has valuable application in many fields of study, most commonly evolutionary and population biology. Before drawing conclusions from phylogenetic trees, it is important to assess how accurate those reconstructions are. This is typically accomplished by examining measures of “clade credibility” (such as bootstrap or posterior probability values), which represent how reproducible relationships are within the tree based on the parameters of the phylogenetic analysis. However, such measures do not necessarily reflect how likely inferred relationships are to have actually occurred in nature. Therefore, using simulated data where relationships are known, we have determined how well several measures of clade credibility correlate with the likelihood that a deduced phylogenetic grouping actually exists in reality. Surprisingly, we found no such correlation, and that the inferred relationships were correctly assigned about as often in cases where clade credibility values were very low as where they were high. This finding suggests that current measures of phylogenetic tree reliability are not useful in predicting whether specific inferred relationships have actually occurred. citationHall BG, Salipante SJ (2007) Measures of clade confidence do not correlate with accuracy of phylogenetic trees. PLoS Comput Biol 3(3): e51. doi:10.1371/journal.pcbi.0030051 ==== Body Introduction Phylogenetic analysis, once the province of systematists and evolutionary biologists, has become a fundamental tool of computational biology and biological disciplines as diverse as biochemistry, epidemiology, and developmental biology. While systematists use phylogenetic analysis of molecular sequences to elucidate the historical relationships among species, other disciplines tend to focus more on the historical relationships of the sequences themselves. The results of phylogenetic analyses are typically presented as phylogenetic trees, diagrams that graphically illustrate those historical relationships. Phylogenetic trees are just estimates of those historical relationships, and it is therefore important to have some way to evaluate the quality and reliability of phylogenetic reconstructions. The most widely used method of estimating the reliability of trees is the nonparametric bootstrap [1]. The bootstrap method addresses the reliability of the tree topology (the branching order) by calculating the bootstrap percentage (BP) for each interior node, or clade, in a tree. In the bootstrap method, the sites in a set of aligned sequences are randomly sampled with replacement to create a pseudo-alignment, and that pseudo-alignment is used to produce an estimated “bootstrap tree.” Typically, 100–2,000 bootstrap trees are estimated, and the BP for a clade on the original phylogenetic tree is the percentage of the bootstrap trees that also include that clade. Thus, confidence in the groupings of taxa can be estimated. A drawback to the bootstrap method is that it can potentially be very time-consuming. For example, maximum likelihood is at present the most widely used statistical phylogenetic method, but because it is computationally intensive, performing a bootstrap analysis on maximum likelihood trees can require prohibitive amounts of time. Recently, a new approach to estimating branch (or clade) support, the approximate likelihood ratio test (aLRT), has been introduced [2]. The aLRT is a fast and accurate method for assessing branch support for maximum likelihood trees. Under conventional LRT, the null hypothesis is that the branch has a length of zero (i.e., it does not exist), and the test statistic is 2(l1 − l0), where l1 is the likelihood of the most likely tree and l0 is the likelihood of the tree in which the branch does not exist. In aLRT, the test statistic is approximated by 2(l1 − l2), where l2 is the likelihood of the second most likely tree, an approximation that enormously decreases computational time and results in a practical and slightly conservative test statistic. The significance of the aLRT test statistic is calculated from a mixed χ2 distribution, with half drawn from zero and half drawn from one degree of freedom. The aLRT approach is implemented in the beta version of PHYML 2.4.5 [3] (http://atgc.lirmm.fr/alrt). The most recent release of the beta version of PHYML also implements an alternative nonparametric Shimodaira–Hasegawa-like (SH-like) procedure that is typically more conservative than the χ2 approach, so PHYML now offers the option of assigning support as the smaller of the values calculated by the two methods. In the last decade, a new method of estimating phylogenetic trees, the Bayesian method, has gained increasing popularity [4–7]. The Bayesian method, as implemented by the program MrBayes [8,9], estimates the posterior probabilities (PPs) of clades by calculating, among the trees with the highest posterior probabilities, the fraction of the time that each clade appears as those trees are visited in proportion to their probabilities. The Bayesian method has the advantage that it calculates PPs during the process of estimating the consensus tree. It is therefore much faster to obtain PP estimates of clade reliability by the Bayesian method than to obtain BPs of clade reliability by maximum likelihood. BP, aLRT, and PP are measures of clade support, but they are often presented as measures of the accuracy of the tree [5,10]. None, however, is a metric of accuracy. aLRT assesses the likelihood that a branch exists on a maximum likelihood tree. BP and PP are simply measures of repeatability; BP measures the repeatability with which a clade occurs among subsamples of the data used to create the original tree, and PP measures the repeatability with which a clade occurs among the set of nearly equally likely trees after the Bayesian process has converged on a set of trees with nearly identical likelihoods. Because of discrepancies between Bayesian posterior probabilities and bootstrapped maximum likelihood percentages, there has been considerable controversy about BP versus PP as measures of clade reliability [11–15]. For real, empirical data, we cannot know the accuracy of a tree because we have no way of knowing the true branching order of the taxa or sequences that are being considered. Simulated datasets, in which the true tree is known, have been used to compare BP and PP with the accuracies of estimated trees. Several such studies have shown that BP underestimates clade reliability (i.e., clades in the estimated tree are more likely to exist in the true tree than is indicated by BP) [10,11,13–16]. There have been conflicting reports about the relationship between PP and accuracy. In general, PP has been found to be less conservative than BP. Some studies [11,12] have concluded that PP is too liberal (i.e., overestimates accuracy), while others [13,14] conclude that PP better reflects accuracy. Another study concluded that BP and PP can be taken as potential upper and lower estimates of accuracy, but that they are not interchangeable and cannot be directly compared [15]. Similarly, Anisimova and Gascuel reported that aLRT using the χ2 approach is similar to posterior probabilities, and their unpublished data suggest that the SH-like approach is more conservative than the χ2 approach (http://atgc.lirmm.fr/alrt). The conclusions of the above studies are only as reliable as the extent to which the simulations mimic real evolutionary processes that generate the empirical data to which we actually apply phylogenetic methods. In all cases, the simulations incorporate specific evolutionary models, the most common being the K2P model [17], to guide the simulation process. The results will be no more realistic than the assumptions and biases of that model. Modeling evolution as a process of substitution confounds two distinct processes, mutation and selection, the outcome of which is the real substitution process [18]. The number of taxa used in the simulations reported in [10–16] ranges from four to 28. Many, and probably most, phylogenetic studies involve many more taxa. Typically, branch lengths are uniform, although some studies included a specific pattern of length variation. Importantly, the simulations only consider base substitutions, not insertions or deletions (indels). The resulting sequences thus need not be aligned. In reality, historical indels necessitate using multiple alignment programs to estimate the homologous characters within. The alignment process strongly affects the reliability of the resulting trees. For coding sequences, the accuracy of a tree is significantly increased by aligning the corresponding protein sequences and using that alignment to place the corresponding gaps into the DNA coding sequences [18]. However, when the average percentage identity of the amino acids is within the “twilight zone” of 20%–30%, only 80% of residues are correctly aligned [19], and when identity is below 10%, less than 50% of residues are correctly aligned [20]. The failure to include indels in the simulation process therefore reduces considerably the confidence we can place in applying conclusions drawn from those simulations to real data. The EvolveAGene simulation program [18,21] was designed to mimic the evolution of sequences in a more biologically realistic fashion. A real sequence is used for the root node of the tree, and a strictly bifurcating bilaterally symmetrical tree is evolved. Branch lengths are randomly varied from zero to a value chosen by the user. Mutation and selection are treated as separate processes. The mutation process is simulated by introducing random mutations, including base substitutions, insertions, and deletions, into the sequences according to the spontaneous mutation spectrum of Escherichia coli. (The mutational spectrum is the experimentally determined relative frequencies with which the various base substitutions and indels of different lengths occur, before selection or drift act on those mutations.) The selection process is simulated by (1) assuming that all frameshift and nonsense mutations are strongly deleterious and thus not accepting those mutations, and (2) accepting nonsynonymous base substitutions with a probability that corresponds to a user-specified nonsynonymous substitution per nonsynonymous site to synonymous substitution per synonymous site (dN/dS) ratio, which can be set to biologically realistic values. The EvolveAGene program has been used to compare accuracies of various phylogenetic methods [18] and to explore the accuracies with which parsimony and Bayesian methods can reconstruct ancestral protein sequences [21]. In this study we are not particularly interested in comparing PP, BP, and aLRT per se. Instead, we are interested in asking two questions: (1) for all measures of clade credibility, how well does the credibility of a clade reflect the probability that that clade really exists on the true tree; and (2) how well does the average clade support reflect the topological accuracy of the tree? Topological accuracy is defined as the fraction of clades on the estimated tree that actually exist on the true tree. In this study, both the true tree and the estimated trees are strictly bifurcating, so the number of interior clades is the same. Thus, the number of false positive errors (clades found on the estimated tree that do not exist on the true tree) is identical to the number of false negative errors (clades on the true tree that are not on the estimated tree). We simulate the evolution of several genes under biologically realistic conditions and find that none of the estimates of clade support correlates with topological accuracy; in other words, clade supports tell us nothing about the likelihood that an inferred clade actually exists. However, we find that the average clade support does correlate well with the topological accuracy of the tree. Results/Discussion Ten simulations were initiated from each of five E. coli K12 coding sequences to assess how well BPs from maximum likelihood trees, aLRT support by the χ2 approach, aLRT support by the minimum of SH-like and χ2 approaches, and posterior probabilities from Bayesian trees corresponded to clade accuracies. The simulation conditions were chosen to generate datasets that were at the practical limits for reliable alignments. Indeed, the typical average Jukes–Cantor [22] distances among the sequences for those datasets was 1.39 ± .02 substitutions per site, well above the limit of 1.0, above which Nei and Kumar [23] state that neighbor-joining trees are unreliable. We define “true clades” as clades in the estimated tree that exist in the true tree, and we define “accuracy” as the percent of the total clades in the estimated tree that are true clades. Tables 1–4 show, respectively, the results for Bayesian trees and for maximum likelihood trees by the bootstrap and the two aLRT approaches. In each case, rows are ranges of clade credibility values. Table 1 Bayesian Trees Table 2 Maximum Likelihood Tree Bootstrap Clade Support Table 3 Maximum Likelihood Tree aLRT Clade Support by χ2 Test of Significance Table 4 Maximum Likelihood Trees aLRT Clade Support by Minimum of SH-Like and χ2 Test of Significance For all methods, accuracy increases as the lengths of the sequences increase. Mean BPs are conservative estimates of mean topological accuracy, and in keeping with [13] and [14] we find that average posterior probabilities are a better estimate of topological accuracy than are BPs. BPs underestimate accuracy, particularly for trees based on the shorter sequences, more than do posterior probabilities or aLRT supports. We do not interpret this finding to mean that BPs should be the “gold standard” measure of reliability; indeed, we find the notion that the less-accurate estimate should be the gold standard to be slightly ludicrous. Our results, however, differ strikingly from those of [13] and [14] with respect to the correspondence between individual clade confidences and the accuracy of those clades. Alfaro et al. [13] found that for most topologies accuracy was higher than either BP or PP when clade confidences were greater than ∼40%, but lower than clade confidences when those measures were less than ∼40%. Hillis and Bull [10] obtained similar results for BP, and Wilcox et al. [14] obtained similar results for PP, but a low crossover point at about 20% for BP. In contrast, we find no significant correlation between individual clade supports and the probability that a clade is correct, whichever method of clade support is used. We regressed the fraction of clades that actually exist within each decile against the midpoint for each decile of clade support; thus, a slope approaching one would be expected for a perfect correlation between those values, whereas a slope of zero would indicate no correlation. Only two out of the 28 plots have slopes that are significantly different from zero (genes nuoK and rplF for bootstrap support of maximum likelihood trees, with p = 0.03 and 0.02, respectively). One of these slopes is slightly positive, and the other is slightly negative; thus, both likely represent outliers. The absence of correlation between clade support and the likelihood that a clade exists means that, whatever the method, clade support values provide no information about, and have no predictive power as to, the likelihood that the clade exists. We attribute the differences between our results and those of others [10,13,14] to our use of a more biologically realistic simulation. It is conceivable that this startling finding is the result of reconstructing relatively large (64 taxon) trees under some false assumption that is unknowingly incorporated into the simulation: perhaps with so many taxa, resulting in an enormous number of possible trees, any clade that has even mild support is likely to be a true clade. We think this possibility is unlikely, because the fraction of false clades that have 81%–100% support is roughly the same as the fraction of false clades that have low (0%–40%) support. Nevertheless, to test the idea that our findings are an artifact of considering large trees, we tested all four methods with 16-taxon datasets of nuoK, replicated ten times each. (The nuoK gene was chosen because it is the shortest gene and the gene for which all methods were the least accurate.) As before, the taxa were a random sample from a 128-taxon dataset. When the number of taxa was reduced from 64 to 16, the quality of the alignments was reduced so that the amino acid identity was <20%, below the zone in which alignments are reliable. The average branch length was therefore reduced from 0.18 to 0.15 substitutions per site to produce alignments that exhibited an average of 24% amino acid identity, well within the “twilight zone” [19]. Table 5 shows that the results are essentially the same as in Tables 1–4: there is no significant correlation between clade support and the fraction of true clades. Both mean accuracy and mean clade support are generally lower than in Tables 1–4, but it remains the case that clade support provides no information about the likelihood that a clade actually exists. Table 5 nuoK (300 bp) Trees Based on 16 Random Sequences Sampled from 128 Evolved Sequences Random sampling of taxa typically results in well-balanced trees (Figure 1), and it is conceivable that our findings apply only to trees with similar topology. To test that possibility, we nonrandomly sampled ten nuoK datasets to generate highly pectinate, unbalanced 16-taxon trees (Figure 2). For the unbalanced trees, the topological accuracies were higher than for the 16-taxon balanced trees, and all methods of clade support again underestimated that accuracy. Again, there was no significant correlation between clade support and the likelihood that a clade existed on the true tree (Table 6). Figure 1 Typical Phylogenetic Trees (A) True 64-taxon tree initiated with nuoK sequences. The arrow indicates a near trichotomy. (B) Bayesian tree estimated from the same data as in (A). Numbers are posterior probabilities of clades whose posterior probabilities are <90%. Arrows indicate the clades that do not exist in the true tree. Figure 2 Topology of a Typical Unbalanced Tree of 16 Taxa Table 6 nuoK (300 bp) Unbalanced Trees Based on 16 Sequences Sampled from 128 Evolved Sequences We conclude that our results are general, and not simply attributable to large trees or to balanced trees. This is one of those good news–bad news stories beloved by comedians. The bad news is that none of the methods of assessing clade support provides any reliable estimation that the clade has been correctly assigned. The good news is that, even with data that are near the practical limits for phylogenetic tree reconstruction, both maximum likelihood and the Bayesian method estimate topologies so well that even when clade support is very low there is a better than 80% chance that the clade is correctly assigned. In addition, for each method, averaged over the 70 datasets, there is a significant correlation between the average branch support and the accuracy of the tree (Table 7), and for all methods except nonparametric bootstrap, the average clade support value is a good, if slightly conservative, estimator of the overall fraction of clades that actually exist. It might be argued that having a good estimate of overall accuracy is not very useful, and that we are generally interested in identifying unreliable branches. Our results show that we simply cannot identify what particular branches are unreliable based on measures of clade support. Thus, with current methods of determining clade credibility we cannot have what we might generally want, and it is important to acknowledge the limitations of those metrics. On the other hand, methods of determining clade confidence do provide a good estimation of the overall reliability of a phylogenetic tree, and permit us to infer how many untrustworthy branches may be present. Just as we can make good predictions about the diffusion of a mass of molecules over time, but not about the motions of individual molecules in that mass, we can make good estimations of the overall topological accuracy of a tree, but not about the accuracy of individual branches. Table 7 Mean Clade Support versus Mean Accuracy Materials and Methods Simulations. Simulations were performed by EvolveAGene [18,21]. Five coding sequences from E. coli K12 were selected from the E. coli genome entirely on the basis of length, and used to initiate the simulations: nuoK, 300 bp, encodes the NADH dehydrogenase subunit K; rplF, 530 bp, encodes 50S ribosomal protein L6; tauB, 763 bp, encodes a taurine transport ATP-binding protein; add, 999 bp, encodes adenosine deaminase; and araB, 1,698 bp, encodes ribulokinase. The genes are not functionally related to each other, and none exhibits detectable homology to another by pairwise BLAST comparisons. For the simulations in Tables 1–4, the average branch length was 0.18 substitutions per site, with lengths ranging from 0 to 0.36 substitutions per site; for Tables 5 and 6 the average branch length was 0.15 substitutions per site, ranging from 0 to 0.30 substitutions per site. The tree was evolved for seven “generations” to give 128 terminal taxa; thus, the average length from the root to the tip was 1.26 substitutions per site. The probability of accepting an indel was 0.02, and the probability of accepting a nonsynonymous base substitution was 0.2. Ten independent simulations were carried out from each of the five root sequences. When all of the terminal sequences descended from the root node were included in the dataset, both Bayesian and maximum likelihood trees included very few nodes with clade confidences <80% (unpublished data). When trees were based on a random subsample of the sequences, both methods produced trees with more low-confidence clades. Datasets used to estimate trees were therefore based on a random sample of 16 or 64 of the 128 evolved sequences. Alignments. Sequences were aligned by ClustalW [24] as implemented by MEGA 3.1 [25]. Sequences were translated to their corresponding protein sequences by MEGA 3.1, aligned with a gap-opening penalty of 3.0 and a gap-extension penalty of 1.8. The average pairwise amino acid identities in the resulting alignment were typically 21%–22%, near the lower boundary of the “twilight zone” below which alignments are not sufficiently reliable to produce valid phylogenetic trees [19,20]. Triplet gaps corresponding to the gaps in the protein alignment were introduced back into the DNA sequences by MEGA 3.1. The resulting DNA sequence alignments were saved in the FASTA format and converted to the PHYLIP format (for input to PHYML) and to the Nexus format (for input to MrBayes) by a Perl script. Estimation of phylogenetic trees. Trees were estimated by two methods: maximum likelihood as implemented by PHYML 2.4.4 [3], and the Bayesian method as implemented by MrBayes 3.1.2 [9]. In both cases, trees were estimated using the GTR + invariants + gamma model. For maximum likelihood trees, clade confidences were estimated from 100 bootstrap replicates. Bayesian trees were estimated from 600,000 generations, sampling every 100 generations, with a heating parameter of 0.15, in two parallel runs. The consensus trees were calculated using the allcompat option (strict consensus) from the final 4,501 trees of each run. Convergence, as judged by the diagnostic average standard deviation of the split frequencies between two parallel runs falling below 0.02, typically occurred before generation 120,000 (1,200 trees). A typical true tree, in this case initiated with the nuoK sequence, is shown in Figure 1A. Note that the true tree includes one near trichotomy, and that the distance from the root to the tips ranges from 0.52 substitutions per site for taxon ZZZZZZZ to 1.55 substitutions per site for taxon PPPPPPPPPP. The corresponding Bayesian estimated tree is shown in Figure 2. Posterior probabilities <90% are indicated. This Bayesian tree is typical in that only 12 of the 61 interior nodes have posterior probabilities <90%, but only four of those low-PP clades (indicated by arrows) are not present in the true tree. Calculation of topological accuracy. A Perl script, InferAcc, was used to compare the estimated trees with the true trees. Clades were sorted into bins as indicated in Tables 1–6, and the clade was scored as existing if it was present in the true tree. Mean accuracy is the fraction of clades in the estimated tree that exist in the true tree averaged over the ten trees in the set. We are grateful to reviewer number two for very helpful comments. Competing interests. The authors have declared that no competing interests exist. Author contributions. BGH and SJS conceived and designed the experiments and analyzed the data. BGH performed the experiments and contributed reagents/materials/analysis tools. BGH and SJS wrote the paper. Funding. SJS is supported by the Poncin Scholarship Fund. Abbreviations aLRTapproximate likelihood ratio test BPbootstrap percentage indelsinsertions or deletions PPposterior probability SH-likeShimodaira–Hasegawa-like ==== Refs References Felsenstein J 1985 Confidence limits on phylogenies: An approach using the bootstrap Evolution 39 783 791 Anisimova M Gascuel O 2006 Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative Syst Biol 55 539 552 16785212 Guindon S Gascuel O 2003 A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood Syst Biol 52 696 704 14530136 Hall BG 2004 Phylogenetic trees made easy: A how-to manual Sunderland (Massachusetts) Sinauer Associates 221 Huelsenbeck JP Larget B Miller RE Ronquist F 2002 Potential applications and pitfalls of Bayesian inference of phylogeny Syst Biol 51 673 688 12396583 Mau B Newton M Larget B 1999 Bayesian phylogenetic inference via Markov chain Monte Carlo methods Biometrics 55 1 12 11318142 Rannala B Yang ZH 1996 Probability distribution of molecular evolutionary trees: A new method of phylogenetic inference J Mol Evol 43 304 311 8703097 Huelsenbeck JP Ronquist F 2001 MrBayes: Bayesian inference of phylogeny Bioinformatics 17 754 755 11524383 Ronquist F Huelsenbeck JP 2003 MrBayes 3: Bayesian phylogenetic inference under mixed models Bioinformatics 19 1572 1574 12912839 Hillis DM Bull JJ 1993 An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis Syst Biol 42 182 192 Suzuki Y Glazko GV Nei M 2002 Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics Proc Natl Acad Sci U S A 99 16138 16143 12451182 Taylor DJ Piel WH 2004 An assessment of accuracy, error, and conflict with support values from genome-scale phylogenetic data Mol Biol Evol 21 1534 1537 15140947 Alfaro ME Zoller S Lutzoni F 2003 Bayes or bootstrap? 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1747633607-PONE-RA-00680R110.1371/journal.pone.0000414Research ArticleCell Biology/Cell Growth and DivisionCell Biology/Developmental Molecular MechanismsCell Biology/Membranes and SortingMolecular Biology/Transcription Initiation and ActivationNephrology/Hereditary, Genetic, and Development NephrologyThe Role for HNF-1β-Targeted Collectrin in Maintenance of Primary Cilia and Cell Polarity in Collecting Duct Cells HNF-1β-Targeted CollectrinZhang Yanling 1 2 Wada Jun 1 * Yasuhara Akihiro 1 Iseda Izumi 1 Eguchi Jun 1 Fukui Kenji 3 Yang Qin 3 4 Yamagata Kazuya 3 Hiesberger Thomas 5 Igarashi Peter 5 Zhang Hong 6 Wang Haiyan 6 Akagi Shigeru 7 Kanwar Yashpal S. 7 Makino Hirofumi 1 1 Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Okayama, Japan 2 Department of Nephrology, Third Hospital Hebei Medical University, Shijiazhuang, China 3 Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan 4 Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America 5 Department of Internal Medicine and Division of Basic Science, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America 6 Renal Division, Institute of Nephrology, Peking University First Hospital, Peking University, Beijing, China 7 Northwestern University, The Feinberg School of Medicine, Department of Pathology, Chicago, Illinois, United States of America Jin Dong-Yan Academic EditorUniversity of Hong Kong, China* To whom correspondence should be addressed. E-mail: [email protected] and designed the experiments: YK JW KY YZ JE. Performed the experiments: PI JW HM KY YZ AY II JE KF QY TH HZ SA. Analyzed the data: PI JW HM YZ JE TH SA. Contributed reagents/materials/analysis tools: JW KY KF QY TH HZ HW SA. Wrote the paper: YK JW HM YZ. 2007 2 5 2007 2 5 e41426 1 2007 10 4 2007 Zhang et al.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1α, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1β and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to γ-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1β and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1β regulated genes. ==== Body Introduction In a previous study, we identified a member of angiotensin converting enzyme (ACE) gene family, collectrin, by its up-regulation in a mouse model of partial nephrectomy [1], which is a long-standing model for the progressive renal diseases. Collectrin is a type I transmembrane protein and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. It is a homologue of ACE2 and identified in immediate proximity of the ace2 locus. ACE2 may be a chimeric protein emerging from the duplication of two genes, having homology with ACE at the catalytic domain and homology with collectrin in the membrane proximal domain[2]. Unlike ACE and ACE2, collectrin lacks N-terminal active dipeptidyl carboxypeptidase catalytic domain, and thus its biology has not been well-established. Recently, two independent studies of targeted disruption of collectrin in mice resulted in a severe and general defects in renal amino acid uptake[3], [4]. Collectrin is demonstrated at the luminal side of brush border membranes of proximal tubules and the deficiency of collectrin is associated with reduction of multiple amino acid transporters, such as B0AT1, rBAT, B0,+AT[4], XT3s1/ST1, XT2, XT3 and EAAC1[3], on luminal membranes. We and others have described the expression of collectrin in pancreatic β cells and collectrin is identified as a target of hepatocyte nuclear factor-α (HNF-1α)[5], [6]. Targeted disruption of HNF-1α resulted in diabetes and a renal phenotype with Fanconi syndrome characterized by glucosuria, phosphaturia, calciuria and aminoaciduria[7]. Localization of HNF-1α in proximal tubules and similar renal phenotype in collectrin and HNF-1α knockout mice suggested that the expression of collectrin in proximal tubules is transcriptionally regulated by HNF-1α. In contrast to HNF-1α, the renal-specific inactivation of HNF-1β develops polycystic kidney disease, and renal cyst formation is accompanied with a drastic defect in the transcriptional activation of several polycystic kidney disease (PKD)-related genes, such as Umod, Pkhd1, Pkd2 and Tg737/Polaris [8]. Similarly, mutations in HNF-1β gene are seen in the autosomal dominant disorder MODY5 (maturity-onset diabetes mellitus of the young, type 5)[9] and MODY5 patients present type 2 diabetes and develop congenital kidney abnormalities including simple cysts, polycystic kidneys, cystic dysplasia and glomerulocystic kidney disease. The expression of collectrin and HNF-1β in collecting duct cells suggested that collectrin is also regulated by HNF-1β and play roles in renal cyst formation or sodium and water handling. Collectrin knockout mice are lacking in the phenotype of diabetes, hypertension and renal cystic formation[3], [4]; however, it can be speculated that collectrin play a role in the pathophysiology of pancreatic β cells and collecting duct cells because many other genes, such as ACE2, may compensate the action of collectrin in the gene disruption studies. For instance, overexpression of collectrin in INS1-E cells and insulin promoter driven collectrin transgenic mice enhanced glucose-induced insulin exocytosis. Collectrin binds to SNARE (soluble N-ethylmaleiamide-sensitive factor attachment protein receptor) complex by interacting with snapin, a SNAP-25 (synaptosomal-associated protein of 25 kDa) binding protein, and facilitates the SNARE complex formation[10]. Thus, collectrin facilitates the insulin excytosis by regulating the SNARE complex formation. Based on line of evidences, we hypothesized that collectrin plays a role in vesicle trafficking of various apical membrane proteins in a polarized manner by which we can easily explain why the recruitment of multiple amino acid transporters at apical membranes is disturbed by collectrin deficiency. Here, we investigated the role of collectrin using collecting duct cell lines (mIMCD-3 cells), Ksp-cadherin promoter driven dominant-negative HNF1β mutant (DN-HNF1β) [11] and HNF-1β deletion mutant lacking C-terminal domain (HNF1βΔC) transgenic mice[12]. We have demonstrated that HNF-1β controls the transcriptional activities of collectrin. Furthermore, collectrin is preferentially associated with primary cilium and is shown to play a role in primary cilium formation, cell polarity and maturation revealed by siRNA experiments. We also demonstrated that the expression of collectrin on vesicles in peri-basal body region and its ability to bind to SNARE complex by interacting with snapin and to form complex with ciliary proteins, such as polycistin-2 and polaris. Collectrin may mediate specific vesicle transport, upon docking deliver the integral membrane proteins to ciliary plasmalemma, and maintain the primary cilia and cell polarity in collecting duct cells. Materials and Methods Animals Ksp-cadherin promoter regulated dominant-negative HNF1β mutant (DN-HNF1β) transgenic mice[11] and HNF-1β deletion mutant lacking C-terminal domain (HNF1βΔC) transgenic mice[12] were produced by the University of Texas Southwestern Transgenic Core Facility. Preparation of stable mIMCD3 cell lines overexpressing collectrin The nucleotide sequence encoding myc epitope, GAACAAAAACTCATCTCAGAAGAGGATCTG, was introduced into human collectrin cDNA in frame distal to the signal sequence by PCR and then subcloned into pcDNA3.1 to generate expression vector pcDNA3.1-myc-collectrin. mIMCD3 cells (ATCC) were transfected with pcDNA3.1-myc-collectrin plasmid using Lipofectamine 2000 reagent (Invitrogen). Stable transformants were selected by the treatment of Geneticin (300 µg/ml, Sigma). Cloning of 5′ flanking promoter region of rat, mouse, and human collectrin and transcriptional elements scanning The 5′ flanking promoter regions of rat, mouse, and human collectrin genes were cloned into pT7 blue vector (Invitrogen) using Genome Walker Kit (Clontech) as described previously[13]. Following which the DNA sequences for transcriptional elements were identified by using TRANSFAC database of weighted matrices of transcription factor binding sites. Generation of collectrin antibodies Polyclonal antibodies were raised in rabbits against the mixture of synthetic peptides derived from mouse collectrin amino acid sequence, AFSMRKVPNREATEISH (55-71) and AAEVQSAIRKNRNRINS (98-114) using custom polyclonal antibody production service (Asahi Techno Glass Corp., Tokyo, Japan). The specific binding to both peptides and the titers were confirmed by ELISA (enzyme-linked immunosorbent assay) (Asahi Techno Glass Corp). Electrophoretic mobility shift assay (EMSA) Plasmids encoding the full length of human HNF-1α and HNF1β were prepared by PCR from human kidney cDNA and cloned into pcDNA3.1(+) vector (Invitrogen). Nuclear proteins were extracted using Nuclear Extract Kit (Active Motif) from mIMCD3 cells with or without pcDNA3.1/HNF-1α transfection. EMSAs were performed with Dig Gel Shift Kit 2nd generation (Roche). Nuclear protein (2 µg) was incubated with digoxygenin-labelled oligonucleotide containing HNF-1 binding site of the human collectrin gene (5′-GATGGGTGTTAATCATTAAACCTTT-3′). The DNA-protein complexes were loaded on 5% polyacrylamide non0denaturing gels. The monoclonal HNF-1α and HNF-1β antibody (Transduction Laboratories) was used for supershift assays. Transient transfection and luciferase-reporter assay Promotor region segments of human collectrin containing putative HNF-1 binding site were generated by PCR and cloned into pGL3 basic reporter vector (Promega)[13]. mIMCD3 cells were co-transfected with 0.5–1.0 µg of pcDNA3.1/HNF-1α and/or pcDNA3.1/HNF-1β vectors, 0.5 µg of pGL3-collectrin reporter and 10 ng of pBIND vector using Lipofectamine 2000 reagent. 48 hours following transfection, the cells were lysed in 500 µl passive lysis buffer (Promega), firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The luciferase activity was expressed as the ratio of firefly and renilla luciferase activity. Western blotting Subcellular fractions of SD rat and CD-1 mouse kidneys were prepared by differential centrifugation as described[14]. Briefly, an initial spin was carried out at 800 g to remove nuclei and incompletely homogenized cellular debris. The supernatant was spun at 17,000 g for 20 min to pellet the plasma membranes (PM); the 17,000 g supernatant was subjected to a high-speed centrifugation (200,000 g) for 60 min in a Beckman 70Ti rotor to sediment the intracellular vesicle membranes (VM). The protein lysates of mIMCD3 cells, whole kidney and its subcellular fraction were subjected to SDS-PAGE and transferred to Nylon membranes. The membranes were treated with specific antibodies (1∶1000 dilution), such as mouse anti-HNF-1α and HNF-1β (Transduction Laboratories), polyclonal anti-SNARE associated protein (snapin) (Synaptic Systems, Göttingen, Germany), rabbit anti-rat aquaporin-2, goat anti-polycystin-2, rabbit anti-epidermal growth factor receptor (EGFR)(Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-collectrin, overnight at 4°C and followed by the treatment with secondary antibodies conjugated with horseradish peroxidase (1∶20000 dilution)[15]. Enzymatic deglycosylation The mouse medullary total protein was extracted and used for N-deglycosylation analysis. N-Glycosidase F Deglycosylation Kit was used following vendor's instructions (Roche). About 500 µg protein was incubated with 10 U of O-Glycosidase (Roche) for 3 h at 37°C. The reaction mixtures were then subjected to Western blot analyses. Yeast two-hybrid screening and tandem affinity tag (TAP) purification Collectrin cDNA encoding intracellular domain was subcloned to pGBKT7 bait vector and mouse kidney library was constructed using pGADT7-Rec vector. Yeast two-hybrid screening was performed according to the manufacturer's protocol (BD Biosciences Clontech). The collectrin cDNA containing full coding region without N-terminal signal sequence was cloned into pNTAP (Stratagene) and transiently transfected into mIMCD3 cells. Final purified proteins were analyzed with SDS-PAGE and Coomassie blue staining. The major bands were isolated and subjected to MALDI-TOF MS (mass spectrometry). Immunoprecipitation studies of mIMCD-3 cell lines mIMCD3 cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1% Triton X-100) with protease inhibitor cocktail (Roche Diagnostics Co., Basel, Switzerland). The lysates were centrifuged at 12,000 g for 30 minutes at 4°C and the supernatants were incubated with the preimmune sera and protein A-Sepharose CL-4B (Amersham Biosciences, GE Healthcare, Uppsala, Sweden). Immunoprecipitations were performed by adding 10 µl of specific sera and 80 µl of protein-A Sepharose CL-4B to 0.5 ml of supernatants further incubated overnight at 4°C on a rocking platform. The immunoprecipitated complexes were dissolved in a gel-loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT, and 0.1% bromophenol blue), subjected to SDS-PAGE under the reducing condition, and electroblotted onto Hybond P PVDF membranes (Amersham Biosciences, Piscataway, NJ). They were immunoblotted with specific antibodies (1∶100 to 1∶1000) overnight at 4°C and followed with the secondary antibodies conjugated with horseradish peroxidase (1∶20000). Finally, the filters were immersed in ECL Plus Western Blotting Detection Reagents (Amersham), and then exposed to Hyperfilm ECL (Amersham). Rabbit anti-rat snapin (Synaptic Systems), goat anti-human SNAP-23 (synaptosomal-associated protein 23 kDa), goat anti-human syntaxin-4, goat anti-polycystin-2 (Santa Cruz), rabbit anti-polaris, rabbit γ-actin (Novus Biologicals, Littleton, CO), rabbit anti-tropomyosin (CHEMICON, Temecula, CA) antibodies were used for immunoprecipitation and Western blot analyses. Normal rabbit and goat IgGs were used for negative controls of immunoprecipitation. Experiments with collectrin specific siRNA siTrio targeting mouse collectrin (NM_020626;5′-CCATAAGAATGAACAGAATT-3′, 5′-GCAGAAGACAAGTGTGAAATT-3′, 5′-CAACAATAGACCACTGAAATT-3′)(Collectrin siRNA) and siTrio negative control cocktail containing double stranded RNAs (5′-ATCCGCGCGATAGTACGTATT-3′, 5′-TTACGCGTAGCGTAATACGTT-3′ and 5′-TATTCGCGCGTATAGCGGTTT-3′)(CON siRNA) were purchased from DHARMACON and transfection of mIMCD3 cells was carried out with Lipofectamine 2000 reagent. Following experiments were performed 24 hours after the transfection. Cells in chamber slides were stained with mouse anti-PCNA monoclonal antibody (BD Transduction Lab) using R.T.U Vectastain Universal Quick kit (Vector Laboratories, Burlingame, CA, U.S.A.), apoptosis was evaluated with the DeadEndTM Colorimetric TUNEL System (Promega), and cell viability was assessed by MTT based colorimetric assay for the cell proliferation and viability (Roche). Western blot analyses and immunofluorescence were carried out using rabbit anti-EGFR (epidermal growth factor receptor) polyclonal antibody (1∶2000 dilution, Santa Cruz). For immunofluorescnce, cells grown in chamber slides were fixed in 4% paraformaldehyde for 30 minutes at 4°C, and then permeabilized with 0.1% Triton X-100 in PBS for 3 min if necessary. After blocking in 8% BSA in PBS for 1 hour at room temperature, cells were incubated at 4°C overnight with the primary antibodies diluted in 1% BSA. After washing for 5 minutes with PBS, cells were incubated for 1 hour with fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies. Rabbit anti-collectrin, mouse γ-tubulin, mouse anti-acetylated α-tubulin, mouse anti-Golgi 58K (Sigma), mouse anti-disulfide isomerase (Stressgen, Bictoria, Canada), rabbit anti-EGFR (Santa Cruz) were used. The cells were then examined by a confocal laser scanning microscope, Laser Scanning System LSM 510 (Carl Zeiss, Jena, Germany). Immunoelectron microscopy was performed using rabbit anti-collectrin antibody (1∶300 dilution) and goat anti-rabbit IgG conjugated with 15 µm colloidal gold (Amersham) as secondary antibody (1∶20 dilution)[16]. Quantification of primary cilium numbers and length in mIMCD3 cells Immunofluorescence images of primary cilia double stained with collectrin and acetylated tubulin were generated using LSM510 confocal microscope system (Zeiss). Percentage of the cells with visually detectable cilia was evaluated in ∼200 mIMCD3 cells and measurements of cilia length were determined using Zeiss LSM image browser software. Organ culture and antisense experiments Embryonic kidneys were harvested from pregnant Crl:CD-1 (ICR) mice at day 13 of gestation (E13)[17]. Antisense-phosphorothioated oligodeoxynucleotides (ODNs) of collectrin, HNF1α, HNF1β and a nonsense ODN were prepared as follows; 5′-CTCTGAAGAGGTATTCTTGATCCGT-3′, 5′-TCAGGTCCCCTCGACTCCACCGCA-3′ and 5′-ATAGTCGTCGCCGTCCTCTGAGCCCTC-3′, and 5′-TAATGATATAATGATTGTAATGATAGTAGT-3′. E13 metanephroi were maintained in an organ culture containing 12.5 µg/ml of epidermal growth factor (EGF). The antisense and nonsense ODNs were added to the culture medium daily at concentrations ranging from 1.5 to 2.5 µM for 4 days, and the metanephroi were subjected to quantitative real-time PCR. PCR was optimized and carried out on an ABI PRISM 7000 sequence detection system (Applied Biosystems) using ABsoluteTM QPCR SYBR Green Mix (ABgene, Rochester, NY). For each gene, the denaturation at 95°C for 15min was followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. Gene specific primers for collectrin (Genbank accession #AF178085), Pkhd1 (NM_153179), Pkd2 (NM_008861), Umod (NM_009470), Tg737/Polaris (NM_009376) and β-actin (NM_007393) were as follows: collectrin forward (5′-GCAATTGCACTACTGGTTCTAT CTG-3′), collectrin reverse (5′-TCCACTCCAGGTGGTCCTTT-3′), Pkhd1 forward (5′-TG ACCTTTTCTAGAT TGGCTGTCTT-3′), Pkhd1 reverse (5′-GT CCTTGATCGAGCTGTAA AATTAG-3′), Pkd2 forward (5′-TGAGCGTGAGCATCAACAGAT-3′), Pkd2 reverse (5′-T GGTAAAGAGCTGT GTTCCAAGTC-3′), Umod forward (5′-AGGTGTCCAGGCCTCAGTG T-3′), Umod reverse (5′-GGAAACAACAGCAGCCAGATG-3′), Tg737/Polaris forward (5′-TGCCT GGGACAGATGAA CCT-3′), Tg737/Polaris reverse (5′-AGGGCCCAGTGGATCCA-3′), β-actin forward (5′-GGCGCT TTTGACTCAGGATT-3′), β-actin reverse (5′-GGGATGTTTGCTCC AACCAA-3′). Data analysis Data were expressed as mean±s.e.m. Multiple groups were compared by analysis of variance (ANOVA). Two-group analysis was performed by t-test. Results Collectrin is transcriptionally regulated by HNF-1β in collecting duct cells To investigate the transcriptional regulation of collectrin gene, ∼500-bp of the 5′ flanking promoter region of rat collectrin gene was cloned. Transcriptional elements and consensus sequence of HNF-1 binding site were identified[13]. Comparison of the rat, human and mouse collectrin genes using GenBank data revealed that HNF-1 binding site was conserved. RT-PCR and Western blot analyses revealed that HNF-1β was expressed in mIMCD3 cells, while HNF-1α was not detected. HNF-1α mRNA and protein were detected in mIMCD-3 cells transfected with pcDNA3.1/HNF-1α ( Figure 1a ). The binding of HNF-1 to the putative promoter elements in mIMCD3 cells was confirmed by electrophoretic mobility shift assay (EMSA) ( Figure 1a ). The incubation of DNA probes of putative HNF-1 binding site with nuclear extracts from non-transfected or pcDNA3.1/HNF-1α transfected mIMCD3 cells revealed specific DNA-protein complex formation. The collectrin gene promoter was inserted into promoterless pGL3-Basic vector, where the transcription of the luciferase gene was driven by the collectrin promoter. This construct was cotransfected into mIMCD3 cells with pcDNA3.1/HNF-1α and/or pcDNA3.1/HNF-1β vectors. As shown in Figure 1b , cotransfection with pcDNA3.1/HNF-1α and/or pcDNA3.1/HNF-1β vectors accentuated luciferase activity in a dose dependent manner. These results indicated that HNF-1α and HNF-1β regulate the transcription of collectrin gene via HNF-1 binding consensus sequence present in its promoter. 10.1371/journal.pone.0000414.g001Figure 1 Transcriptional regulation of collectrin gene by HNF-1β. Panel a: Western blot analysis, RT-PCR and electrophoretic mobility shift assay (EMSA). Endogenous mRNA expression of HNF-1β but not HNF-1α in mIMCD3 cells is detected by Western blot analysis and RT-PCR. The specific binding of HNF-1β and HNF-1α to putative binding site is indicated by antibody mediated supershift (arrow). Panel b: Luciferase reporter gene assay. The mIMCD3 cells are co-transfected with pcDNA3.1/HNF-1α and/or pcDNA3.1/HNF-1β with pGL3-collectrin reporter and pBIND vector. A dose dependent increase in the reporter activity is observed (n = 6, p<0.05 and p<0.01 versus pcDNA3.1(+) treated control) Panel c: Western blot analyses of collectrin using whole kidney lysates of DN-HNF1β transgenic mouse. In two transgenic mouse founders, the expression of DN-HNF1β mutant transgene tagged with V5 and FLAG epitope is indicated by percentage of highest expression, 6% and 100%. (Western blot analysis using anti-FLAG antibody, n = 3 separate measurements, p<0.05 and *p<0.01 versus non-transgenic littermate indicated by 0%) Panel d: Localization of collectrin and HNF-1β deletion mutant protein in epithelia lining the cyst shown by immunofluorescence microscopy. Collectrin is visualized as red, HNF-1β deletion mutant with V5 tag as green, and nuclei as blue. Arrows indicate the absence of collectrin expression on the apical surface of epithelial cells where HNF-1β deletion mutant product is expressed in the nucleus. Scale bar, 20 µm. Dominant negative mutations of HNF-1β inhibited collectrin expression in vivo HNF-1β is expressed in renal collecting duct cells both in tissues and cell lines. Whole kidney membrane fractions from dominant-negative HNF-1β mutant (DN-HNF1β) transgenic mice[11] were extracted to confirm transcriptional regulation of collectrin by HNF-1β. DN-HNF1β transgenic founders with lower expression of transgene had smaller decrease in collectrin, and the transgenic founder with higher transgene expression exhibited remarkable decrease in collectrin revealed by Western blot analysis using whole kidney lysates ( Figure 1c ). Next, we examined the expression of collectrin in collecting ducts in HNF-1β deletion mutant lacking C-terminal domain tagged with V5 epitope (HNF1βΔC) transgenic mice[12]. The kidney tissue sections from transgenic founders, chimeric mice with wild type HNF-1β and HNF-1β deletion mutant, were stained with collectrin antibody (red) and V5 antibody (green) ( Figure 1d ) and nuclei were counterstained blue with 4′-6-diamidino-2-phenylindole. The luminal expression of collectrin was absent on the epithelial cells (arrows) in which HNF1βΔC was highly expressed in the nuclei (green); while collectrin was apparently expressed in surrounding normal collecting duct cells that did not express HNF1βΔC transgene product (blue). Collectrin localizes to primary cilia Plasma and vesicle membrane fractions from mouse and rat renal medulla were associated with aquapirin-2 and snapin, respectively ( Figure 2a ). The molecular weight of collectrin, ∼40 kDa, was similar in mouse, rat and human, both in plasma and vesicle membrane fractions. Collectrin is a glycosylated protein since the predicted molecular weight from amino acid sequence was ∼26 kDa. Deglycosylation with N- and O-glycosidase resulted in the reduction in molecular weight, i.e. 30 kDa and 35 kDa, respectively ( Fig. 2b ). Next, subcellular localization of collectrin by immunofluorescence was investigated. Focal planes of single mIMCD3 cell were obtained; the nuclear focal plane indicated that collectrin localized in cytoplasm, and the apical focal plane revealed the presence of collectrin on apical plasma membrane ( Figure 2c ). 10.1371/journal.pone.0000414.g002Figure 2 Subcellular localization of collectrin in kidney tissues and mIMCD3 cells. Panel a: Western blot analyses of snapin, aquaporin-2 and collectrin, isolated from plasma membrane (PM) and vesicle membrane (VM) fractions. Collectrin antibody detected a single band of ∼40 kDa. Panel b: N-glycosidase (N) and O-glycosidase (O) treatment generates ∼30 kDa and ∼35 kDa respective bands. Panel c: Immunoreactivity of collectrin is detected by confocal microscopy; five layers (1 to 5) are shown and collectrin is seen in cytoplasm (2 to 5 cuts) and apical membrane (1 cut). Panel d: Apical view of mIMCD3 cells. Collectrin (green) and acetylated α-tubulin (red), a primary ciliary marker, are depicted. Panel e: Higher magnification shows that acetylated α-tubulin (red) and collectrin (green) co-localize. Panel f: Collectrin (green) is seen surrounding the γ-tubulin, a marker for the basal body (red). Panel g: Immunoelectron microscopy shows gold particles localized mainly in microvesicles around the basal body of the primary cilium (arrow heads), whereas few gold particles are observed at the peripheral region of proximal centriole. Scale bars, 20 µm for panels c, d, e, and f and 500 nm for panel g. Scanning the mIMCD3 cells using confocal microscopy from basal cell attachment to apical top and re-construction of three-dimensional images indicated that primary cilia displayed intense immunoreactivities. Double staining of anti-acetylated α-tubulin, marker for primary cilia, and anti-collectrin antibodies confirmed that collectrin expression was concentrated in primary cilia ( Figure 2d ). Higher magnification of the apical focal plane clearly showed the co-localization of collectrin and acetylated α-tubulin on primary cilium ( Figure 2e ). Collectrin immunoreactivities (green) were seen surrounding the ciliary basal body, the latter is visualized with anti-γ-tubulin antibody (red)( Figure 2f ). Similarly, immunoelectron microscopy revealed that gold particles were associated with the vesicles adjacent to the basal bodies, while they were not integrated into the basal body microtubule structures. Gold particles were also located at ciliary membranes surrounding the microtubules, whereas few gold particles were observed at the peripheral region of the centriole ( Figure 2g ). Cilia formation is disrupted by collectrin siRNA treatment To demonstrate the role of collectrin in cilia formation, stable mIMCD3 cell lines over-expressing collectrin (collectrin stable) were generated. All 4 cell lines revealed ∼3-fold over-expression compared with control pcDNA3.1 stably transfected mIMCD3 cells (mIMCD3 cells) ( Figure 3a ). The siRNA treatment significantly suppressed collectrin expression in mIMCD3 cells, while the control siRNA had no effect ( Figure 3b ). Confocol microscopy also revealed that collectrin was over-expressed in collectrin stable cells and knocked down by siRNA revealed by confocol microscopy ( Figure 3c ). The fully polarized mIMCD3 cells were associated with single primary cilia, and the cilia formation was not disturbed in control siRNA treated cells or collectrin stable cell lines ( Figure 3d ). However, primary cilia were absent in ∼40% of the cells treated with collectrin siRNA ( Figures 3d and 3e ). In addition, morphometric analyses of the existing primary cilia indicated that mIMCD3 cells transfected with siRNA had shorter or stunted primary cilia compared with those of control siRNA treated cells ( Figures 3d and 3f ). By electron microscopy ( Figure 3g ), the treatment of siRNA resulted in various abnormalities of primary cilia, e.g. disorganization of 9+0 microtubular structure of basal bodies (arrow head), bulging basal bodies (arrow), and stunted shaft projection (asterisk). 10.1371/journal.pone.0000414.g003Figure 3 Primary cilium formation in mIMCD3 cells. Panel a: Expression of collectrin in mIMCD3 cells over-expressing collectrin (n = 4; clone 11, 13, 15 and 18) and mIMCD3 cells stably transfected with pcDNA3.1(+) (n = 2; clone c1 and c2)(mIMCD3 cells). (p<0.05 versus mIMCD3 cells) Panel b: Western blot analysis of collectrin in mIMCD3 cells treated with control siRNA (CON-siRNA) or Collectrin siRNA. (Data represent 3 independent experiments, p<0.01 versus CON-siRNA) Panel c: Immunofluorescence of collectrin in mIMCD3 cells, CON-siRNA and Collecrin siRNA treated cells, and Collectrin stable cells. Panels d & e: mIMCD3 cells double-stained with collectrin (green) and acetylated α-tubulin (red). Cilia are present in every polarized cell, but ∼40% of cells are without detectable cilia with the treatment of collectrin siRNA. AV, apical view. (n = 300 from separate three experiments, p<0.05 versus CON-siRNA) Panel f: Cilium length is also reduced in cells after collectrin siRNA transfection (∼4.5 µm) compared with CON-siRNA group (∼7.8 µm). (n = 300 from three independent experiments, p<0.05 versus CON-siRNA) Panel g: Electron micrographs of mIMCD3 cells treated with CON-siRNA and Collectrin siRNA. Cilium with disorganized microtubule structure (arrow heads), bulging cilium (arrows), and stunted cilium (asterisk) are seen in mIMCD3 cells transfected with Collectrin siRNA. Scale bars, 20 µm for panels c and d and 500 nm for panel g. Colocalization of polycystin-2 and collectrin and effects of collectrin siRNA Since polycyctin-2 reveals ciliary localization and the intracellular trafficking of polycystin-2 is regulated both at the level of the endoplasmic reticulum and the trans-Golgi network, subcellular expression of collectrin and polycystin-2 in mIMCD3 cells was assessed. Inhibition of collectrin expression by siRNA showed negative impact on polycystin-2 protein expression in mIMCD3 cells ( Figure 4a ). Double staining showed that collectrin was fully co-localized with Golgi marker and also partially localized in endoplasmic reticulum (ER) ( Figure 4b ). As expected, collectrin and polycystin-2 colocalized in the cytoplasm of mIMCD3 cells ( Figure 4c ). Apical view by confocal microscopy indicated that polycystin-2 was highly expressed on primary cilia and in the cytoplasm of mIMCD3 cells, whereas polycystin-2 disappeared both in the cilia and cytoplasm of most of the cells transfected with collectrin siRNA ( Figure 4d ). 10.1371/journal.pone.0000414.g004Figure 4 Expression and localization of polycystin-2 in mIMCD3 cells treated with collectrin siRNA. Panel a: Western blot analyses show down regulation of polycystin-2 in mIMCD3 cells transfected with collectrin siRNA (Collectrin siRNA). Collectrin expression is not altered in mIMCD3 cells treated with control siRNA (CON-siRNA) and mIMCD3 cells over-expressing collectrin (Collectrin stable). (Data represent 3 independent experiments, p<0.05 versus CON-siRNA) Panel b: mIMCD3 cells double-stained with anti-collectrin antibody and organella markers. Golgi 58K protein (58K-9) (red) and collectrin (green) are co-localized in cytoplasm of mIMCD3 cells. Endoplasmic reticulum (ER) marker, protein disulfide isomerase (red), and collectrin (green) show partial colocalization. Panel c: mIMCD3 cells double-stained with polycystin-2 (green) and collectrin (red) showing co-localization. Panel d: mIMCD3 cells double-stained with polycystin-2 and acetylated α-tubulin. Polycystin-2 is highly expressed in primary cilia and the apical membrane of mIMCD3 cells, CON-siRNA treated and Collectrin stably transfected cells, whereas polycystin-2 is not detected in the cilia of most of the cells transfected with collectrin siRNA. AV, apical view. Scale bars, 10 µm for panels b, c, and d. Collectrin forms complexes with proteins related to vesicle transport and fusion Further insight into the role of collectrin was investigated by examining protein:protein interactions with ciliary proteins, such as polycyctin-2 and polaris[18]. Co-precipitation of collectrin and polycystin-2 or collectrin and polaris ( Figure 5a ) indicated it forms a complex with polycystin-2 and polaris. Next, collectrin-interacting molecules were identified by yeast two-hybrid system. After screening more than 107 independent clones using intracellular domain of collectrin as bait, snapin, SNAP-25 (synaptosomal-associated protein of 25 kDa) binding protein, was isolated. In a previous study, snapin was also isolated by the yeast two-hybrid system using full-length of collectrin as bait[13]. Snapin is a component of SNARE (soluble N-ethylmaleiamide-sensitive factor attachment protein receptor) and immunoprecipitation study showed collectrin co-precipitated with snapin. In mIMCD3 cells, collectrin also co-precipitated with SNARE proteins, SNAP-23 and syntaxin-4, suggesting collectrin binds to SNARE complex by interacting with snapin ( Fig. 5b ). Further screen of the protein complexes using tandem affinity tag purification and mass spectrometry was performed. Collectrin cDNA lacking N-terminal signal sequence was cloned into pNTAP and transiently transfected into mIMCD3 cells. Purified proteins were analyzed with SDS-PAGE that revealed two major bands. MALDI-TOF MS identified them as nonmuscle myosin heavy chain II-A (myosin heavy polypeptide 9) and γ-actin. Co-immunoprecipitation study revealed collectrin forms the protein complex with γ-actin, myosin II-A, tropomyosin, and polycystin-2 ( Figure 5c ). Although collectrin knockdown alter the intensity and pattern of acetylated α-tubulin, collectrin siRNA treatment did not alter the expression and staining pattern of γ-actin, myosin II-A, and tropomyosin revealed by Western blot analysis and immunofluorescence study (data not shown). 10.1371/journal.pone.0000414.g005Figure 5 Collectrin and interacting proteins. Panel a: Immunoprecipitation indicates interaction between collectrin and polycystin-2, and between collectrin and polaris. Panel b: Collectrin co-precipitates with snapin, SNAP-23, and syntaxin-4. Panel c: Tandem affinity tag purification using collectin as a bait followed by SDS-PAGE and MALDI TOF MS analysis revealed that the major two bands are nonmuscle myosin heavy chain II-A and γ-actin. Immunoprecipitation study shows collectrin, myosin II-A, γ-actin, tropomyosin, and polycystin-2 forms protein complex. Collectrin regulates cell proliferation, apoptosis, and polarity PCNA immunostaining and 5-bromo-2′-deoxyuridine (BrdU) assays showed an increased proliferation activity in mIMCD3 cells transfected with collectrin siRNA, while the stable over-expression of collectrin had no notable effect on cell proliferation ( Figures 6a, 6b and 6c ). To examine the effects of collectrin on apoptosis, TUNEL assay was performed in mIMCD3 cells. The number of apoptotic cells notably increased in mIMCD3 cells transfected by collectrin siRNAs compared with negative control siRNA treated cells. Over-expression of collectrin did not show any alterations in percentage of apoptotic cells ( Figures 6d and 6e ). Although collectrin siRNA-treated mIMCD3 cells underwent apoptosis and revealed proliferation activity, overall viable cell numbers were not altered shown by MTT assay ( Figure 6e ). 10.1371/journal.pone.0000414.g006Figure 6 Proliferation, apoptosis, and mislocalization of epidermal growth factor receptor (EGFR) in mIMCD3 cells treated with collectrin siRNA. Panels a & b: Cellular proliferation assessed by proliferating cell nuclear antigen (PCNA) staining. PCNA positive cells increase in mIMCD3 cells treated with collectrin siRNA compared with control siRNA treated cells (CON-siRNA) and mIMCD3 cells over-expressing collectrin (Collectrin stable). Panel c: The increased proliferation of cells is observed in collectrin siRNA group by BrdU assay. Panels d & e: Apoptosis assay using TdT-mediated dUTP nick end labeling (TUNEL) method. Increased number of apoptotic cells is observed in collectrin siRNA group. Panel f: The viability of the cells is not altered in all groups revealed by MTT assay. Panel g: Imunofluoresence and confocal micrographs of EGFR. EGFR is observed in basolateral membrane of mIMCD3 cells, control siRNA group, and collectrin stable cells, whereas apical membrane expression of EGFR is detected in collectrin siRNA group. Panel h: Western blot analyses of EGFR. EGFR is up-regulated in collectrin siRNA group and down-regulated in cells stably transfected with collectrin. Scale bars, 20 µm for panel f. N = 300 from separate three experiments for panels b, c and e. Data are from 3 independent experiments for panel g, p<0.05, *p<0.01 versus CON-siRNA. Since up-regulation and mis-polarization of epidermal growth factor receptor (EGFR) is linked to abnormal cell proliferation, the expression of EGFR was investigated. EGFR was normally expressed on lateral membrane in mIMCD3 cells, and transient transfection with control siRNA or stable trasfection of collectrin gene did not alter the distribution. Besides lateral membrane expression, the EGFR was also seen mis-localized on the apical membrane in the cells treated with collectrin siRNA, a characteristic feature observed in tubular epithelia lining the cysts in human and animal polycystic kidneys[18], [19] ( Figure 6g ). The Western blot analysis revealed that over-expression of collectrin is associated with reduced EGFR, in turn, the repression of collectrin resulted in up-regulated EGFR expression ( Figure 6h ). Collectrin antisense ODN treatment of metanephric organ culture Addition of EGF (12.5 µg/ml) and nonsense ODN resulted in hypertrophy of the fetal kidney without apparent cyst formation ( Figure 7a-B ) compared with nonsense ODN treated control explants ( Figure 7a-A ). Inclusion of collectrin antisense ODN was associated with formation of cleft-like structure (arrows) in the ureteric bud branches in a dose-dependent manner ( Figures 7a-C and -D ). Addition of HNF-1α antisense ODN into the metanephric organ culture media also resulted in the formation of multiple cleft-like cysts ( Figures 7a-E-F ), while the number or size of the cyst formation were relatively more in the metanephroi with the treatment of HNF1-β antisense ODN ( Figures 7a-G and H ). The mRNA expression of collectrin and the genes under the control of HNF-1α and HNF-1β was investigated. Collectrin antisense ODN suppressed collectrin gene expression along with that of HNF-1, regulator of Pkd2 and Umod. Notably, the expression of collectrin was inhibited by HNF-1β antisense ODN, however, inhibitory effect was not seen in HNF-1α antisense ODN treated group ( Figure 7b ). The addition of HNF-1β antisense ODN resulted in global inhibitory effects on HNF-1 regulated genes compared with HNF-1α antisense ODN treated metanephroi except Tg737/Polaris. 10.1371/journal.pone.0000414.g007Figure 7 Collectrin antisense experiments in mouse metanephric organ culture system. Panels aA & aB: Addition of EGF (12.5 µg/ml) (EGF) into culture media induces hypertrophy of the fetal kidney without cyst formation compared with nonsense ODN (oligodeoxynucleotide)-treated control explants (CON). Panels aC & aD: The formation of cleft-like cysts (arrows) in the ureteric bud branches are seen in collectrin antisense ODN treated groups at concentrations of 1.5 µM (COLL-1.5) and 2.5 µM (COLL-2.5). Panels aG & aH: The formation of multiple cleft-like cysts is accentuated in metanephroi treated with HNF-β antisense ODN at concentrations of 1.5 µM (HNF-β-1.5) and 2.5 µM (HNF-β-2.5). Panels aE & aF: Similar effect, although somewhat less, is seen in HNF-1α antisense ODN treated group at concentrations of 1.5 µM (HNF-α-1.5) and 2.5 µM (HNF-α-2.5). Panel b: mRNA expression of collectrin and the genes regulated by HNF-1α and HNF-1β including Pkhd1, Pkd2, Umod, and Tg737/Polaris. Scale bars, 200 µm for panel a. Collectrin expression decreased in polycystic kidneys To investigate collectrin expression and localization in cystic kidneys, we performed immunochemistry on the kidneys from HNF1βΔC transgenic mice. Transgenic mice at postnatal day-52 (P52) and P100 were compared with a non-transgenic littermate at P52. The kidney-specific expression of HNF1βΔC was associated with renal cyst formation and extensive interstitial fibrosis throughout the parenchyma of the kidney ( Figure 8a ). Collectrin was expressed in cytoplasm and apical membrane throughout collecting duct cells in the non-transgenic mice. In HNF1βΔC transgenic mice, weak apical expression was detected in morphologically normal collecting ducts, while the signals were barely detectable in single-layered or mutilayered cysts ( Figure 8b ). Collectrin in the kidney tissues from ADPKD patients was seen localized in morphologically normal tubules, whereas the epithelial cells lining single cell- and multi-layered cysts showed weak or lacked collectrin expression ( Figure 8c ). 10.1371/journal.pone.0000414.g008Figure 8 Collectrin expressions in polycystic kidneys. Panel a: Kidney tissues from nontransgenic littermates at postnatal day 52 (P52), HNF1βΔC transgenic mice at P52, and HNF1βΔC transgenic mice at P100. Numerous renal cysts are formed in kidneys of HNF1βΔC transgenic mice. (Masson-Trichrome) Panel b: Immunoperoxidase staining of collectrin. Abundant expression of collectrin is seen in nontransgenic mice P52. Collectrin immunoreactivity is readily seen in normal portion of collecting duct cells (arrow head) but not seen in the epithelia lining the cysts (arrow) in HNF1βΔC transgenic mice. Panel c: Collectrin immunoperoxidase staining in kidney tissues form normal human and ADPKD patients. Abundant expression of collectrin is seen in normal collecting duct cells. In ADPKD patients, collectrin expression is observed in normal collecting duct cells (arrow head); however immunoreactivity of collectrin is not seen in both single-layered (arrow) and multilayered cysts. Scale bars, 1 mm for panel a, 50 µm for panels b and c. Discussion In the kidney, HNF-1β, a transcriptional factor, has been shown to modulate activities of several target genes in tubular epithelia, namely Umod, Pkhd1, Pkd2 and Tg737/Polaris. Mutations in these genes have been described in various forms of cystic diseases. Although HNF-1α and HNF-1β bind to the target site with similar affinities, either as homo- or hetero-dimers, the findings of the present study suggest that collectrin is another novel target of HNF-1β in the kidney. In the kidney tissues, HNF-1α was expressed exclusively in the nuclei of renal proximal tubular cells, while HNF-1β was expressed in all segments of the nephron, including collecting ducts[7]. Interestingly, the RT-PCR analyses revealed that both HNF-1β and collectrin are co-expressed in collecting duct and respective cell lines, including mIMCD-3, mIMCD-K2, RCCD1, RCCD2, and M-1 cells, suggesting a potential functional association between these two molecules. Besides kidney, collectrin is also co-expressed with HNF-1β in pancreatic islets[13], suggesting that the transcriptional activities are perhaps under tight and specific control of HNF-1β. Many of HNF-1β driven proteins linked to PKD in mouse and human localize to the primary cilium[20], [21]. Cilia are rod-like organelles originating from the basal body and related to the centriole. Their basic structure consists of a central axoneme composed of 9 doublets of microtubules and a ciliary membrane that is continuous with plasmalemma. One to two primary cilia are present on the surface of most vertebrate cells and also on the apical surface of tubular epithelia of mammalian kidney[22]. A series of discovery began from an insertional mutagenesis screen in mice and the mutation in Tg737 gene was found to cause recessive form of PKD in orpk mice[23]. The protein product of Tg737, polaris, is localized in the axoneme and basal body. It seems to be essential for ciliary assembly and intra-flagellar transport[24], [25]. In addition to structural abnormalities of cilia, functional defects were noted in Invs [26] and Cys1 [27], and both mouse mutants revealed ARPKD phenotype. Later studies revealed that the PKD1 product, polycyctin-1, as well as polycystin-2, polaris, and cystin are all found to be localized to primary cilia in cultured mouse cortical collecting duct cells[28]. Interestingly, defects in primary cilia are seen in Bardet-Biedl syndrome (BBS), characterized by mental retardation, pigmentary retinopathy, polydactyly, obesity, renal cyst formation and hypogenitalism. Among 8 causal genes, BBS8 protein localizes to centrosomes and basal bodies and co-localizes with γ-tubulin and BBS4[29], [30]. Although ciliary functions in renal tubules are incompletely understood, this organelle most likely functions as a flow-sensitive mechanosensor to luminal fluid flow, modulates flow-sensitive ion transport and possibly regulates cell proliferation and differentiation[31]. Like other genes involved in cystogenesis, it is reasonable to speculate that the HNF-1β-driven collectrin plays a role in ciliary functions and thereby in cystogenesis in PKD, since collectrin co-localized with acetylated α-tubulin, maker for primary cilium. In peri-basal body region of primary cilium, collectrin was seen surrounding basal body marker, γ-tubulin, and it revealed immunoreactivities on the microvesicle revealed by immunoelectron microscopy ( Figures 2 and 9 ). Cilia and flagella are unable to synthesize the various ciliary proteins, and thus all non-membrane-bound proteins needed for elongation of the primary cilium are transported to the ciliary tip to endow it with functional properties[32], [33]. The process is accomplished by a transport apparatus known as the intraciliary or intraflagellar transport (ICT or IFT) system. In Chalamydomonas, the IFT particles are composed of at least 17 subunits; the IFT polypeptides move in antrograde direction to the flagellar tip by kinesin-II and move in the retrograde direction to the base of the flagellum by cytoplasmic dynein 1b[34]. However, it is not clear how integral membrane proteins are transported within cilia and flagella. Since some membrane proteins are concentrated in the ciliary membranes, the barrier has to exist at the base of cilium to prevent backward diffusion out of the cilium or one-way barrier facilitates the transportation of integral proteins from the apical plasma membranes[35] ( Fig. 8 ); the former is rather conceivable and the base of the cilium would be expected to serve as an initial docking station[36]. 10.1371/journal.pone.0000414.g009Figure 9 Models for the trafficking of cilliary integral membrane proteins. Snapin is a 15 kDa protein containing predicted coiled-coil domain in its C-terminal half[37] and it has been reported to bind to synaptic SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) complex via a direct interaction with SNAP-25 and SNAP-23 and thus is believed to be involved in vesicle fusion and trafficking[38]. Our previous studies suggest a direct binding between collectrin and snapin by yeast two hybrid system and GST-pull-down assay[13]. Furthermore, immunoprecipitation studies in pancreatic β cell line, INS-1 cell, indicated that collectrin binds to SNARE complex, consisting vesicle-associated membrane protein-2 (VAMP-2), syntaxin-1 and SNAP-25, via direct interaction with snapin[13]. Incidentally, the presence of SNARE proteins such as SNAP-23, syntaxin-4 and VAMP-2 has been reported in collecting duct cells[39]. The current studies extended the observations and demonstrated that collectrin co-immunoprecipitated with snapin and SNARE proteins, SNAP23 and syntaxin-4 in collecting duct cells. In addition, complex formation of collectrin with γ-actin, myosin II-A (non-muscle heavy chain II-A), and tropomyosin was elucidated. The interaction of actin with non-muscle myosin II-A assembled around aggregates of secretory vesicles facilitate their exocytosis in lacrimal acinar epithelial cells[40]. In other cell types, myosin II is involved in vesicle budding from Golgi[41], intracellular vesicle movement or trafficking[42]–[45], and membrane docking[40], [46]. In view of these processes described in other cell types and elucidated in this investigation, it would suggest that collectrin in association with SNARE and actin-myosin complex mediates specific vesicle transport and docking to drive the integral membrane proteins to ciliary membranes. In support of such a contention, the application of collectrin siRNA to mIMCD3 cells resulted in severe defects in cilium formation and subsequent loss of polycystin-2 on cilia, increased cell proliferation and apoptosis, and overexpression of EGFR and its mislocalization. This phenomenon is consistent with the facts that the presence of a cilium is associated with the establishment of polarity and differentiation of the cells in the stationary or G0 phase of the cell cycle. In many cells, the entry into the cell cycle is preceded by ciliary resorption; while exit from mitosis is accompanied by cilium formation[47]. Another significant finding of our study is that collectrin forms complex with polycystin-2 and Tg737/Polaris. Polycystin-1 is a G protein-coupled transmembrane receptor that acts as a ciliary mechanosensor and regulates calcium channel activities of polycystin-2. Polycystin-1 and -2 forms a hetrerodimer complex in primary cilium and polycyctin-2 responds to signals from polycystin-1 induced by the ciliary flexing to generate influx of calcium[31]. Polycystin-1 and polycystin-2 was co-localized both in apical and basolateral membranes including primary cilium, cell-cell adherens junction, and cell-matrix focal adhesion. In contrast to polycystin-1, the presence of polycystin-2 in endoplasmic reticulum (ER) and Golgi apparatus suggests that ER and Golgi are recycling pool or the site of polycystin-2 activity[48]. The trafficking of proteins to specific subcellular compartments is governed by multiple protein-protein interaction following post-translational modifications[49]. Polycystion-2 is phosphorylated on Ser812 by protein kinase (CK2), binds to phosphofurin acidic cluster sorting protein/Golgi-derived coat protomer I (PACS2/COPI), and is retrieved on the ER. Dephosphorylation of polycystin-2 by protein phosphatase 2A (PP2A) facilitates polycystin-2 translocation to the plasma membrane by releasing from the interaction with PACS proteins[50]. Similarly, the retrieval to the Golgi is mediated by PACS-1/adaptin. Other molecules that may modulate these events include CD2-associated protein (CD2AP) which participates in endocytosis of polycystin-2, while PIGEA-14 is involved in forward trafficking from the ER to the Golgi[51]. The above literature information can be interfaced with the current findings suggesting collectrin in association with polycystin(s) modulate the ciliary pathobiology. The application of collectrin siRNA into mIMCD3 cells reduced polycystin-2 expression and resulted in selective loss of polycystin-2 from primary cilium, apical membrane and cytoplasm. The collectrin expression in the Golgi apparatus and ER suggests that it may also be involved in the ER-Golgi-plasma membrane trafficking of polycyctin-2. Defective trafficking of the latter may be case in mice with Tg737orpk mutation where polycystin-2 is seen accumulated in the stunted cilium, since Tg737/Polaris is not required for moving polycystin-2 into cilia but is needed to recycle it to the cell body[52]. The findings of the present investigation indicating an association of collectrin and Tg737/Polaris may also suggest a possible role of collectrin in such intraciliary recycling events of integral transmembrane proteins. In summary, it seems that collectrin is necessary for the maintenance of primary cilium to supply the primary cilia-specific integral membrane proteins, including polycystin-2, and it is involved in multiple membrane trafficking processes; vesicle transport, docking to base of primary cilia, ER-Golgi trafficking, and intraciliary recycling. Credence to this notion comes from the studies where suppression of collectrin expression resulted in severe defects in cilium formation in mIMCD3 cells and slit like cyst formation in mouse metanephric organ culture system. Furthermore, the reduction of collectrin expression in cystic tubular epithelia in HNF1βΔC transgenic mice as well as in human ADPKD patients would be supportive of the role of collectrin in cystogenesis. Lack of phenotype of cyst formation in collectrin knockout mice suggested that ACE2 may compensate of collectrin action. The findings of this study that collectrin plays a central role in pathways common to cystogenesis of various hereditary cystic diseases should yield an impetus to develop new therapeutic intervention for the treatment of PKD with collectrin being a novel target. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by Grant-in-Aid for Scientific Research (16-04241) to Y.Z and Y.Z. is a recipient of JSPS (Japan Society for the Promotion of Science) fellow. Grant-in-Aid for Scientific Research, Ministry of Education, Science and Culture, Japan (17590829) to J.W. and Uehara Memorial Foundation, Grant-in-Aid for Scientific Research, Ministry of Education, Science and Culture, Japan (18390249) to H.M., NIH grants (R01DK42921 and R01DK67565) to P.I. and NIH grants (DK28492 and DK60635) to Y.S.K. We thank Dr. Bradley K. Yoder in University of Alabama at Birmingham for the Polaris antibody. ==== Refs References 1 Zhang H Wada J Kanwar YS Tsuchiyama Y Hiragushi K 1999 Screening for genes up-regulated in 5/6 nephrectomized mouse kidney. Kidney Int 56 549 558 10432394 2 Zhang H Wada J Hida K Tsuchiyama Y Hiragushi K 2001 Collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regulated in embryonic kidneys. 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==== Front PLoS BiolPLoS BiolpbioplbiplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1753511110.1371/journal.pbio.005014607-PLBI-RA-0115R2plbi-05-06-08Research ArticleNeuroscienceNeuroscienceDrosophilaPER-TIM Interactions with the Photoreceptor Cryptochrome Mediate Circadian Temperature Responses in Drosophila CRY Promotes Circadian Temperature ResponsesKaushik Rachna 123¤Nawathean Pipat 123Busza Ania 456Murad Alejandro 45Emery Patrick 45Rosbash Michael 1231 Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts, United States of America 2 National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts, United States of America 3 Department of Biology, Brandeis University, Waltham, Massachusetts, United States of America 4 University of Massachusetts Medical School, Worcester, Massachusetts, United States of America 5 Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America 6 MD/PhD Program, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Schibler Ueli Academic EditorUniversity of Geneva, Switzerland To whom correspondence should be addressed. E-mail: [email protected] 2007 29 5 2007 29 5 2007 5 6 e14622 1 2007 26 3 2007 © 2007 Kaushik et al.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cryb. The period mutant perL manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of perL flies, because a perL; cryb strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase. Author Summary Circadian rhythms profoundly affect the physiology and behavior of most organisms. These rhythms are generated by a self-sustained molecular clock, which is largely conserved between fruit flies and mammals and synchronizes to the day/night cycle. This synchronization is achieved in most organisms by a daily resetting caused by light and/or temperature fluctuations. The molecular mechanisms underlying light synchronization are reasonably well understood, but an understanding of how temperature affects the circadian clock is lacking. This study demonstrates a striking and unanticipated relationship between light and temperature resetting mechanisms in Drosophila. An interaction between the circadian photoreceptor CRYPTOCHROME (CRY) and a complex composed of the key circadian regulators PERIOD (PER) and TIMELESS (TIM) are critical for circadian temperature responses as well a circadian light responses. Moreover, the data not only indicate that light and temperature reset the clock through similar mechanisms but also that these two inputs can act synergistically. An interaction between light and temperature may fine-tune the dawn and dusk response of the clock and even contribute to seasonal adaptation of clock function, an emerging area of research in circadian biology. Temperature-dependent changes in circadian rhythms are mediated by interactions between the photoreceptor cryptochrome (CRY) and the proteins period (PER) and timeless (TIM). citationKaushik R, Nawathean P, Busza A, Murad A, Emery P, et al. (2007) PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila. PLoS Biol 5(6): e146. doi:10.1371/journal.pbio.0050146 ==== Body Introduction Most organisms have circadian rhythms of gene expression and behavior that are controlled by endogenous clocks. A few studies have verified that these systems increase fitness and help organisms adapt to the physical and ecological environment in which they live [1]. At the molecular level, the central pacemaker of animals is proposed to consist of auto-regulatory feedback loops that regulate the expression of key clock genes [2]. An admittedly simplified view of the Drosophila central clock posits a core system of four interacting regulatory proteins. A circadian cycle begins when a CLOCK (CLK) and CYCLE (CYC) heterodimer activates the expression of two other proteins, PERIOD (PER) and TIMELESS (TIM). PER and TIM levels slowly accumulate over time, and these two proteins also heterodimerize. At some point, PER-TIM complexes enter the nucleus and inactivate CLOCK-CYCLE activity, slowing their own production and signaling the end of a cycle. Importantly, kinases and phosphatases modify PER, TIM, and CLK and play critical roles in circadian rhythms [3–7]. Endogenous periods are usually different from the precise 24-h rotation of Earth. Nonetheless, circadian clocks keep precise 24-h time under normal conditions and are reset every day by environmental signals like light and temperature, which are the dominant entraining cues in nature. In Drosophila, circadian light perception is well-understood, and a major fraction of it is mediated by the circadian photoreceptor molecule cryptochrome (CRY) [2,8]. Cryptochromes are related to photolyases, a family of blue-light–sensitive DNA repair enzymes, and also play important roles in photoreception and circadian rhythms of other animals as well as plants [9,10]. Drosophila CRY is prominently expressed in pacemaker neurons [11–13]. Moreover, a mutant cry strain (cryb) manifests severe molecular and behavioral problems. These include a lack of PER and TIM molecular cycling in peripheral tissues under light-dark cycles and an inability to undergo phase resetting in response to short light pulses [14]. cryb flies are also rhythmic in constant light, i.e., the characteristic arrhythmicity of Drosophila and many other animals in constant light is absent [15]. Finally, there is strong evidence that CRY contributes to standard entrainment by light-dark cycles [16]. At the biochemical level, photon capture by CRY leads to an interaction with TIM or with the PER-TIM complex [17–20]. CRY also interacts with and blocks the function of the PER-TIM complex in a light-dependent manner in an S2 cell-based assay [17]. The current view is that the CRY:TIM interaction leads to TIM degradation, which results in phase-resetting in response to a light pulse [21–25]. In addition to light, other factors such as social interactions, activity, and especially temperature can modulate free-running rhythms. Indeed, temperature is generally regarded as secondary only to light as an entrainment cue [26]. Circadian clocks can be highly sensitive to temperature changes; e.g., clocks can be entrained by a regular temperature cycle that oscillates by only 1–2 degrees in some insects, lizards, and vertebrates [27–29]. It has also been shown that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PER and TIM in constant light, a situation that normally leads to molecular and behavioral arrhythmicity [30]. Also relevant to the relationship between temperature and circadian rhythms is temperature compensation: the free-running period in many different circadian systems, including Drosophila, is generally insensitive to alterations in (constant) incubation temperature, i.e., Q10 (the relative rate enhancement corresponding to a 10 °C rise in temperature) ≈ 1.0 [31]. It is believed that temperature compensation is integral to circadian clock function and critical to maintaining dependable time keeping despite fluctuations in ambient temperature. We found a surprising relationship between the response of the Drosophila clock to heat pulses and light pulses as well as between heat pulses and temperature compensation; the connector is the photoreceptor CRY. The heat-induced phase delays that take place in a wild-type strain are paralleled by a physical interaction between CRY and PER-TIM. In perL mutant flies, heat-phase shifts are more robust and occur at lower temperatures, which are mirrored by parallel CRY:PERL-TIM interactions. perL phase shifts are also severely reduced by the addition of cryb to the genetic background. Remarkably, these perL; cryb double-mutant flies have largely restored temperature compensation. The results indicate that a more potent interaction between CRY and PERL-TIM causes most of the temperature compensation defects of perL as well as the more robust heat-mediated phase shifts of these mutant flies. Results Heat Pulse–Mediated Phase Delays of Wild-Type Flies Require 37 °C To investigate the effect of heat on Drosophila locomotor activity rhythms, we first compared a heat phase response curve (PRC) to a standard light PRC. In both cases, the pulses lasted for 30 min, either with saturating light or with a shift from 25 °C to 37 °C. We used a modified PRC protocol, called the anchored PRC (APRC; [32–34]: the pulses are applied to wild-type flies during the night half of a light-dark cycle (zeitgeber time [ZT]12–24) and then during the first 12 h of the subsequent “day” in constant darkness (circadian time [CT]0–12). Locomotor activity phases were then measured after several subsequent days in constant darkness. A typical PRC was obtained for light, with maximum phase delays of about 3.5 h in the early night and maximum phase advances of about 2.5 h in the late night. For heat, early night delays were more modest, about 2.5 h, whereas late night advances were very small or absent (Figures 1A and S1). The data are essentially indistinguishable from the only published examples of 37 °C heat pulse–mediated phase shifts in Drosophila [35,36]. Moreover, there was little or no behavioral phase shift after 30 °C or 34 °C heat pulses (Figure 1A) [36]. Figure 1 Heat Pulse–Mediated Phase Delays of Wild-Type Flies Require 37 °C Phase response curves for CS flies after heat (circle) versus light pulse (square). Flies were entrained for 3 d in 12 h:12 h LD cycles and pulsed for 30 min of light and 30 °C, 34 °C, and 37 °C heat pulse (HP) during the last night of the LD entrainment cycle, after which the flies were released in constant darkness for 5 d. Phase changes were calculated by comparing behavioral offsets of light or HP treated flies 3 d after the pulse to the behavior of the control group of the same genotype that did not receive a pulse. The calculations were made by MATLAB software using previously described methods [46]. Phase delays and phase advances are plotted (± SEM) as negative and positive values respectively. In all cases, the experiments were repeated at least twice with similar results. Data were pooled from the following number of flies (each pair of values referring to wild-type light-pulsed and wild-type 37 °C heat-pulsed): control: 32, 32; pulse at ZT12: 32, 22; pulse at ZT15: 32, 26; pulse at ZT18: 23, 26; pulse at ZT21: 24, 19; and pulse at ZT24: 29, 27. CRY:PER-TIM Heat-Dependent Interactions Parallel the Behavioral Responses The weak heat-mediated delay and absence of a substantial advance makes it uncertain whether there is a relationship between the heat and light PRCs. We therefore assayed the biochemical effects of a heat pulse and compared them to those of a light pulse. The strategy was based on the interaction of CRY with TIM and/or PER, which is a light-dependent event (e.g., [20]). There is also substantial evidence that these events are crucial to clock resetting after short light pulses [19,37,38]. To assay CRY interactions in flies, we used a previously described strain that expresses N-terminal MYC-tagged CRY [20] . We subjected flies to either light or heat pulses and then assayed CRY complexes via immunoprecipitation with anti-MYC anti-sera. Remarkably, an interaction between CRY and PER-TIM was observed at ZT15 after a 37 °C heat pulse as well as after a light pulse. There was no detectable interaction if the ZT15 heat pulse was at 30 °C (Figure 2A), nor was there a robust 37 °C heat-mediated interaction at ZT21, despite a canonical light-mediated interaction at this time (Figure 2B). These results mirror the behavioral observations, namely, a 37 °C phase shift and no 30 °C phase shift at ZT15, with no 37 °C phase shift at ZT21 (Figure 1) [35]. The data indicate that a CRY:PER-TIM interaction correlates with heat-mediated phase shifts and suggest that it might underlie the behavioral phase shifts. Figure 2 CRY Interacts with PER/TIM in a 37 °C Heat Pulse–Dependent Manner at ZT15 but Not at ZT21 Heat- and light-dependent interactions among CRY, TIM, and PER were measured three times with similar results. (A) TMC flies (Myc-CRY) were subjected to standard 12:12 LD conditions referred as control, pulsed with 37 °C (2), pulsed with 30 °C (3), light-pulsed (4), or not (1) for 30 min at ZT15, collected, and frozen. Head extracts (HE) were immunoprecipitated with antibody to MYC (IP), all as previously described [20]. CRY, PER, and TIM levels were measured by Western blotting. (B) Exactly as above but pulses were at ZT21. CRY Is Required for Heat-Mediated Phase Shifts These results predict that heat PRCs should be affected in the severe loss-of-function mutant cryb. Indeed, these flies show little to no response to a heat pulse, i.e., an almost flat PRC (Figure 3A). The results are very similar to those observed for a light PRC in cryb [14]. Figure 3 37 °C Heat Pulse–Mediated Phase Responses of cryb and Rescued Strains (A) Phase response curves for wild-type (circle) flies and cryb (square) mutant flies. The experiment was performed as described in Figure 1. Phase delays and phase advances are plotted (± SEM) as negative and positive values, respectively. Data were pooled from the following number of flies (each pair of values referring to wild-type heat pulsed and cryb heat pulsed): control: 32, 32; pulse at ZT12: 22, 39; pulse at ZT15: 26, 40; pulse at ZT18: 26, 39; pulse at ZT21: 19, 36; and pulse at ZT24: 27, 15. (B and C). Bottom left and right panels show the phase changes observed at ZT15 (B) and ZT21(C), respectively. On the x-axis, the Zeitgeber 37 °C heat (HP) or light pulse (LP) is indicated. Phase delays and advances are described in Figure 1 and plotted on the y-axis (± SEM) as negative and positive values, respectively. The genotype of the flies is indicated on the x-axes: the first row shows the transgenes present (plus sign corresponds to a chromosome without a transgene), whereas the second row indicates the genetic background (wild-type [WT] or cryb). To verify that this result is not due to a strain differences unrelated to the cry locus, we rescued the cryb mutation by expressing CRY in clock-pacemaker cells using pdf-GAL4 [13,39,40]. pdf-GAL4–mediated CRY expression partially rescued the cryb heat delay at ZT15 (Figure 3B) as well as the cryb light delay as previously described [13]. We also compared the response of these strains to heat and light pulses at ZT21 (Figure 3C). As predicted from the wild-type heat PRC pattern (Figure 3A), the addition of pdf-GAL4–mediated CRY expression to the cryb background had no effect on the essentially nonexistent heat-phase shift at ZT21, whereas it rescued the cryb ZT21 light-phase shift (Figure 3C) [13]. In contrast, tim-GAL4–mediated CRY-B expression was unable to rescue either light- or heat-mediated cryb phase shifts (Figure S2), consistent with the strong hypomorphic cryb mutation. Taken together with the heat-mediated physical interaction between CRY and PER-TIM (Figure 2), the results indicate that CRY is important for circadian clock heat responses as well as light responses. perL Flies Are Hypersensitive to Heat The perL genotype shows aberrant temperature compensation, with dramatically increased periods at elevated constant temperatures [41,42]. We speculated that this phenomenon might be related to heat-pulse responses and even light pulse–mediated phase shifts. To examine this possibility, we first assayed a standard light PRC of perL flies. It is very similar to that for wild-type flies, except that the perL curve is delayed by several hours (Figure 4A) [34]. There are essentially indistinguishable phase delays 18 h after the last DL (dark-light) transition for perL flies and 15 h after the last DL transition for wild-type flies. Moreover, there are similar phase advances, about 26 h after the last DL transition in perL and 21 h after the last DL transition in wild-type (compare Figure 4A with Figure 1). Figure 4 perL Is Hypersensitive to Heat Pulses Phase response curves are shown for perL flies, after a heat pulse at 37 °C (square), after a 30 °C heat pulse (diamond) or after a light pulse (circle). The experiment was performed as described in Figure 1A and repeated twice with similar results. Phase delays and phase advances are plotted (+/- SEM) as negative and positive values, respectively. Data were pooled from the following number of flies (each set of values referring to perL light pulsed, heat pulsed at 30 °C or 37 °C): control: 32, 32, 32; pulse at ZT18: 26, 32, 29; pulse at ZT21: 19, 32, 31; pulse at ZT24: 27, 31, 27; pulse at CT02: 30, 31, 22; pulse at CT04: 32, 29, 21. (B) perL; TMC flies (see above) were entrained to 12:12 light:dark conditions and heat pulsed at 37 °C (2) heat pulsed at 30 °C (3), light pulsed (LP) (4) or not (1) for 30 min all at CT02, collected, and frozen. CRY, PER and TIM levels were measured by Western blotting after anti-MYC immunoprecipitation (IP) from head extracts (HE). Heat- and light-dependent interactions among CRY, TIM, and PER were assayed three times with essentially indistinguishable results. Consistent with the notion that perL flies are more heat sensitive than wild-type flies, there is essentially no difference between the perL heat and light PRCs in the delay zone (Figure 4A), in contrast to the magnitude of the wild-type heat-mediated delay, which is clearly less than that of the wild-type light-mediated delay (Figure 1) [35]. Even more impressive is the heat-mediated advance for perL flies, which is indistinguishable from the light-mediated maximal advance (Figure 4A); there is little or no heat-mediated advance in wild-type flies (Figure 1). Finally, perL flies are sensitive to a 30 °C heat pulse, whereas wild-type flies are insensitive even to a 34 °C pulse (Figures 1 and 4B) [36]. The heat-mediated phase advance of perL flies suggested that there might be an interaction between CRY and PERL-TIM at these times, e.g., at CT2 (ZT26 = CT2). Indeed, we confirmed such an interaction after a 30 °C as well as a 37 °C heat pulse (Figure 4B). With minor differences, the interaction was similar to that elicited by a light pulse at this same time, and no interaction was observed without a heat or a light pulse (Figure 4B). There is no detectable heat-mediated interaction between CRY and wild-type PER-TIM in the advance zone or at 30 °C (Figures 1 and 2), i.e., the interactions between CRY and PERL-TIM correlate well with the behavioral observations (Figure 4A) and further indicate that they are important for the observed heat-mediated phase shifts. perL Heat-Mediated Phase Shifts, CRY, and Temperature Compensation To verify that the CRY:PERL-TIM interaction is functionally relevant, we generated perL; cryb double mutant flies. They have a long free running period of ~28 h, characteristic of perL, and are rhythmic in light-light (LL), characteristic of cryb (Figure 5A). These flies also show much smaller phase shifts in response to 37 °C heat pulses in the delay zone at ZT18 as well as in the advance zone at ZT26 (ZT26 = CT2; Figure 5B). The exaggerated perL heat-mediated phase shifts are therefore CRY dependent. Figure 5 perL; cryb Flies Show Reduced Heat Phase Shifts and Better Temperature Compensation (A) Behavior in DD and LL cycles was monitored for per L and per L; cryb flies. The data were collected at a constant temperature of 25 °C. The average activity plots for 16 flies are shown. Adult male flies of 1–3 d old were entrained for 3 d (12 h: 12 h LD) and released in DD (as indicated by shading throughout the actogram in the left columns for each genotype) for 6 d followed by LL (as indicated by lack of shading throughout the actogram in the left columns for each genotype) for 4 d. Within each actogram, a given row shows two consecutive days of activity; the second such day is replotted in the left half of the next row down (thus, consecutive days of locomotion can be viewed both horizontally and vertically); heights of bars within a given actogram row reflect varying amounts of locomotion per half hour data collection bin. In the column next to the actograms, autocorrelation plots for these behavioral records are shown. The autocorrelation plot indicates rhythmicity and gives a measure of rhythm intensity (RI). In LL, the per L flies become arrhythmic after 1 d; per L; cryb remains rhythmic. The data were analyzed as described in Materials and Methods [46]. For details on autocorrelation, also see Material and Methods. (B) Phase response to 37 °C heat pulse in per L and per L; cryb flies. Left and right panels show the phase changes observed at ZT18 and CT02, respectively. On the x-axis, the Zeitgeber 37 °C heat pulse is shown. Phase delays and advances are calculated as described in Figure 1A and are plotted on the y-axis (± SEM) as negative and positive values, respectively. For each genotype, an average phase shift from 15–32 flies is shown. (C) Period in hours was calculated for cryb (circle and blue), per L (squares and red), or per L; cryb (diamonds and yellow) at 15 °C, 18 °C, 25 °C, and 29 °C in constant darkness. The average period is determined from three independent experiments. For each experiment, the average period was calculated using MESA for individual flies and then combined to obtain an average period length. For details on MESA, see Materials and Methods section. The average period (T) and SEM of the period (in hours) are as follows: 15 °C cryb (23, 0.5), 15 °C per L (24.8, 0.5), 15 °C per L; cryb (27.2, 0.5), 18 °C cryb (23.3, 0.58), 18 °C per L (26.9, 1.2), 18 °C per L; cryb (27.5, 0.6), 25 °C cryb (23.3, 0.3), 25 °C per L (29.02, 0.2), 25 °C per L; cryb (27.8, 0.7). 29 °C cryb (23.3, 0.5), 29 °C per L (31.7, 0.5), 29 °C per L; cryb (28, 0.5). Period lengths for the three genotypes at a given temperatures were found to be significantly different using analysis of variance (ANOVA) (p < 0.0001). Finally, to establish a link between the exaggerated heat-mediated phase shifts and the temperature compensation defect of perL flies, we assayed the free-running period of perL; cryb double mutant flies at constant temperatures (Figure 5C). The results indicate that this genotype shows much less period change with temperature, in striking contrast to perL flies. This indicates that a temperature-sensitive CRY:PERL-TIM interaction underlies most of the perL loss of temperature compensation. It also connects the free-running period phenotype assayed at constant temperatures with the response to a heat pulse. Indeed, there is also a CRY:PERL-TIM interaction after incubation of perL flies at a constant temperature of 29 °C (Figure S3). Moreover, the fact that the perL strain has an altered period compared to the perL; cryb double mutant strain at 15 °C (Figure 5C) suggests that even at low temperatures, the PERL-TIM complex interacts with CRY. We suggest that advances predominate (an aggregate shorter period) at 15 °C, whereas delays predominate (an aggregate longer period) at temperatures ≥ 25 °C. A Model These data suggest that the perL missense mutation facilitates a PER-TIM conformational change (Figure 6A; 1 → 2). Heat facilitates the same change in wild-type PER-TIM, although higher temperatures are required and a smaller fraction of PER-TIM is affected. If CRY interacts predominantly with TIM, then the per mutation and heat must also help promote a TIM conformational change (Figure 6A; 3). We imagine that this altered PER-TIM conformation could also facilitate an interaction with active CRY, which is a conformational state similar to that promoted by illumination, i.e. by CRY photon capture (Figure 6A; CRY). The key phase-shifting complex can then be promoted by increasing the concentration of either component, activated PER-TIM by temperature/mutation or activated CRY by light (Figure 6A; 2–3 or CRY, respectively). Importantly, a temperature-sensitive PERL-TIM complex is consistent with a slightly longer average period (~1 h) of the perL; cryb strain at 29 °C relative to 15 °C (Figure 5C). Figure 6 CRY Links Temperature and Light Responses (A) Model picturing the role of CRY in heat-mediated phase shifts. See text for details. (B) Wild-type flies are more rhythmic in constant light at lower temperature. Behavioral LL cycles of wild-type flies were monitored. The data were collected at a constant temperature of 25 °C (lower panels) or 15 °C (top panels). Average activity plots are shown. For details on autocorrelation and the actogram, see Figure 5 legend. Adult male 1–3-d-old wild-type flies were entrained for 3 d (12 h: 12 h LD) followed by 6 d of LL as indicated on top of each column, either for 10 lux (25 °C, n = 26 flies and %R = 23; 15 °C, n = 26 and %R = 65) or 100 lux (25 °C, n = 27 and %R = 18; 15 °C, n = 29 and %R = 50). Only the LL data from days 2–6 are shown. n, number of flies analyzed; %R, percentage of rhythmic flies. Light and Temperature Can Act Together on Wild-Type Fly Rhythms These observations suggest that even in wild-type flies, temperature and light can synergize to affect CRY:PER-TIM complex formation at physiologically normal temperatures. To test this hypothesis, we subjected Canton-S (CS) flies to constant illumination at 10 and 100 lux. Constant light even at low intensities render most flies arrhythmic at a standard incubation temperature of 25 °C (Figure 6B) [41], and constant light arrhythmicity requires CRY [15]. The results and model (Figure 6A) suggest that low temperatures might reduce complex formation and arrhythmicity, and constant light arrhythmicity has not been assayed at 15 °C. Indeed, we observed substantially larger numbers of arrhythmic flies at 25 °C than at 15 °C, at 100 as well as at 10 lux of constant light (Figure 6B). We interpret the result to indicate more CRY:PER-TIM complex formation at 25 °C than at 15 °C, indicating that light and temperature can act together in wild-type flies at physiologically relevant temperatures. The convergence induces phase shifts as well as causes arrhythmicity in constant light. We speculate that it also serves to fine tune the dawn and dusk response of the clock when light and temperature increase and decrease together. Discussion We show here that the photoreceptor CRY and its interaction with the PER-TIM complex is critical for heat shock–mediated phase shifts as well as for the loss of temperature compensation in the perL mutant strain. Heat-induced phase delays take place in a wild-type strain, and they are severely reduced in the cry loss-of-function mutant cryb. Moreover, there is a physical interaction between CRY and PER-TIM at circadian times that correspond to phase delays. More robust heat-mediated phase delays and even phase advances occur in perL mutant flies. The perL behavioral results are mirrored by CRY:PERL-TIM interactions, which occur in the advance zone and also in response to 30 °C temperature pulses. perL phase shifts like wild-type phase shifts are severely reduced by the addition of cryb to the perL background. These perL; cryb double mutant flies also have largely restored temperature compensation, indicating that an interaction between CRY and PER-TIM is responsible for the loss of temperature compensation in the perL strain as well as for heat-mediated phase shifts of wild-type as well as perL flies. The similarity between heat-mediated and light-mediated phase shifts suggests that light and temperature can synergize to cause phase shifts, and an experiment in wild-type flies supports this notion. The temperature-induced complex formation between CRY and PER-TIM parallels the substantial evidence that a similar interaction is critical for light-mediated phase shifts. Biochemical as well as genetic data indicate that complex formation between light-activated CRY and TIM, or between light-activated CRY and PER-TIM, leads to TIM degradation, which is believed to advance or delay the clock (e.g., [25]). Although some data indicate a physical interaction between CRY and PER, most observations indicate that physical contact is predominantly between CRY and TIM; for example, PER usually requires the presence of TIM to interact with CRY, but a TIM:CRY interaction can take place without PER (e.g., [20]). Because much of TIM is in complex with PER, especially in the early night [22], a CRY-TIM interaction is effectively a CRY:PER-TIM interaction. All of this begins with CRY photon capture, which activates CRY by causing a conformational change and a subsequent interaction with PER-TIM. Indeed, experimental studies on Drosophila CRY as well on other related proteins provide a coherent view of a CRY-centric light-initiation event [10]. Although a connection between light pulse– and heat pulse–initiated interactions appeared enigmatic, previous studies in wild-type flies suggested that heat phase shifts are like light pulses and are due to posttranscriptional events that influence PER and/or TIM [36]. The failure to elicit a phase shift with a 34 °C pulse (Figure 1) indicates that a heat shock may be required [43]. This is accompanied by numerous changes in cell physiology and gene expression, which could perturb the dynamics of an oscillatory system [44]. However, perL flies show robust phase shifts and CRY:PER-TIM complex formation after a 30 °C heat pulse, making it unlikely that a heat-shock response is generally required for heat pulse–mediated phase shifts in Drosophila. Extrapolation to wild-type flies makes two assumptions: (i) perL flies do not have an unprecedented heat-shock response triggered at much lower temperatures and (ii) the failure to observe 30 °C behavioral phase shifts and biochemical interactions in wild-type reflects quantitative rather than a qualitative differences between 30 °C and 37 °C and between wild-type and perL genotypes. Indeed, the convergence of light and temperature on wild-type fly behavior at physiological temperatures (Figure 6B) suggests that these CRY:PER-TIM interactions are normally difficult to detect at lower temperatures, because they are quantitatively minor. The perL behavioral and biochemical results indicate that the missense mutation causes a large increase in the fraction of PERL-TIM interacting with CRY at normal temperatures (Figure 4B). This suggests that the PERL-TIM structure is temperature sensitive (Figure 6A), an interpretation consistent with the period of the perL; cryb double mutant strain being somewhat temperature sensitive (Figure 5C; see below). Moreover, this strain has a substantially longer period than the perL single mutant strain at 15 °C (Figure 5C), suggesting that PERL-TIM manifests an enhanced interaction with CRY at all physiologically relevant temperatures. These experiments cannot definitively rule out CRY as the temperature-sensitive component; in this case, the perL mutation would only cause an increased interaction between PER-TIM and CRY. In either case, the close correspondence between the 37 °C perL heat and light PRCs (Figure 4A) indicates that the CRY photocycle is inessential for CRY:PER-TIM interactions and behavioral phase shifts in Drosophila. We speculate that heat activation of PER-TIM causes the same CRY conformational change as does light—albeit indirectly (Figure 6A). The heat-induced interactions between PERL-TIM and CRY as well as the perL; cryb phenotype make a strong link between the circadian response to temperature pulses and incubations at constant temperatures, analogous to nonparametric and parametric light entrainment, respectively. This is because a persistent CRY:PERL-TIM interaction affects the perL period like the enhanced phase-shift response of perL to a heat pulse. This recalls the hypersensitivity of perL to incubation at constant low light intensities, which lengthen the perL period more severely and at lower intensities than is required to lengthen wild-type periods [41]. Our results explain this observation and suggest that the more CRY-interactive PERL-TIM requires less CRY light activation than does wild-type PER-TIM. Moreover, the similarities between light and heat inspired the experiment suggesting that light and temperature function together, even on wild-type flies (Figure 6B). This synergy might fine-tune the dawn and dusk response of the clock and even contribute to seasonal adaptation of clock function [45]. The circadian problem of temperature compensation has gained little traction since the discovery more than 15 y ago that the per missense mutants manifest aberrant temperature compensation [41]. Our results here suggest that the timSL allele suppresses the temperature compensation defect of perL by failing to interact with CRY [42]. The observations suggest that the same PERL-TIM structure that facilitates a CRY interaction in response to a phase-shifting perturbation (heat- or light-mediated CRY activation) keeps time in a temperature-sensitive manner under constant conditions. Characterization of this altered PERL-TIM structure is an important goal for the near future. Materials and Methods Drosophila genetics. Wild-type CS, perL, and cryb flies were used for average activity and phase response analyses (see below) and as controls for the locomotor activity analyses. The perL mutation was combined with cryb to generate perL; cryb flies. The pdf-GAL4 and UAS-cry transgenic flies have been described previously [13]. The y w; tim-GAL4 UAS-myccry/CyO line (TMC) was previously described [20]. The TMC transgenes were introduced into perL to obtain perL; tim-GAL4 UAS-myccry (abbreviated as perL; TMC). The UAS-cry and pdf-GAL4 transgenes were introduced in cryb backgrounds to produce y w; pdf-GAL4/UAS-cry; cryb flies. Phase shift protocol and behavioral analysis. In all experiments unless stated otherwise, CS males were collected at 1–3 d old and reared in LD 12:12 at 25 °C for 3 d. In the APRC protocol, flies were given a 10-min saturating white light pulse (2000 lux) during the third dark phase of the cycle, at the indicated times during the night and the following subjective day. A separate control group of flies was not given a pulse. Flies were then put into constant darkness for another 5 d. For the heat pulse PRCs, flies were placed in activity monitors in LD 12:12 at 25 °C for 3 d. During the third dark phase of the cycle, one monitor of untreated flies was retained as a control. For the heat treatment, behavior tubes containing flies were removed from the monitors, held upright, and an elastic band placed around them to hold them tightly together. The entire package was then placed in a 50-ml conical tube, so that the tubes would stay upright and be in a water-tight environment but small enough for efficient heat transfer from the water bath to the tubes. The top of the activity tubes were always an inch below the top of the 50-ml conical tube, so the water level would be above the tubes. Incubation was in the water bath for 30 min at 37 °C. The 50-ml tube was then removed, and the behavior tubes placed back in the monitors. Each tube had been marked on the top with a number and then placed back in the same monitor channel. A second control set of flies was handled identically except that they were just kept upright (with the elastic band) in 50-ml tubes in the incubator but not placed in a water bath. In all cases, the experiments were repeated at least twice with essentially identical results. For each genotype an average phase shift from 15–32 flies is shown. Locomotor activities of individual flies were monitored using Trikinetics Drosophila activity monitors (TriKinetics Inc, Waltham, Massachusetts, United States). The analysis was done with a signal processing toolbox implemented in MATLAB (Mathworks; http://www.mathworks.com) as described [46]. Autocorrelation it is a measure of how well a signal matches a time-shifted version of itself as a function of the amount of time shift. In our analysis, autocorrelation and spectral analysis were used to assess rhythmicity and to estimate period. The phase information was obtained with circular statistics [46]. The column in Figure 5A labeled autocorrelation shows correlograms for the data. Correlation coefficients are plotted on the ordinate with a range of values from −1 to 1. The gray region centered around 0 describes a 95% confidence interval. The lag of the autocorrelation function is plotted on the abscissa. An asterisk is placed above the third peak of the autocorrelation function. The value at that point defines the rhythmicity index (RI), an estimate of the strength of rhythmicity. When the asterisk is not present, the autocorrelation function indicates a lack of rhythmicity. Values for the RI appear in the lower left corner of these plots along with a related number called the rhythmicity statistic (RS). The RS value is the ratio of the RI to the absolute value of the confidence line. This metric indicates that the rhythmicity described by the correlogram is statistically significant when the value is ≥ 1 [46] . The MESA analysis is a spectral analysis of the data that provides an estimate of period. Spectral density is given in arbitrary units on the ordinate, and the range of assessed periods is shown on the abscissa. Asterisks are placed over the highest peak shown in a range between 18–30 h. Although this value is generally taken as the estimate of circadian period, there may be other periodicities present within the horizontal range (the width) of the peak or elsewhere on the plot, and these additional rhythmic components are also present in the data. Absence of an asterisk indicates either the absence of a peak or that a peak within the plot occurs outside the circadian range. Note that the autocorrelation plot is used to determine rhythmicity, and mesa is used to provide an estimate of the period only when warranted by correlogram [46]. Immunoprecipitation. About 250 adult flies were entrained to a 12-h-light: 12-h-dark cycle for 3 d. At ZT15, ZT21, or CT02, they were pulsed with bright white light for 15 min and 30 °C or 37 °C for 30 min before being collected and frozen. Head extracts were prepared and homogenized in Extraction Buffer (20 mM Hepes, pH 7.5, 100 mM KCl, 1mM Dithiothreitol, 5% glycerol, 0.05% Nonidet P40, 1× Complete Protease Inhibitor [Roche; http://www.roche.com]). Protein G sepharose fast flow beads (Amersham; http://www.amersham.com) were coated with anti-MYC antibody (2 μl; Santa Cruz Biotechnologies; clone 9E10; http://www.scbt.com] plus 20 μl beads/sample) for 1 h. The beads were then washed twice and incubated with the head extracts for 4 h at 4 °C. Pulled-down beads were washed four times with 750 μl extraction buffer before being resuspended in 40 μl 1× SDS loading buffer for Western blot analysis. Head homogenization, incubation, and immunoprecipitation for the light-pulsed samples were done under normal laboratory lighting, whereas the nonpulsed and heat-pulsed samples were processed under red light (700 nm) and incubated in the dark. Protein extracts and Western blots. Fly heads extracts were prepared and Western blots were performed as described [22]. Equal loading and quality of protein transfer were first verified by Ponceau Red staining and then by the intensity of cross-reacting bands on the Western blots, or by reprobing the membrane with a monoclonal α-tubulin antibody (clone DM1A, Sigma, 1:1000 dilution; http://www.sigmaaldrich.com). The anti-CRY rabbit antibody was used at 1:500 dilution [47]. The anti-PER antibody is previously described and used at 1:1500 dilution, whereas the anti-TIM antibody was made in rat and used at 1:3000 dilution [22]. Supporting Information Figure S1 37 °C Heat Pulses Result in Robust Phase Shifts of Wild-Type and perL Flies but Not of cryb or perL; cryb Flies (A) Circular analysis figures of locomotor behavior in wild-type CS and cryb flies after a 37 °C heat pulse (HP). (B) The same circular analyses for perL and perL; cryb after a 37 °C HP. On these plots, time moves forward in a counter-clockwise direction. The behavioral phase estimates for each rhythmic specimen are plotted just outside the unit circle and a mean vector summarizes the phase of the group. The direction of the vector indicates the behavioral phase, whereas its length reflects the dispersion (variability) of the individual estimates (see [46] for more details). The Rayleigh's test was used to determine whether each vector is significantly different (p < 0.05) from the null vector (random distribution). Then, the Watson-Williams-Stevens test was used to obtain an F-statistic that determined whether the two vectors obtained from the nonpulsed control and the experimental group of flies are significantly different, (p < 0.01) Statistically significant differences were found for CS flies at ZT12 and ZT15 and for perL at ZT18 and CT2. cryb and perL; cryb flies did not significantly shift their phase at any time points. For the estimates of the phase differences see Figure 1. (5.0 MB TIF) Click here for additional data file. Figure S2 Functional CRY Is Required for HP-Mediated Phase Delays Top and bottom panels show the phase changes observed at ZT15 (A) and ZT21(B), respectively. On the x-axis, the zeitgeber 37 °C HP or light pulse (LP) is indicated. Phase delays and advances are described in Figure 1A and plotted on the y-axis (± SEM) as negative and positive values, respectively. The genotype of the flies is indicated on the x-axis: the first row shows the transgenes present (a plus sign corresponds to a chromosome without a transgene), whereas the second row indicates the genetic background (wild-type [WT] or cryb). (1.2 MB TIF) Click here for additional data file. Figure S3 CRY Forms a Complex with PERL/TIM at a Constant Temperature of 29 °C but Not with Wild-Type PER/TIM (A) TMC flies (MYCCRY); per+ (PER+), and TMC flies (Myc-CRY); perL (PER-L) were subjected to standard 12:12 light:dark conditions at 29 °C. Samples were collected at either ZT15 (lane 1) or ZT21(lane 2) for PER+ flies and at ZT18 (lane 3) and CT02 (lane 4) for PER-L and frozen. Head extracts (HE) were immunoprecipitated with antibody to MYC (IP), all as previously described [20]. CRY, PER, and TIM levels were measured by Western blotting. These heat-dependent interactions among CRY, TIM, and PER were measured twice with similar results. (2.6 MB TIF) Click here for additional data file. We thank Jose Agosto for help with the MATLAB analysis; Dan Stoleru, Emi Nagoshi, Jerome Menet, Jose Agosto, Rebecca Schoer, and Kavita Babu for inspiration and helpful discussions; and Ravi Allada for critical readings of the manuscript. We also thank Heather Felton, Krissy Palm, and Shawn Jennings for administrative assistance. ¤ Current address: Department of Biological Sciences, Columbia University, New York, New York, United States of America Author contributions. RK, PN, and MR conceived and designed the experiments. RK and PN performed the experiments. AB contributed to experiments in Figure 6. RK, PN and MR analyzed the data. RK, PN, AM, PE, and MR contributed reagents /materials /analysis tools. RK and MR wrote the paper. Funding. The work was supported in part by NIH grants to MR (R01 GM66778; P01 NS44232; P30 NS45713) and to PE (R01 GM066777). Competing interests. The authors have declared that no competing interests exist. 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==== Front PPAR ResPPAR ResPPARPPAR Research1687-47571687-4765Hindawi Publishing Corporation 1771011010.1155/2007/48242Research ArticlePPARs Expression in Adult Mouse Neural Stem Cells: Modulation of PPARs during Astroglial Differentiaton of NSC Cimini A. Cristiano L. Benedetti E. D'Angelo B. Cerù M. P. Department of Basic and Applied Biology, University of L'Aquila, 67100 L'Aquila, ItalyA. Cimini: [email protected] by Jeffrey M. Gimble 2007 10 7 2007 2007 482421 3 2007 1 4 2007 Copyright © 2007 A. Cimini et al.2007This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.PPAR isotypes are involved in the regulation of cell proliferation, death, and differentiation, with different roles and mechanisms depending on the specific isotype and ligand and on the differentiated, undifferentiated, or transformed status of the cell. Differentiation stimuli are integrated by key transcription factors which regulate specific sets of specialized genes to allow proliferative cells to exit the cell cycle and acquire specialized functions. The main differentiation programs known to be controlled by PPARs both during development and in the adult are placental differentiation, adipogenesis, osteoblast differentiation, skin differentiation, and gut differentiation. PPARs may also be involved in the differentiation of macrophages, brain, and breast. However, their functions in this cell type and organs still awaits further elucidation. PPARs may be involved in cell proliferation and differentiation processes of neural stem cells (NSC). To this aim, in this work the expression of the three PPAR isotypes and RXRs in NSC has been investigated. ==== Body 1. INTRODUCTION Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily [1]. After the isolation of PPARα (NR1C1) as the receptor mediating peroxisome proliferation in rodent hepatocytes in 1990 [2], two related isotypes, PPARβ/δ (NR1C2; referred to as PPARβ herein) and PPARγ (NR1C3), have been characterized [3]. PPARs exhibit a broad but isotype-specific tissue expression pattern which can account for the variety of cellular functions they regulate. PPARα is expressed in tissues with high fatty acid catabolism such as the liver, the heart, the brown adipose tissue, the kidney, and the intestine. The two PPARγ isoforms γ1 and γ2 act in the white and brown adipose tissues to promote adipocyte differentiation and lipid storage [4] while only the expression of PPARγ1 extends to other tissues such as the gut or immune cells. PPARβ has a broad expression being detected in all tested tissues but important functions have been assigned to this isotype in the skeletal muscle, the adipose tissue, the skin, the gut, and the brain. PPARs are sensors capable of adapting gene expression to integrate various lipid signals. The diversity of functions in which they are implicated is also reflected by the diversity of ligands that can be accommodated within their ligand binding pocket. Indeed, PPARs are activated by a wide range of naturally occurring or metabolized lipids derived from the diet or from intracellular signaling pathways, which include saturated and unsaturated fatty acids and fatty acid derivatives such as prostaglandins and leukotriens [5, 6]. In contrast to steroid hormone receptors which act as homodimers, PPARs activate the transcription of their target genes as heterodimers with retinoid X receptors (RXR, NR2B) [7, 8]. The three RXR isotypes (α, β, and γ) can dimerize with PPARs, and specific association with each isotype seems to influence the recognition of target gene promoters [9]. However, very little is known on the specificity of RXR isotype utilized by the different PPARs in vivo. The observation that 9-cis retinoic acid and synthetic RXR agonists can promote the transcription of PPAR target genes leads to a model of permissive transcriptional activation where PPAR/RXR heterodimers can induce transcription in response to PPAR or RXR activation [10, 11]. Moreover, concomitant treatment with both PPAR and RXR agonists potentiates the effects observed with each ligand alone. However, the molecular mechanisms underlying transcriptional permissivity and synergy are not well understood in terms of cofactor recruitment by each partner of the heterodimer. Finally, the interplay between PPAR and RXR pathways is further illustrated by PPAR target gene activation in response to RXR homodimers [12]. Cellular proliferation allows the renewal of tissues by providing a pool of undifferentiated cells or progenitors from stem cells. All three PPAR isotypes are involved in the regulation of cell proliferation, death, and differentiation, with different roles and mechanisms depending on the specific isotype and ligand and on the differentiated, undifferentiated, or transformed status of the cell. Thus, proliferative and antiapoptotic or antiproliferative, prodifferentiating and proapoptotic effects, and even procarcinogenic effects have been reported for PPARs [13]. Differentiation stimuli are integrated by key transcription factors which regulate specific sets of specialized genes to allow proliferative cells to exit the cell cycle and acquire specialized functions. The main differentiation programs known to be controlled by PPARs both during development and in the adult are placental differentiation, adipogenesis, osteoblast differentiation, skin differentiation, and gut differentiation. PPARs may also be involved in the differentiation of macrophages, brain, and breast [14]. However, their functions in this cell type and organs still await further elucidation. In astroglial cells, we have demonstrated the involvement of PPARα in astrocytic differentiation [14]. The expression of PPARβ in the brain peaks between days 13.5 and 15.5 of rat embryonic development [15]. The role of PPARβ in the development of the central nervous system is further illustrated by the myelination defects of the corpus callosum observed in PPARβ null mice [16]. However, the outputs in terms of brain development and the mechanisms regulating the potential implication of PPARβ in the differentiation of cerebral cells are unknown. Recently we have demonstrated that PPARβ expression and activation are increased during neuronal in vitro maturation, thus suggesting a role for this transcription factor in this process [17]. Moreover, we have demonstrated that PPARβ agonists trigger neuronal differentiation in a human neuroblastoma cell line [18]. Very recently we found that PPARβ activation by the synthetic agonist GW0742 leads to early neuronal maturation and BDNF increase, thus suggesting a role for PPARβ in neuronal plasticity (Benedetti et al., manuscript in preparation). On the basis of the previous evidences, we hypothesize that PPARs may be involved in cell proliferation and differentiation processes of neural stem cells (NSC). To this aim, the expression of the three PPAR isotypes and RXRs in NSC has been investigated. 2. MATERIALS AND METHODS 2.1. Materials CD1 mice were from Charles River (Harlan, Lecco, Italy); fetal bovine serum (FBS) and Earl's balanced salt solution (EBSS) were obtained from Invitrogen SRL (Milan, Italy); papain was from Worthington Biochemical (Lakewood, NJ, USA); the culture media was a kind gift of Dr Rosella Galli SCRI-DIBIT (Milan, Italy); EGF and bFGF were from Peprotech (Rocky Hill, NJ, USA); matrigel basement membrane matrix-GFR was from Becton Dickinson (Lincoln Park, NJ, USA); BCA protein detection kit from Pierce (Rockford, Ill, USA); antinestin (RAT 401) antibody was from Developmental Studies Hybridoma Bank (DSHB) (University of Iowa, Iowa City, Iowa, USA); mouse anti-PLP and-A2B5 antibodies were from Chemicon International Inc. (Temecula, Calif, USA); mouse anti-β-tubulin III antibody was from Promega (Mannheim, Germany); rabbit polyclonal anti-PPAR α, β/δ, γ antibodies were both from Affinity Bioreagents Inc. (Golden, Colo, USA) and from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif, USA); ECL kit was from Amersham Life Sciences (Little Chalfont, Buckinghamshire, UK); vectashield mounting medium from Vector Laboratories (Burlingame, Calif, USA); trizol reagent and platinum Taq DNA polymerase were from Invitrogen. Kit Gene Specific Relative RT-PCR was from Ambion (Austin, Tex, USA). All other chemicals were from Sigma Aldrich (St. Louis, Mo, USA). 2.2. Primary culture and culture propagation differentiation Adult CD1 Swiss-Albino mice were killed by cervical dislocation and their brains removed and placed into PBS with penicillin and streptomycin (0.1 mg/mL). The tissues containing the forebrain periventricular region SVZ were dissected and incubated in Earl's balanced salt solution (EBSS) containing papain (1 mg/mL), EDTA (0.2 mg/mL), and cystein (0.2 mg/mL) at 37°C for 1 hour. The pieces of tissue were collected by centrifugation at 200 g for 5 minutes and resuspended in 1 mL of the DMEM/ F12 containing 0.7 mg of ovomucoid inhibitor. The cells were dissociated using a fire-polished Pasteur pipette and were collected by centrifugation at 300 g for 5 minutes. The cellular pellets were resuspended in DMEM/F12 containing HEPES buffer (5 mM), glucose (0.6%), sodium bicarbonate (3 mM), L-glutamine (2 mM), insulin (25 mg/mL), putrescine (60 μM), apotransferrin (100 μM), progesterone (6.3 ng/mL), sodium selenite (5.2 ng/mL), heparin (2 μg/mL), EGF (20 ng/mL), and bFGF (10 ng/mL), counted and plated in uncoated 25 cm2 flask at 8 × 103 cells/cm2. Neurospheres were passaged by harvesting them by centrifugation (200 g for 5 minutes) and triturating them in 200 μL of medium with an automatic pipetter (P200 Gilson). 2.3. Differentiation of stem cell progeny and immunofluorescence For differentiation, neurospheres were plated onto Matrigel basement membrane matrix-coated (100 μg/mL) well in the medium described above with addition of FBS (10%) without EGF and bFGF for 5 days (S10). Indifferentiated (S0) and differentiated (S10) neurospheres grown on Matrigel GFR glass coverslips were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes at room temperature (RT) and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at RT. Nonspecific binding sites were blocked with 10% bovine serum albumin (BSA); in PBS, for 10 minutes at RT. This procedure was performed prior to incubation with primary antibodies, except when the A2B5 or the O4 mouse monoclonal antibodies were used. In this case, fixation followed incubation. For single immunofluorescent staining, cells were incubated with either of the following primary antibodies: 1:5 mouse monoclonal antinestin, 1:200 mouse monoclonal antiglial fibrillary acidic protein (GFAP), 1:300 mouse monoclonal anti-β-tubulin III, 1:30 mouse monoclonal PLP, 1:100 rabbit polyclonal anti-PPARα, β/δ, γ, and with 1:200 antimouse monoclonal A2B5 and O4 overnight at 4°C. All the slides were then incubated with fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG, antimouse IgG, or antimouse IgM antibodies (1:100), for 30 minutes at RT. Both primary and secondary antibodies were diluted with PBS containing 10% BSA. Controls were performed by substituting the primary antibody with PBS-BSA, containing or not rabbit nonimmune serum. Double immunofluorescence with anti-A2B5 and anti-GFAP antibodies was performed as described. Briefly, cells were first incubated with 1:100 anti-A2B5, then fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), and incubated with 1:100 secondary FITC-conjugated goat anti-IgM antibodies. Subsequently, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at RT and incubated with 1:200 mouse monoclonal antiglial fibrillary acidic protein (GFAP), followed by 1:100 secondary tetramethylrhodamine isothiocyanate (TRITC)-conjugated antirabbit IgG. The nuclei were stained with 0.5 μm/mL Hoechst 33258 diluted in each secondary antibodies mixture. Coverslips were mounted with Vectashield mounting medium and examined in a Zeiss Axioplan 2 fluorescence microscope. 2.4. Immunocytochemistry oil red O staining Indifferentiated (S0) and differentiated (S10) neurospheres grown on Matrigel GFR glass coverslips were fixed with 10% formaline in PBS for 10 minutes at room temperature (RT) and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at RT. Nonspecific binding sites were blocked with PBS containing 10% BSA for 30 minutes at RT. Immunocytochemistry staining was performed with mouse antinestin (1:5) and anti-GFAP 1:2000 in PBS containing 10% BSA for 1 hour at RT and then with peroxidase-conjugated antimouse IgG secondary antibodies (1:200 in PBS containing 10% BSA) for 30 minutes at RT; the immunoreactivity was detected with the 3,3′diaminobenzidine (DAB) reaction. Subsequently, the oil red O staining was performed by the method of Diascro et al. (1998), with minor modifications. Briefly, the cells were stained with 0.35% oil red O, for 1 hour at RT. The working solution of oil red O was prepared as described by Ramirez-Zacarias et al. [19]. After washing with distilled water, cells were counterstained with Mayer's hematoxylin and allowed to air dry. Coverslips were mounted with Kaiser's glycerol gelatin and observed with a Leitz Wetzlar Ortholux light microscope. 2.5. Protein detection For cell lysis, 107 cells were suspended in 150 μL of RIPA lysis buffer containing NaF [100 mM], Na4P2O7 [2 mM], Na3VO4 [2 mM], NP-40 [1%], SDS [0.1%], EDTA [5 mM], DOC [0.5%], protease inhibitor cocktail, in PBS 1x solution. The lysates were cleared by centrifugation at 12000 rpm for 20 minutes. Protein concentration was determined by BCA protein assay kit, using bovine serum albumin as a standard. Samples (20/50 μg protein) were run on 10%–15% polyacrylamide denaturing gels according to Laemmli [20]. Protein bands were transferred on polyvinylidene difluoride (PVDF) sheets by wet electrophoretic transfer according to Towbin et al. [21]. Nonspecific binding sites were blocked for 1 hour at room temperature with 5% nonfat dry milk in Tris-buffered saline containing 0.25% Tween 20 (TBS-T). Membranes were incubated with the primary antibody at the appropriate dilutions [1:50 for mouse antinestin, 1:1000 mouse anti-GFAP, 1:2000 rabbit antiactin, rabbit anti-PPARα, β, γ] overnight at +4°C in blocking solution, followed by incubation with HP-conjugated secondary antibody (antirabbit; antimouse), at the appropriate dilution (1:2000 in blocking solution), for 1 hour at 4°C. After rinsing, the specific immune complexes were detected by ECL method. Band relative densities were determined and normalized using a semiquantitative densitometric analysis and values are given as relative units. 2.6. RT-PCR Total cellular RNA was extracted by trizol reagent (Invitrogen) according to the manufacturer's instructions. The total RNA concentration was determined spectrophotometrically in RNAase-free water and 1 μg aliquots of total RNA were reverse transcribed into cDNA using Kit Gene Specific Relative RT-PCR. After RT 2 μL of the cDNA was used as template in 20 μL of PCR mixture and Taq platinum. The number of cycles was obtained empirically by sampling the PCR amplification of positive control between 22 and 40 cycles and selecting the approximate midpoint of a linear amplification. Table 1 reports primers sequences and amplification conditions for each gene studied. β-Actin was used as internal control and used for normalization. PCR products were separated by electrophoresis on 2% agarose gels containing ethidium bromide (0.5 μg/mL) in Tris-borate EDTA buffer. A molecular weight marker was run in parallel and bands of the expected molecular size were detected under UV light. The relative densities of the PCR fragments were determined and normalized using a semiquantitative densitometric analysis and values are given as relative units. 2.7. Statistics Statistical analysis for multiple comparisons was performed by one-way ANOVA followed by Scheffe's post hoc test. All statistical calculations were performed using SPSS software. P values <.05 were considered statistically significant. 3. RESULTS In Figure 1, contrast phase microscopy of neural stem cells growing in neurospheres (Figure 1(a)) and after BrdU incorporation (Figure 1(c)) are shown. Nuclear staining with Hoechst 33258 (Figure 1(b)) clearly shows that almost all cells appear positive for BrdU indicating that they are mitotic in our experimental conditions. Since the proliferation ability is not only exclusive of stem cells, but is shared with progenitors of different lineages, markers of indifferentiated status have also been investigated. The immunolocalization of nestin (Figure 2(b)) as compared with Hoechst nuclear staining (Figure 2(a)) shows that almost all cells are immunopositive for nestin, which is asymmetrically concentrated in the perinuclear region. Proteolipid protein (PLP) immunolocalization Figure 2(e), membrane protein of indifferentiated status, shows that almost all cells appear immunopositive for PLP (compare with Figure 2(d)). Only few cells are immunopositive for A2B5, marker of astroglial restricted precursors (Figure 2(c)). GFAP, β tubulin III, and O4, markers of astrocytes, neurons, and oligodendrocytes, respectively, are not expressed (not shown). Figure 3 shows the immunolocalization of the three PPAR isotypes in neurospheres. Nuclear staining of the same fields is shown in Figures 3(a), 3(b), and 3(c). All the three PPARs are present, almost exclusively localized in the nuclei. See Figures (3(b), 3(d), and 3(f)). Western blotting analysis for nestin, GFAP, PPARα, β, and γ, and RXRs in neurosphere cell lysates confirms the presence of the three PPARs and shows that the only RXR isotype detectable in these cells is the RXRβ (Figure 4). To assess the possible quantitative/qualitative variations of the receptors during differentiation, neurospheres were cultured in absence of growth factors and in the presence of 10% FBS for 5 days (S10). Figure 5 shows the immunolocalization of the above-mentioned differentiation markers in S10 neurospheres. Nestin is still expressed, but with lower Fuorescence intensity (Figures 5(a) and 5(b)). Moreover, the protein is no more concentrated in the perinuclear region, but unifromely localized throughout the cytoplasm, including the cellular processes; the number of A2B5 immunopositive cells appears slightly increased (Figures 5(b) and 5(e)), while a clear immunofluorescence for GFAP (Figures 5(c) and 5(f)) is observed in many S10 cells. β-Tubulin III and O4 are absent (not shown). These results demonstrated that, in our differentiating conditions, S10 neurospheres are mainly composed by differentiated astrocytes and their A2B5 precursors. In Figure 6, double immunofluorescence staining for GFAP and PPARs in S10 neurospheres is shown. In these cells the PPARs are still present but with different fluorescence intensity. In particular, PPARα immunostaining (Figure 6(a)) is stronger, while PPARβ appears weaker than in S0 neurospheres (Figure 6(b)); PPARγ appears unchanged (Figure 6(c)). Figure 7 shows the western blotting analysis for nestin, GFAP, PPARs, and RXRs in S0 and S10 neurosphere cell lysates. In S10 cells, nestin is significantly decreased, while GFAP is strongly expressed. Interestingly, RXRα, not present in S0 neurospheres, is now detected while RXRβ is unchanged. In agreement with the immunofluorescence data, PPARβ is strongly decreased and PPARγ appears unchanged; concerning PPARα, no significant quantitative differences are observed. The RT-PCR analysis of PPAR mRNAs in S0 and S10 neurospheres (Figure 8) shows that, during astroglial differentiation, PPARα is significantly increased while PPARβ expression is significantly decreased. PPARγ appears unchanged. Figure 9 shows the double staining of oil red positive lipid droplets and nestin in S0 (Figure 9(a)) and oil red/GFAP in S10 (Figure 9(b)) neurospheres. Nuclei were counterstained with Mayer heamallume. In S0 neurospheres, almost all immunoreactive nestin cells show several lipid droplets in their cytoplasm, some of which being very large. In S10 GFAP-positive cells, lipid droplets are no more observed. 4. DISCUSSION In this paper, the presence of all three isotypes of PPARs in mouse adult neural stem cells has been established for the first time. Moreover, we demonstrated that PPARs are subjected to both quantitative and qualitative variations during astroglial differentiation. The proliferative and undifferentiated status has been demonstrated by immunofluorescence and western blotting. BrdU incorporation demonstrates that almost all cells of the neurospheres are proliferative and the presence of nestin and PLP, in the absence of markers of differentiation such as GFAP, β-tubulin III, and O4, is cosistent with the undifferentiated status and allows to conclude that the cellular population of our neurospheres is constituted by undifferentiated cells [22]. The strongly polarized immunolocalization of nestin suggests that the cells are dividing by asymmetric divisions. In fact, recent studies have demonstrated that, in stem cells, some proteins exhibit different distribution according to their division modality [23, 24]. The result that neural stem cells possess all three PPAR isotypes is new and unexpected. In fact, one would have hypothesized that PPARβ could be the most abundant owing to its relevant presence and early expression during brain development [15] and owing to its involvement in cell proliferation and in the first stages of cellular differentiation [25–27]. Our results demonstrate that all three PPARs are expressed and that they have a nuclear localization in agreement with their function as transcription factors. It is known that PPARs act in heterodimeric form with RXRs. The immunoblotting data reveal that in neural stem cells only RXRβ is present. This finding is in agreement with previous results demonstrating this isotype as the mainly present in rodent brain [28, 29] and suggests that one or more PPAR isotypes may heterodimerize with RXRβ. The simultaneous presence of the three PPARs in the nucleus does not indicate that they are all transcriptionally active; in fact it has been proposed that unliganded PPARβ may act as potent inhibitor of the transcriptional activity of the α and γ isotypes [30]. It is possible to hypothesize that in neural stem cells PPARβ contributes to the maintenance of the undifferentiated, proliferative status, by regulating both genes involved in cell cycle control, as observed in other cell types [18, 31, 32], and inhibiting the activity of the other PPARs, which may be, in turn, involved in cellular differentiation [13, 14]. The finding of large lipid droplets in the cytoplasm of NSC is new and suggests a role for PPARγ in this phenomenon. In fact, the importance of this transcription factor is well known in adipocyte differentiation as well as in cellular types where lipidogenesis occurs, such as oligodendrocytes and macrophages [33, 34]. In agreement with this hypothesis, the PPARγ appears to be strongly expressed both at mRNA level and at protein level in undifferentiated NSC. When NSC were subjected to astroglial differentiation, as expected, GFAP was highly expressed and the nestin was significantly decreased. Moreover, its intracellular distribution is completely different from S0 neurospheres, with the asymmetrical concentration of the protein in the juxtanuclear region being no more observed. The persistance of nestin in these differentiated cells is consistent with data from other authors that have reported a coexpression of GFAP and nestin in astrocytes in culture from postnatal animals; this coexpression, which is not observed in vivo, is induced by in vitro conditions and in vivo during astrogliosis [14, 35]. In the S10 cells, PPARs undergo quantitative modifications. A modulation of PPARs both at protein and mRNA levels is observed. The observed strong decrease of PPARβ is particularly interesting, since it could indicate the removal or reduction of its inhibitory effect on the other PPARs [30]. In this respect, PPARβ might be considered as inhibitor of astroglial differentiation [30, 36]. PPARγ does not vary, both at mRNA and protein levels, while PPARα is significantly increased only at mRNA level. This might be due to the fact that the RT-PCR and western blotting analyses were performed after 5 days of differentiation in vitro. Probably, to observe a significant increase of the protein, a longer time should be tested. However, the increase of PPARα suggests a role for this transcription factor in astroglial differentiation, supported by our previous findings on astrocyte in in vitro differentiation [14]. Moreover, the appearance of RXRα, its heterodimeric pattern [29], is in agreement with this suggestion. As regards RXRs, during NSC astroglial differentiation, the data obtained demonstrate that RXRγ is never expressed, in agreement with its restricted localization in adult brain [29, 37], RXRβ remains unchanged, while RXRα is expressed de novo by differentiated cells. Thus, a downregulation of PPARβ, accompanied by PPARα and RXRα increase may be a condition for the differentiation toward astroglial lineage. As regards PPARγ, the fact that this receptor is not modified may indicate that it is not crucial for astrocyte differentiation, at least concerning the differentiation of type I astrocytes. However, the presence of some A2B5/GFAP immunopositive cells may indicate that, in our experimental conditions, differentiation toward type II astrocytes may also occur. Since type II astrocytes share a common progenitor with oligodendrocytes, the O2A cells, the persistence of PPARγ in differentiating neurospheres could indicate that it may be involved in the oligodendrocyte differentiation pathway. Regarding the presence of lipid droplets in undifferentiated cells, their disappearance during differentiation may be in agreement with the hypothesis that in our experimental conditions, the differentiation toward type I astrocytes is preferred. In fact, differentiated astrocytes are able to utilize lipids as energy fuel [38] through catabolic lipid pathways requiring PPARα and not PPARγ activity, involved instead in lipidogenesis. Overall, the data presented in this work indicate that the decrease of PPARβ and the concomitant increase and/or activation of PPARα together with RXRα are involved in astroglial differentiation of NSC. In our opinion, however, it should be underlined that the regulation of different differentiation pathways and/or the maintenance of undifferentiated status are more affected by the quantitative ratios existing among the receptors isotypes (both PPARs and RXRs) rather than by the absolute amounts of each one of them. ACKNOWLEDGMENT This work has been supported by MIUR PRIA 2007 (Professor A. Cimini). Figure 1 Contrast phase microscopy of neural stem cells growing in neurospheres (a). In (c), BrdU incorporation is shown. Hoechst nuclear staining of the same field is shown in (b). Bar = 40 μm. Figure 2 Immunolocalization in S0 neurospheres of nestin (b) and PLP (e). Nuclear staining of the same field is shown in (a) and (d), respectively. Double A2B5/Hoechst immunostaining is shown in (c). Bar = 70 μm. Figure 3 PPARs immunolocalization in S0 neurospheres. (b) PPARα, (d) PPARβ, (f) PPARγ. Hoechst nuclear staining is shown in (a), (b), and (c), respectively. Bar = 20 μm. Figure 4 Western blotting and relative densitometric analysis in S0 neurosphere cell lysates. An example of western blotting is shown. Densitometric data are means ± SD of 5 different experiments. Figure 5 Immunolocalization of nestin, A2B5, and GFAP in S10 neurospheres. In (a), (b), and (c), double immunostaining of nestin/Hoechst, A2B5/Hoechst, and GFAP/Hoechst is shown, respectively. In (d), (e), and (f), the single immunostaining is shown. Bar = 40 μm. Figure 6 Double immunofluorescence staining for GFAP/PPAR in S10 neurospheres is shown. (a) PPARα, (b) PPARβ, (c) PPARγ. Bar = 30 μm. Figure 7 Western blotting and relative densitometric analysis in S10 neurosphere cell lysates. An example of western blotting is shown. Densitometric data are means ± SD of 5 different experiments. * P < .05; P < .001. Figure 8 RT-PCR analysis in S0 and S10 neurospheres. An example of RT-PCR is shown. Densitometric data are means ± SD of 5 different experiments. Semiquantification has been performed against the housekeeping gene β-actin. P < .001. Figure 9 Double oil red/nestin in S0 neurospheres (a) and oil red/GFAP in S10 (b) neurospheres. Bar = 20 μm. Table 1 Primers and PCR cycling. The adopted sequences of specific primers and relative cycling conditions of each RT-PCR are indicated. Gene Gene bank number Size (bp) Sequence Annealing (°C) Cicles PPAR α Gazouli et al., 2002 741 F 5′ggtcaaggcccgggtcatactcgcagg3′ 69 40 R 5′tcagtacatgtctctgtagatctct3′ PPAR β Gazouli et al., 2002 130 F 5′gtcatggaacagccacaggaggagacccct3′ 69 40 R 5′gggaggaattctgggagaggtctgcacagc3′ PPAR δ Gazouli et al., 2002 421 F 5′gagatgccattctggcccaccaacttcgg3′ 69 40 R 5′tatcataaataagcttcaatcggatggttc3′ β-Actin NM_031144 661 F 5′tgacggggtcacccacactgtgcccatcta3′ 65 28 R 5′ctagaagcattgcggtggacgatggaggg3′ ==== Refs 1 Nuclear Receptors Nomenclature Committee A unified nomenclature system for the nuclear receptor superfamily Cell 1999 97 2 161 163 10219237 2 Issemann I Green S Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators Nature 1990 347 6294 645 650 2129546 3 Dreyer C Krey G Keller H Givel F Helftenbein G Wahli W Control of the peroxisomal β -oxidation pathway by a novel family of nuclear hormone receptors Cell 1992 68 5 879 887 1312391 4 Escher P Wahli W Peroxisome proliferator-activated receptors: insight into multiple cellular functions Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2000 448 2 121 138 10725467 5 Krey G Braissant O L'Horset F Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay Molecular Endocrinology 1997 11 6 779 791 9171241 6 Berger J Moller DE The mechanisms of action of PPARs Annual Review of Medicine 2002 53 409 435 7 Wolfrum C Borrmann CM Börchers T Spener F Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors α - and γ -mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus Proceedings of the National Academy of Sciences of the United States of America 2001 98 5 2323 2328 11226238 8 Keller H Dreyer C Medin J Mahfoudi A Ozato K Wahli W Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers Proceedings of the National Academy of Sciences of the United States of America 1993 90 6 2160 2164 8384714 9 Gearing KL Gottlicher M Teboul M Widmark E. 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==== Front Mol ImmunolMol. ImmunolMolecular Immunology0161-58901872-9142Pergamon Press S0161-5890(07)00231-310.1016/j.molimm.2007.05.011ArticleThe mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility Donev Rossen M. [email protected]⁎Sivasankar Baalasubramanian Mizuno Masashi Morgan B. Paul [email protected]⁎Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK⁎ Corresponding authors. Tel.: +44 2920744001; fax: +44 2920744001. [email protected]@cardiff.ac.uk1 1 2008 1 2008 45 2 534 542 18 4 2007 13 5 2007 14 5 2007 .2007Elsevier LtdOpen Access under CC BY 3.0 licenseIn mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function. Keywords MouseCD59aCD59bComplementReproductive immunologyAcrosome ==== Body 1 Introduction CD59, a broadly distributed glycosyl phosphatidylinositol (GPI)-anchored protein, is the principal regulator of complement membrane attack complex (MAC) assembly on cell membranes (Meri et al., 1990). A decade ago, we identified the mouse analogue of CD59 and showed that it was broadly distributed on cells and tissues (Powell et al., 1997). Deletion of the gene encoding mouse CD59 caused minimal disturbance in unchallenged animals but markedly enhanced susceptibility to complement-driven pathologies (Holt et al., 2001; Turnberg et al., 2003, 2004; Lin et al., 2004; Mead et al., 2004; Williams et al., 2004). Others demonstrated that the gene encoding CD59 is duplicated in the mouse; the protein products of these two genes were then termed CD59a for the first described, and CD59b for the product of the new gene (Qian et al., 2000). In this first report, it was stated that CD59b message was significantly expressed only in testis. We developed monoclonal antibodies specific for CD59a and CD59b, enabling us to confirm the broad distribution of CD59a and testis restricted expression of CD59b at the protein level; we concluded that CD59a was the principle regulator of MAC assembly in mouse tissues (Harris et al., 2003). This conclusion was challenged in a re-analysis of the pattern of distribution of mRNA encoding the two forms of CD59; these studies suggested that both CD59a and CD59b were broadly expressed in the mouse and contended that the latter was the major regulator of MAC in the mouse (Qin et al., 2003). These contentions were bolstered by the demonstration that deleting the gene encoding CD59b in mice caused a severe, spontaneous phenotype with anaemia, platelet dysfunction and fertility problems. In light of these data we reassessed the distribution patterns at protein and mRNA levels using a combination of specific monoclonal antibodies and shared primers for PCR in high sensitivity assays (Baalasubramanian et al., 2004). These analyses confirmed our earlier work showing that CD59a was broadly expressed while significant expression of CD59b was restricted to testis. Trace amounts of CD59b message and protein were detected in erythrocytes but quantitative assays of protein expression showed that CD59b expression on erythrocytes was less that 5% that of CD59a. Further, we confirmed that CD59a was dominant in protection of erythrocytes from MAC lysis. The findings of these painstaking studies have recently been challenged yet again (Qin et al., 2006). The appropriateness of the techniques used and specificity of probes and primers have all been questioned. These authors concluded that CD59b was broadly expressed and an important regulator of MAC assembly on erythrocytes and in tissues. We, and others, are using CD59a knockout mice in disease models to explore roles of MAC based on our evidence that CD59a is the principal regulator of the MAC in most tissues. If CD59b is indeed widely distributed then the value of studies in CD59a knockouts is in question. It is therefore essential that we test the evidence. Here using multiple methods we revisit these published studies and undertake new analyses to explore the expression patterns of CD59a and CD59b at the mRNA and protein level. We conclude that expression of CD59b at the mRNA and protein level is essentially absent in all tissues other than testis. Low level expression on blood cells was confirmed and trace detection of mRNA in tissues was shown to be likely due to blood contamination. We further analysed expression of CD59b in testis and showed that expression coincided with onset of puberty and was restricted to spermatozoa and their immediate precursors. Ligation of CD59b on spermatozoa with monoclonal antibody markedly inhibited sperm motility, suggesting a specific role in reproductive function. 2 Materials and methods 2.1 Mice Adult (8–16 weeks) and infant (1–5 weeks; pre-puberty) male mice, and embryos (day 5, 10 and 15) from C57BL/6(H-2b) background mice were used in our investigation. All experimental procedures were performed in compliance with Home Office and local ethics committee regulations. cd59a−/− mice generated as described previously (Holt et al., 2001), and back-crossed 10 generation onto the same background were used as controls. 2.2 Antibodies and reagents Rat anti-mouse CD59a (CD59a.1; IgG1) was generated in house (Harris et al., 2003). Mouse anti-mouse CD59b (CD59b.2; IgG1) was also made and characterized in house (Baalasubramanian et al., 2004). Separate aliquots of CD59a.1 and CD59b.2 were labelled with NHS-biotin (Sigma–Aldrich, Gillingham, Dorset, UK) and FITC-NHS (Perbio Science UK Ltd., Cramlington, UK) according to manufacturer's protocols. As negative controls, rat IgG1 and mouse IgG1 (purified in-house) were labelled in the same manner. FITC-labelled streptavidin was purchased from DAKO (Ely, Cambridgeshire, UK). HRPO-labeled donkey anti-rat IgG and HRPO-labeled donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Europe (Newmarket, Suffolk, UK). 2.3 Semi-quantitative RT-PCR Total RNA was purified from all investigated mouse tissues using GenElute kit (Sigma–Aldrich) and controlled for DNA contamination by RT-PCR without using reverse transcriptase and employing a β-actin specific primer pair (Table 1). Only samples that did not show amplification were used in all analyses. Aliquots of these RNAs (1 μg each) were reverse transcribed using random hexamers and multiscribe reverse transcriptase according to the manufacturer's instructions (Applied Biosystems, Warrington, UK). Primer pairs specific for either CD59a or CD59b were as described by Qin et al. (2006), and a new primer pair common for both CD59a and b and amplifying a sequence in exon 4 comprising 199 bp for CD59a and 162 bp for CD59b, were used in the subsequent amplifying reactions for detection of CD59a and CD59b mRNAs (Table 1). Either 25 or 35 amplification cycles were used, except where attempting simultaneous detection of both mRNAs when 40 cycles were performed. Amplified products were separated either in 1% agarose or in 5% poly-acrylamide gels (PAAG). To obtain semi-quantitative data for expression of the two genes, we used as templates either 2 μl of the original cDNA, or a series of dilutions as indicated in figures. Gels were scanned and band intensity was measured by densitometry using Quantity One 4.3.0 software (BioRad, Hemel Hempstead, Hertfordshire, UK). The quantifications were carried out in triplicate and means and errors calculated. 2.4 Quantitative real-time PCR analysis The cDNAs from testis and liver prepared as described for the semi-quantitative RT-PCR, were subject to a Taqman assay as described previously (Baalasubramanian et al., 2004). One of the primer pairs was as described previously (Baalasubramanian et al., 2004), a perfect match for the sequence of CD59a mRNA and with a single internal nucleotide mismatch to CD59b mRNA; the second set was a perfect match for the same sequence in CD59b and with a single internal nucleotide mismatch to CD59a (Table 1). To measure the relative number of mRNA copies for CD59b in liver, perfused liver and testis we used the primer pair reported by Qin et al. (2006) to specifically amplify CD59b in a quantitative PCR (QPCR). Because these primers were not designed specifically for this assay, they did not meet the optimal annealing temperature requirements. To correct for this anomaly, the standard amplification programme was modified to include a pre-annealing step at 56 °C for 20 s prior to the extension phase. Primers were designed to β-actin (Table 1) as an internal control for normalization of starting cDNA levels. Quantitative PCR was performed on ABI PRISM 7000 using either TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix according to the manufacturer's instructions (Applied Biosystems) with 50 cycles of amplification. 2.5 Preparation of tissue lysates To obtain tissue lysates, freshly harvested mouse organs were immediately chilled and homogenized with ice-cold lysis buffer (PBS containing 2% NP40, 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 1 μg/ml leupeptin, and 1 μg/ml pepstatin; 1 g of tissue/1.5 ml of buffer) and incubated for 60 min on ice. Insoluble debris was removed by centrifugation (5000 × g, 15 min at 4 °C) and the supernatants stored in aliquots at −80 °C until use. In some experiments the mouse was perfused with saline at the time of sacrifice to reduce blood contamination in harvested organs. 2.6 SDS-PAGE and Western blot analysis Lysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under non-reducing conditions in 15% gels. Separated proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, London, UK), and membranes blocked with 5% (w/v) non-fat milk in PBS (PBS-M). Membranes were then probed with the primary mAb diluted in PBS-M, washed in PBS containing 0.1% Tween-20 (PBS-T), then probed with HRPO-conjugated donkey anti-rat Ig in PBS-M to detect the rat anti-CD59a or HRPO-conjugated donkey anti-mouse Ig (Jackson) to detect the mouse anti-CD59b. After further washing in PBS-T, bands were developed using ECL (Perbio Science UK Ltd.) and captured on autoradiographic film (Kodak Ltd., Hemel Hempstead, Hertfordshire, UK). 2.7 Spermatozoa preparation and analysis Motile cauda epididymal spermatozoa were obtained by a ‘swim-up’ technique as previously described (Mizuno et al., 2004, 2005). Briefly, two cauda epididymi from an adult C57BL/6 mouse were roughly minced in 1 ml of DMEM (Invitrogen, Paisley, UK). This suspension was carefully overlayered with 1 ml DMEM and incubated at room temperature for 15 min. The upper layer was removed and spermatozoa pelleted by centrifugation (1000 × g) for 5 min at room temperature. Cells were washed twice by gentle centrifugation as above. For immunofluorescence studies, the re-suspended cells were smeared onto glass slides, air-dried immediately, fixed in acetone at room temperature for 1 min and stored at −20 °C until use. To prepare lysate, the swim-up spermatozoa from 4 cauda epididymi were pelleted and incubated with mixing in 100 μl of lysis buffer for 30 min on ice. Insoluble debris was removed by centrifugation (15,000 × g, 15 min at 4 °C) and the supernatant stored in aliquots at −80 °C. To compare CD59b expression in unactivated and acrosome-reacted spermatozoa, the acrosome reaction was induced essentially as described (Mizuno et al., 2004). Briefly, swim-up spermatozoa (106 cells/ml of DMEM) were incubated for 1 h at 37 °C with the calcium ionophore A23187 (Sigma–Aldrich) at 1 μM to induce the acrosome reaction. Control cells were incubated without ionophore. Acrosome-reacted and control spermatozoa were smeared on glass slides and immediately air-dried. To observe CD59b distribution, the smears were incubated with FITC-labeled CD59b.2. Nuclei were counterstained with DAPI (4′-6-diamino-2-phenylindole-2 HCl; 100 ng/ml final concentration; Sigma–Aldrich). On each slide, at least 100 cells were counted and the assay was carried out in triplicate. 2.8 Functional inhibition assay of CD59a and CD59b in mouse sperm Spermatozoa harvested by swim-up from two epididymi, either from wild type or cd59a−/− mice, were washed and suspended in 2 ml DMEM. Paired aliquots (200 μl) were incubated at 37 °C with mAbs CD59a.1, CD59b.2, or isotype-matched control mAb, each at 10 μg/ml. After 4 h of incubation, the sample was immediately placed on a glass haemocytometer slide and the total number of sperm and percentage remaining motile were counted under light microscopy. This experiment was performed in triplicate. 2.9 Statistical analysis All values are expressed as mean ± standard error (S.E.M.). The statistical analysis was performed by one-way ANOVA. When significant differences were observed, statistical analysis was further carried out using unpaired t-test between two groups. Significance between two groups was claimed when P < 0.05. 3 Results 3.1 CD59b mRNA is highly expressed only in testis We have previously demonstrated that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). However, in a recent work (Qin et al., 2006), abundant expression of CD59b mRNA in a number of tissues was reported. In an attempt to clarify this issue, which is of key importance for complement studies in mice, we repeated some of the experiments carried out by Qin and co-authors using the primer pairs designed by them. Under routine conditions (25 cycles of amplification), CD59a mRNA was detected in all tissues tested while CD59b mRNA was found only in testis (Fig. 1A). Increasing the number of amplification cycles to 35 (Fig. 1B) revealed weak expression of mRNA for CD59b in liver, blood cells, heart and lung. Amplified sequence was confirmed to be CD59b by sequencing. We next re-visited the primers used in our previous quantitative PCR analysis to assess expression of the two CD59 isoforms in different mouse tissues (Baalasubramanian et al., 2004). We had designed a primer pair that annealed to both isoforms and Taqman probes specific for either CD59a or CD59b to enable accurate quantitation. Qin et al. (2006) criticised these experiments on the basis that, while both primers used in our assay matched perfectly CD59a, each had a single internal nucleotide mismatch compared to the CD59b sequence (Table 1). They suggested, without evidence, that this mismatch favoured amplification of CD59a and caused our inability to detect CD59b in tissues other than testis and bone marrow. There is a large primer design literature that shows clearly that internal mismatches, unlike those at the 3′ end, do not significantly influence amplification (Sommer and Tautz, 1989; Kwok et al., 1990; Christopherson et al., 1997; Löffert et al., 1998). Nevertheless, we addressed further this issue by repeating our previous QPCR investigation but including a second primer pair from the same sequence matching perfectly CD59b mRNA but with a single mismatch to CD59a mRNA in the same position as in the original primer set (Table 1). Using the original primer pair that matched perfectly CD59a we obtained very similar results to those we previously published (Baalasubramanian et al., 2004). When we used the pair matching CD59b, there was a small increase in threshold cycle (Ct) for both CD59a and CD59b, likely a result of altered primer annealing temperature, but there was no difference in the calculated relative amounts for CD59a and CD59b mRNA in testis compared to the original primer set (Table 2). Of note, QPCR with either primer set did not detect any CD59b mRNA in liver, suggesting that the high cycle number PCR described above was detecting very small amounts of mRNA in these tissues. In summary, the data shows an 8-fold greater expression of CD59a mRNA in liver compared to testis, a 5-fold greater expression of CD59a mRNA compared to CD59b in testis and undetectable expression of CD59b in liver, regardless of primer set used. In light of these results we developed a semi-quantitative PCR to quantify the low levels of CD59b mRNA detected in tissues. In order to be able directly to compare the expression of both isoforms, we designed a new primer pair within exon 4 (Table 1), which is 100% homologous to both mRNAs. Amplification of DNA-free mRNA with these primers will result in bands of 199 bp for CD59a and 162 bp for CD59b. In testis we detected both isoforms as expected (Fig. 2A). Densitometric comparison of band intensities at higher dilutions of template, when the amplification reaction is in the linear range, showed approximately 5-fold higher expression of CD59a mRNA as compared to CD59b mRNA in testis, a result compatible with the QPCR data (Table 2) and our published results (Baalasubramanian et al., 2004). However, in liver we failed to detect presence of CD59b even after 40 cycles of PCR (Fig. 2B). We reasoned that the large excess of CD59a mRNA in liver out-competes trace amounts of CD59b mRNA for limited reagents in the initial cycles of amplification, thereby reducing the chance for amplification of the low abundance CD59b mRNA. To test this reasoning we performed semi-quantitative PCR in separate tubes, using the primers specific for either CD59a or CD59b mRNA (Qin et al., 2006). Firstly, to optimise the annealing and amplification efficiency for both primer pairs, we performed this reaction for testis mRNA (Fig. 3A). Similar band intensities for CD59a and CD59b were obtained for 5 × 105-fold and 105-fold template dilution respectively (Fig. 3A and B). This indicated an approximate 5-fold higher expression of CD59a mRNA compared to CD59b mRNA, supporting our results described above (Table 2) and published (Baalasubramanian et al., 2004), and confirming similar amplification efficiency for the specific primer pairs. Amplification of liver mRNA using these primers, and comparison of band intensities in dilutions of templates showed that expression of CD59b mRNA in liver was approximately 105-fold less compared to that of CD59a mRNA (Fig. 3C and D similar band intensities for CD59a and CD59b at 5 × 105 and 5-fold dilution respectively). In these latter experiments we used liver perfused with saline at the time of sacrifice to remove the bulk of entrapped blood; nevertheless, the trace mRNA detected might be from residual blood cells, which clearly express CD59b protein at low level (Baalasubramanian et al., 2004; Qin et al., 2006). To clarify this issue, we compared mRNA from perfused and non-perfused liver by QPCR using CD59b-specific primers and found the amount of CD59b mRNA in liver was reduced 12-fold by perfusion (Fig. 3E). For comparison, expression of CD59b in testis was 2200-fold higher than in unperfused liver and approximately 25,000-fold higher than in perfused liver. Taken together these results strongly suggest that the detectable traces of CD59b mRNA in liver and likely other organs are a consequence of contamination with blood cells. 3.2 Expression of CD59b protein is testis restricted We have previously reported, using immuofluorescence staining, that CD59b protein was highly expressed in testis but absent from all other organs tested (including liver, lungs, spleen, kidney and heart) (Baalasubramanian et al., 2004). We here extend these studies by Western blotting of testis, liver, lungs, plus aorta, chosen because of the suggested role of CD59 in vascular disease (Qin et al., 2004) (Fig. 4). Each organ was perfused with saline prior to preparation of protein lysates. CD59a was strongly detected in all the tissues; however, CD59b was detected only in testis lysates. These data confirm our published contention that organ expression of CD59b protein is restricted to testis. 3.3 Expression of CD59b mRNA in testis coincides with puberty and plays a role in spermatozoal motility We previously reported that CD59b expression in testis was restricted to developing and mature spermatozoa (Baalasubramanian et al., 2004). To confirm this germ cell restricted pattern we examined expression of CD59b mRNA in testis from pre-pubertal mice. CD59b mRNA was absent in testes harvested from mice at days 10 and 20 post-partum and appeared only from day 30 on, coincident with puberty (Fig. 5A). In contrast, CD59a mRNA was present in testes at all timepoints. RNA extracted from embryos at days 5, 10 and 17 were all positive for CD59a but negative for CD59b mRNA (Fig. 5A). These observations were confirmed by Western blotting (Fig. 5B). Expression of CD59b protein was highest in spermatozoa, intermediate in adult testis and absent from infant testis. CD59a was present in all the lysates, albeit at low level in spermatozoa and with a reduced apparent molecular mass compared with the testis protein, suggesting modification during sperm maturation. We previously reported that CD59b on spermatozoa was focussed on the head region in a highly granular pattern (Baalasubramanian et al., 2004). Here we have explored the expression pattern of CD59b in more detail. In freshly isolated spermatozoa, more than 80% showed this head/granular staining pattern, a minority showing a more diffuse staining on the head region (Fig. 6). CD59b was weakly expressed on spermatozoal tails and, in about two thirds, strongly in the mid-piece. Initiation of the acrosome reaction (with A23187) caused a precipitous loss of CD59b expression on sperm heads, more than 60% of cells being negative by 30 min and 90% by 180 min post-initiation, indicating that CD59b was shed with the outer acrosomal membrane (Fig. 6). These data suggested that CD59b might play a role in acrosome function, an essential component of sperm capacitation for fertilisation. Therefore, we next investigated whether ligation of CD59b with antibody influenced spermatozoal mobility, a surrogate marker for fertilisation capacity (Fig. 7). Ligation of CD59b caused a significant suppression of motility of spermatozoa harvested from both wild type and cd59a−/− mice. As a control, CD59a was similarly ligated but did not alter spermatozoal motility compared with the effect of a control antibody. The data suggest that CD59b but not CD59a has a role in regulating spermatozoal motility. These findings are of particular relevance in that a major feature of the CD59b knockout mouse was spermatozoal dysfunction that included diminished motility and infertility (Qin et al., 2003). 4 Discussion We have recently examined the distribution patterns of the two isoforms of CD59 in the mouse and concluded, based upon its broad distribution, that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). We were unable to detect the expression of CD59b, either at mRNA or protein level, in brain, lungs, heart, liver, spleen and kidney. These data have recently been questioned by Qin et al. (2006) who first used a BLAST search of the mouse EST database and found ESTs matching the CD59b sequence from several tissues and organs. These in silico data were supported by RT-PCR analyses using primer sets specific for CD59a and CD59b, respectively that showed abundant expression of CD59b mRNA in all tissues tested, indeed, expression in testis was lower than in other organs. The apparent absence of CD59b mRNA in the CD59a knockout mouse in these published data was unexplained. Here we designed a number of quantitative and semi-quantitative assays to comprehensively address this controversial issue. Our data unambiguously demonstrated that expression of CD59b is essentially restricted to testis (Figs. 3 and 4). A comparative quantitative analysis of expression of CD59b mRNA in perfused and unperfused liver (Fig. 3) showed a 12-fold decrease in expression of CD59b following perfusion, strongly suggesting that the trace detection of this mRNA was a consequence of contamination with blood cells. This would explain why Qin et al. (2001) were able to detect CD59b in multiple mouse tissues using Northern analysis in which they loaded 10 μg of mRNA in each lane, a huge excess for this sensitive procedure. The contamination likely also explains the presence in multiple tissues of CD59b-specific EST sequences. We further demonstrated that the testis expression of CD59b coincides with puberty, supporting our findings of expression only on spermatozoa and their immediate precursors (Fig. 5). We found that CD59b was released from spermatozoa heads upon acrosome activation (Fig. 6), an observation that strongly support a role for this protein in functioning of acrosome. Ligation experiments with antibodies showed that CD59b but not CD59a was involved in spermatozoa motility (Fig. 7), providing support for an earlier report describing decreased motility and viability of sperm in cd59b KO mice (Qin et al., 2005). 5 Concluding remarks The data presented here, obtained using a broad panel of reagents and methods designed to give unbiased and unequivocal results, show that CD59b expression is limited. CD59b mRNA is abundantly expressed only on male germ cells and present in trace amounts in bone marrow and blood cells, but is absent from other organs where trace detection of mRNA is likely due to blood contamination. CD59b protein is abundant only on developing and mature spermatozoa. Erythrocytes express CD59b at low levels that we have previously quantified as less than 200 molecules per cell, irrelevant for protection from complement when CD59a is present at 2500 molecules per cell (Baalasubramanian et al., 2004). We show a unique distribution pattern of CD59b protein on spermatozoa, the precipitous loss of this protein with outer acrosomal membranes and effects of CD59b ligation on sperm motility. We conclude that CD59a is indeed the principal regulator of MAC expressed in the mouse and that the CD59a knockout is an appropriate model for studying the roles of MAC and its regulation in disease models. The CD59b knockout mouse might prove of value for studies of fertility; however, it should be noted that the mouse described by Halperin and co-workers is, for unexplained reasons, also markedly deficient in CD59a (Qin et al., 2006), limiting its utility for studying specific roles of CD59b in vivo. Acknowledgements This work was supported by the Wellcome Trust through Programme Grant (068590) funding to B.P.M. We thank Marie-Laure Aknin for technical support and Dr. Claire Harris for advice and support. Fig. 1 RT-PCR detection of CD59a and CD59b in mouse tissues. Primer pairs described in the text and specific for each isoform were used for the PCR step and either 25 (A) or 35 (B) amplification cycles were carried out. Using these primers, an approximately 350 bp product was obtained from CD59a mRNA and was readily detected in all tissues after 25 cycles. CD59b mRNA was detected after 25 cycles in testis only and the product size was approximately 400 bp. After 35 cycles of amplification, this product was detected in all tissues. Fig. 2 RT-PCR analysis of expression of CD59a and CD59b using common primers. A new primer pair was designed from identical sequences within exon 4 of CD59a and CD59b mRNA and used to amplify mRNA from testis (A) and liver (B). Forty amplification cycles were carried out using different dilutions of cDNA (shown above) as template. The amplified products were 199 bp-long for CD59a and 162 bp-long for CD59b. Products were separated in 5% PAAG. Intensity of the bands was measured and the CD59a/CD59b ratio calculated from three independent experiments (given below each band pair in A). This ratio was approximately 1 when the cDNA template was not diluted or diluted 5-fold, indicating that amplification has reached saturation. However, the calculated ratio was around 5 for higher dilutions of the template, suggesting that amplification at the final point was in the linear range. Fig. 3 Semi-quantitative RT-PCR to estimate relative expression of CD59a and CD59b in testis (A) and perfused liver (C). Different primer pairs for each isoform and different dilutions of the cDNA-templates (numbers above) were used. Thirty-five cycles were carried out and reaction products were separated in a 1% agarose gel. The average densitometric intensity for each band calculated from three independent experiments is given. Panels B and D present band intensity for CD59a (♦) and CD59b (■) as function of template dilution for testis and liver, respectively. (E) SYBR Green QPCR analysis of expression of CD59b in liver without and with perfusion using primers specific for CD59b. Number of the CD59b mRNA copies in liver (set as 1 for unperfused) was compared that in testis. Data are mean of two independent experiments ± S.E.M. Fig. 4 Western blot for expression of CD59a and CD59b in different mouse tissues. Lysates from testis, liver, lungs, and aorta were separated by SDS-PAGE, blotted to nitrocellulose membranes, and probed either with the mAb CD59a.1 or with CD59b.2 for detection of CD59a and CD59b, respectively. Fig. 5 Expression profile of CD59a and CD59b during development and testis maturation. Whole embryos were used to purify total RNA and assess expression of CD59a and CD59b. (A) A primer pair that recognizes both isoforms yielding products of 204 bp for CD59a and 237 bp for CD59b was used. EL4 cells transfected with plasmids expressing either CD59a or CD59b were used as controls. (B) Western blots analysis of CD59a and CD59b expression in infant and adult testis and sperm. Equal amounts of proteins were loaded in each lane in both panels. Fig. 6 Distribution of CD59b in spermatozoa and effects of acrosome activation. The right-hand panel shows representative images for each pattern of CD59b staining with CD59b.2 mAb. Arrowheads point to the sperm head showing granular and diffuse staining respectively. Arrows indicate the mid-piece showing dense staining. The left panel shows the effects of acrosome reaction on CD59b expression. Data are means of two independent experiments ± S.E.M. Compared sets are shown by columns with interrelated P values for comparison (P < 0.0001). Fig. 7 Ligation of CD59a and CD59b in mouse sperm with monoclonal antibodies. In wild type (WT) and cd59a−/− mouse sperm, CD59a and/or CD59b were ligated with CD59a.1 or CD59b.2. Rat and mouse IgG1 were used as controls for CD59a and CD59b specific antibodies as appropriate. The percentage of sperm retaining motility at 4 h post-antibody ligation was assessed. Data shown are means of three independent experiments ± S.E.M. Compared sets are shown by columns with interrelated P values for comparison (P < 0.05). Table 1 Primers used for detection of Cd59a and Cd59b mRNA Sequence detected Primer pairs Cd59a + Cd59b by QPCR (sequence matches Cd59a) (Baalasubramanian et al., 2004) 5′-GCCGGAATGCAAGTGTATCA-3′ (F); 5′-GTCCCCAGCAATGGTGTCTT-3′ (R) Cd59a + Cd59b by QPCR (sequence matches Cd59b) 5′-GCCGGAAGGCAAGTGTATCA-3′ (F); 5′-GTCCCCAGCAATGCTGTCTT-3′ (R) Cd59a (Qin et al., 2006) 5′-TGTCTAGAGCAGGATCTAGC-3′ (F); 5′-ATCCGTCACTTTTGTTACAC-3′ (R) Cd59b (Qin et al., 2006) 5′-AGTCACTGGCGATCTGAAAAG-3′ (F); 5′-ATGAGGAAGTTTCTGCGTTG-3′ (R) Cd59a + Cd59b (exon 4) 5′-GTTCTGGTGGCCATTTTGAA-3′ (F); 5′-TGTCCAAGATGTTCAAGTGAAC-3′ (R) Cd59a + Cd59b (expression during testis development) 5′-GATTCCTGTCTCTATGCTGTA-3′ (F); 5′-CAAAATGGCCACCAGAAC-3′ (R) β-Actin 5′-ACGGCCAGGTCATCACTATTG-3′ (F); 5′-AGTTTCATGGATGCCACAGGAT-3′ (R) Table 2 Comparison of data from QPCR analyses obtained by primers matching either Cd59a or Cd59b Primers matching CD59a, Ct Primers matching CD59b, Ct ΔΔCt = ΔCt(b) − ΔCt(a) Relative amount of CD59a (%)a Relative amount of CD59b (%)a CD59a CD59b CD59a CD59b Primers matching CD59a Primers matching CD59b Primers matching CD59a Primers matching CD59b Primers matching CD59a Primers matching CD59b Testis 24.8 ± 0.3 27.1 ± 0.4 26.1 ± 0.4 28.4 ± 0.4 2.3 ± 0.3 2.3 ± 0.4 100 ± 4 100 ± 5 20 ± 1 20 ± 1 Liver 21.8 ± 0.3 UD 23.1 ± 0.3 UD UD UD 822 ± 9 811 ± 8 UD UD UD: undetectable, Ct: threshold cycle, ΔCt(a), ΔCt(b): Ct measured for CD59a or CD59b respectively, standardised by Ct for the housekeeping gene (β-actin). a Data are presented as percent compared to testis. ==== Refs References Baalasubramanian S. Harris C.L. Donev R.M. Mizuno M. Omidvar N. Song W.C. Morgan B.P. CD59a is the primary regulator of membrane attack complex assembly in the mouse J. Immunol. 173 2004 3684 3692 15356114 Christopherson C. Sninsky J. Kwok S. The effects of internal primer-template mismatches on RT-PCR: HIV-1 model studies Nucleic Acids Res. 25 1997 654 658 9016609 Harris C.L. Hanna S.M. Mizuno M. Holt D.S. Marchbank K.J. Morgan B.P. Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: tissue distribution and functional comparison Immunology 109 2003 117 126 12709025 Holt D.S. Botto M. Bygrave A.E. Hanna S.M. Walport M.J. Morgan B.P. Targeted deletion of the CD59 gene causes spontaneous intravascular hemolysis and hemoglobinuria Blood 98 2001 442 449 11435315 Kwok S. Kellogg D.E. McKinney N. Spasic D. Goda L. Levenson C. Sninsky J.J. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies Nucleic Acids Res. 18 1990 999 1005 2179874 Lin F. Salant D.J. Meyerson H. Emancipator S. Morgan B.P. Medof M.E. Respective roles of decay-accelerating factor and CD59 in circumventing glomerular injury in acute nephrotoxic serum nephritis J. Immunol. 172 2004 2636 2642 14764738 Löffert D. Seip N. Karger S. Kang J. PCR optimization: degenerate primers QIAGEN News 2 1998 3 6 Mead R.J. Neal J.W. Griffiths M.R. Linington C. Botto M. Lassmann H. Morgan B.P. Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis Lab. Invest. 84 2004 21 28 14631387 Meri S. Morgan B.P. Davies A. Daniels R.H. Olavesen M.G. Waldmann H. Lachmann P.J. Human protectin (CD59), an 18,000–20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed insertion of C9 into lipid bilayers Immunology 71 1990 1 9 1698710 Mizuno M. Harris C.L. Johnson P.M. Morgan B.P. Rat membrane cofactor protein (MCP; CD46) is expressed only in the acrosome of developing and mature spermatozoa and mediates binding to immobilized activated C3 Biol. Reprod. 71 2004 1374 1383 15215199 Mizuno M. Harris C.L. Suzuki N. Matsuo S. Morgan B.P. Expression of CD46 in developing rat spermatozoa: ultrastructural localization and utility as a marker of the various stages of the seminiferous tubuli Biol. Reprod. 72 2005 908 915 15601919 Powell M.B. Marchbank K.J. Rushmere N.K. van den Berg C.W. Morgan B.P. Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59 J. Immunol. 158 1997 1692 1702 9029105 Qian Y.M. Qin X. Miwa T. Sun X. Halperin J.A. Song W.C. Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59 J. Immunol. 165 2000 2528 2534 10946279 Qin X. Dobarro M. Bedford S.J. Ferris S. Miranda P.V. Song W. Bronson R.T. Visconti P.E. Halperin J.A. Further characterization of reproductive abnormalities in mCD59b knockout mice: a potential new function of mCD59 in male reproduction J. Immunol. 175 2005 6294 6302 16272280 Qin X. Ferris S. Hu W. Ziegeler G. Halperin J.A. Analysis of the promoters and 5′-UTR of mouse cd59 genes, and of their functional activity in erythrocytes Genes Immun. 7 2006 287 297 16541098 Qin X. Goldfine A. Krumrei N. Grubissich L. Acosta J. Chorev M. Hays A.P. Halperin J.A. Glycation inactivation of the complement regulator protein CD59: a possible role in the pathogenesis of the vascular complications of human diabetes Diabetes 53 2004 2653 2661 15448097 Qin X. Krumrei N. Grubissich L. Dobarro M. Aktas H. Perez G. Halperin J.A. Deficiency of the mouse complement regulatory protein mCD59b results in spontaneous hemolytic anemia with platelet activation and progressive male infertility Immunity 18 2003 217 227 12594949 Qin X. Miwa T. Aktas H. Gao M. Lee C. Qian Y.M. Morton C.C. Shahsafaei A. Song W.C. Halperin J.A. Genomic structure, functional comparison, and tissue distribution of mouse CD59a and CD59b Mamm. Genome 12 2001 582 589 11471050 Sommer R. Tautz D. Minimal homology requirements for PCR primers Nucleic Acids Res. 17 1989 6749 16749 2506529 Turnberg D. Botto M. Lewis M. Zhou W. Sacks S.H. Morgan B.P. Walport M.J. Cook H.T. CD59a deficiency exacerbates ischemia-reperfusion injury in mice Am. J. Pathol. 165 2004 825 832 15331407 Turnberg D. Botto M. Warren J. Morgan B.P. Walport M.J. Cook H.T. CD59a deficiency exacerbates accelerated nephrotoxic nephritis in mice J. Am. Soc. Nephrol. 14 2003 2271 2279 12937303 Williams A.S. Mizuno M. Richards P.J. Holt D.S. Morgan B.P. Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis Arthritis Rheum. 50 2004 3035 3044 15457473	17597212	PMC1995235	CC BY	2021-01-05 04:10:41	yes	Mol Immunol. 2008 Jan; 45(2):534-542
==== Front Bioinorg Chem ApplBioinorg Chem ApplBCABioinorganic Chemistry and Applications1565-36331687-479XHindawi Publishing Corporation 10.1155/2007/54562Research ArticleDNA Binding and Photocleavage Studies of Cobalt(III) Polypyridine Complexes: [Co(en)2PIP]3+, [Co(en)2IP]3+, and [Co(en)2phen-dione]3+ Nagababu Penumaka Satyanarayana S. Department of Chemistry, Osmania University, Hyderabad 500 007, Andhra Pradesh, IndiaS. Satyanarayana: [email protected] by Nick Katsaros 2007 25 6 2007 2007 5456218 10 2006 25 1 2007 21 3 2007 Copyright © 2007 P. Nagababu and S. Satyanarayana.2007This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.In this paper, three complexes of type [Co(en)2PIP]3+(PIP=2-phenylimidazo[4,5-f][1,10,] phenanthroline)(1), [Co(en)2IP]3+ (IP = imidazo[4,5-f][1,10,] phenanthroline)(2), and [Co(en)2phen-dione]3+(1,10 phenanthroline 5,6,dione)(3) have been synthesized and characterized by UV/VIS, IR, 1H NMR spectral methods. Absorption spectroscopy, emission spectroscopy, viscosity measurements, and DNA melting techniques have been used for investigating the binding of these two complexes with calf thymus DNA, and photocleavage studies were used for investigating these binding of these complexes with plasmid DNA. The spectroscopic studies together with viscosity measurements and DNA melting studies support that complexes 1 and 2 bind to CT DNA (= calf thymus DNA) by intercalation mode via IP or PIP into the base pairs of DNA, and complex 3 is binding as groove mode. Complex 1 binds more avidly to CT DNA than 2 and 3 which is consistent with the extended planar ring π system of PIP. Noticeably, the two complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA. ==== Body 1. INTRODUCTION The interaction of transition metal complexes with DNA has been extensively studied in the past few years. Metal complexes of the type [M(LL)3]n+ where LL is either 1,10,phenathroline or modified phenanthroline ligand are particularly attractive species to recognize and cleavage DNA [1–4]. Barton demonstrated that tris(phenanthroline) complexes of ruthenium(II) display enantiomeric selectivity in binding to DNA, which can be served as spectroscopic probes in solution to distinguish right- and left-handed DNA helies [5]. The ligands or the metal in these complexes can be varied in an easily controlled manner to facilitate an individual application. The change in the metal ion or ligand would lead to changes in the binding mode and affinity [6, 7]. Much attention has been paid to the complexes of Ru(II) [8–10]. We choose to concentrate our work on cobalt(III) ethylenediamine polypyridyl complexes which have the same interesting characteristics and DNA cleaving properties as Ru(II) complexes. We have chosen ethylenediamine because in classical antitumor agent (cis platin) one of the ligands must be N donor and possessing at least one hydrogen atom attached to nitrogen. In this paper, we report the synthesis and characterization of the two complexes 1 and 2 (see Schemes 1 and 2), and their binding ability to CT DNA, DNA-binding properties are studied by electronic absorption, luminescent spectra, viscosity measurement, and DNA melting curve. The photochemical DNA cleavages of the complexes are also demonstrated. 2. EXPERIMENTAL METHODS 2.1. Materials All materials were purchased and used without further purification unless otherwise noted. The compounds 1,10 phenanthroline-5,6-dione [11], (IP) and (PIP) [12], [Co(en)2phen]Br3 [13] cis-[Co(en)2Cl2]Cl · 3H2O [14], and [Co(en)2L]Br3 were prepared by the procedure given below. The absorption spectra of CoCl2 6H2O, cis [Co(en)2Cl2]Cl, and [Co(en)2PIP]Br3 are shown in Figure 1. All the experiments involving the interaction of the complexes with DNA were carried out in buffer (5 mM tris-HCl, 50 mM NaCl, pH 7.2). A solution of CT DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of about 1.90 indicating that the DNA was sufficiently free of protein [15]. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M−1Cm−1) at 260 nm [16]. 3. SYNTHESIS OF COMPLEXES 3.1. [Co(en)2PIP]Br3 A mixture of cis-[Co(en)2Cl2]Cl (1.43 g) and phenylimi-dazo[4,5-f][1,10,] phenanthroline (1 g) was dissolved in EtOH (6 ml) and NaBr (3.0 g) in H2O (5 ml) was added and heated on a water bath until a dark yellow solution was formed. It was then cooled in ice where the thick crystalline precipitate of [Co(en)2PIP]3+ was collected and recrystallized from water (30 ml). The yield by this method was about 80% (Mol. Wt 715). (Elemental analysis found: C, 37.12; H, 3.2; N, 15. Calc. for C23N8H28Br3Co : C, 38.63; H, 3.95; N, 15.67.) IR stretching frequencies are (C=C); 1458, (C=N); 1508, (Co–N (en)); 779, (Co–N(ligand)); 625.5. 3.2. [Co(en)2IP]Br3 The complex [Co(en)2IP]3+ was prepared according the procedure described above from the mixture of cis-[Co(en)2-Cl2]Cl (1.43 g) and IP (1 g). (Yield: 88%.) (Elemental analysis found: C, 37.28; H, 3.2; N, 15.6. Calc. for C23N8H28-Br3Co: C, 38.63; H, 3.95; N, 15.67.) IR stretching frequencies are (C=C); 1398, (C=N); 1558, (Co–N (en)); 614, (Co–N(ligand)); 573. 3.3. [Co(en)2phen-dione]Br3 The complex [Co(en)2phen-dione]3+ was prepared according to the procedure described above from the mixture of cis-[Co(en)2Cl2]Cl (1.43 g) and phen-dione (1 g). (Yield: 88%.) IR stretching frequencies are (C=C); 1384, (C=N); 1559, (Co–N (en)); 626, (Co–N(ligand)); 615. (Elemental analysis found: H 3.3 C 30. N 13.1 O. Calc. for, C16N6O2H22Br3Co H, 3.53, C, 30.55, N, 13.36.) 4. PHYSICAL MEASUREMENTS 4.1. Elemental analysis and conductivity data Carbon, hydrogen, and nitrogen analyses were obtained from microanalytical Heraeus Carlo Etba 1108 elemental analyser. Chloride analysis was done. The metal contents were estimated from these solutions on atomic absorption spectrometer Perkin-Elmer 23380. The conductivity of metal complexes was measured in freshly prepared DMSO solutions using digital conductivity bridge (model: DI-909) and a dip-type cell calibrated with KCl solution. 5. SPECTRAL ANALYSIS UV/visible spectra were recorded with Elico bio-spectro-photometer, model BL198, IR spectra were recorded on a Hitachi U-3410, KBr phase on Perkin-Elmer FTIR-1605 spectrophotometer, 1H NMR spectra were measured on a Varian XL-300 MHz spectrometer with D2O as a solvent at room temperature and tetramethylsilane (TMS) as the internal standard, magnetic susceptibility measurements for solid samples of the complexes were carried out using Faraday bakany model. Hg [Co(CNS)4] were employed as magnetic susceptibility standards, microanalyses (C, H, and N) were carried out on a Perkin-Elmer 240 of elemental analyzer. For the absorption spectra an equal solution of DNA was added to both complex solutions and reference solution to eliminate the absorbance of DNA itself. Viscosity experiments were carried out using an Ostwald viscometer maintained at a constant temperature at 30.0 ± 0.1°C in athermostatic water bath. Calf thymus DNA samples approximately 200 base pairs in average length were prepared by sonicating in order to minimize complexities arising from DNA flexibility [17]. Data were presented as (η/η 0)1/3 versus the concentration of Co(III) complexes, where η is the viscosity of DNA in presence of complex, η 0 is the viscosity of DNA alone. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t > 100 s) corrected for the flow time of buffer alone (t 0)η = (t − t 0)/t 0, where t is the observed flow time of DNA and t 0 is the flow time of buffer [18]. The DNA melting experiments were carried by controlling the temperature of the sample cell with a Shimadzu circulating bath while monitoring the absorbance at 260 nm. For the gel electrophoresis experiments, super coiled pBR 322 DNA (100 μM) was treated with Co(III) complexes in 50 mM Tris-HCl, 18 mM NaCl buffer, pH = 7.2, and the solution was incubated for 1 hour in the dark, then irradiated at room temperature with a UV lamp (302 nm, at 10 W). The samples were analyzed by electrophoresis for 2.5 hour at 40 V on a 0.8% agarose gel in Tris acetic acid EDTA buffer; pH 7.2. The gel was stained with 1 μg/ml ethidium bromide and then photographed under UV light. 6. SPECTROSCOPIC CHARACTERIZATION The IR spectral data for the complexes were determined; two complexes clearly exhibit a band at 1450 and 1560–1590 cm−1, corresponding to C=C and C=N of the ring, respectively. Bands at around 626 and 579 cm−1 corresponding to Co–N (en) and Co–N of (L=PIP, IP and phen-dione) NH2 (en) bending at around 1650 cm−1 were observed. The UV-visible spectral peaks were observed at 317, 492, 347, 437 nm and fluorescence peaks at 407, 408, and 609 of 1, 2, and 3 complexes, respectively, based on the literatures data on the spectral properties of complexes 2 and 1. Bands appearing in the spectra of Co(III) complexes can be assigned exclusively to MLCT charge transition bands between 400–500 nm, Table 1, [19]. The electronic environment of many aromatic hydrogen atoms (PIP, IP, and phen-dione) are similar and hence their 1H NMR spectra appear in a narrow chemical shift range. In fact the aromatic regions of the spectra of these complexes are complicated due to the overlapping of several signals, which have precluded the identification of individual resonance. In the 1H NMR spectra of the cobalt(III) complexes the peaks due to various H-atoms of PIP, IP, and phen-dione were shifted downfield compared to the free ligands suggesting complexation in Figure 1. As expected the signal for PIP, IP, and phen-dione appeared in the range between 7.5 to 9.2 ppm in agreement with an earlier report [19], CH2 groups of the ethylenediamine gave peaks at 2.72, (4H, en-CH2) and 3.0, (2H, en-CH2). 7. RESULTS AND DISCUSSION 7.1. Absorption spectral studies The application of electronic absorption spectroscopy is one of the most useful techniques for DNA binding studies [20]. Complex binding with DNA through intercalation usually results in hypochromism and bathchromism, due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The extent of the hypochromism commonly parallels the intercalative binding strength. The absorption spectra of complexes 1, 2, and 3 in absence and presence of CT DNA are given in Figures 2, 3, and 4. As the concentration of DNA is increased, it results in hypochromism and moderate bathochromic shift in the UV-visible spectra of three complexes 1, 2, and 3. According to the data presented in Figures 1, 2, and 3, it seems that the change in absorption spectra of the two complexes upon addition of DNA follows: 1 > 2 > 3. These spectral data may suggest a mode of binding that involves a stacking interaction between the complex and the base pairs of DNA. To compare quantitatively the binding strength of the two complexes, the intrinsic binding constants K of the two complexes with CT DNA were determined according to the following equation [21] through a plot of [DNA]/∑b−∑f) versus [DNA] (1) [DNA]∑a−∑f=[DNA](∑b−∑f)+1[K(∑b−∑f)], where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients ∑a, ∑f, and ∑b correspond to A obsd/[Co], the extinction coefficient for the free cobalt complex and the extinction coefficient for the free cobalt complex in the fully bound form, respectively. In plots [DNA]/(∑b − ∑f) versus [DNA], K is given by the ratio of slope to intercept. Intrinsic binding constants K of 1, 2, and 3 were obtained about 5.34 ± 0.2 × 104, 4.575 ± 0.3 × 104, and 3.9 ± 0.1 × 104 M−1 from the decay of the absorbance. The binding constants indicate that complex 1 binds strongly than 2 > 3. This result is expected, since PIP possesses a greater planar area and extended π system than that of IP which will lead to PIP penetrating more deeply into and makes stacking more strongly. Hypochromism was indeed observed in the complexes with the order being 1 > 2 > 3. 7.2. Fluorescence studies The complexes 1, 2, and 3 can emit luminescence in Tris buffer (pH 7.0) at ambient temperature with maxima at 408, 407, and 609 nm. Binding of both complexes to DNA was found to increase the fluorescence intensity. The emission spectra of both complexes in the absence and presence of CT DNA are shown in Figures 5, 6, and 7. The plots of the relative intensity versus the ratio of [DNA]/[Co] are also inserted in Figures 5, 6, and 7. Upon addition of CT DNA, the emission intensity increases steadily. The emission intensity difference between absence of CT DNA and presence of CT DNA is greater for PIP complex than IP and phen-dione complex as shown in Figures 5, 6, and 7. The extent of enhancement increases on going from 3 to 2 to 1 and which is consistent with the above absorption spectra results. The order of increase in emission intensity of complexes is 1 > 2 > 3. These results were strengthened by viscosity studies. This observation is further supported by the emission quenching experiments using [Fe(CN)6]4− as quencher. The method essentially consists of titrating a given amount of DNA binding-metal complexes with increasing the concentration of [Fe(CN)6]4− and measuring the change in fluorescence intensity. The ion [Fe(CN)6]4− has been shown to be able to distinguish differentially bound Cobalt(III) complexes. The positively charged free complex ions should be readily quenched by [Fe(CN)6]4−, where as DNA bound cobalt complex is protected from the quencher, because highly negatively charged [Fe(CN)6]4− would be repelled by the negatively change DNA phosphate backbone. The ferro-cyanide quenching curves for 1, 2, and 3 in the presence and absence of CT DNA are shown in Figure 8. Obviously complex 1 inserts into DNA much deeper than complexes 2 and 3. The absorption and fluorescence spectroscopy studies thus determine the binding of complexes with DNA. 7.3. Viscosity studies The mode of the two complexes binding to DNA was explained by viscosity measurements. Optical photo-physical probes are necessary, but do not give sufficient clues to support a binding model. Hydrodynamic measurements that are sensitive to length change (i.e., viscosity and sedimentation) are regarded as the least ambiguous [18]. For complexes 2 and 1 the viscosity of DNA increases highly with the increasing of the concentration of complex which is similar to that of proven intercaltor EtBr [22]. Both complexes change the relative viscosity of DNA in a manner consistent with binding by intercalation mode shown in Figure 9. This result also parallels the pronounced hypochromism and spectral red shift and emission enhancement of both complexes, whereas this result is comparable with proven classical intercalator EtBr. Viscosity of DNA increases with the increase of the concentration of EtBr. So these two complexes increase DNA helix length. On the basis of the viscosity results, it shows that complexes bind with DNA through intercalation mode. The order of increase in viscosity of complexes follows the order 1 > 2 > 3. 7.4. DNA melting studies Another strong evidence for binding of the complexes 1, 2, and 3 to the double helix of DNA is the melting temperature Tm. The binding of small molecules into the double helix is known to increase the helix melting temperature. Helix melting temperature is the temperature at which the double helix is denatured into single-stranded DNA. The extinction coefficient of DNA bases at 260 nm in the double-helical form is much less than in the single stranded form. Hence melting of the helix leads to an increase in the absorption at this wavelength. Thus the transition temperature from helix to coil can be determined by monitoring the absorbance of the DNA base at 260 nm as a function of temperature. The melting curves of CT DNA in the absence and presence of 1, 2, and 3 are presented in Figure 10. The increase in the melting temperature values of IP and PIP comparable to the value observed (Table 2) with the classical intercalators EtBr. It is clear from these figures that the complexes 1 and 2 are intercalator because the relative absorbance is so high compared to that of the pure DNA sample. The increase in absorbance of complexes follows the order 1 > 2 > 3. 8. PHOTOACTIVATED CLEAVAGE OF pBR 322 DNA BY COMPLEXES There has been considerable interest in DNA endonucleolytic cleavage reactions that are activated by metal ions [23, 24]. The delivery of high concentrations of metal ion to the helix, in locally generating oxygen or hydroxide radicals, leads to an efficient DNA cleavage reaction. DNA cleavage was monitored by relation of supercoiled circular pBR 322 (form I) into nicked circular (form II) and linear (form III). When circular plasimd DNA is subjected to electrophoresis, relatively fast migration will be observed for the supercoiled form (form I). If scission occurs on one strand (nicking), the supercoils will relax to generate a slower-moving open circular form (form II) [25]. If both strands are cleaved, a linear form (form III) will be generated that migrates between forms I and II. Figure 11 shows the gel electrophoretic separations of plasmid pBR 322 DNA after incubation with Co(III) complexes and irradiation at 302 nm. Figure 9 reveals the conversion of Form I and II after 60 min irradiation in the presence of varying concentrations of 1, 2, and 3. It was observed that by increasing the concentration of 1, 2, and 3, form (I) slightly diminishes gradually. The same has been observed with increasing irradiation time. This is the result of single stranded cleavage of pBR322 DNA. It can also be seen in Figure 11 that neither irradiation of DNA at 302 nm without Co(III) nor incubation with Co(III) without light yields significant strand scission. It is most likely that the reduction of Co(III) is the important step leading to DNA cleavage. Further studies are required to find out the path of reaction mechanism. 9. CONCLUSIONS The binding behavior of complexes 1, 2, and 3 with DNA was characterized by absorption titration, fluorescence quenching, and viscosity measurements. The results show that the binding constants followed the order: 1 > 2 > 3 which is consistent with the extended planar and π system of PIP. ACKNOWLEDGMENT The authors are grateful to the UGC, New Delhi, India, for providing financial support in the form of MJRP. ABBREVIATIONS CT DNA:Calf thymus DNA PIP:2-phenylimidazo[4,5-f][1,10,]phenanthroline IP:Imidazo[4,5-f][1,10,]phenanthroline Phen:[1,10,]phenanthroline en:ethylenediamine EtBr:Ethedium bromide. Figure 1 Absorption spectra of CoCl26H2O (1), cis [Co(en)2Cl2]Cl (2), and [Co(en)2L]Br3 (3). Scheme 1 Scheme 2 Scheme 3 Figure 2 Absorption spectra of [Co(en)2PIP]3+ in the absence and presence of CT DNA in Tris-HCl buffer. The absorbance changes upon increasing CT DNA concentrations. (10 μL, 20 μL, 30 μL, 40 μL—of DNA addition), [Co] = 10 μM, [DNA] = 0–126 μM. The arrows show the decrease in intensity up on increasing DNA concentration. Insert: plots of [DNA]/(Σa − Σf) versus [DNA] for the titration of DNA with Co(III) complex. Figure 3 Absorption spectra of [Co(en)2IP]3+ in the absence and presence of CT DNA in Tris-HCl buffer in the absence (top) absorbance decreases upon increasing CT DNA concentrations, (10 μL, 20 μL, 30 μL, 40 μL—of DNA addition), [Co] = 10 μL, [DNA] = 0–126 μL. The arrows show the decrease in intensity up on increasing DNA concentration. Insert: plots of [DNA]/(Σa − Σf) versus [DNA] for the titration of DNA with Co(III) complex. Figure 4 Absorption spectra of [Co(en)2phen-dione]3+ in the absence and presence of CT DNA in Tris-HCl buffer, in the absence (top) absorbance changes upon increasing CT DNA concentrations. (10 μL, 20 μL, 30 μL, 40 μL—of DNA addition), [Co] = 10 μM, [DNA] = 0–126 μM. The arrows show the intensity decrease up on increasing DNA concentration. Insert: plots of [DNA]/(Σa − Σf) versus [DNA] for the titration of DNA with Co(III) complex. Figure 5 Emission spectra of complex of [Co(en)2PIP]3+ in aqueous buffer (Tris 5 mM, pH 7.2) at 298 K in the presence of CT DNA. [Co] = 20 μM, [DNA]/[Co] 0, 5, 10 …. λmex = 407 nm. Arrow shows the intensity change upon increasing DNA concentrations. Insert: plots of relative integrated emission intensity versus [DNA]/[Co]. Figure 6 Emission spectra of complex of [Co(en)2IP]3+ in aqueous buffer (Tris 5 mM, pH 7.2) at 298 K in the presence of CT DNA. [Co] = 20 μM, [DNA]/[Co] 0, 5, 10 …. λmex = 408 nm. Arrow shows the intensity change upon increasing DNA concentrations. Insert: plots of relative integrated emission intensity versus [DNA]/[Co]. Figure 7 Emission spectra of complex of [Co(en)2phen-dione]3+ in aqueous buffer (Tris 5 mM, pH 7.2) at 298 K in the presence of CT DNA. [Co] = 20 μM, [DNA]/[Co] 0, 5, 10 …. λ mex = 608 nm. Arrow shows the intensity change upon increasing DNA concentrations. Insert: plots of relative integrated emission intensity versus [DNA]/[Co]. Figure 8 Emission quenching curves of (A) [Co(en)2phen-dione]3+ in presence of DNA (▪), (B) [Co(en)2IP]3+ (▴) and (C) [Co(en)2PIP]3+ (⧫) alone ([Co] = 2 μmol/cm−3, [DNA]/[Co] = 40). Figure 9 Effect of increasing amount of [Co(en)2PIP]3+ (A), [Co(en)2IP]3+ (B), and [Co(en)2phen-dione]3+ (C) on the relative viscosities of CT DNA at 25 ± 0.1°C. Figure 10 Thermal melting curves of calf thymus DNA alone and in presence of complexes [Co(en)2PIP]3+ (a), [Co(en)2IP]3+ (b), and [Co(en)2phen-dione]3+ (c). Figure 11 Photoactivated cleavage of pBR 322 DNA, lane 1 control plasmid DNA (untreated pBR 322), lanes 2–5 addition of complex 5 μL, 10 μL, 20 μL, 30 μL, and 6th lane +5 μL at 0 time lanes 7, 8 +5 μL complex upon irradiation (λirrd = 302 nm) at 5 minutes, 10 minutes, complex (A) [Co(en)2phen-dione]3+, (B) [Co(en)2IP]3+, and (C) [Co(en)2PIP]3+. Table 1 λmax for different complexes. Complexes Peak Peak CoCl26H2O 229 492 nm cis [Co(en)2Cl2]Cl 346 594 nm [Co(en)2PIP]Br3 317 495 nm [Co(en)2IP]Br3 347 453 nm [Co(en)2phen-dione]Br3 333 445 nm Table 2 Thermal melting temperature (Tm) for CT DNA and CT DNA + various complexes. Compound TM°C CT DNA 60 [Co(en)2phen]3+ 62 [Co(en)2IP]3+ 64 [Co(en)2PIP]3+ 68 ==== Refs 1 Sigman DS Nuclease activity of 1,10-phenanthroline-copper ion Accounts of Chemical Research 1986 19 6 180 186 2 Sigman DS Mazumder A Perrin DM Chemical nucleases Chemical Reviews 1993 93 6 2295 2316 3 Jenkins Y Friedman AE Turro NJ Barton JK Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation Biochemistry 1992 31 44 10809 10816 1420195 4 Carlson DL Huchital DH Mantilla EJ Sheardy RD Murphy WR Jr A new class of DNA metallobinders showing spectator ligand size selectivity: binding of ligand-bridged bimetallic complexes of Ru(II) to calf thymus DNA Journal of the American Chemical Society 1993 115 14 6424 6425 5 Barton JK Tris (phenanthroline) metal complexes: probes for DNA helicity Journal of Biomolecular Structure & Dynamics 1983 1 3 621 632 6400890 6 Barton JK Metals and DNA: molecular left-handed complements Science 1986 233 4765 727 734 3016894 7 Carter MT Rodriguez M Bard AJ Voltammetric studies of the interaction of metal chelates with DNA. 2. Tris-chelated complexes of cobalt(III) and iron(II) with 1,10-phenanthroline and 2,2′-bipyridine Journal of the American Chemical Society 1989 111 24 8901 8911 8 Morgan RJ Chatterjee S Baker AD Strekas TC Effects of ligand planarity and peripheral charge on intercalative binding of Ru(2,2′-bipyridine)2 L2+ to calf thymus DNA Inorganic Chemistry 1991 30 12 2687 2692 9 Sitlani A Long EC Pyle AM Barton JK DNA photocleavage by phenanthrenequinone diimine complexes of rhodium(III): shape-selective recognition and reaction Journal of the American Chemical Society 1992 114 7 2303 2312 10 Nagababu P Latha JNL Satyanarayana S DNA-binding studies of mixed-ligand (ethylenediamine)ruthenium(II) complexes Chemistry and Biodiversity 2006 3 11 1219 1229 17193235 11 Yamada M Tanaka Y Yoshimoto Y Kuroda S Shimao I Synthesis and properties of diamino-substituted dipyrido [3,2-α : 2′, 3′ -c ]phenazine Bulletin of the Chemical Society of Japan 1992 65 4 1006 1011 12 Wu J-Z Ye B-H Wang L Bis(2,2′-bipyridine)ruthenium(II) complexes with imidazo[4,5-f][1,10]-phenanthroline or 2-phenylimidazo[4,5-f][1,10]phenanthroline Journal of the Chemical Society—Dalton Transactions 1997 8 1395 1401 13 Barve AC Ghosh S Kumbhar AA Kumbhar AS Puranik VG DNA-binding studies of mixed ligand cobalt(III) complexes Transition Metal Chemistry 2005 30 3 312 316 14 Bailar JC Jr Fernelius WC Cis & trans dichlorobis - (ethylenediamine) Co (III) chloride Inorganic Syntheses: Volume II 1946 New York, NY, USA McGraw-Hill 15 Marmur J A procedure for the isolation of deoxyribonucleic acid from microorganisms Journal of Molecular Biology 1961 3 208 218 16 Reichmann ME Rice SA Thomas CA Doty P A further examination of the molecular weight and size of desoxypentose nucleic acid Journal of the American Chemical Society 1954 76 11 3047 3053 17 Chaires JB Dattagupta N Crothers DM Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: equilibrium binding studies on interaction of daunomycin with deoxyribonucleic acid Biochemistry 1982 21 17 3933 3940 7126524 18 Satyanaryana S Dabrowiak JC Chaires JB Tris(phenanthroline)ruthenium(II) enantiomer interactions with DNA: mode and specificity of binding Biochemistry 1993 32 10 2573 2584 8448115 19 Barton JK Metals and DNA: molecular left-handed complements Science 1986 233 4765 727 734 3016894 20 Barton JK Danishefsky AT Goldberg JM Tris(phenanthroline)ruthenium(II): stereoselectivity in binding to DNA Journal of the American Chemical Society 1984 106 7 2172 2176 21 Wolfe A Shimer GH Jr Meehan T Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA Biochemistry 1987 26 20 6392 6396 3427013 22 Zhang Q-L Liu J-G Chao H Xue G-Q Ji L-N DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes: [Co(phen)2 IP]3+ and [Co(phen)2 PIP]3+ Journal of Inorganic Biochemistry 2001 83 1 49 55 11192699 23 Hertzberg RP Dervan PB Cleavage of double helical DNA by (methidiumpropyl-EDTA)iron(II) Journal of the American Chemical Society 1982 104 1 313 315 24 Graham DR Marshall LE Reich KA Sigman DS Cleavage of DNA by coordination complexes. Superoxide formation in the oxidation of 1,10-phenanthroline-cuprous complexes by oxygen-relevance to DNA-cleavage reaction Journal of the American Chemical Society 1980 102 16 5419 5421 25 Barton JK Raphael AL Photoactivated stereospecific cleavage of double-helical DNA by cobalt(III) complexes Journal of the American Chemical Society 1984 106 8 2466 2468	18253471	PMC1997276	CC BY	2021-01-05 10:03:07	yes	Bioinorg Chem Appl. 2007 Jun 25; 2007:54562
==== Front Mol NeurodegenerMolecular Neurodegeneration1750-1326BioMed Central 1750-1326-2-191790327410.1186/1750-1326-2-19Research ArticleDopaminergic regeneration by neurturin-overexpressing c17.2 neural stem cells in a rat model of Parkinson's disease Liu Wei-Guo [email protected] Xi-Jing [email protected] Guo-Qiang [email protected] Biao [email protected] Gang [email protected] Sheng-Di [email protected] Department of Neurology & Neuroscience Institute, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China2 Lab of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes of Biological Sciences, Chinese Academy of Science & Shanghai Jiaotong University School of Medicine, Shanghai 200025, China2007 1 10 2007 2 19 19 12 3 2007 1 10 2007 Copyright © 2007 Liu et al; licensee BioMed Central Ltd.2007Liu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Genetically engineered neural stem cell (NSC) lines are promising vectors for the treatment of neurodegenerative diseases, particularly Parkinson's disease (PD). Neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family, has been demonstrated to act specifically on mesencephalic dopaminergic neurons, suggesting its therapeutic potential for PD. In our previous work, we demonstrated that NTN-overexpressing c17.2 NSCs exerted dopaminergic neuroprotection in a rat model of PD. In this study, we transplanted NTN-c17.2 into the striatum of the 6-hydroxydopamine (6-OHDA) PD model to further determine the regenerative effect of NTN-c17.2 on the rat models of PD. Results After intrastriatal grafting, NTN-c17.2 cells differentiated and gradually downregulated nestin expression, while the grafts stably overexpressed NTN. Further, an observation of rotational behavior and the contents of neurotransmitters tested by high-performance liquid chromatography showed that the regenerative effect of the NTN-c17.2 group was significantly better than that of the Mock-c17.2 group, and the regenerative effect of the Mock-c17.2 group was better than that of the PBS group. Further research through reverse-transcriptase polymerase chain reaction assays and in vivo histology revealed that the regenerative effect of Mock-c17.2 and NTN-c17.2 cell grafts may be attributed to the ability of NSCs to produce neurotrophic factors and differentiate into tyrosine hydroxylase-positive cells. Conclusion The transplantation of NTN-c17.2 can exert neuroregenerative effects in the rat model of PD, and the delivery of NTN by NSCs may constitute a very useful strategy in the treatment of PD. ==== Body Background A pathologic feature of Parkinson's disease (PD) is the loss of melanized dopaminergic neurons within the substantia nigra (SN) pars compacta coupled with depletion of striatal dopamine. This is responsible for the major motor features of the disease [1]. Current symptomatic treatments involving dopaminergic replacement therapy and deep brain stimulation (DBS) therapy cannot prevent further neurodegeneration and disease progression [2,3]. In recent years, researchers have searched for preventative and curative strategies, leading to the development of certain novel therapeutic approaches. Most of these approaches are based on strategies of neuroprotection, whereby dopaminergic neurons are prevented from dying, and neuroregeneration, whereby dead or injured neurons are supplemented by transplantation [4-7]. Neurturin (NTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. NTN acts on dopaminergic neurons through a receptor complex composed of ligand-binding subunits and GDNF family receptors a-1 (GFR a-1) and a-2 (GFR a-2) [8,9]. The neuroprotective and neuroregenerative effects of NTN are equipotent to GDNF when tested on developing ventral mesencephalic dopamine (DA) neurons cultured in vitro [10] as well as on damaged nigrostriatal DA neurons in vivo [11]. Based on the neuroprotective effects on nigrostriatal DA neurons, NTN has been suggested as a candidate for the treatment of PD. Recently, we cloned the prepro-NTN cDNA and inserted it into the pcDNA3.1-hygro-NTN plasmid for introduction into a stable NSC line (c17.2) [12]. We have demonstrated that NTN-expressing c17.2 neural stem cells (NSCs) exerted dopaminergic neuroprotection in a rat model of PD [13]. In this study, we transplanted NTN-c17.2 into the striatum of the 6-hydroxydopamine (6-OHDA) model of PD to further determine the regenerative effect of NTN-c17.2 on the rat models of PD. Results Engineering and characterization of NTN expression in NSCs The c17.2 mouse NSCs were transfected with the pcDNA3.1-Hygro-NTN vector to generate NTN-c17.2 cells or the pcDNA3.1-Hygro vector to generate Mock-c17.2 cells. The amount of NTN mRNA and secreted NTN protein in the culture medium were detected by northern and western blots, respectively. Clone 1, the highest expressor, was named NTN-c17.2, and detailed characterization of NTN-c17.2 was described elsewhere [13]. Detection of NTN protein expression in NSCs after intrastriatal grafting in vivo NTN and nestin protein-expressing cells of the corpus striatum were identified by immunohistochemistry 4.5, 30, and 120 d after the animals were transplanted with NTN-c17.2 cells (Fig. 1A–F). A large number of cells expressing NTN and nestin protein were detected at 4.5 d (Fig. 1D). The majority of cells were round and not differentiated. The cells differentiated 30 days after transplantation (Fig. 1E) but only expressed the NTN protein, while the number of cells declined. A small number of NTN-positive cells were still detected 120 d postgrafting (Fig. 1F). Figure 1 In the neuroregeneration study, the engrafted NTN-c17.2 cells survived the grafting procedure well and expressed high levels of the NTN protein in vivo. A-F, Double immunohistochemistry with anti-β-Gal (LacZ, in red) or NTN (red) antibody (no crossreaction with GDNF) and anti-Nestin (green) showed cells expressing the NTN protein in the NTN-c17.2 engrafted striatum 4.5 d, 30 d and 120 d postgrafting; however, with time, the cells differentiated, and the number of cells declined and almost no nestin protein was detected 30 d and 120 d postgrafting. G, Quantification of the number of LacZ+ Nestin+, and NTN+ Nestin+ cells. The number of immune-positive cells were counted in 7 serial sections through the striatum. Values represent the mean ± SEM. #P < 0.01. The striatum grafted with NTN-c17.2 or Mock-c17.2 cells at 4.5 d were compared with 30 d and 120 d postgrafting, which was determined by one-way ANOVA. Scale bars: 250 μm. NTN-c17.2 cells efficiently prevent the loss of nigral dopaminergic neurons in a rat model of PD In our study, tyrosine hydroxylase (TH) immunohistochemistry demonstrated comparable 40% or 36% loss of nigral dopaminergic neurons in animals injected with PBS plus 6-OHDA or Mock-c17.2 plus 6-OHDA, respectively (Fig. 2C,D,E). In contrast, only a 16% loss of nigral dopaminergic neurons was measured in animals grafted with the NTN-c17.2 NSCs (P < 0.05) (Fig. 2B,E). These results suggest that the grafts of NTN-c17.2 cells in the corpus striatum could protect the TH-positive neurons of the ipsilateral SN from 6-OHDA insult. Figure 2 In the neuroprotection study, NTN-c17.2 grafts protected SN dopaminergic neurons in a 6-OHDA model of PD. A-D, TH immunohistochemistry showed the grafting of NTN-c17.2 cells (B) prevented the loss of dopaminergic neurons in the SN, while the grafts of Mock-c17.2 cells (C) and treatment with PBS (D) could not prevent the loss of dopaminergic neurons. E, Quantification of the number of TH-positive neurons in SN under the indicated experimental conditions. The number of TH-positive cells was counted in 7 serial sections of the SN. Values represent the mean ± SEM (n = 5). #P < 0.05. The lesioned SN grafted with NTN-c17.2 cells was compared with samples from animals grafted with Mock-c17.2 cells or injected with PBS, as determined by one-way ANOVA (significant effect of treatment, p < 0.05). Scale bars: (in D) A-D, 250 μm. Behavior observation Intrastriatal grafting of NTN-c17.2 and Mock-c17.2 improves behavioral abnormalities We observed the ethology of experimentally grafted rats for up to 10 months to determine whether NTN-c17.2 cells had a regenerative effect on animal behavioral abnormalities (Fig. 3). The behavioral asymmetry in the NTN-c17.2 group was improved at almost all time points tested as compared with the PBS and Mock-c17.2 groups. A significant statistical difference was noted between the NTN-c17.2 group and PBS group from 2 months to 10 months. Behavioral improvement was also observed from 3 months to 10 months among the NTN-c17.2 and Mock-c17.2 groups. Otherwise, the behavioral asymmetry was greatly improved in the Mock-c17.2 group than in the PBS group from 4 months to 10 months following grafting. Figure 3 In the neuroregeneration study, the NTN-c17.2 and Mock-c17.2 cells survived after transplantation and ameliorated the apomorphine-induced rotational behavior in the rat model of PD. The rat model of 6-OHDA-induced hemiparkinsonism was selected and grafting was performed. Apomorphine-induced circling behavior was tested every month for 10 months after transplantation. From 2 to 10 months, the rats engrafted with NTN-c17.2 cells showed fewer rotations than those injected with Mock-c17.2 cells (#P < 0.05) or PBS (P < 0.05). From 4 to 10 months, rats engrafted with Mock-c17.2 showed fewer rotations as compared with those injected with PBS (P < 0.05). NTN-c17.2 and Mock-c17.2 NSCs exert regenerative effects assayed by high- performance liquid chromatography Ten months after the ethological observation, the animals were sacrificed and examined for the measurement of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) levels by high-performance liquid chromatography (HPLC) (Fig. 4). The contents of DA, DOPAC, and HVA in the corpus striatum were 35%, 42%, and 39%, respectively, in the PBS group; 67% (P < 0.05), 77% (P < 0.05), and 73% (P < 0.05), respectively, in the NTN-c17.2 group; and 52% (P < 0.05), 51%, and 65%, respectively, in the Mock-c17.2 group. Taken together, these results showed that the NTN-c17.2 and Mock-c17.2 groups recovered significantly during the latter period of the transplantation study as compared with the PBS group, and the NTN-c17.2 group recovered better than the Mock-c17.2 group. Figure 4 DA, DOPAC, and HVA concentrations (mean ± SEM) in the striatum as measured by HPLC with electrochemical detection. NSCs expressing c17.2 and their derivatives constitutively secrete neurotrophic factors and partially differentiate into TH-positive cells To further determine the regenerative effect of NTN-c17.2 and Mock-c17.2 transplantation on 6-OHDA-induced Parkinsonism in rats, we first performed a double immunofluorescence assay to test whether NTN-c17.2 and Mock-c17.2 cells transplanted in the corpus striatum could differentiate into TH-positive cells. Simultaneously, c17.2 cells were transplanted into the corpus striatum. We found that only a few cells differentiated into TH-positive cells (<10%) (Fig. 5A–I), and no significant statistical difference was noted in the number of cells among NTN-c17.2, Mock-c17.2, and c17.2. Secondly, we used reverse-transcriptase polymerase chain reaction (RT-PCR) to specifically test Mock-c17.2 and NTN-c17.2 for the expression of the mouse-derived neurotrophic factors GDNF, brain-derived neurotrophic factor (BDNF) (Fig. 5J), nerve growth factor (NGF), and NTN in the transplanted corpus striatum. We identified the expression of these neurotrophic factors in vitro and in vivo. Thus, the regenerative effect of Mock-c17.2 transplantation on the rat PD model may be due, at least in part, to both palliative differentiation and expression of neurotrophic factors, while this effect could be strengthened by NTN-c17.2 transplantation. Figure 5 In the neuroregeneration study, it is possible that the observed neuroregenerative effect from intrastriatal grafting with NTN-c17.2 and Mock-c17.2 cells may be due to partial differentiation of cells into TH-positive cells and the expression of neurotrophic factors by NSCs in vivo. A-I, Double immunohistochemistry of TH (B, E, H), LacZ (A, D), and NTN(G) revealed that some grafted c17.2, NTN-c17.2, and Mock-c17.2 cells could differentiate into TH-positive cells 30 d postgrafting. J, Nonquantitative RT-PCR of neurotrophic factor genes using mouse-specific primers (mouse-specific) demonstrated the expression of these genes in Mock-c17.2 (lane 1), NTN-c17.2 cells (lane 4), rat striatum grafted with Mock-c17.2 cells (lane 2), and NTN-c17.2 cells (lane 5) (30 d after grafting), but this expression was not observed after RT-PCR of the cDNA of control rat cerebrum (lane 3). The endogenous control β-actin was amplified using mouse-specific primers in lanes 1, 2, 4, and 5 with rat-specific primers in lane 3. Discussion Neuroprotective therapy involves postponing or retarding the development of a neurodegenerative disease by inhibiting its pathogenic factors. The exact pathogenesis of PD is not fully understood, and there is no evidence that PD is linked to deficiencies in GDNF and other neurotrophic factors. However, experimental data have shown that oxidative stress, mitochondrial dysfunction, and calcium overload induced by internal and external toxins are associated with the pathogenesis of PD [14-17]. Similar to GDNF, NTN not only can promote the development and function of dopaminergic neurons but also has neuroprotective and regenerative effects on dopaminergic neurons injured by neurotoxins such as 6-OHDA, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methyl amphetamine [11,18]. In our previous experiment, we transplanted an NTN-secreting c17.2 NSC line into the striatum of a 6-OHDA PD model and found that the inverse transfer of the NTN protein into the SN protected dopaminergic neurons from 6-OHDA toxicity. The observation of rotational behavior showed that the improvement in the NTN group was obvious compared with that in the Mock group (data not shown). Only NTN-c17.2 NSCs demonstrated their neuroprotective effects. To further investigate the therapeutic effects of NTN on a 6-OHDA-induced hemiparkinsonian rat model, we transplanted the various NSC cells into the corpus striatum of the model animals and measured their rotational activity in response to apomorphine. We know that the rat model of PD induced by 6-OHDA has a tendency of gradual recovery, which was also observed in our research. After 4 months, the number of rotations in the PBS group decreased gradually. However, the overall number of rotations demonstrated the following order: NTN group < Mock group < PBS group. After 4 months, the NTN and Mock groups showed a similar number of rotations, which was clearly lower than those in the PBS group. The difference between the NTN and Mock groups was statistically significant from 4 to 10 months. Since the model was established by administering 6-OHDA injections into the ventral tegmental area (VTA) and the medial forebrain bundle (MFB), the dopaminergic neurons in the SN and the nigrostriatal dopaminergic pathway were both damaged. Thus, our previous results are explained by the fact that while the NTN protein protects against 6-OHDA toxicity and the resultant inflammatory process, it cannot increase the total number of dopaminergic neurons after the SN is destroyed. We thought that the transplanted NSC cells combined with the host cells in the striatum and improved the symptoms in a hemiparkinsonian rat model 4 months post-transplantation. Further, the study of rotational behavior and DA and its metabolites in the therapeutic study supported this conclusion [19]. Yang et al [20] found that after the c17.2 clonal lines were transplanted into the intact striatum or striatum bearing 6-OHDA-induced lesions, the majority of cells spontaneously expressed the DA biosynthetic enzymes, TH, and aromatic L-amino acid decarboxylase. Further study [21] showed that all the engrafted cells in 65% of the grafts that were obtained only from high confluence cultures and maintained for 12–20 passages expressed TH but not the markers of other neurotransmitter systems. However, in our study, c17.2 was transfected with NTN and Mock, which ranged from 5 to 9 passages and was not passaged at a comparably high confluence. We have shown that NTN-c17.2 and Mock-c17.2 NSCs can differentiate only partially into TH-positive neurons, not as many as observed by Yang. There was no obvious difference in cell numbers between NTN-c17.2 and Mock-c17.2 NSCs (data not shown). These TH-positive neurons and other differentiated cells from transplanted cells improved the symptoms of the hemiparkinsonian rat model. The present RT-PCR experiment revealed that the mRNAs for GDNF, BDNF, and NGF were expressed in NTN-c17.2 and Mock-c17.2 NSCs both in vitro and in vivo. This is consistent with the findings of Lu et al [22] who reported that NSCs constitutively secreted neurotrophic factors. Although the concentrations of these neurotrophic factors are not as high as NTN from NTN-c17.2, their regenerative effects are potent. Thus, NSCs can not only differentiate into distinct cells of the nervous system but also promote the repair of the nervous system by secreting neurotrophic factors. Hence, the regenerative effect of NSC grafts may be related to these 2 factors, while the NTN-c17.2 cell grafts may reinforce the therapeutic effect through high expression of NTN. Conclusion The transplantation of NTN-c17.2 exerted neuroregenerative effects in the rat model of PD. The regenerative effect of NSC grafts may be related to these 2 factors: NSCs can differentiate into distinct cells of the nervous system and constitutively secrete neurotrophic factors, while the NTN-c17.2 cell grafts may reinforce the therapeutic effect through high expression of NTN. Materials and methods Cell culture c17.2 NSCs and their derivatives were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 5% horse serum, 100 mg/L ampicillin, and 100 mg/L streptomycin (all obtained from Life Technologies, Grand Island, NY) at 37°C under a 5% CO2 atmosphere and passaged as described previously [12]. The differentiating cells were grown in an N2 medium consisting of a 1:1 mixture of F12 and DMEM with 1% N2. c17.2 NSCs contained the autonomous genetic marker LacZ in the cell. c17.2 transfected with NTN and Mock was named passage 1. The cells were passaged at 90% confluence. The cells from 5 to 9 passages were collected for transplantation. Construction of the NTN-c17.2 cell line A cDNA fragment encoding prepro-NTN was amplified by RT-PCR using total RNA from mouse testis. The cDNA was further subcloned into pcDNA3.1-Hygro plasmids, which were transfected into c17.2 cells using the Lipofectamine (LF) 2000 reagent (Life Technologies). Briefly, according to the manufacturer's instructions, logarithmically growing cells were transfected with 1 μg of plasmids and 2 μl of LF2000 reagent. At 72 h after transfection, the selective medium containing 1 mg/ml of the antibiotic hygromycin (Life Technologies) was selected. Two weeks later, 5 colonies were picked, propagated, and characterized for mRNA and protein expression. The mRNA assay was detected by northern blot, and protein expression was detected by western blot. The detailed process is described in our previous work [13]. Study design Young adult male Sprague-Dawley rats (Sino-British Sippr/Bk Lab Animal Ltd., Shanghai, China), weighing 180–210 g at the beginning of this experiment, were housed under a 12-h light/12-h dark cycle with free access to food and water. All the experiments were approved by the local ethics committee. A total of 90 male Sprague-Dawley rats were used for the neuroprotection and regeneration studies, as shown in Table 1. For the neuroprotection study, NTN-c17.2, Mock-c17.2, and PBS were injected into the striatum, and striatal lesions were induced using 6-OHDA on the same side after 15 d [13]. In the neuroregeneration study, 16 μg of 6-OHDA was stereostatically injected into MFB and VTA at the following coordinates (in millimeters) with an incisor bar at -2.4: anteroposterior (AP)(bregma), -3.7; lateral (L), 1.7; dorsoventral (DV), -7.8; and AP (bregma), -4.8; L, 1.0; DV, -7.8, respectively. Four weeks post-injection, 45 animals showing rotational behavior of over 7 turns/min in response to 0.5 mg/kg of apomorphine (Sigma) were selected for neuroregeneration study of NTN. Proliferative state Mock-c17.2 and NTN-c17.2 cells were washed twice with serum-free DMEM, detached with a cell lifter (Costar, Cambridge, MA), dissociated with a fire-polished Pasteur pipette, pelleted, and resuspended at a concentration of 50,000 cells/μl. A total of 600,000 cells were injected into 6 locations at the following coordinates (in millimeters) with the incisor bar at 0: AP (bregma), 1.0; L, 3.0; DV (dura), -4.5 and -5.0; AP (bregma), -0.1; L, 3.7; DV (dura), -4.5 and -5.0; and AP (bregma), -1.2; L, 4.5; DV (dura), -4.5 and -5.0. Every month post-transplantation, the animals (n = 15 in each group) were tested for similar rotational behavior at different time points, and biochemical measurements were carried out at the end of the experiments. Table 1 Experimental design of the neuroprotection and regeneration study Protection Study NTN (n = 15) Mock (n = 15) PBS (n = 15) 15 days 6-OHDA lesion 4.5 days or every month (n = 15 in each group) Rotation behavior and Sacrifice for IHC, HPLC Regeneration Study 6-OHDA lesion (n = 45) 4 weeks Rotation Behavior NTN (n = 15) Mock (n = 15) PBS (n = 15) every month (n = 15 in each group) Rotation behavior and Sacrifice for IHC, HPLC NTN-c17.2, Mock- c17.2, and PBS were transplanted into the striatum by the conventional microinjection method. Subsequently, 6-hydroxydopamine (6-OHDA) was infused into the striatum or into the medial forebrain bundle (MFB) and the ventral tegmental area (VTA) in the neuroprotection study or regeneration study, respectively (see Materials and Methods). After transplantation, all the animals were analyzed for apomorphine-induced rotational behavior and then sacrificed for immunohistochemistry (IHC) and high-performance liquid chromatography (HPLC) measurements. Histology Rats were sacrificed and transcardially perfused with 4% paraformaldehyde. Serial cryostat sections (15 μm thick) of the striatum were obtained. The specific cell-type markers used were anti-nestin for NSCs (1:1000; Chemicon, Temecula, CA), anti-NTN for the NTN protein (1:400; Santa Cruz), anti-TH for dopaminergic neurons, and anti-β-Gal for c17.2 or Mock-c17.2 cells (1:1000; Chemicon). The appropriate secondary antibodies were horse anti-mouse fluorescein isothiocyanate-conjugated antibody (1:100; Vector Laboratories, Burlingame, CA), horse anti-mouse Texas Red (1:100; Vector), or rabbit anti-goat fluorescein (1:100; Vector). Double immunostaining of the sections was performed by simultaneous incubation of the sections with the appropriate pairs of primary and secondary antibodies. The number of LacZ+ Nestin+, NTN+ Nestin+, or TH+ cells were counted in the striatum by using an optical fractionator method for unbiased stereological cell counting [23]. The stained slides were examined using a Zeiss microscope (Oberkochen, Germany). RT-PCR for in vitro and in vivo measurements of growth factor mRNA expression Total RNA was isolated from undifferentiated cultured NTN-c17.2 and Mock-c17.2 NSCs, grafted NTN-c17.2 and Mock-c17.2, and contralateral cerebrum (30 d after grafting) using the Trizol reagent. First-strand cDNA was synthesized from 2 μg total RNA using the Reverse Transcription System for RT-PCR Kit (Promega, USA) with oligo (dT) priming, according to the manufacturer's instructions. In order to specifically detect the gene expression of mouse NSC-derived growth factor, mouse-specific primers with at least one 3' end nucleotide mismatch to the rat genes were designed for the genes of the neurotrophic factors NGF, BDNF, GDNF [22], and NTN based on BLAST comparison of the mouse and rat genes. The mouse and rat β-actin genes were amplified to serve as a normalization control. Statistical analysis All data were expressed as mean ± SEM and analyzed using SPSS statistical software. The changes in turning behavior were analyzed using one-way ANOVA with repeated measures for time. Statistical significance was defined as P < 0.05. Acknowledgements The authors would like to thank Dr. Even Y. Snyder (Children's Hospital, Harvard Medical School, Boston, USA) for his generous gift of c17.2 neural stem cells. This work was supported by grants from the National Program of Basic Research of China (No. 2006cb500706), the National Natural Science Foundation of China (No. 30570637 and No. 30471918) and the Shanghai Key Project of Basic Science Research (No. 04DZ14005) and the Program for Outstanding Medical Academic Leader (LJ 06003) ==== Refs McGeer PL Itagaki S Akiyama H McGeer EG Rate of cell death in parkinsonism indicates active neuropathological process Ann Neurol 1988 24 574 576 3239957 10.1002/ana.410240415 Olanow CW Tatton WG Etiology and pathogenesis of Parkinson's disease Annu Rev Neurosci 1999 22 123 144 10202534 10.1146/annurev.neuro.22.1.123 Betchen SA Kaplitt M Future and current surgical therapies in Parkinson's disease Curr Opin Neurol 2003 16 487 493 12869808 10.1097/00019052-200308000-00008 Burton EA Glorioso JC Fink DJ Gene therapy progress and prospects: Parkinson's disease Gene Ther 2003 10 1721 1727 12939638 10.1038/sj.gt.3302116 Dunnett SB Bjorklund A Prospects for new 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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central 1471-2202-8-811790833010.1186/1471-2202-8-81Research ArticleAgmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line Hong Samin [email protected] Jong Eun [email protected] Chan Yun [email protected] Gong Je [email protected] Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea2 Department of Anatomy, Yonsei University College of Medicine, Seoul, Korea2007 2 10 2007 8 81 81 3 4 2007 2 10 2007 Copyright © 2007 Hong et al.; licensee BioMed Central Ltd.2007Hong et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5). RGC-5 cells were cultured in a closed hypoxic chamber (5% O2) with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH) assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38) and nuclear factor-kappa B (NF-κB) were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for retinal ganglion cells. Results After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-κB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-κB, while BDNF suppressed phosphorylation of ERK and p38. Conclusion Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-κB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia. ==== Body Background Agmatine is an endogenous polyamine that is synthesized by the decarboxylation of L-arginine by arginine decarboxylase [1,2]. It is known to be widely but unevenly distributed in the brain and other mammalian tissues [3,4]. Agmatine has been reported to have various biological actions. It stimulates the release of catecholamines from adrenal chromaffin cells [3], insulin from pancreatic islets [5], and luteinizing hormone-releasing hormone from the hypothalamus [6]. Also, it enhances analgesic effects of morphine [7], inhibits inducible nitric oxide synthase (NOS) [8], and contributes to polyamine homeostasis [2,9]. It is known that agmatine is an agonist for α2-adrenergic and imidazoline receptors [3], and an antagonist for the N-methyl-D-aspartate (NMDA) receptor [10]. However, the precise cellular mechanisms by which agmatine acts are not yet well established. Currently, a large body of experimental evidence has demonstrated the neuroprotective effects of agmatine. Agmatine reduces infarct areas and neuronal loss in cerebral ischemic and ischemic-reperfusion injury models [11-13]. It protects neurons from cell death after exposure to NMDA and glutamate [14,15]. It also attenuates the extent of neuronal loss following a spinal cord injury [16,17] and shelters neurons from glucocorticoid-induced neurotoxicity [18] and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-related dopaminergic toxicity [19]. On the basis of these neuroprotective effects, agmatine can be presumed to have similar neuroprotective effects on retinal ganglion cells (RGCs). Several molecules, including α2-adrenergic agonists [20-25], NMDA receptor antagonists [26-28] and NOS inhibitors [29], have been reported to protect RGCs. Agmatine also acts as an α2-adrenergic agonist [3], NMDA receptor antagonist [10], and suppressor of inducible NOS [8]. In the present investigation, we examined the protective effects of agmatine on hypoxia-induced apoptosis of RGCs by using the transformed rat RGCs (RGC-5 cell line) [30-32]. Effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for RGCs [33-35]. In addition, several molecular pathways associated with these neuroprotective effects of agmatine were evaluated. Results Agmatine inhibits hypoxia-induced cell damage of RGC-5 cells We first examined the effects of hypoxia on RGC-5 cells. As shown in Figure 1, exposure to a hypoxic environment for 12, 24, and 48 hours significantly increased release of lactate dehydrogenase (LDH) by 10.17%, 20.04%, and 52.25%, respectively (all p < 0.001), thus demonstrating time-dependent hypoxia-induced neurotoxicity. Figure 1 LDH release in RGC-5 cells. LDH release in RGC-5 cells, illustrating the neuroprotective effects of agmatine and BDNF against hypoxia for (A) 12 hours, (B) 24 hours, and (C) 48 hours. Data are shown as mean ± S.E.M. of 32 measurements. *P < 0.001. Next, we examined the protective effects of agmatine on hypoxia-induced damage in RGC-5 cells. After 12 and 24 hours of hypoxia, agmatine treatment groups did not show significant amounts of LDH release (Fig. 1A and 1B), but there were significant effects after 48 hours of exposure (Fig. 1C). After 48 hours, the addition of 100 μM and 500 μM agmatine decreased hypoxia-induced LDH release by 25.60% and 27.09%, respectively (both p < 0.001). When the protective effects of 100 μM agmatine were compared with those of 10 ng/mL BDNF, agmatine demonstrated a more powerful protective effect than that observed for BDNF (p < 0.001). The neuroprotective effect of agmatine against hypoxia-induced damage to RGC-5 cells was further studied using Hoechst 33342 and propidium iodide (PI) double staining. The control normoxic culture exhibited confluent Hoechst-positive cells with homogeneous and compact nuclear morphology, and sparse numbers of PI-labeled cells (Fig. 2A). Exposure to hypoxia for 48 hours resulted in a significant loss of Hoechst-positive cells and many PI-positive cells with distorted and condensed nuclei (Fig. 2B). These changes were reduced by the addition of 100 μM agmatine (Fig. 2C) or 10 ng/mL BDNF (Fig. 2D) to the cultures, and agmatine had a greater protective effect. Figure 2 Hoechst 33342 and propidium iodide double staining in RGC-5 cells. Agmatine and BDNF reduce the hypoxia-induced cell death in RGC-5. RGC-5 cells were exposed to hypoxia for 48 hours either alone (B) or in the presence of 100 μM agmatine (C) or 10 ng/mL BDNF (D). A control normoxic culture is shown in (A). The cultures were stained with Hoechst 33342 and propidium iodide. The magnification is × 400. Agmatine protects RGC-5 cells from hypoxia-induced apoptosis In order to verify whether agmatine had protective effects on hypoxia-induced apoptotic death of RGC-5 cells, further analyses using annexin V assay were performed. While 12 hours of hypoxic exposure did not change the proportion of apoptotic cells compared with the normoxic culture, there were significant increases in apoptotic cells after 24 hours (Fig. 3B). With the addition of 100 μM agmatine and 10 ng/mL BDNF, the proportion of apoptotic cells decreased (Fig. 3C and 3D). Figure 3 Annexin V assay in RGC-5 cells. Flow cytometric analysis of effects of agmatine and BDNF on the hypoxia-induced apoptosis of RGC-5 cells. Cells were exposed to hypoxia for 24 hours either alone (B) or in the presence of 100 μM agmatine (C) or 10 ng/mL BDNF (D). A control normoxic culture is shown in (A). Cultures were stained with annexin V-FITC and propidium iodide. Cells of high reactivity with FITC and low reactivity with propidium iodide (right lower area) are the apoptotic cells. Specific caspase-3 activity was assessed using a caspase-3 assay, which could measure the cleavage of the caspase-3 specific substrate Ac-DEVD-pNA (Fig. 4). After 24 hours of hypoxic injury, the caspase-3 activity was significantly increased, and it was suppressed by 100 μM agmatine. The results obtained by adding 100 μM agmatine were similar to those seen with 50 μM caspase-3 inhibitor Z-VAD-FMK. Figure 4 Caspase-3 assay in RGC-5 cells. Colorimetric analysis of the effects of agmatine on the caspase-3 activity induced by hypoxic injury in RGC-5 cells. Cells were exposed to hypoxia for 24 hours with or without 100 μM agmatine or caspase-3 inhibitor Z-VAD-FMK (50 μM). Specific activity of caspase-3 was measured by cleavage of the caspase-3 substrate Ac-DEVD-pNA. Selective suppression of JNK activation by agmatine Three mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase, (ERK) and p38 kinase (p38), were investigated using Western immunoblots. The amounts of total and phosphorylated MAPKs and β-actin are shown in Figure 5. Figure 5 Western blot analysis of MAPKs in RGC-5 cells. Western blot analysis showing effects of agmatine and BDNF on mitogen-activated protein kinases (MAPKs). Western immunoblots probed with antibodies against JNK and phospho-JNK (A), ERK and phospho-ERK (B), p38 and phospho-p38 (C), and β-actin (D). Total expression of the three MAPKs (JNK, ERK, and p38) and β-actin were not affected by hypoxic injury. In addition, there were no significant changes after treatment with BDNF or agmatine. Antibodies against phospho-JNKs detected two bands at 54 and 46 kDa, and both bands had a similar tendency. Increases of phospho-JNKs in RGC-5 cells became evident 9 hours after hypoxic injury and remained elevated for 12 hours (Fig. 5A). Agmatine suppressed the hypoxia-induced phosphorylation of JNKs, but BDNF did not influence their phosphorylation. Antibodies against phospho-ERKs also detected two bands at 44 and 42 kDa, and both bands were similar. Phospho-ERKs were not detected in the normoxic cultures. However, they were highly expressed in RGC-5 cells even after 3 hours of hypoxia and remained elevated for 12 hours (Fig. 5B). BDNF completely blocked the phosphorylation of ERKs for 6 hours, but it had no effect thereafter. In comparison, agmatine did not significantly affect the phosphorylation of ERKs. Antibodies against phospho-p38 detected one band at 38 kDa. Phospho-p38 was not detected in normoxic cultures until 12 hours of exposure to hypoxia, but it was evident in hypoxic cultures even after 3 hours and remained elevated for 12 hours (Fig. 5C). BDNF only blocked the phosphorylation of p38 at 6 hours and agmatine had no effect on phospho-p38 levels at any time points. Thus, phospho-MAPKs showed different activation profiles in response to hypoxic injuries in RGC-5 cells; ERK and p38 were activated relatively earlier than JNK. BDNF inhibited the activation of ERK (until 6 hours after hypoxia) and p38 (at 6 hours after hypoxia), while agmatine suppressed the activation of JNK (in significant amounts from 9 hours after hypoxia). Suppression of NF-κB signaling by agmatine Total expression and activation of the nuclear factor-kappa B (NF-κB) from nuclear and cytosolic fractions were evaluated separately. Representative bands from the Western immunoblots are shown in Figure 6. Antibodies against total and phospho-NF-κB bound to their respective bands at 65 kDa. Figure 6 Western blot analysis of NF-κB in RGC-5 cells. Western blot analysis showing the effect of agmatine and BDNF on nuclear factor-kappa B (NF-κB). Western immunoblots probed with antibodies against NF-κB and phospho-NF-κB from nuclear (A) and cytosolic (B) proteins. Histone 3 (A) and β-actin (B) were used as internal controls. In nuclear fraction, total NF-κB and histone 3 were unaffected by hypoxic injury, and there were no changes with the addition of BDNF and agmatine. However, phospho-NF-κB was significantly increased with 1 hour of hypoxia and returned to normal levels after 3 hours. This increase in phospho-NF-κB was suppressed by agmatine but not by BDNF (Fig. 6A). In comparison, in cytoplasmic fraction, there were no significant changes in levels of phospho-NF-κB and β-actin. However, total NF-κB expression increased after 1 hour exposure to a hypoxic environment. This increase was reduced by treatment with agmatine but not BDNF (Fig. 6B). Discussion Our present study demonstrates that agmatine, an endogenous polyamine with a guanidino group, prevents hypoxia-induced LDH release and apoptotic death in cultured transformed rat RGCs (RGC-5 cell line). Release of LDH was detected by LDH assay and the proportions of apoptotic cells were determined by annexin V and caspase-3 assays. Although agmatine cannot completely block cellular damage due to hypoxic injury, it has similar and even more extensive neuroprotective effects than BDNF, a well-known protective neurotrophin for RGCs [33-35]. Many molecules have been studied to rescue RGCs from glaucomatous cell death [20-29,33-35], but there is still no drug which completely shelters RGCs from injury. In this study, undifferentiated RGC-5 cells were used instead of differentiated RGC-5 cells or primary RGCs. These immortalized cells behave differently than original RGCs, and our in vitro hypoxic model does not perfectly replicate in vivo conditions that lead to real glaucomatous injury. However, RGC-5 cells, even if they are undifferentiated, have been widely used to investigate glaucomatous RGC apoptosis as a matter of convenience [36-43]. It has been often stated that RGC-5 cells have similar characteristics to primary RGCs [30-32,44,45]. The present study using RGC-5 cells suggests a solution to the problem, although further investigations using primary cultured RGCs or in vivo glaucoma models are needed. Various functions of agmatine have been reported [3-10], but the precise cellular mechanisms of agmatine are not well established. In the present study, three types of MAPKs and NF-κB signaling pathways were evaluated. With hypoxic injury, phosphorylation of all three MAPKs and NF-κB were increased. Agmatine suppressed the hypoxia-induced activation of JNK and NF-κB, whereas BDNF inhibited the activation of ERK and p38. These differences might be caused by different mechanisms of action of the two molecules. MAPKs are involved in highly conserved signaling pathways that regulate diverse cellular functions including cell proliferation, differentiation, migration, and apoptosis [46-48]. They are activated through phosphorylation by distinct pathways depending on stimulus and cell type. When activated, they can phosphorylate a wide range of substrates, including transcription factors and cytoskeletal proteins, resulting in specific cellular responses. In the present study, agmatine regulated the activation of JNK, but not ERK and p38, in RGC-5 cells after hypoxic injury. Our results are discrepant with those of a previous report using kidney mesangial cells under high-glucose conditions, in which agmatine was involved in the ERK pathway [49]. However, there are no reports about agmatine's effects on MAPKs in the literature, and MAPKs have been known to work differently depending on stimuli and cell types. Furthermore, due to the implications of a report demonstrating that another antagonist of the NMDA receptor MK801 can block the phosphorylation of MAPKs [50], agmatine's actions as an antagonist for the NMDA receptor [10] suggest that it might also regulate the phosphorylation of MAPKs. In the present study, we revealed that there was an activation of NF-κB in RGC-5 cells after hypoxic injury, and agmatine was able to suppress it. Our results are consistent with previous reports that suggest that NF-κB is activated during oxidative stress [51-54]. However, Charles et al. [44] obtained a discrepant result in which the activity of NF-κB was decreased with serum-deprivation-induced apoptosis. While oxidative stress models, including our own hypoxic model, are based on the vascular theory of glaucoma development, the serum deprivation model is based on the mechanical pressure theory [55]. NF-κB signaling is presumed to have various responses according to the type of injury. Perhaps the most significant finding in this study was that both the increases in annexin V-positive cell number and caspase-3 activity produced by exposure of RGC-5 cells to hypoxia were counteracted by the addition of agmatine into the culture medium. This suggests that agmatine may exert a neuroprotective effect by inhibiting apoptosis in the hypoxia-injured RGC-5 cells. To our knowledge, this is the first report regarding the potential anti-apoptotic characteristics of agmatine in RGCs. Even though this study demonstrates that activations of JNK and NF-κB were associated with the agmatine treatment, it is still not certain whether there is a close connection between neuroprotective effects of agmatine and signaling of JNK and NF-κB. However, it is presumed that they are related in some way. The ability of agmatine to regulate JNK and NF-κB pathways may contribute to protecting RGCs against hypoxia-induced cell death. Further studies are needed to elucidate the precise mechanisms by which agmatine blocks apoptosis. A deeper understanding of these mechanisms may facilitate efforts to improve the survival of RGCs from various injuries. Conclusion Agmatine prevents hypoxia-induced LDH release and apoptotic death in transformed RGCs (RGC-5 cells). These neuroprotective effects of agmatine might be associated with the activity of JNK and NF-κB pathways. Methods Chemicals and antibodies Agmatine sulfate and recombinant human BDNF were purchased from Sigma (St. Louis, MO) and R&D System, Inc. (Minneapolis, MN), respectively. Rabbit polyclonal anti-JNK p54/46, anti-ERK p44/42, anti-p38, anti-NF-κB p65, anti-phospho-JNK p54/46, anti-phospho-ERK p44/42, anti-phospho-p38, anti-phospho-NF-κB p65, and anti-histone 3 antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA). Mouse monoclonal anti-β-actin antibody was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Cell culture RGC-5 cell line [30-32], a transformed RGCs developed from post-natal Sprague-Dawley rats, was grown in modified Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA) and 100 U/mL of penicillin and 100 μg/mL of streptomycin (Gibco, Carlsbad, CA). Cells were passaged every 2 to 3 days, and the cultures incubated at 37°C in 5% CO2 and air. During cultivation, cells exhibited the same morphological phenotype. For all experiments, cells were used at an 80% confluence. Hypoxic injury to retinal ganglion cells Cultures were transferred into a closed hypoxic chamber (Forma Scientific Co., Seoul, Korea) in which oxygen level (5% O2, 5% CO2, 90% N2) and temperature (37°C) were automatically controlled. After washing twice with deoxygenated serum-free DMEM, cells remained in the hypoxic chamber for the designated lengths of time. Control cells were not exposed to hypoxia. Agmatine or BDNF were added to the culture medium at the start of injury as indicated. Lactate dehydrogenase assay Cell viability was quantified by measurement of LDH released by injured cells after hypoxic or normoxic culture for 12, 24, and 48 hours [56,57]. LDH release is expressed relative to the value of 100, which represented the maximum LDH release that occurred after freezing overnight at -70°C and subsequent rapid thawing of each culture, which induced nearly complete cell damage. All experiments were performed in at least quadruplicate and repeated at least eight times using cell cultures derived from different platings. Preliminary studies with the LDH assay tested agmatine concentrations from 10 μM to 1 mM and BDNF concentrations ranging from 5 ng/mL to 100 ng/mL. Cell death was reduced significantly at 100 μM and greater concentrations of agmatine and 10 ng/mL and greater concentrations of BDNF, so we used 100 μM agmatine and 10 ng/mL BDNF for subsequent experiments. Hoechst 33342 and propidium iodide staining Apoptotic or necrotic cell death was characterized by the use of Hoechst 33342 and PI double staining [58,59]. Cells were stained with 10 μg/mL Hoechst 33342 and 10 μg/mL PI for 30 min at 37°C. After washing twice with phosphate buffered saline, cells were imaged with a digital camera attached to a fluorescence microscope. Annexin V assay Percentage of cells actively undergoing apoptosis was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. Briefly, cells were harvested and resuspended in binding buffer (106 cells/mL). 105 cells were mixed with 5 μL of annexin V-FITC and 5 μL of PI. After incubating at room temperature for 15 minutes in the dark, analysis was performed by flow cytometry. Measurement of Caspase-3 activity Caspase-3 activity was measured using the CaspACETM colorimetric assay system (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, cells were harvested and resuspended in cell lysis buffer (108 cells/mL). After lysis, 106 cells were mixed with 32 μL of assay buffer and 2 μL of 10 mM DEVD-pNA substrate. After incubating at 37°C for 4 hours, absorbance was measured using a microplate reader at 405 nm. Absorbance of each sample was determined by subtraction of the mean absorbance of the blank from that of the sample. Western blot analysis For extraction of whole cellular proteins, cells were washed twice with ice-cold phosphate buffered saline and then lysed with cell lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin) on ice for 30 minutes. Lysates were sonicated, and the cell homogenates were centrifuged at 15,000 g for 10 minutes (4°C). For fractions of cytosolic and nuclear proteins, cells were lysed with lysis buffer A (10 mM HEPES pH 7.4, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 10 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin) on ice for 15 minutes, and 10% NP-40 was added. After vortexing for 10 seconds, lysates were centrifuged at 15,000 g for 1 minute (4°C). Supernatant was collected from the cytosolic fraction, and pellet was resuspended in lysis buffer C (20 mM HEPES pH 7.4, 400 mM NaCl, 1 mM EDTA, 1% glycerol, 1 mM DTT, 10 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin) on ice for 30 minutes. Lysates were centrifuge at 15,000 g for 15 minutes (4°C), and supernatant was collected from the nuclear fraction. Protein concentrations in the resultant supernatants were determined with the Bradford reagent, and equal amounts of protein (40 μg) were boiled in Laemmli sample buffer and resolved by 10 or 15% SDS-PAGE. The proteins were transferred to polyvinylidene fluoride membranes and probed overnight with antibodies against JNK, ERK p44/42, p38, NF-κB p65, phospho-JNK, phospho-ERK p44/42, phospho-p38, phospho-NF-κB, β-actin and histone 3 as indicated (diluted 1:1000). The immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence. Statistical Analysis Data were analyzed by a two-tailed Student t-test or a one-way ANOVA using the Statistical Package for Social Sciences 12.0 (SPSS). Differences were considered statistically significant at p < 0.05. Authors' contributions GJS and SH designed the experiments and wrote the bulk of the manuscript. SH, JEL and CYK carried out the molecular studies. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1806007007-PONE-RA-02142R210.1371/journal.pone.0001276Research ArticleEvolutionary Biology/Animal BehaviorEvolutionary Biology/Evolutionary EcologyVirology/Animal Models of InfectionInfectious Offspring: How Birds Acquire and Transmit an Avian Polyomavirus in the Wild Viruses, Ectoparasites & BirdsPotti Jaime 1 * Blanco Guillermo 2 Lemus Jesús Á. 3 Canal David 1 1 Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (CSIC), Department of Evolutionary Ecology, Pabellón del Perú, Sevilla, Spain 2 Instituto de Investigación en Recursos Cinegéticos (IREC) Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Castilla-La Mancha (UCLM) - Junta de Comunidades de Castilla-La Mancha (JCCM), Ciudad Real, Spain 3 Department of Evolutionary Ecology, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain Rands Sean Academic EditorUniversity of Bristol, United Kingdom* To whom correspondence should be addressed. E-mail: [email protected] and designed the experiments: JP GB JL DC. Performed the experiments: JP DC. Analyzed the data: JP GB JL DC. Contributed reagents/materials/analysis tools: GB JL. Wrote the paper: JP GB. 2007 5 12 2007 2 12 e12763 9 2007 13 11 2007 Potti et al.2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Detailed patterns of primary virus acquisition and subsequent dispersal in wild vertebrate populations are virtually absent. We show that nestlings of a songbird acquire polyomavirus infections from larval blowflies, common nest ectoparasites of cavity-nesting birds, while breeding adults acquire and renew the same viral infections via cloacal shedding from their offspring. Infections by these DNA viruses, known potential pathogens producing disease in some bird species, therefore follow an ‘upwards vertical’ route of an environmental nature mimicking horizontal transmission within families, as evidenced by patterns of viral infection in adults and young of experimental, cross-fostered offspring. This previously undescribed route of viral transmission from ectoparasites to offspring to parent hosts may be a common mechanism of virus dispersal in many taxa that display parental care. ==== Body Introduction Arthropods are well-characterized vectors of many viruses of plants and animals [1], [2], including arboviruses (a non-systematic grouping of arthropod-borne, mostly RNA, viruses of vertebrates, where viral replication occurs in both the vertebrate and invertebrate hosts [3]). Although patterns of pathogen transmission are central to the evolution of infectious disease and host resistance [4]–[6], including those related to arboviruses [3], most of our knowledge stems from rather loose patterns of virus dispersal from broad, life-cycle perspectives which generally lack detailed information on the realized modes of virus dispersal across hosts at the population level [3]. The main modes of virus dispersal are vertical transmission, from a parent (usually the mother) to the offspring across host generations, and horizontal transmission, such as transmission through contact with infected non-parental individuals or objects in the environment. In a search for the effects that nest ectoparasitic blowflies (Protocalliphora azurea (Fallén)) may have on the biology of a forest passerine migrant species, the European pied flycatcher (Ficedula hypoleuca (Pallas)) [7], we screened birds' blood for the prevalence of several virus groups (including circovirus, polyomavirus, reovirus, smallpox and West Nile virus), and discovered a high degree of association between the presence of blowflies in the nest and polyomaviruses in the nestlings. Polyomaviruses are a group of small, double-stranded DNA viruses best known from mammals and birds [8], though also present in lower vertebrates [9]. They have potential, confirmed pathogenic etiology and morbidity in at least man [10], apes, mice, parrots and finches, among a few other taxa [11]–[13]. Apart from horizontal virus transmission within flocks of caged parrots and other pet bird species there is no published information [8], [11] on how polyomavirus infections are primarily acquired before they jump across individual hosts. The modes of polyomavirus acquisition in this study system are clearly limited by the known biology of its rather specialized, putative vector, as no larvae of Holarctic species of Protocalliphora have ever been recorded parasitizing individuals other than nestling birds. In pied flycatchers, eggs laid in the nest by the adult fly will hatch into larvae that develop to the pupal stage (our sampling unit) by hiding in the nest material. During this period, the larvae will take intermittent blood meals from nestlings, and will then burrow into the nest cup to pupate [14]. Blood-sucking by blowflies has a direct effect upon the nestlings, by rendering them anemic, decreasing their growth, and increasing their risk of mortality [14]–[17]; effects of the larvae on the parents are indirect, such as through causing them to increase their feeding rates to chicks [18]. This life-cycle immediately suggests a role for nestlings as the main agents of virus acquisition and eventual dispersal to the population as a whole. Alternative routes of virus dispersal may, however, complicate the pattern. For instance, vertical transmission from mothers to offspring via the egg, may also exist [19]. There is, furthermore, the possibility of horizontal transmission among related and unrelated individuals or objects in the environment (e.g., other nest parasites). In this study, we evaluated whether the presence of an avian polyomavirus (APV hereafter) infecting nestlings of the pied flycatcher is associated with the presence of the nest ectoparasitic blowfly P. azurea, to assess its potential as vector of the virus. After dissecting the patterns of APV prevalence in adult and young birds in relation to ectoparasite prevalence, we performed an experiment to discard alternative routes of virus transmission across host generations. Results and Discussion Prevalences of infection of APV in pied flycatcher blood were similarly high in nestlings in both study years (63.4%, n = 568 and 48.5%, n = 641 in 2005 and 2006, respectively) and breeding females (46.5%, n = 129 and 61.8%, n = 131, in 2005 and 2006, respectively; nestlings vs. females: χ2 1 = 0.26, P = 0.61, pooling both years and applying Yates' correction for continuity) and lower in breeding males (37.1%, n = 116 and 39.7%, n = 121, in 2005 and 2006, respectively; males vs. females: χ2 1 = 17.47, P<0.001; males vs. nestlings χ2 1 = 22.95, P<0.001) while a sex difference in virus prevalence did not exist at fledging age (χ2 1 = 0.03, P = 0.87, n = 184 male and 188 female nestlings from 2005 sexed with molecular methods [7]). Blowfly prevalence and APV presence in blood were tightly associated in both years, with all nests infested by blowflies containing at least one nestling infected by APV while only one brood out of 89 had a single nestling positive for APV in uninfested nests (Fisher's exact test, P<0.0001). The abundance scores of another nest ectoparasite (Dermanyssus Duges mites [7], [15]) were unrelated to the prevalence of APV in nestlings and adults (broods: χ2 1 = 0.40, P = 0.53, females: χ2 1 = 0.53, P = 0.47; males: χ2 1 = 0.21, P = 0.65, pooling both years and applying Yates' correction for continuity) and all mites were negative for APV (n = 120). The striking association found in both study years suggests that the transmission of the virus occurs from blowflies to offspring, and subsequently from offspring to parents, and not in the opposite direction, as virtually no nestlings from uninfested nests were infected with the virus (one out of 426 nestlings, Fig. 1) despite the infection of parents in many cases (Fig. 1). This discards possible transmission routes such as oral contact or aerosol spreading from infected parents to their nestlings. Furthermore, the likelihood that all nestlings in a brood were infected by APV increased with the number of blowfly larvae present in their nests. Only nestlings with six or less parasitic larvae in their nests could escape APV infection (Fig. 2). Adults, especially females, showed a similar association between APV infection and presence (Fig. 1) and abundance of blowflies in their nests (logistic regressions: 2005: females: B = 0.27, Wald = 22.79, P<0.0001, n = 123: males: B = 0.14, Wald = 12.95, P<0.0001, n = 109; 2006: females: B = 0.36, Wald = 14.68, P<0.0001, n = 116; males: B = 0.02, Wald = 0.40, P = 0.53, n = 96). Broods also resembled their parents, females in particular, as to prevalence of APV (Chi-square tests with Yates' correction for continuity; 2005: females: χ1 2 = 6.22, n = 124, P = 0.013; males: χ1 2 = 6.58, n = 113, P = 0.010; 2006: females: χ1 2 = 7.84, n = 66, P = 0.005; males: χ1 2 = 0.14, n = 59, P = 0.70). 10.1371/journal.pone.0001276.g001Figure 1 Prevalence of APV infections in blood of nestlings and adult pied flycatchers in relation to presence or absence of nest ectoparasitic blowflies. 2005 nestlings: Fisher's exact test, P<0.0001; adult males: χ2 1 = 6.00, P = 0.014; adult females: χ2 1 = 5.99, P = 0.014; 2006 nestlings: χ2 1 = 531.74, P<0.0001; adult males: χ2 1 = 0.80, P = 0.370; adult females: χ2 1 = 19.74, P<0.0001. The Yates' correction for continuity was applied. Numbers above bars are sample sizes (numbers of individuals). 10.1371/journal.pone.0001276.g002Figure 2 Relationship between the number of blowflies in the nest and the prevalence of APV in nestlings in both study years. Logistic regressions; 2005: B = 1.05, Wald = 118.61, P<0.0001, n = 535; 2006: B = 2.55, Wald = 90.22, P<0.0001, n = 625. The correspondence between the prevalences of blowflies and APV, on the one hand, and between APV infections in nestlings and their parents, on the other, points to genetic and/or environmental sources of resemblance between parents and offspring, e.g. in susceptibility or common exposure to APV infections, as potential sources of confusion in the interpretation of the routes of transmission of APV in this system. We therefore performed a cross-fostering experiment of whole clutches to test the hypotheses of parent-offspring similarity in patterns of APV infection due to vertical transmission of APV through the egg [19], or to genetic resistance to APV. The experiment showed that the APV status of infection in cross-fostered broods resembled that of their foster parents, females in particular (females: Fisher's exact test, P = 0.00003, n = 45; males: Fisher's exact test, P = 0.72, n = 35), rather than their genetic parents (females: χ1 2 = 1.03, n = 38, P = 0.31; males: χ1 2 = 0.88, n = 24, P = 0.88). This result indicates that APV is not passed to offspring from the mother, as occurs in mice [20], and discards the notion of strictly vertical (i.e. from parents to offspring) transmission of the virus. Therefore, both natural patterns of infection in parents and offspring and experimental evidence with cross-fostered broods point to a common environmental primary source of infection, most likely the larvae of P. azurea. Analyses of 40 live blowfly larvae whose oral region was dissected and screened for APV presence supported the inferred pattern of virus transmission to nestlings, with 100% of foregut samples of the larvae exhibiting positive evidence for APV presence, most probably due to the likely installation of the virus in the salivary glands of its vector. APV was more rarely detected in the hindgut samples of the same blowfly individuals (15%, n = 40, of which 34 had empty guts and 6 had recently fed). Our results point to the most plausible route of APV infection from nestlings to both their male and female parents as being through the parents' nest sanitation tasks. This is clearly supported by the finding that most APV-positive nestlings from nests infested by blowflies were actively shedding the virus in the sampled feces (85.0%, n = 40 nestlings from 26 nests) while APV was not isolated in feces from nestlings which had no detectable APV presence in blood (0%, n = 27 nestlings from 19 nests; Fisher's exact test, P<0.0001). This excludes the possibility of nestlings showing APV infection in the digestive tract with APV passing unnoticed in blood. Both sexes remove fecal sacs in the pied flycatcher but, as in other bird species [21], females are reported to do so at higher rates than males, in addition to swallowing them up to day three of nestling age [22]. Thus, both female-biased nest sanitation behavior and patterns of APV prevalence in feces support an APV vertical, ‘upwards’ route of transmission from offspring to parents, especially females. Also, a higher intensity of infection (as assessed by absolute quantification of viral gene copies [23]) in nestlings than in adults (Fig. 3) makes it more likely that nestlings were shedding the APV in feces [24], increasing the chance of transmission of APV to adults through their removal of fecal sacs. 10.1371/journal.pone.0001276.g003Figure 3 Intensities of infection by APV in nestlings and adult birds. Shown are mean±SD scores quantifying the number of viral gene copies as assessed by RT-PCR Mann-Whitney U-tests give highly significant differences (P<0.001) for all possible between-group comparisons in both years. Numbers above bars are sample sizes (numbers of individuals). To our knowledge, larvae of P. azurea are the first recorded vector for any polyomavirus, which may in turn be considered, under epidemiological criteria, both as an arbovirus [3] and an enteric virus (i.e., acquired through fecal-oral transmission [25]). African swine fever virus was the only previously known DNA-arbovirus [3], [11], a group to which APV of pied flycatchers should be added henceforth. As in tick arbovirus-vertebrate interactions [3], blowfly larvae surely inject APV through the wounds they produce with their sucking action into the bloodstream of nestling flycatchers during meals. This is consistent with the high intensity of infection displayed in the blood of nestlings, as compared to the reduced intensity of infection in adults (Fig. 3). However, lower intensities of infection in highly immunocompetent adults when compared to naïve, young individuals are also to be expected [3]. Furthermore, a low intensity of infection in adult birds may also reflect the fact that APV reaches adult individuals via a less direct route (through nestling feces) than the simple injection of viruses into the bloodstream by the parasite, as occurs in nestlings. In addition to the persistence of APV after infection in early life, which was confirmed one year later for all individuals already infected as nestlings (with only one exception, n = 13), brooding females could also be directly infected by attacks from blowfly larvae, as seemingly apparent from the increase in APV prevalence in females with the greater number of blowfly larvae counted in their nests. However, neither our long-term hands-on experience (23 years) with flycatchers nor the literature support the possibility that blowfly larvae ever attach to adult birds. Furthermore, excluding common infections as nestlings, a similar but weaker relationship in male flycatchers cannot be explained by the same mechanisms as in females, because males do not incubate or brood nestlings [22] and thus are unexposed to attacks by blowfly larvae. Therefore, common APV infections in adults and nestlings sharing a nest most likely reflect contagion from the nestling to the adult via cloacal shedding [26], with the virus jumping to parents as a result of their nest sanitation behavior, especially to females when swallowing feces. Intensity of infection in females could also be increased due to virus reactivation during breeding as occurs in mice during pregnancy [27], which necessitates further research. The design and results of this study only allow us to speculate on where the APV infection originates, as all blowflies were positive for APV and the virus was only detectable in their primary (and, most probably, exclusive from the point of view of first transmission) host, the nestlings, when and only when their nest was infested by blowflies. However, this circle on the ‘true’ source of virus infection could be addressed at different levels of explanation. One hypothesis is that the non-parasitic, adult female flies may transmit the APV via vertical transmission through their eggs (transovarial transmission), and then throughout the larval (and pupal) stages to the adults (transstadial transmission) as known for some arboviruses [3]. This hypothesis, however, leaves unresolved the issue of the origin of infection by displacing the problem to the former fly generation. Studies of the biology of adult flies, e.g. of their food habits [14], could offer some clues on an earlier, ‘ultimate’ source of infection and also on whether all adult flies are reservoirs for APV. A complementary hypothesis, however, could make superfluous that search for the viral ‘origin’ by addressing the question of whether there has been joint cospeciation of APV of pied flycatchers and the blowfly P. azurea. We think this issue might be most profitably addressed through phylogenetic analysis of APV variants within the family Calliphoridae and, ideally, also within its sister group, flies in the family Sarcophagidae [28], [29]. In conclusion, nest ectoparasites of birds transmit polyomaviruses to nestlings, which in turn pass them on to their parents. To our knowledge, this is the first known natural example of a primary, rather than sporadic, route of upward transmission of a potential pathogen from offspring at an early ontogenetic stage to adult individuals. This route of infection may reveal itself as a common mechanism of virus transmission in the many taxa that exert parental care and/or feed and preen their offspring and thus might be a hitherto unnoticed [30] cost of parental care, with potential differences between the sexes depending on their roles in breeding tasks. Further, the arthropod to offspring to parent hosts route of virus transmission should probably be explored for other viral infections. Finally, given that pied flycatchers share breeding cavities and parasites with many other species [31], the findings reported here may open new research agendas on the evolution of virulence and cospeciation of vectors, virus and vertebrate hosts in the wild [3]–[6], [8], [32], [33], with added important potential implications of concern in conservation biology [34], [35]. Materials and Methods Field methods The study was conducted in 2005 and 2006 in an intensively studied population of pied flycatchers breeding in nest boxes in central Spain [7]. We recorded breeding phenology and reproductive success in all nests (n = 273) and trapped almost all breeding males and females within their nests while they fed nestlings aged 8–11 days. Nestlings were sampled at 13 days of age. A drop of blood was extracted from the brachial vein of all individuals and stored frozen in EDTA for molecular sex determination of nestlings [7] (only in 2005) and virological analyses (both years). Nest contents were removed after breeding and the nestboxes were cleaned again just before the breeding season. Thirty nestlings died in their nests before fledging, of which 12 were positive for APV and 18 negative. Cross-fostering experiment In 2006, we exchanged all eggs in the second day of incubation between matched pairs of nests of the same (±1 d) breeding date and clutch size. Experimental nest dyads (n = 94 nests, i.e. 47 dyads) were at least 1 km apart. Cross-fostered eggs were replaced by the same number of rubber, blue-painted canary egg dummies mimicking size and color of pied flycatcher eggs to avoid desertion by the females during clutch exchanges. In all cases, females ‘incubated’ the egg dummies during the time needed for transportation, as indicated by direct observation or our estimated temperature of egg dummies. Eggs were kept safe and warm in water-heated containers (cotton-coated, commercial hen egg packs) and transported by car in about 20 to 30 min. As a result of our procedure, all pairs of exchanged nests contained broods reared by totally unrelated adult birds. Final sample sizes were unbalanced due to some nests being lost to predation or because we were unable to trap the parent(s). Ectoparasite assessment The abundance of blowflies was assessed by dismantling nest contents just after the young fledged and counting the number of pupae buried in the nest material. The abundance of mites, which can range from zero to thousands [15], was visually estimated as low or high on the day the fledglings were bled. These bimodal scores are highly predictive indices of the intensities of mite infestations, as shown by mite counts in Berlese funnels [15]. Viral detection and quantification Blood samples from adult and nestling pied flycatchers were tested for the presence of APV by means of a sensitive and specific real-time quantitative PCR (RQ-PCR) assay. Following previous work with a murine polyomavirus [23], the target sequences used for quantification of viral and cellular genes included the N termini of the T-antigens of APV (large, middle and small) and sequences of intron 3 within the avian wild-type p53 gene. P53 primers were used as a cellular normalization standard to allow calculations of viral genome copies per cell. Absolute quantification of viral gene copies was determined from standard curves generated by plotting the log10 of the known input gene copy number of the standard dilution series against the CT value observed in the RQ-PCR analysis. Semi quantitative scores (from 1 to 5) were calculated by using 10 increment copies, so 0 is 0 copies detected, 1 from 10 to 102 copies; 2 from 102 to 103 copies, 3 from 103 to 104 copies; 4 from 104 to 105 and, finally, 5 from 105 to 106 copies. Detection of APV in nestling feces, blow fly larvae and Dermanyssus mites was conducted by using a classical PCR assay [36]. A brood was defined as infected by APV when at least one nestling was positive for APV. We thank Inés Valencia and Paola Laiolo for help in the field and A. Baz, M. Carrete, J. Figuerola, F. Hiraldo, R. Jovani, P. Laiolo, C. Manjavacas, R.C. Soriguer, D. Serrano and J.L. Tella for advice, comments and encouragement. Sarah Young checked the English. We also thank Sonia Kleindorfer, Sean Rands and Doug Wilson for constructive comments on the manuscript. Consejería de Medio Ambiente, Comunidad de Madrid and Delegación de Medio Ambiente, Junta de Castilla-La Mancha gave working permissions and authorized the experiment. Competing Interests: The authors have declared that no competing interests exist. Funding: J. Potti and D. Canal were partially supported by projects from the Spanish Ministry of Education (CGL2004-04479/BOS, to J. A. Fargallo and CGL2006-07481/BOS, to J. C. Senar) and Universidad de Castilla-La Mancha (PAC05-006-1, to J. A. Dávila). G. Blanco and J.Á. Lemus were supported by Junta de Comunidad de Castilla-La Mancha Project (PAI-05-051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Gray SM Banerjee N 1999 Mechanisms of arthropod transmission of plant and animal viruses. Microbiol Mol Biol Rev 63 128 148 10066833 2 Carn VM 1996 The role of dipterous insects in the mechanical transmission of animal viruses. 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==== Front Virol JVirology Journal1743-422XBioMed Central 1743-422X-4-921789257610.1186/1743-422X-4-92MethodologyUniversal primers for HBV genome DNA amplification across subtypes: a case study for designing more effective viral primers Zhang Qingrun [email protected] Guanghua [email protected] Elliott [email protected] Shan'gang [email protected] Changqing [email protected] Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101300, China2 Department of Biology, College of Biology and Agriculture, Brigham Young University, Provo UT 84602, USA2007 24 9 2007 4 92 92 28 6 2007 24 9 2007 Copyright © 2007 Zhang et al; licensee BioMed Central Ltd.2007Zhang et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification. Taking advantage of the large number of virus DNA sequences in public databases to select conserved sites for primer design is an optimal way to tackle the difficulties in virus genome amplification. Results Here we use hepatitis B virus as an example to introduce a simple and efficient way for virus primer design. Based on the alignment of HBV sequences in public databases and a program BxB in Perl script, our method selected several optimal sites for HBV primer design. Polymerase chain reaction showed that compared with the success rate of the most popular primers for whole genome amplification of HBV, one set of primers for full length genome amplification and four sets of walking primers showed significant improvement. These newly designed primers are suitable for most subtypes of HBV. Conclusion Researchers can extend the method described here to design universal or subtype specific primers for various types of viruses. The BxB program based on multiple sequence alignment not only can be used as a separate tool but also can be integrated in any open source primer design software to select conserved regions for primer design. ==== Body Background Chronic hepatitis B virus (HBV) infection is a major health problem worldwide, affecting approximately 350 million people, and 500,000 to 1.2 million deaths worldwide per year are attributed to HBV infection. Currently there are eight accepted genotypes (A to H) for HBV, based on one of the following criteria: an inter-group divergence of 8% or greater in the complete genome nucleotide sequence or a 4 ± 1% divergence of the surface antigen gene [1,2]. It has been widely reported that it is possible to have two HBV genotypes or recombinant types in one infected individual [3-5]. The HBV reverse transcriptase (RT) is an error-prone enzyme as a result of lacking 3'-5'-exonuclease proofreading capacity. HBV, like other viruses such as HIV, HCV and poliovirus, has a high mutation rate of 2 × 10-5/site/year [6,7]and quasispecies distribution in infected individuals [8]. This means that HBV circulates as a complex mixture of genetically distinct but closely related variants that are in equilibrium at a certain time point of infection in a given circumstance. A mixture of HBV quasispecies is in fact a mixture of HBV haplotypes, which is a more important concept to researchers, such as in drug resistant mutant studies-different haplotypes of HBV may represent different types of drug resistance [9-12]. Because of the existence of quasispecies, the only way to obtain HBV haplotype sequences is through full length genome amplification and clone-sequencing instead of assembling the PCR sequences of several amplified fragments of the genome. However, the partially double stranded characteristic of HBV DNA structure causes the instability of exposed HBV DNA and the low efficiency of whole genome amplification. Günther et al. developed a set of primers for full length HBV genome amplification, with a restriction enzyme site for further cloning and function study [13,14]. The success rate reported in this paper is one out of eight genomes (12%) amplified with Taq polymerase, and seven out of seventeen genomes amplified with Taq-Pwo polymerases (41%). Further studies showed similar success rate (40%). In our laboratory, 141 of 420 genomes amplified with Takara-LA polymerases (34%) using this method. Tellier et al. developed two pairs of primers for nested PCR. Those primers can amplify nearly full length of HBV (3.12 kb), yet the whole process is complicated, time consuming and may introduce risk of cross contamination [15,16]. So it is not widely used and no success rate has been reported. The considerable number of HBV isolates with rather divergent nucleotide sequences and the partially double-stranded characteristic of HBV impose the need for extreme care in the choice of primers for both full length and fragment amplification. In order to identify optimal sites for primer design, we utilized 1020 whole genome sequences in public databases (NCBI, EMBL and DDBJ) and 103 sequences in our laboratory, and developed a program BxB to select conserved regions as candidates for primer design. We testified those primer designs in silico by e-PCR and real polymerase chain reactions. One set of primers for nearly full length HBV genome amplification (3181 bp, 40 bp shorter than full length) and four sets of walking primers for fragment amplification were finally obtained. These primer sequences are within areas that are highly conserved across all genome sequences available in public databases, therefore the use of such primers makes it unlikely that HBV strains are missed due to sequence variations and allows further search for quasispecies as well as unknown HBV genotypes and other subtypes. Results Identification of candidate regions for primer design by BxB We analyzed 1123 sequences, 1020 from public databases (Additional file 1) and103 sequences identified in our laboratory, with the BxB program. 10 regions were selected according to BxB analysis (Table 1). Candidate regions were defined as sites within the desired locations that had 17+ bases from the 3' end and with a frequency of 0.90+ in the BxB. The output of BxB analysis was designated as a FASTA format, which could be illuminated in sequence analysis software interface such as ClustalX software to facilitate primer selection (Figure 1). Table 1 Candidate regions selected by BxB for primer design Candidate Regions* Sequence ORF located in 8~26 ACCTCTGCCTAATCATCTC X/preC 40~68 ACTGTTCAAGCCTCCAAGCTGTGCCTTGG preC 591~616 GCCGCGTCGCAGAAGATCTCAATCTC Terminal Protein 993~1018 GGGTCACCATATTCTTGGGAACAAGA Terminal Protein 1450~1469 CCTGCTGGTGGCTCCAGTTC pre-S2 1571~1592 TCCTAGGACCCCTGCTCGTGTT S 1657~1679 ACTTCTCTCAATTTTCTAGGGGG S 2131~2159 TATATGGATGATGTGGTATTGGGGGCCAA S 2491~2516 TTCTCGCCAACTTACAAGGCCTTTCT RT 3199~3221 CACCAGCACCATGCAACTTTTT X/preC Here we select "CTTTTTC" of X ORF as the start point Figure 1 Output of BxB illiminated in ClustalX software. When the ratio of the most frequently presented nucleotide is larger than current cutoff value, the program outputs this nucleotide, otherwise outputs a '-'. The cutoff was set to (0.05, 1), and the step length is 0.05. The frequency is listed in the left box and the nucleotides are in the right box. Primer selection One set of full length genome primers, four sets of walking primers were designed with the aid of Primer 3 (Figure 2, Table 2). Degenerate sites were also considered when there were sites yielding low BxB frequency in selected primers. All these primers gave negative result when they were tested in UCSC in silico PCR to see whether primers would amplify human DNA. Table 2 Primers for full length genome amplification and fragment amplification Primers Sequence Location Length GC % Tm(°C) Amplicon Size (bp) WA-L ACTGTTCAAGCCTCCAAGCTGTGC 40 24 54.2 60.6 3181 WA-R AGCAAAAAGTTGCATGGTGCTGGT 3221 24 45.8 60.7 FA1-L TTTCACCTCTGCCTAATCATCTC 4 23 43.5 52.0 1014 FA1-L' TTT ACCTCTGCCTAATCATCTC 4 22 40.9 47.5 FA1-R TCTTGTTCCCAAGAATATGGTG 1018 22 40.9 51.0 FA2-L GCGTCGCAGAAGATCTCAAT 593 20 50.0 51.9 1074 FA2-R TTGAGAGAAGTCCACCACGAG 1667 21 52.4 51.7 FA3-L CTGCTGGTGGCTCCAGTT 1451 18 61.1 50.6 1059 FA3-R GCCTTGTAAGTTGGCGAGAA 2510 20 50.0 52.2 FA4-L GTATTGGGGGCCAAGTCTGT 2416 20 55.0 52.8 1072 FA4-L' GTATTGGGGGCCAAATCTGT 2416 20 50.0 52.8 FA4-R AAAAAGTTGCATGGTGCTG 3218 19 42.1 48.5 *Here we select "CTTTTTC" of X ORF as the start point. FA1-L' and FA4-L' are degenerate primers Figure 2 Diagram of HBV ORFs and designed primers. WA-L and WA-R in blue arrows represents the primers for full length genomic DNA amplification. FA1-L/FA1-L' and FA1-R (amplicon size: 1014 bp), FA2-L and FA2-R(amplicon size: 1074 bp), FA3-L and FA3-R (amplicon size: 1059 bp), FA4-L/FA4-L' and FA4-R (amplicon size: 1072 bp) in red arrows represent the four sets of walking primers for fragment amplification. FA2-L and FA2-R Here we select "CTTTTTC" of X ORF as the start point. FA1-L' and FA4-L' are degenerate primers. Experiment verification All primers, including one set of primers for full length (3181 bp) amplification and four sets of primers for fragment amplification, demonstrated a good efficiency in real polymerase chain reactions (Figure 3). Figure 3 Agarose gel analysis of HBV genomes amplified by the newly designed primers. Sample 1 and sample 2 are for fragment amplification primers testing. 1, 2, 3, 4 in the figure represent: FA3-L and FA3-R (amplicon size: 1059 bp), FA1-L/FA1-L' and FA1-R (amplicon size: 1014 bp), FA4-L/FA4-L' and FA4-R (amplicon size: 1072 bp), FA2-L and FA2-R (amplicon size: 1074 bp) primer pairs respectively. Sample 3~7 are for full length genome amplification primers (WA-L and WA-R) testing (amplicon size: 3181 bp). Discussion Using an alignment of 1123 complete genomes from public databases and our laboratory, we selected primers from several highly conserved regions of HBV genomes. These primers are situated in the sequences encoding X, preC, terminal protein, pre-S2, S and reverse transcriptase regions. Sequences of the primers are sufficiently conserved in all HBV genotypes and are believed to be conserved in quasispecies. All these primers were shown to be very efficient in real polymerase reaction. The advantage of such approach is that it utilized all HBV sequences available and a simple Perl program to precisely select optimal regions in HBV genome for amplification. Such approach is unlikely to produce significant bias towards any one genotype when there is no bias in the multiple sequences alignment which the approach was based on. These primer designs make it possible to efficiently amplify quasispecies and allow further search for unknown HBV genotypes/subtypes. We used two methods to estimate HBV genotype distribution in the public databases were: counting the genotyped sequences with a simple Perl program and calculating the percentages; or using BLAST[17] to align eight HBV genotype reference sequences from NCBI with those from public databases to get all sequences of different HBV genotypes and then to calculate the percentage. Both methods yielded similar results: Genotype C counts most (about 1/3 in the databases); Genotype B, A, D in descending order. These four genotypes represent about 80~90% in the databases and the rest are E, F, G and H. Besides, there are also a few CD and GC recombinants. The primer design described in this study is based on sequences from the public databases and our laboratory which are genotype B and C. Therefore, it would only give bias when the genotype distribution in the databases does not reflect the actual HBV genotype distribution in reality. Since this method is based on multiple sequences, it can be much more reliable when target amplifications are within one genotype or within a certain group. In such occasions, sequences of one genotype or a given group should be used and analyzed with the BxB program to obtain genotype-specific or group-specific candidate regions and primer sequences. Recently, based on full length sequences in our laboratory most of which are from Beijing, we successfully selected optimal primers for HBV in Beijing regions using this approach. Further research of this approach should be done on other genotypes like A, D, E, F etc. to testify its specificity, either through sequences of one genotype or sequences of mixtures. The amplified full length genome with our method is 3181 bp which is only 40 bp shorter than the full length of HBV genome. It is not applicable in functional study but much valuable in genomic study. The set of primers were proved to have a good PCR efficiency. The four sets of fragment primers are also based on the most conserved regions from public sequences. These primers are walking primers covering the whole HBV genome. They should be very useful in amplifying certain regions of the genome. In future research on this method, both full length amplification primers and fragment amplification ones should be testified in samples with different viral titers to check its sensitivity. The BxB program we utilized in this study was a simple Perl script, which can be easily integrated in any primer design software and online tools. What BxB demands is just a multiple sequences alignment of the target sequences FASTA format, and outputs a description of conserved sites for primer design in FASTA format. It not only can be used as a separate tool but also can be integrated in any open source primer design software[18] to select conserved sites based on the alignments. The highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification. Taking advantage of the large number of virus DNA sequences in public databases to select conserved sites for primer designing should be an optimal way to tackle the difficulties in virus genome amplification. DNA sequences in public databases are on the increase. Take HIV and Hepatitis viruses for example, up to March 2007, the number of full length genome DNA sequences in public databases (Additional file 1) are ranges from about 40 to more than 2000: HIV is 2005; HCV is 183; HBV is 1020; HEV is 78; HAV is 35 and HDV is 83. This amount of data makes it possible to easily select conserved sites for primer design in different scale, genome regions, subtypes and groups. Conclusion Utilizing the HBV sequence in public databases and our laboratory, and a Perl program, we selected optimal regions for primer design. Those primers designed were verified in silico by e-PCR and polymerase chain reactions. One set of primers for full length HBV genome amplification and four sets of walking primers for fragment amplification were proved to be efficient. The use of such primers makes it unlikely that HBV strains are missed due to sequence variations and allows furthermore search for quiasispecies as well as genotype-unknown HBV strains. Our approach of primer design is simple, efficient and is totally applicable to other viruses, such as HIV, HCV etc. when multiple sequences alignments are available and efficient amplification in a heterogeneous mixture is needed. Methods HBV sequence data Initially in the study all complete genome sequences of HBV available in March 2007 from GenBank, EMBL, and DDBJ were downloaded. 1020 public sequences together with 103 sequences from our laboratory were aligned in ClustalW. The alignment was manually corrected by shifting sequences in places, for some sequences possessed large spans of unique deletions or insertions which threw off the alignment algorithm. Finally, as the start point of the sequences in databases were different, most of which were the EcoR I restriction enzyme cutting site, a unanimous start point was selected and the alignment was corrected to begin at the same location. Here we select "CTTTTTC" of X ORF as the start point. Selection of highly conserved genome regions for primer design The term "conserved genome regions" used here is defined as genome regions that have most frequently presented nucleotide sequences. To identify the highly conserved regions for primer design in HBV genome, Perl script[19] was used to write a program BxB (Base by Base) to scan through the alignment of the 1123 sequences base by base. BxB demands a multiple sequences alignment of the target sequences in FASTA[20] format. It is to detect the most frequently presented base in the same coordinate for all sequences of the alignment. Different cutoff values were tested to identify a best one for the alignment scan. If the ratio of the most frequently presented nucleotide is larger than current cutoff value, the program outputs this nucleotide, otherwise outputs a '-'. Finally, the cutoff was set to (0.05, 1), and the step length is 0.05. The output is a FASTA file which could be easily illuminated in sequence analysis software such as ClustalX[21], with which conserved region selection and primer design could be much facilitated in a user friendly interface. Primer design With the aid of the BxB, candidate regions were selected for primer design. Candidate regions were defined as sites within the desired locations that had 17+ bases from the 3' end and with a frequency of 0.90+ in the BxB. Using Primer 3 [22], primers were selected within the candidate regions, taking target regions, primer length and sequence, GC content and Tm etc. into consideration. In silico primer testing All primers were tested in University of California Santa Cruz (UCSC) in silico PCR to see whether primers would amplify human DNA. Experiment verification Clinical material Serum samples were collected from seven patients with hepatitis B surface antigen (HBsAg)-positive chronic hepatitis (serum HBsAg positive for at least 6 months). Five of them were genotype C and two were genotype B. All patients were seronegative for hepatitis C virus. The serum samples were stored at -20°C until analysis. Extraction of serum HBV DNA Serum viral DNA was extracted by using commercially available kits (QIAamp DNA Blood Mini Kit, QIAGEN, Inc., Valencia, CA). Polymerase chain reaction Full length amplification The PCR was performed in a 96-well cycler (GeneAmp PCR System 9700; Applied Biosystems) and in a 10 μl reaction volume containing 0.5 U LA Taq (TaKaRa). The primers were WA-R and WA-L (Table 2). The cycling conditions were initial denaturation at 95°C for 2 min 30 s, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min 30 s and extension at 72°C for 3 min, finally extension at 72°C for 10 min. Amplicons (1 μl) were analyzed by electrophoresis on 1.5% agarose gel, stained with ethidium bromide and observed under UV light. Fragment amplification For fragment amplification, the primers were FA1-R, FA1-L/FA1-L', FA2-R, FA2-L, FA3-R, FA3-L, FA4-R, FA4-L/FA4-L' (Table 2). The cycling conditions were initial denaturation at 95°C for 2 min 30 s, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min, finally extension at 72°C for 10 min. Amplicons (1 μl) were analyzed by electrophoresis on 1.5% agarose gel, stained with ethidium bromide and observed under UV light. Competing interests The author(s) declare that they have no competing interests. Authors' contributions QRZ conceived of the study, designed the experiment, performed the multiple sequence alignment and selected the primers. GHW did the experiments and wrote the manuscript. ER corrected the multiple sequence alignment, wrote the Perl scripts and performed in silico testing SGJ did some of the experiments. CZ supervised the whole research. Supplementary Material Additional file 1 Sequence IDs of HBV full length genome entries in GenBank, EMBL and DDBJ (March 2007). The data provided represent all available HBV genome sequences when the study was conducted. Click here for file Acknowledgements This study was supported by CAS National Knowledge Innovation Program (KIP) (KSCX2-SW-207), Beijing Municipal Education Commission Funds Program (KM20070025024) and Beijing Integrated Traditional and Western Medicine Key Disciplines. ==== Refs Norder H Courouce AM Magnius LO Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes Virology 1994 198 489 503 8291231 10.1006/viro.1994.1060 Okamoto H Tsuda F Sakugawa H Sastrosoewignjo RI Imai M Miyakawa Y Mayumi M Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes J Gen Virol 1988 69 2575 2583 3171552 Chen BF Kao JH Liu CJ Chen DS Chen PJ Genotypic dominance and novel recombinations in HBV genotype B and C co-infected intravenous drug users J Med Virol 2004 73 13 22 15042642 10.1002/jmv.20051 Chen BF Liu CJ Jow GM Chen PJ Kao JH Chen DS Evolution of Hepatitis B virus in an acute hepatitis B patient co-infected with genotypes B and C J Gen Virol 2006 87 39 49 16361416 10.1099/vir.0.81357-0 Wang Z Liu Z Zeng G Wen S Qi Y Ma S Naoumov NV Hou J A new intertype recombinant between genotypes C and D of hepatitis B virus identified in China J Gen Virol 2005 86 985 990 15784891 10.1099/vir.0.80771-0 Okamoto H Imai M Kametani M Nakamura T Mayumi M Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission Jpn J Exp Med 1987 57 231 236 3430800 Orito E Mizokami M Ina Y Moriyama EN Kameshima N Yamamoto M Gojobori T Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences Proc Natl Acad Sci USA 1989 86 7059 7062 2780562 10.1073/pnas.86.18.7059 Blum HE Hepatitis B virus: significance of naturally occurring mutants Intervirology 1993 35 40 50 8407249 Alexopoulou A Dourakis SP Genetic heterogeneity of hepatitis viruses and its clinical significance Curr Drug Targets Inflamm Allergy 2005 4 47 55 15720236 10.2174/1568010053622867 Ngui SL Teo CG Hepatitis B virus genomic heterogeneity: variation between quasispecies may confound molecular epidemiological analyses of transmission incidents J Viral Hepat 1997 4 309 315 9310929 10.1046/j.1365-2893.1997.00066.x Ohishi W Chayama K Rare quasispecies in the YMDD motif of hepatitis B virus detected by polymerase chain reaction with peptide nucleic acid clamping Intervirology 2003 46 355 361 14688452 10.1159/000074992 Yim HJ Hussain M Liu Y Wong SN Fung SK Lok AS Evolution of multi-drug resistant hepatitis B virus during sequential therapy Hepatology 2006 44 703 712 16941700 10.1002/hep.21290 Gunther S Li BC Miska S Kruger DH Meisel H Will H A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients J Virol 1995 69 5437 5444 7636989 Gunther S Sommer G Von Breunig F Iwanska A Kalinina T Sterneck M Will H Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations J Clin Microbiol 1998 36 531 538 9466771 Tellier R Bukh J Emerson SU Miller RH Purcell RH Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes J Clin Microbiol 1996 34 3085 3091 8940452 Tellier R Bukh J Emerson SU Purcell RH Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon Proc Natl Acad Sci USA 1996 93 4370 4373 8633073 10.1073/pnas.93.9.4370 Altschul SF Gish W Miller W Myers EW Lipman DJ Basic local alignment search tool J Mol Biol 1990 215 403 410 2231712 Thompson JD Gibson TJ Plewniak F Jeanmougin F Higgins DG The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 1997 25 4876 4882 9396791 10.1093/nar/25.24.4876 Rozen S Skaletsky H Primer3 on the WWW for general users and for biologist programmers Methods Mol Biol 2000 132 365 386 10547847	17892576	PMC2099425	CC BY	2021-01-04 16:48:05	yes	Virol J. 2007 Sep 24; 4:92
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1819725407-PONE-RA-02457R110.1371/journal.pone.0001444Research ArticleEcology/Conservation and Restoration EcologyInfectious Diseases/Antimicrobials and Drug ResistanceInfectious Diseases/Bacterial InfectionsInfectious Diseases/Epidemiology and Control of Infectious DiseasesInfectious Diseases/Fungal InfectionsPathology/HistopathologyPublic Health and Epidemiology/Environmental HealthPublic Health and Epidemiology/Infectious DiseasesAntibiotics Threaten Wildlife: Circulating Quinolone Residues and Disease in Avian Scavengers Antibiotics in WildlifeLemus Jesús Á. 1 Blanco Guillermo 2 * Grande Javier 2 Arroyo Bernardo 2 García-Montijano Marino 3 Martínez Felíx 2 1 Departamento de Ecología Evolutiva, Museo de Ciencias Naturales, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain 2 Instituto de Investigación en Recursos Cinegéticos (IREC), Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Castilla-La Mancha (UCLM), Junta de Comunidades de Castilla-La Mancha (JCCM), Ciudad Real, Spain 3 GIR Diagnostics SL, Madrid, Spain Carter Dee Academic EditorUniversity of Sydney, Australia* To whom correspondence should be addressed. E-mail: [email protected] and designed the experiments: GB JL. Performed the experiments: GB JL FM JG BA MG. Analyzed the data: GB JL. Contributed reagents/materials/analysis tools: GB JL FM JG BA MG. Wrote the paper: GB JL. 2008 16 1 2008 3 1 e144410 10 2007 18 12 2007 Lemus et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Antibiotic residues that may be present in carcasses of medicated livestock could pass to and greatly reduce scavenger wildlife populations. We surveyed residues of the quinolones enrofloxacin and its metabolite ciprofloxacin and other antibiotics (amoxicillin and oxytetracycline) in nestling griffon Gyps fulvus, cinereous Aegypius monachus and Egyptian Neophron percnopterus vultures in central Spain. We found high concentrations of antibiotics in the plasma of many nestling cinereous (57%) and Egyptian (40%) vultures. Enrofloxacin and ciprofloxacin were also found in liver samples of all dead cinereous vultures. This is the first report of antibiotic residues in wildlife. We also provide evidence of a direct association between antibiotic residues, primarily quinolones, and severe disease due to bacterial and fungal pathogens. Our results indicate that, by damaging the liver and kidney and through the acquisition and proliferation of pathogens associated with the depletion of lymphoid organs, continuous exposure to antibiotics could increase mortality rates, at least in cinereous vultures. If antibiotics ingested with livestock carrion are clearly implicated in the decline of the vultures in central Spain then it should be considered a primary concern for conservation of their populations. ==== Body Introduction Antibiotics (formally antimicrobials) are one of the biomedical revolutions of the 20th century. Nonetheless, their misuse has led to an increase in diseases in humans and domestic animals worldwide [1]. Huge quantities of antibiotics are used annually in livestock farming operations throughout the world, but the eventual fate of their residues and their potential damage to environmental health generally remains unknown [2], [3]. Withdrawal periods and residue controls are conducted in slaughterhouses to prevent harmful drug residuals in food that humans consume [4]. However, these waiting periods do not apply to carcasses and other wastes disposed of in dumps exploited by scavengers. Therefore, scavengers may consume drug residues in livestock carrion. The use of antibiotics and other drugs in livestock may directly damage the health and survival of scavengers if ingested as toxic residues [5]–[7]. Indirect effects on health may include the acquisition of antibiotic-resistant bacteria [4]–[8] and the alteration of normal protective flora through the acquisition of transient flora that may include pathogenic bacteria [8]–[11]. Furthermore, antibiotic residues ingested by avian scavengers may select for antibiotic resistance, the emergence, dissemination and persistence of which represents a major health problem in human and veterinary medicine worldwide [1], [4]. The “vulture crisis” on the Indian subcontinent caused by diclofenac, of which the use on livestock is banned in the European Union, demonstrates the potential of veterinary drugs to cause massive wildlife mortalities [7]. In Spain, after the bovine spongiform encephalopathy crisis, the ban on abandoning carcasses of free-range livestock in the countryside closed most traditional disposal sites for livestock carcasses used by avian scavengers as food sources [12]. Since then the diet of avian scavengers has been mainly composed of intensively farmed livestock carrion (swine and poultry) treated with antibiotics and other veterinary drugs, which represents the only livestock carrion available for scavengers [8], [9]. Thus, antibiotic residues from treated livestock could pass to scavengers and reduce their populations [8], [12], [13]. We investigated the possible transmission of antibiotic residues from livestock carcasses to nestlings of three vulture species in central Spain (griffon vulture, Gyps fulvus, cinereous vulture, Aegypius monachus and Egyptian vulture, Neophron percnopterus). We also searched for antibiotic residues in liver samples from dead cinereous vultures in the same area, and assessed their potential effects in this and other organs by histopathological analyses. The potential relationships between circulating antibiotic residues and the presence of bacterial and fungal pathogens causing severe disease were also evaluated in the three vulture species. Finally, we assessed whether disease-mediated mortality was associated with the presence of circulating antibiotics in cinereous vultures. Results Antibiotic residues in livestock carrion Results of the Four Plate Test of bacterial growth inhibition to detect antibiotic residues (see Material and methods) showed their presence in 21 of 29 samples (72%) from several tissues of swine carcasses available to vultures, varying from 40% of liver to 100% of kidney samples. This suggests that vultures may ingest antibiotics when feeding on livestock carrion. Circulating antibiotic residues We found a high proportion of nestlings carrying circulating antibiotics, especially in Egyptian and cinereous vultures (Table 1). In the three species, enrofloxacin and its metabolite ciprofloxacin showed the highest prevalences and concentrations alone or in combination with other antibiotics (Table 1). 10.1371/journal.pone.0001444.t001Table 1 Antibiotic residues in nestlings of three vulture species in central Spain and their concentrations in plasma. Number of samples with antibiotics (%) Concentration (µg/ml) mean±SD (range) n Griffon vulture (n = 50) Antibiotic residues (total)a 6 (12) Quinolones (total)b 6 (12) Enrofloxacin 1 (2) 0.16±0.028 (0.14–0.18) 2 Ciprofloxacin 3 (6) 0.077±0.056 (0.025–0.17) 5 Amoxicillin 0 (0) 0.005* 1 Oxytetracycline 0 (0) Enrofloxacin+Ciprofloxacin 1 (2) Ciprofloxacin+Amoxicillin 1 (2) Cinereous vulture (n = 49) Antibiotic residues (total)a 28 (57) Quinolones (total)b 26 (53) Enrofloxacin 7 (14) 0.073±0.076 (0.0005–0.21) 12 Ciprofloxacin 14 (29) 0.095±0.059 (0.025–0.21) 16 Amoxicillin 2 (4) 0.027±0.037 (0.005–0.07) 3 Oxytetracycline 0 (0) 0.005±0.000* 2 Enrofloxacin+Ciprofloxacin 2 (4) Enrofloxacin+Amoxicillin 1 (2) Enrofloxacin+Oxytetracycline 2 (4) Egyptian vulture (n = 25) Antibiotic residues (total)a 10 (40) Quinolones (total)b 6 (24) Enrofloxacin 2 (8) 0.104±0.116 (0.0005–0.28) 5 Ciprofloxacin 0 (0) 0.078±0.026 (0.04–0.10) 4 Amoxicillin 2 (8) 0.061±0.112 (0.005–0.23) 4 Oxytetracycline 1 (4) 0.005±0.000* 2 Enrofloxacin+Ciprofloxacin 3 (12) Ciprofloxacin+Amoxicillin 1 (4) Amoxicillin+Oxytetracycline 1 (4) * Values corresponding to the half of the detection limit. a Pooling all different antibiotics. b Pooling enrofloxacin and ciprofloxacin. Eight additional fledglings and one adult cinereous vulture from the same colony (as identified by leg rings previously applied) which had not been sampled for blood in their nests, were treated in rehabilitation centres in 2002 and 2005. All had one to three antibiotics in plasma (ciprofloxacin = 67%, enrofloxacin 89%, amoxicillin 11%, oxytetracycline 11%) at high mean concentrations (ciprofloxacin = 0.15±0.066 µg/ml, n = 6, enrofloxacin = 0.089±0.049 µg/ml, n = 8, amoxicillin = 0.09 µg/ml, n = 1, oxytetracycline = 0.005 µg/ml, n = 1). Residues of both enrofloxacin and ciprofloxacin were found in all samples of liver tissue from nine dead cinereous vultures at mean concentrations of 0.18±0.11 µg/g (range 0.08–0.21 µg/g) and 0.09±0.04 µg/g (range 0.03–0.07 µg/g) respectively. Relationships between circulating antibiotics, disease and mortality Variable prevalences of bacterial and fungal pathogens involved in severe disease were found in the sampled nestlings (Table 2). A proportion of cinereous (16.3%, n = 49) and Egyptian (4.0%, n = 25) vultures were infected with two or more of these pathogens. 10.1371/journal.pone.0001444.t002Table 2 Prevalence (% of infected individuals) of each pathogen in nestlings of three vulture species from central Spain. Egyptian vulture n = 25 Cinereous vulture n = 49 Griffon vulture n = 50 Salmonella sp 32 16 0 typhimurium 50 25 - enteritidis 25 75 - gallinarum 12.5 - - pullorum 12.5 - - Mycobacterium avium (serotype 7) 4 8 0 Candida albicans 0 26.5 6 Aspergillus fumigatus 9 16 0 Total 32 53 6 Prevalence of each Salmonella serotype was calculated from the total positive isolates in each vulture species (n = 8 for both Egyptian and cinereous vultures). The presence of antibiotic residues was clearly associated with the presence of pathogens in the three vulture species (Fig. 1). Pathogen prevalence was only related to the presence of quinolones in the vulture species (cinereous and Egyptian) in which there were a sufficient number of samples with several antibiotics (log-linear analyses, both P<0.00001, goodness-of-fit, both P>0.855). 10.1371/journal.pone.0001444.g001Figure 1 Relationships between prevalence of pathogens (% of individuals with pathogens) and the presence or absence of antibiotics in plasma of nestlings of three vulture species from central Spain. Differences were statistically significant in the griffon (Fisher exact tests, P = 0.001), cinereous (G-tests, G = 18.198, d.f. = 1, P<0.0001) and Egyptian (G-tests, G = 11.778, d.f. = 1, P = 0.001) vultures. Sample size is shown above bars. Monitoring until fledging detected the deaths of several nestling griffon (1 of 50) and cinereous (4 of 49) vultures. Necropsy and post-mortem investigations revealed that nestling cinereous vultures that died in the nests were severely infected by the same pathogens found during sampling (n = 4, prevalence of Candida albicans = 100%, Aspergillus fumigatus 25%, Salmonella sp. 50%, total prevalence = 100%). Five more cinereous vultures that had been sampled in their nests were found dead or ill in the countryside at various times after fledging. Dead and sick fledgling cinereous vultures were examined in wildlife rehabilitation centres and the latter were treated for severe infections by C. albicans (prevalence = 100%, n = 5), from which they probably would have died without human intervention. All the sick or dead griffon (n = 1) and cinereous (4 nestlings and 5 fledglings) vultures showed antibiotics in plasma when they were sampled in their nests. This suggests an association between fatal disease and the presence of antibiotics in cinereous vultures (Fig. 2). 10.1371/journal.pone.0001444.g002Figure 2 Relationships between the presence of antibiotics in plasma of nestling cinereous vultures and their subsequent survival (Fisher exact test, P = 0.006). Sample size is shown above bars. In addition, nine other cinereous vultures from the same colony that had not been sampled for blood in their nests had one to three antibiotics in their plasma when presented to the rehabilitation centre (see above) and showed severe disease due to the tested pathogens (prevalence of C. albicans 78%, A. fumigatus 22%, total prevalence = 100%). Post-mortem findings and histopathology External examination of nine cinereous vulture carcasses revealed cachexia, dehydration and poor development. Cultures in standard fungal media showed all had multiple oral, pharyngeal and oesophageal necrotic mucosal plaques caused by C. albicans, as well as upper digestive tract congestion and swelling. Macroscopically the liver was enlarged, congested and friable, and the kidney was enlarged and pale pink with white, “chalky” deposits. Histopathological examinations revealed lesions in the liver and kidney and severely depleted lymphoid organs. Seven out of nine individuals (78%) had vacuolar degeneration of the liver parenchyma and deformed trabeculae. Five individuals (56%) had hyperplasia and fibrosis of the bile ducts with mononuclear infiltrates. All nine individuals had glomerulonephritis and glomerulonephrosis, mucosal hyperplasia and mild heterophilic inflammation in their renal pelvises and proximal ureters. Six individuals (67%) had mononuclear infiltrates in their renal tissues and a clearly visible tubular epithelium with areas of degeneration and necrosis. Large white aggregates obscured the renal architecture (glomeruli, distal convoluted tubules and collecting tubules) in all individuals but inflammation was minimal. There were extensive diffuse white precipitates on the surface and within the renal parenchyma, consistent with visceral gout, in seven individuals (78%). Discussion Antibiotic bacterial resistance in wildlife has been highlighted as evidence of the impact of increasing human intrusions on wildlife habitats [6], [14]. In this study we demonstrated residues of four different antibiotics in three species of wild birds. To our knowledge, this is the first report of circulating antibiotic residues in wildlife. This striking finding furthers our knowledge about the impact of human activities on environmental health through the potential detrimental effects of circulating antibiotics on wildlife, including the selection and dissemination of antibiotic resistant bacteria. Avian scavengers ingested antibiotics present in the livestock carrion upon which they feed. Veterinary antibiotics are used in large, although regionally variable quantities throughout the world [15], [16]. Therefore, the potential impact of antibiotic residues on scavenging wildlife may be widespread but the severity is likely to vary with the features of livestock operations and practices of eliminating livestock residues in different regions [8], [9]. The use of different antibiotics to treat livestock in the study area and their variable kinetics may explain their varying presence in each vulture species. Both amoxicillin and oxytetracycline were displaced by quinolones as the drugs of choice alone or with other antibiotics [17], [18], which may explain their high presence and concentrations. The detection of quinolones with other antibiotics in several samples may indicate their combined use and the metabolic transformation of enrofloxacin to ciprofloxacin in livestock and vultures. Differing prevalences and concentrations of antibiotics in the three vulture species may reflect their different feeding habits and physiology, especially the pH of the digestive tract. Griffon vultures evolved as social consumers of entire corpses of large herbivores [19], which has been argued to be associated with the evolution of a very acidic gastric pH (between 1 and 2) to minimise infection by pathogens from rotten meat [20]. Cinereous and Egyptian vultures evolved as scavengers and opportunistic predators of small vertebrates, and preferentially feed upon small fragments of livestock carcasses, especially tendons, skin and viscera [19] which tend to concentrate these antibiotics [21]. The differing ecological and evolutionary strategies for carrion exploitation may explain the different impacts of antibiotics on the health of griffon vs. cinereous and Egyptian vultures. Griffon vultures would degrade more antibiotics and pathogens with such a highly acidic gastric pH. This may explain the lower prevalence of antibiotics and pathogens despite the species' greater dependence on livestock carrion. The less acidic gastric pH of cinereous and Egyptian vultures may make them more susceptible to the direct effects of ingested antibiotics despite their higher dependence on wild prey [19]. The mean quinolone concentrations found in vulture plasma may equal that expected in birds treated with a therapeutic dose 24–48 h before testing [22]–[24]. Such high antibiotic concentrations in vultures may indicate an intensive use of these drugs in farming operations in the study area. Antibiotic ingestion is likely to vary with the frequency of use of carrion from different livestock species or wild prey by parental vultures and the concentrations of antibiotics in different livestock carcasses. Nestling vultures may be fed every several days and antibiotics may be metabolised and excreted by birds over a similar time period. This may explain the lack of antibiotics in several nestlings despite the consumption of livestock carrion by all individuals. The most striking result of this study was the clear association between antibiotic residues, especially quinolones, and disease in the three vulture species. Potential negative health effects of direct antibiotic ingestion with livestock meat include immunosuppression, toxicity, allergy and bacterial flora alteration which may temporarily reduce host resistance to pathogens [1], [23], [24]. The presumably discontinuous ingestion of different antibiotics at different concentrations may lead to sideeffects similar to health problems resulting from the misuse of antibiotics in humans and domestic animals [1], [22]–[24]. For instance, severe infections with C. albicans or A. fumigatus may be important causes of morbidity and mortality among individuals ingesting antibiotics which enhance the invasiveness of these and other pathogens by altering the normal flora and by depressing the host defences [1], [24]. The impact of antibiotics on the health of cinereous vultures was clearly illustrated by post-mortem findings and histopathology of both liver and kidney samples. All liver samples from dead cinereous vultures contained both enrofloxacin and ciprofloxacin. Although there is no detailed information about the pathology caused by antibiotics in birds, lesions and tissue damage in the liver and kidney are consistent with the expected direct toxic effects of antibiotics in these organs in humans [25]–[28], especially because in birds they concentrate these drugs [21]–[22]. In addition, the severe depletion of lymphoid organs may indicate immunosuppression directly by antibiotics [23]–[24] which may be related to the acquisition and proliferation of the recorded pathogens, even causing severe lesions as in the case of C. albicans. The detrimental effects of ingested antibiotics including the acquisition of pathogens may decrease the health of vultures with a lethal potential, especially in nestlings and fledglings as reported in cinereous vultures. This effect may be associated with the recent steady decline of vulture populations in the study area, especially of cinereous and Egyptian vultures [12], [29]. Therefore, the ingestion of antibiotics in livestock carrion represents a major concern for the conservation of these species. Antibiotic residues in meat in livestock dumps used by scavengers should be regulated to avoid damaging the health and conservation of vultures. The intensive use of antibiotics worldwide and their presence in wild birds have global implications for conservation and in generating and spreading resistant pathogens throughout the environment. Materials and Methods Fieldwork We sampled nestlings of the three vulture species breeding in central Spain (primarily in Segovia Province) from 2003 to 2005. In this area a large population of avian scavengers depends on livestock farming providing carrion [8], [9], [29], especially in Segovia province, which has the highest concentration and number of fattening pigs in Spain. Vulture nests were accessed by climbing and nestlings were sampled at 60–80 days of age, depending on the species. A sample of blood (5 ml) was taken from the brachial vein, centrifuged and the plasma frozen until analysed. Bacterial microflora were sampled from the cloaca, choana and nares of nestlings with sterile microbiological swabs and Amies transport medium. Samples were transported in a chilled container to the laboratory within 12 h after collection and were processed within one to two hours of arrival. Nestlings were monitored with telescopes until fledging to assess their survival in the nest. Several of these nestlings were found dead in the countryside one or two months after fledging or they were found ill and admitted to wildlife rehabilitation centres where their health status was assessed, including determining the presence of bacterial and fungal pathogens and of antibiotics in plasma. In addition, eight fledglings and one adult cinereous vulture from the same colony as the sampled nestlings (as demonstrated by their rings), which had not been sampled for blood in their nests were found ill in the countryside and admitted to a rehabilitation centre in Madrid between 2002 and 2005. Similar samples were also taken from these individuals. Necropsy and histopathological examination Nine dead nestling cinereous vultures found in or around nests were necropsied and samples of lesions and selected tissues (liver, kidney, lungs, thymus, spleen, gonads, bursa of Fabricius and heart) were collected and fixed in 10% neutral buffered formalin for histopathological examination. Liver samples from each were also taken and frozen at −20°C for the determination of quinolone residues. Antibiotic residues in livestock carrion The Four Plate Test of bacterial growth inhibition to detect antibiotic residues was used on swine tissues (liver, muscle, kidney, oral mucosa, rectum) from seven carcasses found in several livestock refuse dumps where vultures usually forage. This test is extremely sensitive to antibiotics and therefore is routinely performed in slaughterhouses to quickly confirm the presence of antibiotics in food animals [30]. Samples were collected with sterile microbiological swabs, transported in a chilled container to the laboratory within 12 h after collection and processed within one to two hours of arrival. The test was performed in plates with Bacillus subtilis spore suspension and Kocuria rhizophila bacterial suspension (Merck). Media used to test for residues included test agar pH 6.0 (Merck, dehydrated medium 10663), test agar pH 7.2 with the addition of trimetoprim (Merck, dehydrated medium 15787), and test agar pH 8.0 (Merck, dehydrated medium 10664). Media were prepared according to the manufacturer's instructions. After cooling the agar to 45–55°C, cell and spore suspensions were added to the appropriate media. Sterile standard Petri dishes were filled with 8 ml of the inoculated media and stored at 2–5°C for a maximum of five days. Antibiotic residue determination The presence and concentrations of antibiotic residues in plasma were determined using HPLC techniques and standard protocols [31]–[33]. Briefly, for enrofloxacin and ciprofloxacin, plasma samples (300 µl) were added with an internal standard (75 µl ofloxacin), mixed and shaken with chloroform (4.5 ml). After centrifugation the organic phase was collected and dried under nitrogen. The extracted sampled was injected directly into the HPLC (UV) apparatus (Spectra System AS1000 Autosampler, Thermo Separation Products, Florida USA). These antibiotics were detected using ultraviolet spectrophotometry at 279 nm. The limits of quantification of both molecules were 0.005 µg/ml and the method was linear up to 30 mg/L. The mean percentage recoveries of enrofloxacin and ciprofloxacin were 93% and 90%, respectively. The inter- and intra-assay reproducibility was below 4%. Liver samples used to determine quinolone residues were homogenised in methanol and centrifuged for pellet debris. Three millilitres of the supernatant were then passed through a solid phase extraction cartridge. Elution was concentrated to a volume of 1 ml. Quinolone concentrations were determined using the same methodology as for plasma samples. The recovery, limit of detection, accuracy and precision of this method were evaluated at concentrations from 0.025 to 250 µg/g. The method was validated and shown to be linear in the range of 0.01–50 µg/g. Spike recoveries for liver prepared at 4 spiking levels ranged from 81% to 98%. The coefficient of variation for recovery as a measure of relative variability was between 3% and 8% and the relative standard deviation was <11%. The limits of quantification were 0.1 µg/g for enrofloxacin and 0.25 µg/g for ciprofloxacin. For amoxicillin determination, 50 µl of plasma were mixed with 50 µl of perchloric acid using a vortex mixer and then centrifuged to precipitate plasma proteins. The clear supernatant was then injected into the HPLC. Amoxicillin was eluted with a mobile phase consisting of 6% methanol plus phosphate buffer with pH adjusted to 3.2. The concentration of amoxicillin was scanned at a wavelength of 227 nm, and the injection volume was 20 µl. The limit of quantification was 0.05 µg/ml in plasma. The absolute recovery of amoxicillin was 93%. The intra- and inter-assay coefficients of variation were 2% and 3%, respectively. To determine oxytetracycline concentrations, 9.5g of Mueller-Hinton medium were dissolved in 250 ml of distilled water and autoclaved at 121°C for at least 15 minutes. The solution was cooled to 50°C in a water bath and 0.4 ml of spore solution (1 ml of B. cereus spores in 50 ml sterile saline) was added. After the agar solidified 90 µl wells were cut into the bioassay plates. Plasma samples were deproteinised by adding 20 µl of a 30% trichloroacetic acid solution to 40 µl of plasma. The mixture was gently vortexed and centrifuged and the supernatant was assayed. Ninety microlitres of standards, controls and samples were pipetted into duplicate wells in the bioassay plates. Assay plates were incubated at room temperature overnight and the zones of inhibition were measured in micrometres using electronic digital callipers. The plasma concentration was calculated from a standard curve. The limit of quantification was 0.05 µl/ml. Quality control samples were spiked at 0.2, 0.8 and 8 ppm and assayed on each plate. Pathogen determination We determined the presence of two bacterial (Mycobacterium avium, Salmonella sp.) and two fungal (C. albicans, A. fumigatus) species due to their known severe pathogenicities in birds [34] and their potential to proliferate following the ingestion of antibiotics through the alteration of normal flora [1], [9], [34]. To culture M. avium, cloacal and tracheal samples taken with sterile swabs were plated on Lowenstine-Jenssen media and incubated for three months. Ziehl-Nielsen and auramine rhodamine acid-fast stains and PCR techniques were used to identify any Mycobacterium grown [35], [36]. The presence of a mycobacterium was confirmed if both cultures and molecular techniques were positive. C. albicans and A. fumigatus were cultured in standard fungical media (Agar Sabouraud) at 37°C for 48 h. Only clinical C. albicans was considered, which was determined by the presence of lesions in the oral cavity. For Salmonella, samples were cultured and identified to serotype following standard methods described in detail elsewhere [9]. We thank J.C. Rincón, O. Frías, L. Mateus, A. Tejedor, J. de la Puente and N. Baniandrés for their help with the fieldwork. We also thank two anonymous reviewers for constructive comments and corrections on the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: The authors have no support or funding to report. ==== Refs References 1 Levy SB 2002 The antibiotic paradox: how the misuse of antibiotics destroys their curative powers. Cambridge HarperCollins Publishers 296 2 Daughton CG Temes TA 1999 Pharmaceutical and personal care products in the environment: Agents of subtle change? 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Appl Environ Microbiol 42 1123 1124 7032423 11 Rodrigues L Macedo L Robert J Fernandes MC Santos Meira AT de Lima LA 2003 Dominant culturable bacterial microbiota in the digestive tract of the American black vulture (Coragyps atratus Bechstein 1793) and search for antagonistic substances. Brazilian J Microbiol 34 218 224 12 Tella JL 2001 Action is needed now, or BSE crisis could wipe out endangered birds of prey. Nature 410 408 13 Martínez F Arroyo B Lemus JA Blanco G 2004 El declive del alimoche en Segovia da la pista sobre la situación actual de la especie. Quercus. 227 69 14 Gilliver MA Bennett M Begon M Hazel SM Hart CA 1999 Antibiotic resistance found in wild rodents. Nature 401 233 234 10499578 15 Aarestrup FM 2005 Veterinary drug usage and antimicrobial resistance in bacteria of animal origin. 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Rev Sci Techn Off Int Epiz 20 180 203	18197254	PMC2186382	CC BY	2021-01-05 15:59:01	yes	PLoS One. 2008 Jan 16; 3(1):e1444
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1825350107-PONE-RA-02994R110.1371/journal.pone.0001554Research ArticleNeuroscience/Neuronal Signaling MechanismsAnesthesiology and Pain Management/Basic Science of Pain ManagementHigh-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence pM Naloxone Binds Filamin AWang Hoau-Yan 1 * Frankfurt Maya 1 Burns Lindsay H. 2 1 Department of Physiology and Pharmacology, City University of New York Medical School, New York, New York, United States of America 2 Pain Therapeutics, Inc., San Mateo, California, United States of America Steinhardt Richard Academic EditorUniversity of California, Berkeley, United States of AmericaE-mail: [email protected] and designed the experiments: HW. Performed the experiments: HW. Analyzed the data: HW. Contributed reagents/materials/analysis tools: MF. Wrote the paper: LB. 2008 6 2 2008 3 2 e155410 12 2007 10 1 2008 Wang et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor–Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence. ==== Body Introduction Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia and attenuate tolerance and dependence, with a mechanism long hypothesized as a blockade of excitatory signaling opioid receptors [1]–[4]. Ultra-low-dose opioid antagonists can also reverse hyperalgesia caused by acute, low-dose opioids to produce analgesia [5]. Additionally, ultra-low-dose naltrexone has recently been shown to attenuate opioid reward or addictive properties in conditioned place preference [6] and self-administration and reinstatement paradigms [7]. In a recent clarification of the mechanism of action of ultra-low-dose opioid antagonists, we showed that co-treatment with 10 ng/kg naloxone (NLX) prevented a chronic morphine-induced, Gi/o-to-Gs switch in G protein coupling by the mu opioid receptor (MOR) as well as a coincident interaction of the Gβγ dimer with adenylyl cyclase II and IV [4]. While opioid receptors preferentially bind Gi and Go proteins to inhibit adenylyl cyclase [8], chronic morphine induces MOR–Gs coupling [4], [9]. Although Chakrabarti et al (2005) also demonstrated some MOR–Gs coupling in the opioid naïve state while we did not, we believe this difference may be due to their use of non-immobilized anti-Gα antibodies producing some background binding by Fc receptors. First postulated as the sole mediator of excitatory effects of opiates [10], the Gβγ interacting with adenylyl cyclases originates from the Gs protein coupling to MOR and not from MOR's native G proteins [11]. Ultra-low-dose opioid antagonists were initially thought to preferentially bind a subset of MORs [1], and a Gs-coupling MOR subpopulation was again recently proposed [9]. While it is difficult to estimate the relative proportion of MORs signaling via Gs versus Gi/o during tolerance, it seemed unlikely that the ultra-low doses of NLX or naltrexone influencing opioid agonist effects would be sufficient to selectively antagonize such a subpopulation. Based on saturation binding studies [12], which incorporate NLX's affinity to MOR, we estimate receptor occupancy of 10 ng/kg NLX as no more than 1%. More importantly, our co-immunoprecipitation data showed that ultra-low-dose NLX co-treatment reduces MOR–Gs coupling while restoring levels of coupling to MOR's native Gi/o proteins; further, in spinal cord of co-treated rats, MOR–Gi/o coupling levels greatly surpassed those of opioid-naïve rats [4]. If NLX were selectively antagonizing a subpopulation of “Gs-coupled” MORs, coupling to native G proteins would likely be unaffected. Since NLX prevents MOR–Gs coupling at concentrations well below its affinity for MOR and by influencing the coupling behavior of MORs, we considered proteins that interact with MOR and MOR-associated G proteins as the most likely targets, particularly those able to interact with multiple MORs. We first examined proteins that co-immunoprecipitated with MOR during activation. We identified a 300-kDa protein co-immunoprecipitating with MOR as FLNA and then demonstrated specific, high-affinity binding by NLX to FLNA. Best known for cross-linking cytoplasmic actin into dynamic scaffolds to control cell motility, filamins are large cytoplasmic proteins increasingly found to regulate cell signaling by interacting with over 30 different receptors and signaling molecules [13], [14], including MOR [15]. We deduced the precise binding site on FLNA by using overlapping peptides within the c-terminal, since c-terminal FLNA was shown to interact with MOR using a yeast-two hybrid [15]. To assess the functional significance of this high-affinity interaction, we used peptide fragments containing the binding site to prevent NLX from binding full-length FLNA in organotypic striatal slice cultures. Our findings suggest that FLNA interacts with ultra-low-dose NLX and naltrexone to prevent chronic morphine-induced MOR–Gs coupling, possibly by preventing a critical MOR–FLNA interaction. This high-affinity binding site in c-terminal FLNA therefore appears to underlie the paradoxical enhancement of opioid analgesia and prevention of analgesic tolerance and dependence by ultra-low-dose opioid antagonists. In identifying the binding site though which ultra-low-dose opioid antagonists prevent MOR–Gs coupling, our data also reveal an important regulation of MOR–G protein coupling by filamin A. Results Identification of NLX-binding protein in MOR immunoprecipitates In previous co-immunoprecipitation experiments of MOR and G proteins, we noted a protein with molecular weight at approximately 300-KDa in Gαi/o immunoprecipitates in an amount that closely paralleled the amount of MORs in these immunoprecipitates, suggesting a functional interaction. A battery of antibodies against various cytoskeletal proteins, preliminarily identified this protein that co-immunoprecipitated with MORs and their associated G proteins as FLNA. Using striatal tissue of rats treated chronically with vehicle, morphine, ultra-low-dose NLX or morphine+ultra-low-dose NLX, we performed a two-tiered co-immunoprecipitation with anti-Gα followed by anti-MOR antibodies. The final probing with a specific anti-FLNA antibody showed that FLNA associates with Go-coupled MOR and not with Gs-coupled MOR (Fig. 1 A,B). These blots also demonstrate the morphine-induced G protein coupling switch by MOR and its attenuation by co-treatment with ultra-low-dose NLX. The blots were stripped and re-probed with antibodies to MAP1B and yaotio to illustrate the absence of these cytoskeletal proteins in these immunoprecipitates (Fig. 1C). The exclusive presence of FLNA in the MOR/Go complexes led us to hypothesize that FLNA is the target through which ultra-low-dose NLX blocks the chronic morphine-induced switch from normal MOR–Gi/o coupling to Gs coupling. 10.1371/journal.pone.0001554.g001Figure 1 FLNA associates with Go-coupled MORs and not Gs-coupled MORs. Neuronal membranes were prepared from striata of rats chronically treated with vehicle, morphine, or ultra-low-dose NLX alone or combined with morphine. After stimulation by in vitro morphine or not, membranes were solubilized and immunoprecipitated first with immobilized anti-Gα. Anti-Gα immunoprecipitates were then immunoprecipitated with immobilized anti-MOR before final Western blot detection with anti-FLNA. Densitometric quantitation (B) of Western blots of both MOR in the second immunoprecipitate and FLNA in the final (A) demonstrates that FLNA is associated with MORs coupling to Go but not MORs coupling to Gs. These blots and their quantitation also show that NLX co-treatment prevented the chronic morphine-induced Go-to-Gs coupling switch. Solid bars indicate basal coupling, and hatched bars indicate coupling after receptor stimulation by in vitro morphine. n = 4. p<0.05, *p<0.01 compared to respective basal coupling level. # p<0.01 compared to respective value in vehicle or morphine+NLX groups. Blots were stripped and re-probed with antibodies to yaotio, MAP2, H-neurofilament and MAP1B (C). NLX binding in FLNA-expressing cells and affinity measurement To validate the binding of NLX to FLNA, we assessed binding of [3H]NLX to membranes prepared from the human melanoma cell line M2 that lacks filamin and to membranes from its FLNA-transfected subclone A7. We first confirmed FLNA expression in A7 cells and absence in M2 cells by Western blotting using a specific anti-FLNA antibody (Table 1). FLNA was also detected in the human neuroblastoma SK-N-MC cell line and in rat cortical membranes (Table 1). Importantly, [3H]NLX bound to A7 membranes and this binding was robustly displaced by naltrexone but not by morphine, illustrating that NLX and naltrexone bind to a novel site distinct from MOR (Fig 2A). Negligible [3H]DAMGO binding to A7 cells suggested that these cells (and presumably their M2 parent line) do not express MORs (Fig. 2B). Finally, the filamin-deficient M2 cells do not express molecules that bind [3H]NLX (Fig. 2C). 10.1371/journal.pone.0001554.g002Figure 2 NLX target is distinct from MOR but also bound by naltrexone. A, Naltrexone but not morphine markedly reduced [3H]NLX binding in FLNA-expressing A7 membranes. n = 6. B, The absence of [3H]DAMGO binding using twice the amount of A7 membranes shows these lines do not express MOR. n = 4. C, Parent M2 melanoma cells do not express NLX targets. n = 4. p<0.05 compared to control. 10.1371/journal.pone.0001554.t001Table 1 FLNA expression (Optical intensity of Western blot bands, arbitrary units) A7 cells M2 cells SK-N-MC cells Rat cortex 15 µg 825.8±69.6 0±0 351.3±63.9 230.3±29.4 30 µg 2454.8±259.9 0±0 983.8±89.0 664.8±67 Solubilized proteins (15 or 30 µg) from membrane preparations were size-fractioned by 7.5% SDS-PAGE and electrophorectically transferred onto nitrocellulose for Western blotting using specific anti-FLNA antibodies. n = 4. In an affinity assessment, a competition (displacement) curve for the inhibition of [3H]NLX binding to A7 cell membranes was performed using 16 concentrations of naltrexone. Analysis by nonlinear regression showed two saturable sites (R2 = 0.9788) with an IC50-H of 3.94 picomolar and an IC50-L of 834 picomolar (Fig. 3). 10.1371/journal.pone.0001554.g003Figure 3 NLX binds A7 membranes with picomolar affinity. A competition (displacement) curve for the inhibition of [3H]NLX binding by naltrexone to membranes from FLNA-expressing A7 cells shows two affinity states with IC50-H of 3.94 picomolar and IC50-L of 834 picomolar. A nonlinear curve-fit analysis was performed using a competition equation that assumed two saturable sites for the naltrexone curve comprising of 16 concentrations ranging from 0.1 pM to 1 µM. Data are derived from 6 experiments each using a different set of A7 cells. Precise NLX binding site determined using overlapping peptides To deduce the precise binding domain within FLNA where NLX binds, several overlapping peptide sequences derived from the carboxy-terminus where FLNA intersects with synaptic membranes were used to absorb [3H]NLX. Both FLNA2556–2565 and FLNA2561–2570 markedly attenuated [3H]NLX binding to A7 cell membranes and to purified human FLNA (Fig. 4). This result was confirmed using FLNA purified from FLNA- and MOR-expressing SK-N-MC cells. At 500 pM concentration, [3H]NLX binds to immunoaffinity-purified FLNA in the presence or absence of irreversible MOR antagonist, β-FNA. These data together suggest that NLX binds with high affinity to FLNA with the binding site located at FLNA2561–2565. 10.1371/journal.pone.0001554.g004Figure 4 FLNA2561–2565 is the binding site for [3H]NLX. A7 cell membranes or purified FLNA was incubated with [3H]NLX in the presence of various overlapping FLNA c-terminal peptide fragments. Peptides containing FLNA2561–2565 markedly reduced [3H]NLX binding to A7 membranes (A) and to purified FLNA (B, C). The reduction of [3H]NLX binding to purified FLNA was not affected by the presence of the irreversible MOR antagonist β-FNA (C). n = 6. * p<0.01 compared to vehicle. Confirmation of pentapeptide binding and alanine scan To confirm that FLNA2561–2565 binds NLX and to deduce the critical amino acid(s) within the NLX interacting FLNA2561–2565 region, we generated 4 alterations of FLNA2561–2565 each with 1 amino acid replaced by alanine (alanine scan). Using FLNA2561–2565 and the alanine-replaced pentapeptides to compete with [3H]NLX binding to FLNA in A7 cell membranes, we show that the lysine residue at FLNA2663 is critical to the NLX–FLNA interaction. While the FLNA2561–2565 displacement of [3H]NLX binding was only slightly attenuated by alanine substitutions at the first, fourth and fifth amino acid residues, substitution of the lysine completely prevented it (Fig. 5). The notion that NLX binds to the extreme carboxyl terminus of FLNA that tethers to the internal potion of cell membranes is supported by our data illustrating that FLNA2561–2565 (VAKGL) prevented the [3H]- and FITC-NLX labeling that localized internally in intact A7 but not M2 cells (Fig. 6). This labeling occurred without membrane permeabilization, indicating that NLX can easily penetrate cell membranes to access FLNA. 10.1371/journal.pone.0001554.g005Figure 5 Mid-point lysine of FLNA2561–2565 is critical for NLX binding. An alanine scan of the FLNA2561–2565 pentapeptide (VAKGL) shows that residue of FLNA2561–2565 is critical for NLX binding. All other individual alanine substitutions only mildly attenuated [3H]NLX binding to FLNA in A7 membranes. n = 6. * p<0.01 compared to vehicle. 10.1371/journal.pone.0001554.g006Figure 6 [3H]- and FITC-NLX binding to intact A7 cells is blocked by FLNA2561-2565. Intact A7 and M2 cells were incubated with 0.5 nM [3H]NLX or 10 nM FITC-NLX in the presence or absence of 10 nM VAKGL or VAAGL. [3H]NLX bound to A7 but not to M2 cells and VAKGL markedly reduced this binding to A7 cells (A). Likewise, FITC-NLX labelled A7 (B.I) but not M2 cells (B.II), and co-treatment with VAKGL (B.III) but not VAAGL (B.IV) abolished this FITC-NLX labelling in A7 cells (B). n = 4. * p<0.01 compared to vehicle. Peptide binding site interferes with NLX's prevention of MOR–Gs coupling induced by chronic morphine To confirm that NLX binding to FLNA prevents the chronic morphine-induced Gi/o-to-Gs coupling switch, we utilized organotypic rat brain striatal slice cultures. We previously showed that in vivo chronic morphine induced a switch to MOR–Gs coupling in rat striatum, periaqueductal gray and dorsal spinal cord from Gi or Go coupling in these latter two regions but from an exclusive Go coupling in striatum [4]. To mimic the in vivo chronic morphine treatment and resulting opioid tolerance in that study, rat striatal slices were treated for 7 days twice daily for 1 hr with 100 µM morphine in serum-free culture medium. This morphine treatment induced a robust Go-to-Gs coupling switch, similar to that previously observed following in vivo chronic morphine, that was also similarly blocked by co-treatment with ultra-low-dose (10 pM) NLX (Fig. 7). The addition of FLNA2561–2570 but not FLNA2550–2560 or FLNA2566–2575 abolished NLX's prevention of the Go-to-Gs coupling switch, presumably by preventing NLX from binding FLNA in the tissue (Fig. 7). Densitometric quantitations of blots from slices treated with NLX or FLNA peptides alone did not differ from those of vehicle and are omitted from Fig. 7B for simplification. 10.1371/journal.pone.0001554.g007Figure 7 FLNA2561-2565 blocks 10 pM NLX's prevention of the chronic morphine-induced Go-to-Gs coupling switch. Striatal slices were chronically treated with vehicle, morphine, NLX, morphine+NLX, or with one of the three FLNA peptides alone or in combination with morphine+NLX. Coupling between MOR and Gs/Go proteins was assessed by Western blot (A) and analyzed by densitometric scanning (B). Chronic morphine exposure caused a Go-to-Gs coupling switch that was blocked by NLX co-treatment. NLX's suppression of this coupling switch was blocked by FLNA2561-2570 but not by FLNA2566–2575 or FLNA2550–2560, illustrating that NLX's protective effect occurs via its binding to FLNA within FLNA2561–2570. Solid bars indicate basal coupling; hatched bars indicate coupling after in vitro morphine stimulation. n = 6. p<0.01 compared to Kreb's Ringer; +p<0.01 compared to morphine; #p<0.01 compared to morphine+NLX. Consistent with the G protein coupling switch, morphine treatment also increased basal cAMP levels, caused DAMGO to stimulate cAMP production, and attenuated the DAMGO-mediated reduction in forskolin-stimulated cAMP accumulation (Fig. 8). As with the coupling switch, the addition of FLNA2561–2570 but not FLNA2550–2560 or FLNA2566–2575 abolished NLX's prevention of these cAMP indices of morphine tolerance. Treatment with NLX or FLNA peptides alone did not alter cAMP levels from those of vehicle (data not shown). These data together suggest that ultra-low-dose NLX prevents analgesic tolerance in response to chronic morphine by binding to a specific region of FLNA. 10.1371/journal.pone.0001554.g008Figure 8 FLNA2561–2565 blocks 10 pM NLX's prevention of chronic morphine-induced cAMP accumulation. After chronic treatments, slices were stimulated with DAMGO, forskolin or DAMGO+forskolin before solubilizing tissues and measuring cAMP levels. In accordance with the Go-to-Gs coupling switch, chronic morphine increased basal cAMP production by 24%, caused DAMGO to stimulate basal cAMP production, and reduced the DAMGO-mediated inhibition of the forskolin effect from 35% in vehicle/NLX groups to 7%. NLX co-treatment blocked these morphine-induced effects, and the protective effect of NLX was blocked by FLNA2561–2570 but not by FLNA2566–2575 or FLNA2550–2560, again demonstrating that NLX's protection occurs through binding to FLNA within FLNA2561–2570. n = 4. p<0.01 compared to forskolin alone. +p<0.01 compared to vehicle. ##p<0.05, #p<0.01 compared to basal level. Discussion We have identified a high-affinity binding site for NLX in the carboxyl-terminal region of the scaffolding protein FLNA that appears to mediate ultra-low-dose NLX's prevention of the chronic opioid-induced G protein coupling switch by MOR. This finding further elucidates the mechanism of action of certain ultra-low-dose opioid antagonists in enhancing opioid analgesia and preventing opioid tolerance and dependence [4]. We measure the binding affinity of NLX or naltrexone to FLNA in cell membranes as 4 picomolar, i.e. approximately 200-fold higher than their binding affinity for MOR [16], [17]. To our knowledge, this is the first demonstration of picomolar binding by a psychoactive compound that is not to a cell surface receptor. NLX binds FLNA tightly with critical involvement of a 5-amino acid segment that is intracellular but near its c-terminal transmembrane domain. The presence of FLNA in MOR signalplex in native brain tissues demonstrated here by co-immunoprecipitation agrees with previous data using yeast-two hybrid and co-transfection methods [15]. The dependence of [3H]NLX binding on FLNA expression in cell lines, the absence of MOR in these cell lines, and the displacement by naltrexone but not morphine clarified FLNA as a novel target for NLX and naltrexone. The interaction was confirmed using immunopurified FLNA from A7 or SK-N-MC cells. Since FLNA2556–2565 and FLNA2561–2570 both markedly reduced [3H]NLX binding to A7 membranes or to purified FLNA proteins, we assumed that FLNA2561–2565, within the 24th repeat, is the NLX-binding site on FLNA. We confirmed FLNA2561–2565 as the binding site by showing NLX binding to this pentapeptide, and an alanine scan revealed that the lysine at FLNA2563 is critical for binding. We cannot easily explain a modest enhancement in NLX binding caused by FLNA2575–2583, but this could be the result of reducing steric constraints around the NLX binding site on FLNA. Our demonstration that FLNA2561–2565 (VAKGL) but not VAAGL abolished [3H]- and FITC-NLX binding to intact A7 cells again confirmed that this pentapeptide is a NLX binding site and demonstrated that NLX can access this intracellular target. Finally, the blockade of NLX's protective effects on both Gs coupling and cAMP accumulation by peptides containing FLNA2561–2565 provides evidence that ultra-low-dose NLX may block MOR–Gs coupling and associated opioid tolerance and dependence by binding to FLNA at approximately FLNA2561–2565. Although MOR desensitization, i.e. the decrease in coupling to native G proteins, is more commonly thought to underlie opioid tolerance and dependence [18], [19] than a switch to Gs coupling, our present data indicate that a decrease in MOR efficacy can not alone mediate chronic opioid effects. Specifically, the increase in cAMP accumulation caused by DAMGO without forskolin following chronic morphine treatment illustrates that MOR has not merely desensitized, losing its ability to inhibit cAMP production, but that its stimulation actually augments cAMP production. The detection of FLNA in immunoprecipitates containing Go-coupled MOR and not Gs-coupled MOR appears counterintuitive since NLX prevents MOR–Gs coupling via a tight binding to FLNA. We propose that repeated MOR stimulation may force a particular conformation of the MOR-FLNA complex that weakens the entire signalplex. MOR may subsequently release from both its native G protein and from FLNA itself leading to association with Gs without FLNA in the complex. By binding to FLNA, NLX could block this particular MOR–FLNA interaction and stabilize the MOR-FLNA-G protein complexes, thereby reducing the morphine-induced release of MORs and their subsequent coupling with Gs in response to receptor stimulation. MOR and FLNA might be forced into an altered conformation or interaction during opioid tolerance by proteins involved in MOR recycling and internalization such as β-arrestins [20]. Additionally, as exogenous GM1 ganglioside has been shown to mimick opioid tolerance in vitro [21] and since blocking GM1 ganglioside via cholera toxin B subunit also blocks the excitatory effects of opiates in vitro [22] and in vivo [23], GM1 ganglioside may also influence MOR–FLNA interactions. Interestingly, although we observed FLNA in MOR signalplexes containing Go proteins in native brain tissue, the prior data in co-transfected cells indicates that FLNA is not required for the native coupling state of MOR; in fact, its absence actually enhances MOR function [15]. This prior study by Onoprishvili et al., the first to show that FLNA interacts with the c-terminal of MOR, proposed a role for FLNA in receptor regulation and trafficking [15]. The authors reported that MOR functioned normally in cells lacking FLNA, but that the agonist DAMGO was unable to desensitize MOR, as measured by the decreased ability of DAMGO to inhibit forskolin-mediated cAMP accumulation following prolonged DAMGO exposure. In agreement, our present slice culture data show that ultra-low-dose NLX, by binding to FLNA, prevents this morphine-induced desensitization measure, as well as the upstream MOR–Gs coupling that we previously showed to underlie tolerance and dependence in vivo [4]. Rather than desensitization, Onoprishvili et al. actually noted enhanced inhibition by DAMGO of cAMP accumulation in cells lacking FLNA [15], a finding that also agrees with the idea that in signaling complexes that do not contain FLNA, MOR does not release from its Gi/o protein to interact with Gs. This increased Gi/o recruitment leading to heightened DAMGO-induced cAMP inhibition also concurs nicely with the increased Gi/o coupling we previously observed in spinal cord of animals treated with morphine+ultra-low-dose NLX [4], as well as with the enhanced analgesia that follows these co-treatments [2]. It is possible that the ultra-low-dose opioid antagonist attenuation of opioid addictive properties [6], [7] may also be mediated by this high-affinity binding to FLNA. While the enhancement of opioid analgesia and reduction of analgesic tolerance is “paradoxical,” and occurs only at “ultra-low” doses of NLX and naltrexone (since higher doses of these opioid antagonists also antagonize opioid receptors), the attenuation of rewarding or addictive properties of opioids by ultra-low-dose naltrexone is not paradoxical. Hence, one would expect a continuous suppression of reward as increasing doses of naltrexone are combined with the opioid. Yet, in the conditioned place preference paradigm, while both ultra-low (0.03 and 0.3 ng/kg) and higher (30 ng/kg) naltrexone doses blocked the acute rewarding effects of oxycodone, an interim dose (3 ng/kg) was without effect [6]. Similarly, in a self-administration paradigm, while co-self-administering 10 or 1 pg/kg/infusion both attenuated measures of reinstatement when oxycodone was not available, only the lower dose altered oxycodone's rewarding potency during self-administration [7]. The blunting of opioid rewarding effects by ultra-low-dose naltrexone first suggests that Gs coupling by MOR may contribute to the rewarding or addictive properties of opioids, possibly by cAMP activation of PKA and subsequent CREB phosphorylation. However, opioid inhibitory effects, such as the proposed disinhibition of VTA dopamine neurons via inhibition of GABA interneurons [24], may also contribute to opioid reward. The loss of effect at intermediate naltrexone doses may indicate such complexities of the neural mechanisms contributing to opioid reward and addiction. Alternatively, the fact that the attenuation of rewarding effects is diminished as the naltrexone dose increases could also suggest an upper limit of an effective ultra-low dose range for disrupting the FLNA–MOR interaction and consequent MOR–Gs signaling. In summary, here we identify a specific c-terminal region of FLNA as the high-affinity binding site of NLX and naltrexone in their suppression of MOR signaling alterations that result from chronic opioid treatment. This work therefore provides a molecular target for ultra-low-dose NLX through which ultra-low-dose opioid antagonists enhance opioid analgesia and decrease opioid tolerance and dependence. We propose that repeated MOR stimulation leads to a particular conformation of MOR–FLNA that weakens Gi/o–MOR–FLNA complexes and allows MORs to release to interact with Gs upon subsequent stimulation by morphine. By binding to FLNA, NLX and its analogs prevent this altered MOR–FLNA interaction, thereby preventing the release from the complexes and the resultant altered coupling. There are multiple signaling consequences of the switch to Gs coupling by MORs chronically exposed to opioids, and each may contribute differently to the various behavioral effects of long-term opioid administration such as analgesic tolerance, physical dependence and the possibility of addiction. This notion may explain the multiple beneficial effects of ultra-low-dose opioid antagonist co-treatment, shown to preserve the normal G protein coupling profile of MOR [4]. By identifying the target and binding site of ultra-low-dose NLX and naltrexone, we further elucidate their mechanism of action when combined with opioids. Finally, these findings create an opportunity to formulate a new generation of pain therapeutics that may provide long-lasting analgesia with minimal tolerance, dependence and addictive properties. Methods Animals Male Sprague Dawley rats (200-250 g) purchased from Taconic (Germantown, NY) were housed two per cage and maintained on a regular 12-hr light/dark cycle in a climate-controlled room with food and water available ad libitum. For identification of NLX-binding protein in MOR immunoprecipitates, four groups of 4 rats were treated twice daily for 7 days with vehicle, NLX (10 ng/kg, s.c.), morphine (10 mg/kg, s.c.), or morphine+NLX. These animals were sacrificed by decapitation 16 hr after the last injection, and whole striatum was harvested on ice immediately. For organotypic striatal slice cultures, brain slices harvested from treatment-naïve rats were maintained and treated in vitro as described below. All procedures in this protocol are in compliance with the City College of New York IACUC on the use and care of animals. Identification of FLNA in MOR immunoprecipitates Neuronal membranes (200 µg) were prepared as described previously [4], [25] from striata of rats treated as described above. Membranes were incubated with 1 µM morphine for 5 min at 37°C in Kreb's Ringer solution before solubilization in immunoprecipitation buffer (25 mM HEPES, pH 7.5; 200 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 0.2% 2-mercaptoethanol, 50 µg/ml leupeptin, 25 µg/ml pepstatin A, 0.01 U/ml soybean trypsin inhibitor, 0.04 mM phenylmethylsulfonyl floride [PMSF]) containing 0.5% digitonin, 0.2% sodium cholate and 0.5% NP-40 at 4°C with end-over-end shaking for 60 min. The supernatant was collected after centrifugation at 50,000 X g for 5 min to remove insoluble debris before immunoprecipitation. MOR and its associated scaffolding proteins and G proteins were immunopurified together using anti-Gαs/olf or -Gαo antibodies that were immobilized to prevent interference from immunoglobulins. Anti-Gα antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were covalently cross-linked to protein A conjugated resin in a Seize-X protein A immunoprecipitation kit (Pierce-ENDOGEN, Rockford, IL) according to manufacturer's instructions. MOR-G protein-scaffolding protein complexes in solubilized brain lysate were first isolated by immunoprecipitation in which 200 µg solubilized brain membrane extract from striatum was incubated with immobilized anti-Gα-protein A-resin at 4°C overnight. After centrifugation and three washes with phosphate-free Kreb's-Ringer (pH 7.4) containing mixtures of protease and protein phosphatase inhibitors at 4°C, the MOR-G protein-scaffolding protein complexes were eluted with 200 µl of neutral pH gentle antigen elution buffer, diluted 5-fold with immunoprecipitation buffer, and immunoprecipitated with anti-MOR at 4°C for 4 hr followed by 50 µl protein A/G-conjugated beads (Santa Cruz Biotechnology) for 2 hr. The immunoprecipitates containing MOR-G protein-scaffolding protein complexes were collected by centrifugation and washed twice with phosphate-free Kreb's-Ringer. The washed immunocomplex was re-suspended in 75 µl phosphate-free Kreb's-Ringer and solubilized by combining with 75 µl of 2X PAGE sample preparation buffer (62.5 mM Tris-HCl, pH6.8; 20% glycerol, 4% SDS; 10% 2-mercaptoethanol, 0.1% bromophenol blue) and boiled for 5 min. The levels of selective MOR-associated scaffolding proteins were determined by Western blotting using specific antibodies directed against various cytoskeletal and scaffolding proteins including FLNA, MAP1B and yaotio. NLX binding to FLNA-expressing cells To confirm FLNA as the high-affinity target of NLX, we assessed [3H]NLX binding to the human melanoma cell line M2 subclone that was stably transfected with human filamin A cDNA (obtained from Drs. Stossel and Ohta at Harvard Medical School). Membranes prepared from A7 cells (100 µg) were incubated in binding medium (50 mM Tris HCl, pH7.5; 100 mM NaCl; and protease and protein phosphatase inhibitors) with 500 pM [3H]NLX in the presence of 10 µM of either morphine or NTX at 37°C for 30 min. Total incubation volumes were 500 µl. Non-specific binding was defined by 1 µM NTX. Membranes prepared from the parent M2 cells (200 µg) that do not express FLNA served as a negative control for [3H]NLX binding, and A7 cell membranes (200 µg) incubated with 2 nM [3H]DAMGO illustrated the lack of MOR in both cell lines. Reactions were terminated by rapid filtration through 3% BSA-treated GF/B membranes under vacuum. Filters were washed twice with 5 ml ice-cold binding medium, and [3H]NLX retained on the filters was measured by liquid scintillation spectrometry. In a separate experimental series, whole cell binding of [3H]NLX in intact A7 and M2 was performed. A7 and M2 cells were grown on 35-mm dishes until 80% confluent. After two washes with PBS (37°C), [3H]NLX (0.5 nM) was added to cells and incubated for 30 min at 37°C in the absence or presence of 10 µM pentapeptide FLNA fragment, VAKGL. Following removal of [3H]NLX containing binding medium, cells were washed twice with PBS (37°C), collected and [3H]NLX in cells were counted by scintillation spectrometry. To visualize NLX binding, A7 and M2 cells were grown on chambered slides (Nalge Nunc, Naperville, IL) to 80% confluency. FITC-NLX (10 nM, Invitrogen) was added to the cells in 0.5% FBS-containing culture medium and incubated for 30 min at 37°C in the presence or absence of 10 µM VAKGL or VAAKGL. Following incubation, medium was removed and cells were washed three times with warm PBS. Cells were then fixed in 10% formalin-PBS overnight at 4°C and coverslipped. The bound FITC-NLX was visualized with a fluorescence microscope. Affinity measurement A competition (displacement) curve was generated for the inhibition of [3H]NLX binding by naltrexone to membranes prepared as described above from FLNA-expressing A7 cells. A nonlinear curve-fit analysis was performed using competition equation that assumed two saturable sites for naltrexone curve comprising of 16 concentrations ranging from 0.1 pM to 1 µM. Six experiments each using a different set of A7 cells were included in the analysis. Determination of binding site using FLNA peptides To determine the NLX binding site on FLNA, various overlapping peptides encoding the c-terminal section of FLNA were used to compete for [3H]NLX binding to FLNA using either A7 cell membranes or purified FLNA from A7 or SK-N-MC cells. Peptides were generated by Sigma-Genosys (The Woodlands, TX). The reaction mixture consisted of 100 µg A7 membranes or 2 µg purified FLNA, 500 pM [3H]NLX, and 10 µM of either FLNA2550–2560, FLNA2556–2565, FLNA2561–2570, FLNA2566–2575 or FLNA2576–2581 in 500 µl binding medium. With FLNA purified from SK-N-MC cells, 1 µM of the irreversible MOR antagonist β-funaltrexamine (β-FNA) was used to demonstrate binding independent of MOR. The reactions were carried out at 37°C for 30 min and terminated by rapid filtration through 3% BSA-treated GF/B membranes under vacuum. Filters were washed twice with 5 ml ice-cold binding medium, and [3H]NLX retained on the filters was measured by liquid scintillation spectrometry. Alanine scan of pentapeptide on FLNA binding To determine the essential amino acid(s) within the NLX-interacting pentapeptide, four additional pentapeptides, each with one amino acid replaced by alanine, were used along with the correct FLNA2561-2565 pentapeptide to compete for [3H]NLX binding to FLNA using A7 cell membranes. Peptides were generated by Sigma-Genosys. The reaction mixture consisted of 500 pM [3H]NLX, 200 µg A7 membranes, and 10 µM pentapeptide (AAKGL, VAKGL, VAAGL, VAKAL or VAKGA) in 500 µl binding medium. Reactions were conducted at 37°C for 30 min and terminated by rapid filtration through 3% BSA-treated GF/B membranes under vacuum. Filters were washed twice with 5 ml ice-cold binding medium, and [3H]NLX retained on the filters was measured by liquid scintillation spectrometry. Organotypic striatal slice cultures In this set of experiments, rat brain slice organotypic culture methods were modified from those published previously [26], [27]. Striatal slices (200 µM thickness) were prepared using a McIlwain tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK). Slices were carefully transferred to sterile, porous culture inserts (0.4 µm, Millicell-CM) using the rear end of a glass Pasteur pipette. Each culture insert unit contained 2 slices and was placed into one well of the 12-well culture tray. Each well contained 1.5 ml of culture medium composed of 50% MEM with Earl's salts, 2 mM L-glutamine, 25% Earl's balanced salt solution, 6.5 g/l D-glucose, 20% fetal bovine serum, 5% horse serum, 25 mM HEPES buffer, 50 mg/ml streptomycin and 50 mg/ml penicillin. The pH was adjusted to 7.2 with HEPES buffer. Cultures were first incubated for 2 days to minimize the impact of injury from slice preparation. Incubator settings throughout the experiment were 36°C with 5% CO2. To induce tolerance, culture medium was removed and the culture insert containing the slices was gently rinsed twice with warm (37°C) phosphate-buffered saline (pH 7.2) before incubation in 0.1% fetal bovine serum-containing culture medium with 100 µM morphine for 1 hr twice daily (at 9–10 AM and 3–4 PM) for 7 days. To assess the effect of ultra-low-dose of NLX on the chronic morphine-induced signaling switch, some slices were exposed to 100 µM morphine plus 10 pM NLX. To determine whether NLX's protective effect occurs by binding to FLNA at FLNA2561–2565, slices were incubated with morphine plus NLX with the addition of 10 µM FLNA2550–2560, FLNA2561–2570, or FLNA2566–2575. Slices were returned to culture medium with normal serum after each drug exposure. Tissues were harvested 16 hr after the last drug exposure by centrifugation. MOR–Gs coupling in slice cultures For determination of MOR–G protein coupling, slices were homogenized to generate neuronal membranes. Membranes (400 µg), prepared as described above, were incubated with either 1 µM morphine or Kreb's-Ringer solution for 10 min before solubilization in 250 µl of immunoprecipitation buffer (25 mM HEPES, pH7.5; 200 mM NaCl, 1 mM EDTA, 50 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml soybean trypsin inhibitor, 0.04 mM PMSF and mixture of protein phosphatase inhibitors). Following centrifugation, striatal membrane lysates were immunoprecipitated with immobilized anti-Gαs/olf or -Gαo conjugated with immobilized protein G-agarose beads. The level of MOR in anti-Gαs/olf or -Gαo immunoprecipitates was determined by Western blotting using specific anti-MOR antibodies. cAMP accumulation in slice cultures To measure the magnitude of MOR-mediated inhibition of cAMP production, brain slices were incubated with Kreb's-Ringer (basal), 1 µM DAMGO, 1 µM forskolin or 1 µM DAMGO+1 µM forskolin for 10 min at 37°C in the presence of 100 µM of the phosphodiesterase inhibitor IBMX. Tissues were homogenized by sonication and protein precipitated with 1M TCA. The supernatant obtained after centrifugation was neutralized using 50 mM Tris, pH 9.0. The level of cAMP in the brain lysate was measured by a cAMP assay kit (PerkinElmer Life Science, Boston, MA) according to manufacturer's instructions. Data analysis All data are presented as mean±standard error of the mean. Treatment effects were evaluated by two-way ANOVA followed by Newman-Keul's test for multiple comparisons. A two-tailed Student's t test was used for post hoc pairwise comparisons. Data are presented as means±s.e.m. The threshold for significance was p<0.05. Competing Interests: This study was supported by Pain Therapeutics, Inc. and LHB is an employee of this company. Funding: This study was supported by Pain Therapeutics, Inc. ==== Refs References 1 Crain SM Shen K-F 2000 Antagonists of excitatory opioid receptor functions enhance morphine's analgesic potency and attenuate opioid tolerance/dependence liability. Pain 84 121 131 10666516 2 Crain SM Shen K-F 1995 Ultra-low concentrations of naloxone selectively antagonize excitatory effects of morphine on sensory neurons, thereby increasing its antinociceptive potency and attenuating tolerance/dependence during chronic cotreatment. 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==== Front BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central 1472-6947-7-341799607410.1186/1472-6947-7-34Research ArticleEstimation of progression of multi-state chronic disease using the Markov model and prevalence pool concept Shih Hui-Chuan [email protected] Pesus [email protected] Chi-Ming [email protected] Tao-Hsin [email protected] Department of Nursing, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan2 Community Medicine Research Center and Institute of Public Health, National Yang-Ming University, Taipei, Taiwan3 Department of Medical Research and Education, Cheng Hsin Rehabilitation Medical Center, Taipei, Taiwan4 Faculty of Public Health, School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan2007 9 11 2007 7 34 34 13 2 2006 9 11 2007 Copyright © 2007 Shih et al; licensee BioMed Central Ltd.2007Shih et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background We propose a simple new method for estimating progression of a chronic disease with multi-state properties by unifying the prevalence pool concept with the Markov process model. Methods Estimation of progression rates in the multi-state model is performed using the E-M algorithm. This approach is applied to data on Type 2 diabetes screening. Results Good convergence of estimations is demonstrated. In contrast to previous Markov models, the major advantage of our proposed method is that integrating the prevalence pool equation (that the numbers entering the prevalence pool is equal to the number leaving it) into the likelihood function not only simplifies the likelihood function but makes estimation of parameters stable. Conclusion This approach may be useful in quantifying the progression of a variety of chronic diseases. ==== Body Background While the relationship between exposure and outcome is explored in traditional epidemiology, the status of the disease in question is usually expressed as a dichotomous state: disease and non-disease. Categorizing the disease of interest into two states, more often than not, may not only widen the gap between epidemiologists, who are interested in the occurrence of disease, and clinicians, who are concerned with the prognosis of disease, but also limit investigation of the disease progression for the majority of chronic diseases. As a matter of fact, chronic diseases usually have a multi-state property for which a dynamic progression from the early stage to the late stage proceeds under the influence of a range of internal and external risk factors. In order to elucidate the mechanism of disease progression quantifying the multi-state natural history of the disease becomes important in the new era of epidemiology. Multi-state models are increasingly used to model the progression of chronic diseases [1,2]. Such models are useful for study of both natural history and progression of the related disease [3,4]. Examples include the estimation of transition rates of growth, spread of breast cancer [4], and outcomes of cardiac transplantation [2]. Quantifying the progression of chronic diseases from mild state to advanced state is also relevant to prevention and screening. The multi-state model traditionally associated with chronic diseases has three states: no disease, preclinical but screen-detectable disease, and symptomatic clinical disease. In the context of screening for chronic diseases, the estimations of progression rates based on mathematical models are usually complicated and computationally intensive. For example, Day and Walter (1984) used screening results of breast cancer to simultaneously estimate false negative cases (cases missed at screen) and the mean sojourn time (the average duration of the screen-detectable phase (PCDP), abbreviated as MST hereafter) based on prevalent screen-detected cases and interval cases (clinical cases occurring between screens) [5]. Duffy et al. (1995) and Chen et al. (1996) also applied stochastic models to estimate parameters of breast tumor progression on the basis of screen-detected and interval cancers. Although these methods had their strengths, some major problems still arose [1,6]. Firstly, time to pre-clinical screen-detectable phase for prevalent screen-detected cases (identified in the first screen) is more uncertain than that for incident screen cases (identified in later screens) because prevalent screen-detected cases are treated as a left-censored mode whereas incident screen-detected cases are classified an interval-censored mode in the context of survival analysis. The latter usually provide more information on the occurrence of event than the former. To simplify the estimation of parameters, previous methods often assume that occurrence of prevalence cases as in exponential distribution which has a property of constant pre-clinical incidence. Secondly, estimation of parameters in previous methods needs interval cases. However, it may be difficult to obtain interval cases in countries with incomplete registration; one may be concerned with whether estimation of parameters lacking of this information could bias the result. Although a previous study on quantifying the progression of breast cancer demonstrates that estimation of parameters using interval-censored data may yield an unbiased result consistent with those estimates using interval cases it is uncertain whether data on screening for other chronic diseases has the same result. How to treat the missing information on interval cases while relevant parameters are estimated will be considered in this study. Thirdly, the progression of a multi-state disease may be affected by a set of risk factors or covariates. For example, the onset of Type 2 diabetes may vary by sex, age, obesity and other relevant risk factors. Previous studies on quantifying the progression of chronic diseases either did not take relevant risk factors into account [1,5,6] or considered covariates based on computationally intensive method [7,8]. Fourthly, since certain disease states could not be directly observed, there may be difficulty estimating the model parameters as the models may not be identifiable. This issue is aggravated by a lack of interval cases (cases diagnosed between screens). We find the application of Rothman prevalence pool concept and its extension plus E-M algorithm approach can not only simplify the likelihood function but make estimation of parameters become stable [9]. Missing information on interval cases could be also taken into account. In this study, a three-state Markov model and an illness-and-death Markov model are proposed to model the progression of multi-state disease natural history. The prevalence pool concept proposed by Rothman is applied to prevalent screen-detected cases to estimate parameters dispensing with the exponential assumptions used in previous studies. To tackle the identifiable problem, an E-M algorithm (Expectation-Maximum likelihood estimate) approach, is proposed to take the prevalence pool equation and its extension to death as expectation equations. Accordingly, these expectation equations in combination with the above two Markov models are then used to estimate relevant parameters. An E-M approach was first advocated by Dempster in 1977 [10]. Since then, an E-M algorithm had been extensively used in handling missing data and dealing with latent variables. The major tenet of this approach is to build up a complete likelihood function as if missing information or latent variables are known. Then, parameters generated from expectation equations are further applied to simplify the likelihood function. This iterative procedure is also used to demonstrate the convergence of parameters. As above, the aim of this study is to demonstrate how to estimate parameters with respect to multi-state disease progression based on a three-state Markov model plus Rothman prevalence pool concept or an illness-and-death Markov model plus the extension of Rothman prevalence pool concept under the context of an E-M algorithm approach. A Type 2 diabetes screening regime in Taiwan is used as an illustration. The remainder of this study is organized as follows. We first present how to define disease natural history models for Type 2 diabetes, i.e. a three-state Markov model and an illness-and-death Markov model, and then delineate how to apply Rothman prevalence pool concept and an E-M algorithm approach to estimate parameters. Second, an illustration is given using data from a type 2 diabetes screening regime in Taiwan. Third, numerical results and discussion are given respectively. Methods Markov model specification A three-state Markov model Suppose the natural history of a chronic disease can be defined by three states, including normal (no detectable disease), asymptomatic (preclinical screen-detectable disease) and symptomatic (clinical disease). The progression rates are expressed as in Figure 1, where λ1 represents the incidence rate of asymptomatic cases and λ2 the progression rate from asymptomatic to symptomatic phase. The inverse of λ2 is the mean sojourn time (MST). Figure 1 A three-state Markov model for a disease natural history. We assume that there is no possibility of regression from the asymptomatic phase to normal, or from the symptomatic phase to the asymptomatic phase. This assumption has been extensively used in chronic disease screening models [8,11,12]. Given transition parameters λ1 and λ2, one can develop transition probabilities for each possible transition during time t on the basis of the forward Kolomogorov equations [13]. The transition probabilities for the above three-state model are expressed in equation (1): State123123[P11(t)P12(t)P13(t)0P12(t)P23(t)001]=[e−λtλ1(e−λ2t−e−λ1t)λ1−λ21−e−λt−λ1(e−λ2t−e−λ1t)λ1−λ201−e−λ2te−λ2t001] MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqbaeaabiGaaaqaaiabdofatjabdsha0jabdggaHjabdsha0jabdwgaLbqaauaabeqaceaaaeaafaqabeqaeaaaaeaaaeaacWaGaYC=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T7aSnaaBaaabaGaeGymaedabeaacqGHsislcqWF7oaBdaWgaaqaaiabikdaYaqabaaaaaGcbaGaeGymaeJaeyOeI0Iaemyzau2aaWbaaSqabeaacqGHsislcqWF7oaBcqWG0baDaaGccqGHsisljuaGdaWcaaqaaiab=T7aSnaaBaaabaGaeGymaedabeaacqGGOaakcqWGLbqzdaahaaqabeaacqGHsislcqWF7oaBdaWgaaqaaiabikdaYaqabaGaemiDaqhaaiabgkHiTiabdwgaLnaaCaaabeqaaiabgkHiTiab=T7aSnaaBaaabaGaeGymaedabeaacqWG0baDaaGaeiykaKcabaGae83UdW2aaSbaaeaacqaIXaqmaeqaaiabgkHiTiab=T7aSnaaBaaabaGaeGOmaidabeaaaaaakeaacqaIWaamaeaacqaIXaqmcqGHsislcqWGLbqzdaahaaWcbeqaaiabgkHiTiab=T7aSnaaBaaameaacqaIYaGmaeqaaSGaemiDaqhaaaGcbaGaemyzau2aaWbaaSqabeaacqGHsislcqWF7oaBdaWgaaadbaGaeGOmaidabeaaliabdsha0baaaOqaaiabicdaWaqaaiabicdaWaqaaiabigdaXaaaaiaawUfacaGLDbaaaaaaaa@BA3F@ An illness-and-death Markov model When death is taken into account, we further formulate a four-state Markov model as Figure 2. The transition probabilities for a four-state illness-and-death Markov can be derived in a similar manner. The detailed algebra for transition probabilities is given in Appendix A. Figure 2 A four-state illness-and-death Markov model. Prevalence pool concept The concept of the prevalence pool was firstly used by Rothman and Greenland (1998) [9]. Brookmeyer (1995) applied this concept to estimate progression rates associated with HIV and AIDS [14]. It states that, in a steady population, the number of people entering the prevalence pool is balanced by the number exiting from it. That is, Inflow (to prevalence pool) = outflow (from prevalence pool) Rothman used this concept to derive the relationship between prevalence and incidence. This concept can be extended to any equilibrium state with respect to disease progression. In the above three-state model, for example, a linear relationship between the asymptomatic and symptomatic phase, in the context of screening, can be defined as follows. The first screen in a screening regime contains prevalent asymptomatic cases. If the total number of subjects attending the screen is N and the prevalence pool (number of asymptomatic phase cases) is P, then the size of population at risk that fed the prevalence pool is N-P. During a very small time interval Δt, the number of subjects who enter the prevalence pool is λ1Δt (N-P), where λ1 is the incidence rate of asymptomatic phase. During the same interval Δt, the outflow from the prevalence pool is λ2Δt P, where λ2 is the rate of exiting from the prevalence pool, i.e., the hazard rate of surfacing to the symptomatic phase. According to the above prevalence pool concept, a linear relationship between λ1 and λ2 is obtained as follows: Inflow = λ1Δt (N-P) = outflow = λ2Δt P λ2=N−PP×λ1 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGH9aqpjuaGdaWcaaqaaiabd6eaojabgkHiTiabdcfaqbqaaiabdcfaqbaakiabgEna0kab=T7aSnaaBaaaleaacqaIXaqmaeqaaaaa@39F8@ This forms what we will call hereafter the expectation equation. The Markov model in combination with the prevalence pool concept enables us to estimate the parameters using an E-M algorithm approach. In a similar way, the prevalence pool concept can be applied to an illness-and-death model which includes death as an absorbing state. Taking death into account, we extend the prevalence pool concept to derive the relationship between λ2 and λ3. If P asymptomatic phase cases are detected and the follow-up period J is relatively short, the expected symptomatic phase cases (CE) if the screen has not taken place is: CE = λ2 × J × P The above expression assumes deaths from asymptomatic cases are rare. Despite early intervention, some asymptomatic cases will progress to symptomatic disease and then to death. Assuming an average time of progression to symptomatic disease midway through the period J, the total number of expected death from symptomatic disease is approximately: DE = λ3 × J/2 × CE This gives the relationship between λ2 and λ3: λ3=2DEJ2×P×λ2 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aaSbaaSqaaiabiodaZaqabaGccqGH9aqpjuaGdaWcaaqaaiabikdaYiabdseaenaaBaaabaGaemyraueabeaaaeaacqWGkbGsdaahaaqabeaacqaIYaGmaaGaey41aqRaemiuaaLaey41aqRae83UdW2aaSbaaeaacqaIYaGmaeqaaaaaaaa@3E2B@ In a steady population, the relationship between λ1 and λ3 via prevalence pool equation (2) is therefore: λ3=2DEJ2×(N−P)×λ1 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aaSbaaSqaaiabiodaZaqabaGccqGH9aqpjuaGdaWcaaqaaiabikdaYiabdseaenaaBaaabaGaemyraueabeaaaeaacqWGkbGsdaahaaqabeaacqaIYaGmaaGaey41aqRaeiikaGIaemOta4KaeyOeI0IaemiuaaLaeiykaKIaey41aqRae83UdW2aaSbaaeaacqaIXaqmaeqaaaaaaaa@41ED@ An E-M algorithm approach The E-M algorithm is an iterative method for estimating parameters in two steps: The E-step (expectation step) and the M-step (maximization step) [10]. Let Y represent the observed data and Z missing data or latent variables (in our case, Z represents subjects who dropped out after the first screen). The E-step augments the observed data Y with the latent data Z. Doing so can simplify the likelihood function in order to obtain a maximum likelihood estimate in the M-step. Formally, we define the E-M algorithm in the same way as Tanner (1996) [15]. Let λi represent the current guess to the mode of observed posterior P(λ\|Y). The observed data Y includes the first screen (Y1), the second screen (Y2), and deaths (D). For the sake of brevity, we let Y'1 denote a vector including Y1 and D. Thus, P (λ\|Y2, Y'1, Z) denotes the augmented and simplified posterior distribution and P (Z, Y'1\|Y2, λi) denotes the conditional predictive distribution of missing data Z and Y'1, conditional on the current guess to the posterior mode. In the E-step, the computation is as follows: Q(λ,λi)=∫∫log⁡[P(λ\|Z,Y1',Y2)]P(Z,Y1'\|λi,Y2)dZdY1' MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=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@65F7@ In the M-step, parameters are estimated by: ∂Q(λ,λi)∂λ\|λ=0 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqcfa4aaSaaaeaacqGHciITcqWGrbqucqGGOaakiiGacqWF7oaBcqGGSaalcqWF7oaBdaahaaqabeaacqWGPbqAaaGaeiykaKcabaGaeyOaIyRae83UdWgaaOGaeiiFaW3aaSbaaSqaaiab=T7aSbqabaGccqGH9aqpcqaIWaamaaa@3F41@ In addition to missing data on interval cases, we simplify the likelihood by indirectly estimating parameters via the prevalence pool equation (2) and the illness-death equation (4) using data from Y1' MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaemywaK1aa0baaSqaaiabigdaXaqaaiabcEcaNaaaaaa@2F01@. Instead of estimating λ1 and λ2 simultaneously in a three-state Markov model, we augment the observed data and simplify the likelihood function in this study by only estimating λ1 in the M-step, given the expected λ2, which is derived from the prevalence pool equation. In other words, we use observed data from the first screen in combination with the prevalence pool equation to simplify the likelihood function based on data from the second screen. A similar procedure is also applied to the illness-and-death Markov model. Since subjects may attend the first screen but may be lost to follow up we therefore perform one analysis based on complete data only and one estimating missing data in the E-M algorithm. Complete data analysis For a three-state model, suppose we only have data on two rounds of screening. Let N1 and N2 represent subjects attending the first screen and the second screen, respectively. The corresponding asymptomatic phase cases in each screen are P1 and P2, respectively. Let x be the time interval in years between the first and second screen. The likelihood function for data from the second screen is developed using the transition probabilities in (1). The transition probabilities for asymptomatic phase cases and screen negative cases are P11(t) and P12(t), respectively. Recall that we estimate λ1 given the expected λ2, which is estimated on the basis of the prevalence pool equation. For a three-state model, the application of expressions (2) and (5) to this data yield the following E-step computation: Q(λ1,λ1i)=E((N2−P2)×log⁡[P11(x)]+P2×log⁡[P12(x)])=((N2−P2)×log⁡(e−λ1x)+P2×log⁡[(λ1λ1−E(λ2\|λ1i,Y1))×(e−E(λ2\|λ1i,Y1)x−e−λ1x)] MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqbaeWabmqaaaqaaiabdgfarjabcIcaOGGaciab=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T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKcaaOGaeiykaKIaey41aqRaeiikaGIaemyzau2aaWbaaSqabeaacqGHsislcqWGfbqrcqGGOaakcqWF7oaBdaWgaaadbaGaeGOmaidabeaaliabcYha8jab=T7aSnaaDaaameaacqaIXaqmaeaacqWGPbqAaaWccqGGSaalcqWGzbqwdaWgaaadbaGaeGymaedabeaaliabcMcaPiabdIha4baakiabgkHiTiabdwgaLnaaCaaaleqabaGaeyOeI0Iae83UdW2aaSbaaWqaaiabigdaXaqabaWccqWG4baEaaGccqGGPaqkcqGGDbqxaaaaaa@BC43@ where E(λ2\|λ1i,Y1)=E(λ2\|λ1i,P1,N1)=N−P1P1λ1i MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaemyrauKaeiikaGccciGae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGG8baFcqWF7oaBdaqhaaWcbaGaeGymaedabaGaemyAaKgaaOGaeiilaWIaemywaK1aaSbaaSqaaiabigdaXaqabaGccqGGPaqkcqGH9aqpcqWGfbqrcqGGOaakcqWF7oaBdaWgaaWcbaGaeGOmaidabeaakiabcYha8jab=T7aSnaaDaaaleaacqaIXaqmaeaacqWGPbqAaaGccqGGSaalcqWGqbaudaWgaaWcbaGaeGymaedabeaakiabcYcaSiabd6eaonaaBaaaleaacqaIXaqmaeqaaOGaeiykaKIaeyypa0tcfa4aaSaaaeaacqWGobGtcqGHsislcqWGqbaudaWgaaqaaiabigdaXaqabaaabaGaemiuaa1aaSbaaeaacqaIXaqmaeqaaaaakiab=T7aSnaaDaaaleaacqaIXaqmaeaacqWGPbqAaaaaaa@59C3@ For an illness-and-death model, if the number of deaths in P (= P1 + P2) asymptomatic phase cases is denoted as D we used the relationship between λ1 and λ3 in expression (4) to obtain the expected λ3 for simplifying the likelihood function. This is in addition to using the prevalence pool equation to determine the relationship between λ1 and λ2. Since time of death is exactly known in principle, an instantaneous rate, dP24(t), is required. Censored cases, surviving to time t are modelled by 1-P24(t). The computation in the E-step is: Q(λ1,λ1i)=E[(P2×log⁡[P12(X)]+(N2−P2)×log⁡[P11(X)]+∑i=1Dlog⁡dP24(ui)+∑j=P−DPlog⁡(1−P24(vj))]=(N2−P2)×log⁡(e−λ1t)+P2×log⁡[(λ1X1−E(λ2\|λ1i,Y1))×(e−E(λ2\|λ1i,Y1)X−e−λ1X)+∑i=1Dlog⁡[(E(λ2\|λ1i,Y1)E(λ3\|λ1i,Y1,D))×(e−E(λ3\|λ1i,Y1,D)t−e−E(λ2\|λ1ii,Y1)t)(E(λ2\|λ1i,Y1)−E(λ3\|λ1i,Y1,D))]+∑j=P−d1Plog⁡(e−E(λ2\|λ1i,Y1)t+E(λ2\|λ1i,Y1)×e−E(λ3\|λ1i,Y1)−e−E(λ2\|λ1i,Y1)E(λ2\|λ1i,Y1)−E(λ3\|λ1i,Y1,D)) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqbaeWabiqaaaqaaiabdgfarjabcIcaOGGaciab=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T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKcaaOGaeiykaKIaey41aqRaeiikaGIaemyzau2aaWbaaSqabeaacqGHsislcqWGfbqrcqGGOaakcqWF7oaBdaWgaaadbaGaeGOmaidabeaaliabcYha8jab=T7aSnaaDaaameaacqaIXaqmaeaacqWGPbqAaaWccqGGSaalcqWGzbqwdaWgaaadbaGaeGymaedabeaaliabcMcaPiabdIfaybaakiabgkHiTiabdwgaLnaaCaaaleqabaGaeyOeI0Iae83UdW2aaSbaaWqaaiabigdaXaqabaWccqWGybawaaGccqGGPaqkaeaaaeaacqGHRaWkdaaeWbqaaiGbcYgaSjabc+gaVjabcEgaNjabcUfaBjabcIcaOiabdweafjabcIcaOiab=T7aSnaaBaaaleaacqaIYaGmaeqaaOGaeiiFaWNae83UdW2aa0baaSqaaiabigdaXaqaaiabdMgaPbaakiabcYcaSiabdMfaznaaBaaaleaacqaIXaqmaeqaaOGaeiykaKIaemyrauKaeiikaGIae83UdW2aaSbaaSqaaiabiodaZaqabaGccqGG8baFcqWF7oaBdaqhaaWcbaGaeGymaedabaGaemyAaKgaaOGaeiilaWIaemywaK1aaSbaaSqaaiabigdaXaqabaGccqGGSaalcqWGebarcqGGPaqkcqGGPaqkcqGHxdaTjuaGdaWcaaqaaiabcIcaOiabdwgaLnaaCaaabeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeG4mamdabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiilaWIaemiraqKaeiykaKIaemiDaqhaaiabgkHiTiabdwgaLnaaCaaabeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXiabdMgaPbqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKIaemiDaqhaaiabcMcaPaqaaiabcIcaOiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKIaeyOeI0IaemyrauKaeiikaGIae83UdW2aaSbaaeaacqaIZaWmaeqaaiabcYha8jab=T7aSnaaDaaabaGaeGymaedabaGaemyAaKgaaiabcYcaSiabdMfaznaaBaaabaGaeGymaedabeaacqGGSaalcqWGebarcqGGPaqkcqGGPaqkaaGccqGGDbqxaSqaaiabdMgaPjabg2da9iabigdaXaqaaiabdseaebqdcqGHris5aaGcbaaabaGaey4kaSYaaabCaeaacyGGSbaBcqGGVbWBcqGGNbWzcqGGOaakcqWGLbqzdaahaaWcbeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaameaacqaIYaGmaeqaaSGaeiiFaWNae83UdW2aa0baaWqaaiabigdaXaqaaiabdMgaPbaaliabcYcaSiabdMfaznaaBaaameaacqaIXaqmaeqaaSGaeiykaKIaemiDaqhaaOGaey4kaScaleaacqWGQbGAcqGH9aqpcqWGqbaucqGHsislcqWGKbazdaWgaaadbaGaeGymaedabeaaaSqaaiabdcfaqbqdcqGHris5aOGaemyrauKaeiikaGIae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGG8baFcqWF7oaBdaqhaaWcbaGaeGymaedabaGaemyAaKgaaOGaeiilaWIaemywaK1aaSbaaSqaaiabigdaXaqabaGccqGGPaqkcqGHxdaTjuaGdaWcaaqaaiabdwgaLnaaCaaabeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeG4mamdabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKcaaiabgkHiTiabdwgaLnaaCaaabeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKcaaaqaaiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKIaeyOeI0IaemyrauKaeiikaGIae83UdW2aaSbaaeaacqaIZaWmaeqaaiabcYha8jab=T7aSnaaDaaabaGaeGymaedabaGaemyAaKgaaiabcYcaSiabdMfaznaaBaaabaGaeGymaedabeaacqGGSaalcqWGebarcqGGPaqkaaGccqGGPaqkaaaaaaaa@DD8E@ where P = P1 + P2 D is the number of deaths P-D is the number of censored cases ui: exact death time vj: censored time Note that λ2 and λ3 are repeatedly estimated by E(λ2\|λ1i,Y1)=E(λ2\|λ1i,P1,N1)=N−P1P1λ1iE(λ3\|λ1i,Y1,D)=E(λ3\|λ1i,P1,N1,D)=2DJ2×(N1−P1)×λ1 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqbaeaabiqaaaqaaiabdweafjabcIcaOGGaciab=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T7aSnaaDaaaleaacqaIXaqmaeaacqWGPbqAaaGccqGGSaalcqWGqbaudaWgaaWcbaGaeGymaedabeaakiabcYcaSiabd6eaonaaBaaaleaacqaIXaqmaeqaaOGaeiilaWIaemiraqKaeiykaKIaeyypa0tcfa4aaSaaaeaacqaIYaGmcqWGebaraeaacqWGkbGsdaahaaqabeaacqaIYaGmaaGaey41aqRaeiikaGIaemOta40aaSbaaeaacqaIXaqmaeqaaiabgkHiTiabdcfaqnaaBaaabaGaeGymaedabeaacqGGPaqkcqGHxdaTcqWF7oaBdaWgaaqaaiabigdaXaqabaaaaaaaaaa@9F91@ In the M-step, λ1 is estimated iteratively by equation (6). Missing data analysis As stated earlier, some subjects drop out after the first screen. We also use the E-M algorithm to estimate parameters taking this missing information into account. Following the principle of handling missing data proposed by Longford et al. (2000) in diaries of alcohol consumption, E-M algorithm and multiple imputations are used to handle missing data on interval cases [16]. The procedure is described as follows. If there are W dropouts after the first screen, these subjects could have been in three possible states, normal, asymptomatic phase or symptomatic phase, with respective numbers, W1, W2, and W3, between the first screen and second screen. The W follows a multinomial distribution with the corresponding probabilities: P11(x), P12(x), and P13(X) for W1, W2, and W3, given a total of subjects W. The expected values for the corresponding three states are calculated as: μi = W × P1i(X), i = 1, 2 and 3 Computation in the E-step is now: Q(λ1,λ1i)=E((N2−P2+W1)×log⁡[P11(X)]+(P2+W2)×log⁡[P12(X)]+W3×log⁡[P13(X)]))=((N2−P2+μ1)×log⁡(e−λ1x)+(P2+μ2)×log⁡[(λ1λ1−E(λ2\|λ1i,Y1)×(e−E(λ2\|λ1i,Y1)x−e−λ1x)]+(μ3×log⁡[1−e−λ1x−(λ1λ1−E(λ2\|λ1i,Y1))×(e−E(λ2\|λ1i,Y1)x−e−λ1x)]) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqbaeaabiqaaaqaaiabdgfarjabcIcaOGGaciab=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X7aTnaaBaaaleaacqaIYaGmaeqaaOGaeiykaKIaey41aqRagiiBaWMaei4Ba8Maei4zaCMaei4waSLaeiikaGscfa4aaSaaaeaacqWF7oaBdaWgaaqaaiabigdaXaqabaaabaGae83UdW2aaSbaaeaaieaacqqFXaqmaeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaabaGaeGOmaidabeaacqGG8baFcqWF7oaBdaqhaaqaaiabigdaXaqaaiabdMgaPbaacqGGSaalcqWGzbqwdaWgaaqaaiabigdaXaqabaGaeiykaKcaaOGaey41aqRaeiikaGIaemyzau2aaWbaaSqabeaacqGHsislcqWGfbqrcqGGOaakcqWF7oaBdaWgaaadbaGaeGOmaidabeaaliabcYha8jab=T7aSnaaDaaameaacqaIXaqmaeaacqWGPbqAaaWccqGGSaalcqWGzbqwdaWgaaadbaGaeGymaedabeaaliabcMcaPiabdIha4baakiabgkHiTiabdwgaLnaaCaaaleqabaGaeyOeI0Iae83UdW2aaSbaaWqaaiabigdaXaqabaWccqWG4baEaaGccqGGPaqkcqGGDbqxaeaaaeaacqGHRaWkcqqGOaakcqWF8oqBdaWgaaWcbaGaee4mamdabeaakiabgEna0kGbcYgaSjabc+gaVjabcEgaNjabcUfaBjabigdaXiabgkHiTiabdwgaLnaaCaaaleqabaGaeyOeI0Iae83UdW2aaSbaaWqaaiabigdaXaqabaWccqWG4baEaaGccqGHsislcqGGOaakjuaGdaWcaaqaaiab=T7aSnaaBaaabaGaeGymaedabeaaaeaacqWF7oaBdaWgaaqaaiabigdaXaqabaGaeyOeI0IaemyrauKaeiikaGIae83UdW2aaSbaaeaacqaIYaGmaeqaaiabcYha8jab=T7aSnaaDaaabaGaeGymaedabaGaemyAaKgaaiabcYcaSiabdMfaznaaBaaabaGaeGymaedabeaacqGGPaqkaaGccqGGPaqkcqGHxdaTcqGGOaakcqWGLbqzdaahaaWcbeqaaiabgkHiTiabdweafjabcIcaOiab=T7aSnaaBaaameaacqaIYaGmaeqaaSGaeiiFaWNae83UdW2aa0baaWqaaiabigdaXaqaaiabdMgaPbaaliabcYcaSiabdMfaznaaBaaameaacqaIXaqmaeqaaSGaeiykaKIaemiEaGhaaOGaeyOeI0Iaemyzau2aaWbaaSqabeaacqGHsislcqWF7oaBdaWgaaadbaGaeGymaedabeaaliabdIha4baakiabcMcaPiabc2faDjabcMcaPaaaaaaaaa@2C14@ As above, λ1 is estimated in the M-step by iteration according to the score function as in the complete data analysis. λ2 is estimated by iteration using the prevalence pool equation. Estimation of parameters The program for estimating parameters in M-step is written using Mathematica software version 3.0 [17]. The details of iteration between E-step and M-step are as follows. For the three-state Markov model, λ2 (0) is first guessed and an estimate of λ1 (1) is obtained on the basis of (6) and (7). 1. Substitution of λ 1 (1) into the prevalence pool equation (2) yields a new estimate of λ 2 (1) 2. Repeat procedures (1) and (2) until λ1 and λ2 converge to four decimal points. A similar procedure is applied to the illness-and-death model. An E-M approach taking covariates into account The E-M algorithm approach can be extended to estimate parameters making allowance for covariates affecting the progression rates. For instance, suppose preclinical incidence (λ1) increases with age. Two approaches are used to consider this problem. The first is based on a stratified analysis by age, in which two separate E-M estimations are performed in age groups < 50 and 50+. This yields independent estimates of λ1 and λ2 for each age group. Another method to take covariates into account is the use of exponential hazard regression to model the effects of covariates on the relevant progression rates. Let age, dichotomized by two groups as in the above, be considered as a covariate and labeled by x = 1 for age over 50 years and x = 0 otherwise. The exponential hazard regression with respect to the preclinical incidence rates for the two groups is written as follows: λ12 = λ11 exp (β1x) The progression rates from the asymptomatic to symptomatic phase for two age groups (λ21 and λ22) are estimated using the prevalence pool equation stratified by age. Thus we have a single E-step estimating both λ11 and β, and two M-steps at each iteration. Variance estimation As λ1 is estimated given λ2 in the three-state model, and given λ2 and λ3 in the illness-and-death model, the variance of λ1 calculated through the inverse of the second derivative of the likelihood function in the expression (7) or (8) will be underestimated in that this is a conditional, rather than an unconditional, estimate. Details of calculating the unconditional variance for λ1, λ2 and λ3 are given in Appendix B. Results The above method is applied to data on Type 2 diabetes screening for subjects aged over 30 years in Taiwan. The details of the study design and execution have been described in full elsewhere [18]. In brief, three rounds of screening were conducted between 1987 and 1995 with an approximate 4-year inter-screening interval. All overnight fasting and 2 h serum and plasma samples (preserved with EDTA and NaF) were collected and kept frozen (-20°C) until analysis. Fasting plasma glucose concentrations were determined using the hexokmase-glucose-6-phosphate dehydrogenase method with a glucose (HK) reagent ldt (Gilford, Oberlin, OH). Three-fixed cohorts, 1987, 1991 and 1995, were identified according to when subjects attended their first screen. Because few subjects attended the third screen in 1995 as a first screen, we excluded them from analysis. Subjects who did not take the oral glucose tolerance test (OGTT) were also excluded from the analysis. For the 1987 cohort, 66 (8.9%) of the 678 patients tested had asymptomatic Type 2 diabetes. Among 678 subjects, only 237 (35%) subjects attended the second screening (1991) with complete information on OGTT test. Of these, 10 had newly diagnosed asymptomatic Type 2 diabetes. For the 1991 cohort, 39 (8.2%) of the 475 subjects were detected as having asymptomatic Type 2 diabetes at the time of the first screening. Thus, a total of 105 (39 + 66) asymptomatic Type 2 diabetes cases were ascertained at first screen. To ascertain deaths from Type 2 diabetes (ICD code 250), the above 115 asymptomatic Type 2 diabetes patients were followed until Dec 1997. Of the 115 subjects, 8 had died of Type 2 diabetes. The average follow-up was 8.29 years. Table 1 summarizes the observed transitions and corresponding transition probabilities used in the three-state Markov model and the illness-and-death Markov model. Table 1 Descriptive results of early detection of Type 2 diabetes for two fixed cohorts in Puli, Taiwan Number of Transition Type of Transition Transition probability (1) First screen Asymptomatic Type 2 diabetes 105 (1 → 2, age at first screen(A)) P12(A) Negative 1114 (1 → 1, age at first screen(A)) P11(A) Total 1219 (2) Second screen Asymptomatic Type 2 diabetes 10 (1 → 2, 4 year) P12(X) Negative 227 (1 → 1, 4 year) P11(X) Total 237 Death 8 (1 → 4, time to death(t)) dP21(X) Table 2 shows the estimated results for a three-state Markov model. After three iterations the convergence of λ1 and λ2 was met. We started from the guessed value of λ2(0) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabikdaYaqaaiabcIcaOiabicdaWiabcMcaPaaaaaa@314D@ equal to 11.76% using the inverse of the ratio of prevalence (8.6136%), estimated by cases at first screen, to the incidence rate (1.0132%) estimated by cases at second screen divided by 987 person-years. Given this rate, λ1(1) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabigdaXaqaaiabcIcaOiabigdaXiabcMcaPaaaaaa@314D@ was estimated as 0.107594 according to the above method. The prevalence pool equation was further applied to estimate λ2(1) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabikdaYaqaaiabcIcaOiabigdaXiabcMcaPaaaaaa@314F@ as: 11.4152%(=(N−P)×0.107594P=(1219−105)×0.107594105) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaeGymaeJaeGymaeJaeiOla4IaeGinaqJaeGymaeJaeGynauJaeGOmaiJaeiyjauIaeiikaGIaeyypa0tcfa4aaSaaaeaacqGGOaakcqWGobGtcqGHsislcqWGqbaucqGGPaqkcqGHxdaTcqaIWaamcqGGUaGlcqaIXaqmcqaIWaamcqaI3aWncqaI1aqncqaI5aqocqaI0aanaeaacqWGqbauaaGccqGH9aqpjuaGdaWcaaqaaiabcIcaOiabigdaXiabikdaYiabigdaXiabiMda5iabgkHiTiabigdaXiabicdaWiabiwda1iabcMcaPiabgEna0kabicdaWiabc6caUiabigdaXiabicdaWiabiEda3iabiwda1iabiMda5iabisda0aqaaiabigdaXiabicdaWiabiwda1aaakiabcMcaPaaa@5DDC@. The annual preclinical incidence rate of λ1 was estimated as 1.08% (95% CI: 0.45%–2.58%). The annual progression rate of λ2 was estimated as 11% (95% CI: 0.06–0.21). The inverse of λ2 yielded approximately 8 years of mean sojourn time (MST). The stratified analysis in combination with E-M algorithm gave 1.51% (95% CI: 0.49%–4.70%) and 0.75% (95% CI: 0.19%–3.00%) of annual preclinical incidence rate of (λ1), and 9% and 19% of annual progression rate of λ2 for aged over 50 years and aged under 50 years, respectively (Table 2). Subjects aged over 50 years had a two-fold risk of occurrence of asymptomatic Type 2 diabetes compared with those aged under 50 years. The approach based on exponential hazard regression yielded similar results (Table 3). The rate ratio of λ1 for aged over 50 years against aged under 50 years was estimated as 2.01 (e0.70). The estimates of λ1 and λ2 based on exponential regression were very close to those based on stratified analysis in Table 2. Table 2 The E-M iteration results for a three-state Markov model Parameter Iteration λ1(95% CI) λ2(95% CI) Overall ----- ---------- 0.1176 1 0.0108 0.1141 2 0.0108 0.1141 3 0.0108 0.1142 (0.0045~0.0258) (0.0614~0.2122) ≥ 50 yrs ----- --------- 0.1176 1 0.0151 0.0926 2 0.0151 0.0926 3 0.0151 0.0926 (0.0049~0.0470) (0.0416~0.2062) <50 yrs ----- ---------- 0.1176 1 0.0075 0.1934 2 0.0075 0.1933 3 0.0075 0.1933 0.0075 0.1933 (0.0019~0.0300) (0.0732~0.5099) λ1:normal → asymptomatic λ2:asymptomatic → symptomatic Table 3 The E-M iteration results for a Three-state Markov model taking age as a covariate in proportional hazard regression model Parameter Iteration β (≥ 50 yrs) (< 50 yrs) (95% CI) λ11 λ21 λ12 λ22 ----- --------- ---------- 0.0926 ---------- 0.1934 1 0.7008 0.0151 0.0926 0.0075 0.1933 2 0.7008 0.0151 0.0926 0.0075 0.1933 3 0.7008 0.0151 0.0926 0.0075 0.1933 (0.0681~7.2133) λ11 &λ12:normal → asymptomatic. λ21 &λ22:asymptomatic → symptomatic Table 4 shows the estimated results taking the missing data on interval cases into account. These estimates were very similar to those not allowing for missing data in Table 2. This suggests that missing information did not affect the point estimates of λ1 and λ2 although the confidence intervals allowing for missing data were narrower than those obtained without taking missing information into account. Table 4 The E-M iteration results for a Three-state Markov model taking missing data on interval cases into account Parameter Iteration λ1(95% CI λ2(95% CI) Overall 0 0.0103 0.1089 1 0.0104 0.1107 5 0.0107 0.1135 6 0.0107 0.1135 (0.0064~0.0180) (0.0786~0.1639) ≥ 50 yrs 0 0.0151 0.1176 1 0.0151 0.0926 11 0.0151 0.0926 12 0.0151 0.0926 (0.0078~0.0294) (0.0579~0.1482) < 50 yrs 0 0.0075 0.1176 1 0.0075 0.1934 15 0.0075 0.1933 16 0.0075 0.1933 (0.0029~0.0192) (0.0993~0.3761) λ1:normal → asymptomatic. λ2:asymptomatic → symptomatic The estimated results for the four-state illness-and-death model are presented in Table 5. As in the three-state Markov model, λ2(0) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabikdaYaqaaiabcIcaOiabicdaWiabcMcaPaaaaaa@314D@ and λ3(0) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabiodaZaqaaiabcIcaOiabicdaWiabcMcaPaaaaaa@314F@ were first guessed and λ1(1) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabigdaXaqaaiabcIcaOiabigdaXiabcMcaPaaaaaa@314D@ was estimated as 0.0107594. Again, λ2(1) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabikdaYaqaaiabcIcaOiabigdaXiabcMcaPaaaaaa@314F@ was estimated on the basis of the prevalence pool equation. λ3(1)(=2×8(8.29)2×(1219−105)×0.0107594) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaacciGae83UdW2aa0baaSqaaiabiodaZaqaaiabcIcaOiabigdaXiabcMcaPaaakiabcIcaOiabg2da9KqbaoaalaaabaGaeGOmaiJaey41aqRaeGioaGdabaGaeiikaGIaeGioaGJaeiOla4IaeGOmaiJaeGyoaKJaeiykaKYaaWbaaeqabaGaeGOmaidaaiabgEna0kabcIcaOiabigdaXiabikdaYiabigdaXiabiMda5iabgkHiTiabigdaXiabicdaWiabiwda1iabcMcaPiabgEna0kabicdaWiabc6caUiabicdaWiabigdaXiabicdaWiabiEda3iabiwda1iabiMda5iabisda0aaakiabcMcaPaaa@5559@ was estimated via the illness-and-death equation. The estimates of λ1 and λ2 were close to those obtained from Table 2. The annual rate of death from Type 2 diabetes was estimated as 1.94%. Results from the stratified analysis also showed that older subjects had an approximately four-fold for risk for death from Type 2 diabetes compared with younger subjects. Table 5 The E-M iteration results for the illness-and-death Markov model Parameter Iteration λ1(95% CI) λ2(95% CI) λ3(95% CI) Overall ----- --------- 0.1176 0.0100 1 0.0108 0.1142 0.0194 2 0.0108 0.1142 0.0194 3 0.0108 0.1142 0.0194 (0.0045~0.0258) (0.0614~0.2122) (0.0063~0.0600) ≥ 50 yrs ----- --------- 0.1176 0.0100 1 0.0151 0.0926 0.0258 2 0.0151 0.0926 0.0258 3 0.0151 0.0926 0.0258 (0.0049~0.0469) (0.0416~0.2062) (0.0064~0.1033) < 50 yrs ----- -------- 0.1176 0.0100 1 0.0075 0.1934 0.0068 2 0.0075 0.1933 0.0068 3 0.0075 0.1933 0.0068 (0.0019~0.0300) (0.0725~0.5149) (0.0005~0.0906) λ1:normal → asymptomatic ; λ2:asymptomatic → symptomatic. λ3:symptomatic → death of Type 2 diabetes Discussion Markov chain models are a natural approach to take when modeling the transitions of patients between discrete health states over time. Welton and Ades (2005) provided a unified Bayesian approach to propagation of uncertainty from both fully and partially observed event history data to Markov model parameters [19]. In this study, we propose a new approach, based on the E-M algorithm, to estimate the progression of a multi-state chronic disease using the prevalence pool concept and Markov process models. From the methodological viewpoint, one limitation of our approach is that the prevalence pool concept is only appropriate in a population where rates of disease are assumed to be at a steady state and this assumption may not necessarily apply to diabetes today, given the recent rapid increase in incidence of diabetes in some countries today. Furthermore, our population data sample was restricted only to subjects with complete OGTT data, and thus some selection bias might have occurred. Finally, Type 2 diabetes occurs in older populations for whom death is a significant competing risk; both subjects without disease and those with asymptomatic disease may also die from other causes. We did not have enough data information to formulate a more complete model which includes competing mortality. Further studies are needed to explore how competing risks could influence the parameters of natural history. Nevertheless, there are several strengths of this approach. Firstly, it is not as computationally intensive as a single stage estimation using the traditional Markov model. The parameter estimation is simplified by integrating the illness and death equation into the likelihood function. The traditional three-state model usually estimates λ1 and λ2 simultaneously using a full likelihood function. Therefore, the likelihood function in the traditional method is more complicated than that in the present study. In addition, simultaneous estimation of λ1 and λ2 may encounter a collinearity problem due to a high correlation between two parameters. This phenomenon may be observed when there is no data on interval cases, which are sometimes unavailable for unregistered conditions such as Type 2 diabetes. That is, it is hard to disaggregate the overall rate into distinct rates for each individual state transition if we have little information on the intermediate states. Moreover, our E-M algorithm approach can also take account of missing data on interval cases. This has not been considered in previous studies when interval cases were not available. Secondly, the previous parametric method of modeling the first screen data usually required the assumption of constant pre-clinical incidence over all ages, which may be unrealistic. Our approach can dispense with this assumption and can estimate λ1 in the E-step using an age-specific prevalence rate. Thirdly, results from our approach can be readily applied to design of studies. Suppose we wish to design a randomized trial of screening in this population. We estimate λ1, λ2 and λ3 as 0.011, 0.114 and 0.019 (Table 5). From equation (2) we would expect the prevalence at first screen to be λ1/(λ1 + λ2) = 88/1000. These cases could be expected to have 5-year cumulative death rate, that is, DE = λ3 × 5/2 × λ2 × 5 = 0.027. Clinical type 2 diabetes arising spontaneously would be expected to have a different mortality rate λ4 from those arising from progression of asymptomatic screen-detected cases. Mortality from spontaneous symptomatic cases can be estimated from the case series of Chen et al. (1999) in which there were 131/766 = 0.17 deaths in an average follow-up of 3.5 years [20]. This gives an estimate for λ4 of 0.054. We would therefore expect deaths from spontaneous interval cases of ∫05λ1e−λ1t∫05−tλ2e−λ2v∫05−t−vλ4e−λ4ududvdt=(1−e−5λ1)−λ1λ4(e−5λ1−e−5λ2)(λ4−λ2)(λ2−λ1)+λ1λ2(e−5λ1−e−5λ4)(λ4−λ2)(λ4−λ1) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaqbaeaabiqaaaqaamaapedabaacciGae83UdW2aaSbaaSqaaiabigdaXaqabaGccqWGLbqzdaahaaWcbeqaaiabgkHiTiab=T7aSnaaBaaameaacqaIXaqmaeqaaSGaemiDaqhaaaqaaiabicdaWaqaaiabiwda1aqdcqGHRiI8aOWaa8qmaeaacqWF7oaBdaWgaaWcbaGaeGOmaidabeaaaeaacqaIWaamaeaacqaI1aqncqGHsislcqWG0baDa0Gaey4kIipakiabdwgaLnaaCaaaleqabaGaeyOeI0Iae83UdW2aaSbaaWqaaiabikdaYaqabaWccqWG2bGDaaGcdaWdXaqaaiab=T7aSnaaBaaaleaacqaI0aanaeqaaOGaemyzau2aaWbaaSqabeaacqGHsislcqWF7oaBdaWgaaadbaGaeGinaqdabeaaliabdwha1baaaeaacqaIWaamaeaacqaI1aqncqGHsislcqWG0baDcqGHsislcqWG2bGDa0Gaey4kIipakiabdsgaKjabdwha1jabdsgaKjabdAha2jabdsgaKjabdsha0bqaaiabg2da9iabcIcaOiabigdaXiabgkHiTiabdwgaLnaaCaaaleqabaGaeyOeI0IaeGynauJae83UdW2aaSbaaWqaaiabigdaXaqabaaaaOGaeiykaKIaeyOeI0scfa4aaSaaaeaacqWF7oaBdaWgaaqaaiabigdaXaqabaGae83UdW2aaSbaaeaacqaI0aanaeqaaiabcIcaOiabdwgaLnaaCaaabeqaaiabgkHiTiabiwda1iab=T7aSnaaBaaabaGaeGymaedabeaaaaGaeyOeI0Iaemyzau2aaWbaaeqabaGaeyOeI0IaeGynauJae83UdW2aaSbaaeaacqaIYaGmaeqaaaaacqGGPaqkaeaacqGGOaakcqWF7oaBdaWgaaqaaiabisda0aqabaGaeyOeI0Iae83UdW2aaSbaaeaacqaIYaGmaeqaaiabcMcaPiabcIcaOiab=T7aSnaaBaaabaGaeGOmaidabeaacqGHsislcqWF7oaBdaWgaaqaaiabigdaXaqabaGaeiykaKcaaOGaey4kaSscfa4aaSaaaeaacqWF7oaBdaWgaaqaaiabigdaXaqabaGae83UdW2aaSbaaeaacqaIYaGmaeqaaiabcIcaOiabdwgaLnaaCaaabeqaaiabgkHiTiabiwda1iab=T7aSnaaBaaabaGaeGymaedabeaaaaGaeyOeI0Iaemyzau2aaWbaaeqabaGaeyOeI0IaeGynauJae83UdW2aaSbaaeaacqaI0aanaeqaaaaacqGGPaqkaeaacqGGOaakcqWF7oaBdaWgaaqaaiabisda0aqabaGaeyOeI0Iae83UdW2aaSbaaeaacqaIYaGmaeqaaiabcMcaPiabcIcaOiab=T7aSnaaBaaabaGaeGinaqdabeaacqGHsislcqWF7oaBdaWgaaqaaiabigdaXaqabaGaeiykaKcaaaaaaaa@BBB0@ after a little algebra. Substituting for λ1, λ2, and λ4, the above is equal to 0.0011. We would therefore expect, per thousand screened and then followed up for 5 years, 88 × 0.027 + 912 × 0.0011, i.e., 3.4 deaths per thousand. In an unscreened control group, one would expect the number of death to be the number from progression of those in the prevalence pool plus the number from new cases arising, i.e., 88×∫05λ2e−λ2t∫05−tλ4e−λ4vdvdt+912×0.0011 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaeGioaGJaeGioaGJaey41aq7aa8qmaeaaiiGacqWF7oaBdaWgaaWcbaGaeGOmaidabeaakiabdwgaLnaaCaaaleqabaGaeyOeI0Iae83UdW2aaSbaaWqaaiabikdaYaqabaWccqWG0baDaaaabaGaeGimaadabaGaeGynaudaniabgUIiYdGcdaWdXaqaaiab=T7aSnaaBaaaleaacqaI0aanaeqaaaqaaiabicdaWaqaaiabiwda1iabgkHiTiabdsha0bqdcqGHRiI8aOGaemyzau2aaWbaaSqabeaacqGHsislcqWF7oaBdaWgaaadbaGaeGinaqdabeaaliabdAha2baakiabdsgaKjabdAha2jabdsgaKjabdsha0jabgUcaRiabiMda5iabigdaXiabikdaYiabgEna0kabicdaWiabc6caUiabicdaWiabicdaWiabigdaXiabigdaXaaa@5ED3@ =88×(1−e−λ25−λ2(e−5λ2−e−5λ4)(λ2−λ4))+1.0032 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaeyypa0JaeGioaGJaeGioaGJaey41aqRaeiikaGIaeGymaeJaeyOeI0Iaemyzau2aaWbaaSqabeaacqGHsisliiGacqWF7oaBdaWgaaadbaGaeGOmaidabeaaliabiwda1aaakiabgkHiTmaalaaabaGae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGGOaakcqWGLbqzdaahaaWcbeqaaiabgkHiTiabiwda1iab=T7aSnaaBaaameaacqaIYaGmaeqaaaaakiabgkHiTiabdwgaLnaaCaaaleqabaGaeyOeI0IaeGynauJae83UdW2aaSbaaWqaaiabisda0aqabaaaaOGaeiykaKcabaGaeiikaGIae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGHsislcqWF7oaBdaWgaaWcbaGaeGinaqdabeaakiabcMcaPaaacqGGPaqkcqGHRaWkcqaIXaqmcqGGUaGlcqaIWaamcqaIWaamcqaIZaWmcqaIYaGmaaa@5CD7@ = 88 × 0.0586 = 6.2 per thousand. To have 50% power to detect the difference between the 5-year death of 6.2 and 3.4 as significant, we would need 1,718 subjects per arm. The above assumes 100% compliance and perfect sensitivity. To cope with some anticipated non-compliance and imperfect sensitivity, we might expect to have, say, 70% of the 88 per thousand in the prevalence pool. The remaining 30% would then arise as interval cases, and the expected death rate in the study arm over 5 years would be 62 × 0.027 + 26 × 0.0586 + 912 × 0.0011 = 4.2 per thousand. For 90% power in this case, we would require 5,177 subjects per arm. This method can also be adapted to take into account covariates affecting the progression rates of the disease by use of stratified analysis or proportional hazard regression model. Although the only covariate used in this study was age, the approach can accommodate a set of covariates if necessary. Also, the E-M algorithm used in this study was extended to estimate missing information on interval cases. To check whether parameters estimated from the proposed method were valid, a goodness of fit test was performed to check the adequacy of models. As Table 6 shows, there were no significant differences between the observed and expected rates for an illness-and-death Markov model. A similar finding was observed for the three-state model (data not shown). This suggests a good fit of the model for empirical data. Table 6 Results for the goodness of fit for the illness-and-death Markov model Parameter Observed Expected Residual Overall Negative of first screen 1114 1102 11.998 Positive of first screen 105 117 -11.998 Negative of second screen 227 227.02 -0.016 Positive of second screen 10 8.04 1.9569 Death 8 6.07 1.9254 χ2 = 2.4473 P = 0.2941 ≥ 50 yrs Negative of first screen 496 483.471 12.5289 Positive of first screen 81 93.529 -12.5289 Negative of second screen 96 96.011 -0.0109 Positive of second screen 6 4.995 1.0046 Death 7 5.447 1.5534 χ2 = 2.6481 P = 0.2661 < 50 yrs Negative of first screen 618 617.084 0.9157 Positive of first screen 24 24.916 -0.9157 Negative of second screen 131 131.007 0.0073 Positive of second screen 4 2.775 1.2246 Death 1 0.728 0.2715 χ2 = 0.6765 P = 0.7130 Conclusion In conclusion, a simple E-M algorithm approach using the prevalence pool concept and its extension in conjunction with the Markov model was proposed to estimate parameters pertaining to progression rates of chronic disease. This approach may be useful to quantify the multi-state natural history of certain chronic diseases and to evaluate disease screening strategies. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HC and CM participated in the design of the study and performed the statistical analysis. P and TH conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript. Appendix A Transition probabilities in the four state model The transition probabilities for an illness-and-death model are: P=12341234[P11(t)P12(t)P13(t)P14(t)0P22(t)P23(t)P24(t)00P33(t)P34(t)0001] MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=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@801C@ where states 1, 2, 3 and 4 represent no disease, asymptomatic disease, symptomatic disease and death, respectively, and: P11(t)=e−λ1tP12(t)=λ1(e−λ1tλ2−λ1+e−λ2tλ1−λ2)P13(t)=λ1λ2(e−λ1t(λ1−λ2)(λ1−λ3)−e−λ2t(λ1−λ2)(λ2−λ3)−e−λ3t(λ1−λ3)(λ3−λ2)P14(t)=1−λ1λ2λ3(e−λ1tλ1(λ1−λ2)(λ1−λ3)+e−λ2tλ2(λ1−λ2)(λ2−λ3)+e−λ3tλ3(λ1−λ3)(λ3−λ2)P22(t)=e−λ2tP23(t)=λ2(e−λ2tλ3−λ2+e−λ3tλ2−λ3)P24(t)=1+λ3e−λ2tλ2−λ3−λ2e−λ3tλ2−λ3P33(t)=e−λ3tP34(t)=1−e−λ3t MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=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jHiTKqbaoaalaaabaGaemyzau2aaWbaaeqabaGae8NeI0Iae43UdW2aaSbaaeaacqaIYaGmaeqaaiabdsha0baaaeaacaqFOaGae43UdW2aaSbaaeaacaqFXaaabeaacqWFsislcqGF7oaBdaWgaaqaaiaa9jdaaeqaaiaa9LcacaqFOaGae43UdW2aaSbaaeaacqaIYaGmaeqaaiab=jHiTiab+T7aSnaaBaaabaGaeG4mamdabeaacqGGPaqkaaGccqWFsisljuaGdaWcaaqaaiabdwgaLnaaCaaabeqaaiab=jHiTiab+T7aSnaaBaaabaGaeG4mamdabeaacqWG0baDaaaabaGaeiikaGIae43UdW2aaSbaaeaacqaIXaqmaeqaaiab=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jHiTKqbaoaalaaabaGae43UdW2aaSbaaeaacqaIYaGmaeqaaiabdwgaLnaaCaaabeqaaiab=jHiTiab+T7aSnaaBaaabaGaeG4mamdabeaacqWG0baDaaaabaGae43UdW2aaSbaaeaacqaIYaGmaeqaaiab=jHiTiab+T7aSnaaBaaabaGaeG4mamdabeaaaaaakeaacqWGqbaudaWgaaWcbaGaeG4mamJaeG4mamdabeaakiabcIcaOiabdsha0jabcMcaPaqaaiabg2da9aqaaiabdwgaLnaaCaaaleqabaGae8NeI0Iae43UdW2aaSbaaWqaaiabiodaZaqabaWccqWG0baDaaaakeaacqWGqbaudaWgaaWcbaGaeG4mamJaeGinaqdabeaakiabcIcaOiabdsha0jabcMcaPaqaaiabg2da9aqaaiabigdaXiabgkHiTiabdwgaLnaaCaaaleqabaGae8NeI0Iae43UdW2aaSbaaWqaaiabiodaZaqabaWccqWG0baDaaaaaaaa@A4C5@ Appendix B B.1 The three-state Markov model Two parameters, λ1 and λ2, were estimated in this model. As stated in the text, the variance of λ1 was a conditional rather than unconditional estimate. In this case, we should re-calculate the unconditional variance of λ1 as follows: Var(λ1) = Var(E(λ1 \| λ2) + E(Var(λ1 \| λ2) If the asymptotic theory held, E(Var(λ1\|λ2)) can be assumed to be equal to the observed Var (λ1\|λ2), which was obtained from the inverse of the second derivative of the likelihood function given the estimates of λ1 and λ2 in Table 2. The first component via the prevalence pool equation in (B.1) was: Var(E(λ1\|λ2)=Var(PN−P×λ2)=(PN−P)2Var(λ2) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaemOvayLaemyyaeMaemOCaiNaeiikaGIaemyrauKaeiikaGccciGae83UdW2aaSbaaSqaaiabigdaXaqabaGccqGG8baFcqWF7oaBdaWgaaWcbaGaeGOmaidabeaakiabcMcaPiabg2da9iabdAfawjabdggaHjabdkhaYjabcIcaOKqbaoaalaaabaGaemiuaafabaGaemOta4KaeyOeI0IaemiuaafaaOGaey41aqRae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGGPaqkcqGH9aqpcqGGOaakjuaGdaWcaaqaaiabdcfaqbqaaiabd6eaojabgkHiTiabdcfaqbaakiabcMcaPmaaCaaaleqabaGaeGOmaidaaOGaemOvayLaemyyaeMaemOCaiNaeiikaGIae83UdW2aaSbaaSqaaiabikdaYaqabaGccqGGPaqkaaa@5CE5@ where P and N are numbers of positive cases and attendants. Given P and N, an unconditional variance of λ2 is needed to calculate Var(E(λ1\|λ2)). However, it is very difficult to obtain unconditional variance of λ2 unless one has other external data. We used an approximation method to calculate an unconditional variance of λ2 as follows. Suppose the occurrence of asymptomatic phase cases follows a Poisson distribution, the likelihood function based on the second screen data in Table 1 is: L(λ1')=(1−e−λ1'×4)10(e−λ1'×4)227 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaemitaWKaeiikaGccciGae83UdW2aa0baaSqaamXvP5wqSXMqHnxAJn0BKvguHDwzZbqegqvATv2CG4uz3bIuV1wyUbaceaGaa4xmaaqaaiaa+DcaaaGccqGGPaqkcqGH9aqpcqGGOaakcqaIXaqmcqGHsislcqWGLbqzdaahaaWcbeqaaiabgkHiTiab=T7aSnaaDaaameaacaGFXaaabaGaa43jaaaaliabgEna0kabisda0aaakiabcMcaPmaaCaaaleqabaGaeGymaeJaeGimaadaaOGaeiikaGIaemyzau2aaWbaaSqabeaacqGHsislcqWF7oaBdaqhaaadbaGaa4xmaaqaaiaa+DcaaaWccqGHxdaTcqaI0aanaaGccqGGPaqkdaahaaWcbeqaaiabikdaYiabikdaYiabiEda3aaaaaa@5C62@ The MLE of λ'1 based on the score function was estimated 0.011. The variance of λ'1 from the inverse of the second derivative of the above likelihood function was estimated as 0.000011. An unconditional variance of λ'2 via the prevalence pool equation was therefore estimated as 0.000011 × (11142/1052). We believe that such an approximation may not be unreasonable because the estimate of λ'1 using the likelihood function in (B.3) was very close to λ1 using the joint likelihood of λ1 and λ2 in Table 2. B.2 The illness-death Markov model If we assume λ1 conditionally independent of λ3(i.e., E(λ1\|λ3, λ2) = E(λ1\| λ2)), the unconditional variance of λ1 and λ2 can be calculated as above. To calculate the unconditional variance of λ3, we assumed λ3 was conditionally independent of λ1. The unconditional variance of λ3 was: Var(λ3) = Var(E(λ3 \| λ2) + E(Var(λ3 \| λ2) As above, E(Var(λ3\|λ2)) can be assumed to be equal to the observed Var(λ3\|λ2), obtained from the inverse of the second derivative of the likelihood function based on MLE estimate of λ1 conditional on λ2 and λ3 (see Table 5). Also, using equation (3), Var(E(λ3\|λ2)=Var(2DP×J×λ2)=(2DP×J)2=(2DP×D)2×1λ24Var(λ2) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=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@7311@ A similar procedure was applied to calculate the variance of the regression coefficient β, assuming λ21 independent of λ22. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1828619808-PONE-RA-0330810.1371/journal.pone.0001643Research ArticleBiochemistryMolecular BiologyOncologyCell Biology/Cell SignalingBiochemistryCell Biology/Cell SignalingMolecular BiologyOncologyA Cross-Talk between TrkB and Ret Tyrosine Kinases Receptors Mediates Neuroblastoma Cells Differentiation TrkB Transactivates RetEsposito Carla Lucia 1 3 D'Alessio Amelia 2 de Franciscis Vittorio 1 Cerchia Laura 1 * 1 Istituto per l'Endocrinologia e l'Oncologia Sperimentale "G. Salvatore", Consiglio Nazionale delle Ricerche (CNR), Naples, Italy 2 Cell Biology and Preclinical Models Unit, INT-Fondazione Pascale, Naples, Italy 3 Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano”, Università di Napoli “Federico II”, Naples, Italy Abraham Edathara Academic EditorUniversity of Arkansas, United States of America*E-mail: [email protected] and designed the experiments: Vd LC. Performed the experiments: AD CE. Analyzed the data: Vd LC CE. Contributed reagents/materials/analysis tools: Vd LC. Wrote the paper: Vd LC. 2008 20 2 2008 3 2 e164315 1 2008 24 1 2008 Esposito et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs) to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA)-treatment of neuroblastoma (NB) cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process. ==== Body Introduction Receptor tyrosine kinases (RTKs) are transmembrane proteins that in response to growth factors stimulate intracellular pathways that control most fundamental cellular processes including the cell cycle, cell migration, cell metabolism and survival, as well as cell differentiation and proliferation. All known RTKs become activated by the selective binding of dimeric ligands to their extracellular domains, resulting in their dimerization and autophosphorylation [1], [2]. However, there is accumulating evidence that enlarges the traditional view of growth factor receptors activation in that the receptor may be transactivated and, thus, tyrosine-phosphorylated by ligands which specifically bind to other membrane receptors. For example, it has been widely demonstrated the transactivation of the epidermal growth factor receptor (EGFR) by agonists for G protein-coupled receptors (GPCRs) and other extracellular stimuli unrelated to EGF-like ligands [3], [4]. Similar to EGFR, it has been shown that activation of Trk (tropomyosin-related kinase) receptor tyrosine kinases TrkA (P04629 Swiss-Prot accession number) and TrkB (Q16620 Swiss-Prot accession number) can also occur via a GPCR mechanism, without involvement of nerve growth factor (NGF, P01138 Swiss-Prot accession number) or brain-derived neurotrophic factor (BDNF, P23560 Swiss-Prot accession number) [5], [6], [7] thus indicating alternative modes of stimulating trophic functions in neuronal cells by linking different receptor signalling pathways. As first example of intracellular cross-activation between RTKs, it has been shown that in cultures of sympathetic neurons the NGF receptor, TrkA, acts on Ret (rearranged during transfection, P07949 Swiss-Prot accession number) kinase by an intracellular mechanism that transactivates its catalytic domain whereas doesn't require ligand expression [8]. As demonstrated for brain tumor glioblastoma multiforme and other solid tumours, the co-activation of RTKs limits the efficacy of therapies targeting single receptors [9], thus a deeper insights in the inter-RTK signalling pathways is needed to find combination of therapeutic agents with increased anti-cancer efficacy. Neuroblastoma is the most common solid tumor of childhood outside the central nervous system; it is made of neural crest cells that can occur at any point along the sympathetic ganglia or the adrenal medulla. Expression of neurotrophin receptors of Trk family is an important prognostic factor in NB and activation of different Trk receptors leads to variable clinical presentation and behaviour of the tumor [10], [11]. Differences in the expression pattern of the receptors or their downstream signalling components could in part explain the differences in favourable (in the case of TrkA) or unfavourable (in the case of TrkB) tumors. However, the cross-talk between the Trk receptors and other receptors on cell surface may influence the outcome of the NB cellular response. In NB cells it has been shown that the stably transfection of TrkB receptor results in enhancing neuroblastoma invasiveness via upregulating the expression of hepatocyte growth factor and its receptor TK, c-Met [12]. The need of cooperation between TrkA and Ret pathways has been shown in the HTLA230 neuroblastoma cells as critical to promote neuronal differentiation [13]. Furthermore, there is evidence in literature indicating that the Ret and TrkB signal pathways are associated with the process of NB differentiation [14]. Previous studies have shown that RA induces the neuronal differentiation of many human neuroblastoma cell lines. It has been reported that RA alone induces neurite extension in NB cells (SH-SY5Y, LAN-5 and KCNR cells) which have endogenous expression of BDNF, via induction of TrkB expression [15], [16]. The existence of an autocrine loop sustained by RA-treatment and involving increase of TrkB expression and its activation, has been demonstrated for KCNR cells [15]. Furthermore, in all these cell lines RA also induces Ret tyrosine kinase receptor expression [17] and we have recently demonstrated that in SK-N-BE cells, the RA-treatment induces a positive autocrine loop that sustains Ret activation and downstream signalling and depends on glial-derived neurotrophic factor (GDNF, P39905 Swiss-Prot accession number) expression and release. Ret stimulation in turn is crucial to mediate cell differentiation [18]. However, even if collectively these data indicate a central role of TrkB and Ret receptors in RA-induced cell differentiation, a clear understanding of their respective biological need is still lacking and the mechanism for their involvement in this process has been never clarified. Here, we investigated the mechanism of Ret and TrkB involvement in differentiation of malignant NB cells. Our results demonstrate that the RA-induced differentiation of human NB cells depends on the signalling interplay by TrkB and Ret receptors. By promoting Ret phosphorylation in a GDNF-independent manner, the BDNF-stimulated TrkB, causes the induction of the neurite outgrowth and expression of differentiation-specific molecular markers. This is the first report that describes the need of a functional transactivation between two unrelated RTKs to cause the final cell phenotype. Results Effects of Ret expression and activation in RA-induced differentiation of human NB cell lines In order to dissect the respective roles played by TrkB and Ret receptors in RA-dependent NB cells differentiation we first analysed the effects of disabling the function of Ret by small interfering RNA (siRNA) in SK-N-BE, LAN-5 and SH-SY5Y cells. The treatment of the three cell lines with 10 µM RA for 3 days caused a dramatic increase in Ret protein and autophosphorylation levels that were reduced to basal upon transfecting with a siRNA targeting human Ret (Figure 1A, compare lanes 1 and 4 to lane 2, for the three cell lines), whereas a non-silencing control siRNA had no effect (compare lane 3 to 2). Interestingly, inhibiting Ret expression hampered the process of differentiation induced by RA as assessed by monitoring the levels of two proteins whose expression is up-regulated upon RA-induced differentiation in several NB cell lines, the nerve growth factor inducible protein (VGF) and the growth associated protein 43 (GAP-43) [18], [19], [20]. As shown in Figure 1B, silencing of Ret resulted in a drastic inhibition of the RA-dependent induction of VGF and GAP-43 (between 60% to 90–100% of inhibition for the three cell lines), as expected no effect was observed with the negative control siRNA. To further confirm that the Ret silencing was correlated with a significant impairment of the RA-induced NB cells differentiation, we determined the expression levels of tissue transglutaminase (tTG) an enzyme whose increase has been reported to mediate RA-induced cell differentiation in NB cells [18], [21], [22]. As shown (Figure 1B), even upon RA-treatment, in the Ret-interfered cells the levels of tTG were almost undetectable as in the absence of RA-treatment (compare lanes 4 to 1). In agreement with our previous findings in SK-N-BE [18] these results demonstrate that Ret is generally required to the RA-induced differentiation of human NB cells. 10.1371/journal.pone.0001643.g001Figure 1 Ret activity is involved in RA-induced NB cell differentiation. (A) and (B) SK-N-BE (left panels), LAN-5 (middle panels) and SH-SY5Y (right panels) cells were either left untreated (lane 1) or RA-treated (lane 2); RA-treated cells were transfected with Ret specific siRNA (siRNARet in lane 4) or a non-silencing control siRNA (siRNAns in lane 3). C, mock-treated cells. (A) Cell lysates were immunoblotted with anti-pRet or anti-Ret antibodies. To confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies, as indicated. Quantitations are done on the sum of the two Ret-specific enhanced chemiluminescence bands of 170 and 150 kDa corresponding to different glycosylation states of Ret. Intensity of bands have been calculated using the NIH Image Program on at least two different expositions to assure the linearity of each acquisition. Fold values are expressed relative to the reference points, arbitrarily set to 1 (labelled with asterisk). (B) To follow biochemical cell differentiation lysates were immunoblotted with anti-VGF, anti-GAP-43 or anti-tTG antibodies. The filters were blotted with anti-αtubulin? antibodies to normalize the loaded proteins, as indicated. Quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see A). Blots shown in (A) and (B) are representative of at least four independent experiments. RA induces the expression of BDNF and its receptor TrkB that mediates NB cell differentiation In apparent discrepancy with the results reported above, in human KCNR neuroblastoma cells an autocrine loop involving the activation of TrkB by its ligand BDNF mediates RA-induced differentiation [15]. Therefore, we determined whether TrkB receptor was involved in SH-SY5Y, LAN-5 and SK-N-BE cells responsiveness to RA. To this aim, we first evaluated the expression levels of TrkB and its primary ligand BDNF in the three cell lines. As shown in Figure 2A, the cells express the full-length TrkB and BDNF RNA following RA-treatment (left panel). Furthermore, SH-SY5Y, LAN-5 and SK-N-BE cells express low levels of TrkB protein and their ability to express high levels of the receptor is regulated by RA (right panel). As previously reported for KCNR cells, we found that in SH-SY5Y and in LAN-5, the RA-treatment leads to an increase of TrkB phosphorylation, suggesting that the endogenous BDNF expressed by these cell lines is sufficient to stimulate the activity of its own receptor (Figure 2B, compare lane 2 to l, in left and middle panels). At difference, in SK-N-BE the increase in the level of the protein following RA-treatment was not accompanied by detectable stimulation of its activity (compare lane 2 to 1 in right panel). Furthermore, in agreement with previously report [15], the TrkB receptor in SH-SY5Y and LAN-5 cells is competent for exogenous BDNF stimulation (Figure 2C, compare lane 2 to 1 and lane 4 to 3, respectively). In contrast, in SK-N-BE, phosphorylation of TrkB was undetectable even in the presence of an acute stimulation with exogenous BDNF (Figure 2C, compare lane 6 to 5) thus indicating that in this cell line RA induces the expression of a non-functional TrkB receptor. It has been widely reported that, in vivo, TrkB is spliced to generate truncated receptors that lack the cytoplamic kinase domain and that are expressed at different degrees in NB cells and tumors [23]–[26]. 10.1371/journal.pone.0001643.g002Figure 2 RA-dependent induction of expression and activation of TrkB in NB cells. (A) Left, RNA isolated from RA-treated SH-SY5Y, LAN-5 and SK-N-BE cells was reverse transcribed and amplified as described in Materials and Methods. Samples loaded on the gel are as follows: lanes 1, 2 and 3 amplification of TrkB in SH-SY5Y, LAN-5 and SK-N-BE, respectively; lanes 4, 5 and 6 amplification of BDNF in SH-SY5Y, LAN-5 and SK-N-BE, respectively; lane 7 markers IX (Roche); lanes 8 to 13, RNA samples as in lanes 1 to 6 but in the absence of RT as negative controls. Right, Lysates from SH-SY5Y, LAN-5 and SK-N-BE, left untreated or RA-treated, were immunoblotted with anti-TrkB antibodies against the extracellular domain of the receptor. The molecular weights of full-length TrkB and hypothetical TrkB.T1 isoform are indicated. The membranes were stripped and re-probed with anti-αtubulin? antibodies to control equal loading. Blots shown are representative of at least five independent experiments. (B) SH-SY5Y (left panels), LAN-5 (middle panels) and SK-N-BE (right panels) cells were left untreated or treated with RA in the absence or in the presence of TrkB siRNA (siRNATrkB) or the negative control siRNAns, as indicated. Cell lysates were immunoprecipitated with anti-panTrk antibodies and analysed by Western blotting with anti-pTyr or anti-TrkB antibodies. Blots shown are representative of at least four independent experiments. (C) RA-treated SH-SY5Y, LAN-5 and SK-N-BE cells were left un-stimulated (lanes 1, 3 and 5) or stimulated with BDNF (lanes 2, 4 and 6). Cell extracts were immunoprecipitated with anti-panTrk antibodies and immonoblotted with anti-pTyr or anti-TrkB antibodies. Blots shown are representative of at least three independent experiments. In (A), (B) and (C), quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to Figure 1A). The truncated isoforms have been shown to act as dominant inhibitors on BDNF signalling by sequestering the full-length receptor in non-functional heterodimers [25]. It is plausible that a similar mechanism of competitive TrkB inhibition by a truncated isoform could occur in SK-N-BE cells. Whether the 95 kDa-molecular weight, clearly visible in the immunoblot with TrkB antibodies (Figure 2A, right panel ), corresponds to the truncated TrkB.T1 isoform remains to be determined. Next, we wondered whether the RA-mediated TrkB up-regulation and activation mediate cell differentiation. To this aim we treated the cells with a specific siRNA for TrkB that completely blocked the expression of TrkB protein in the three cell lines (Figure 2B, lanes 4). As shown in Figure 3A, blocking TrkB expression resulted in the suppression of RA-dependent biochemical differentiation in SH-SY5Y and LAN-5 cells (left and middle panels) as monitored by the levels of GAP-43, tTG and VGF. In agreement with the observation that SK-N-BE cells express a non-functional TrkB (see Figure 2B and C), silencing had no effect in this cell line (right panel). Furthermore, both in SH-SY5Y and LAN-5 the extent of inhibition of RA-dependent induction of these same differentiation markers was comparable to that observed by interfering with Ret (compare Figure 1B to Figure 3A). 10.1371/journal.pone.0001643.g003Figure 3 TrkB mediates the RA-dependent differentiation in SH-SY5Y and LAN-5. SH-SY5Y (left panels), LAN-5 (middle panels) and SK-N-BE (right panels) cells were left untreated or treated with RA in the absence or in the presence of siRNATrkB or the negative control siRNAns, as indicated. (A) Cell lysates were immunoblotted with anti-GAP-43, anti-tTG or anti-VGF antibodies. Equal loading was confirmed by immunoblotting with anti-αtubulin antibodies. Quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to Figure 1A). Blots shown are representative of at least four independent experiments. (B) to (K): Following RA-treatment, the percentage of neurite outgrowth was calculated and reported as histogram (K). Data are percentage of neurite bearing cells/total cells analyzed. Columns, average of three independent experiments. Microphotographs of cells are shown (B to J). The same behaviour as referred to the biochemical differentiation was also observed by monitoring the neurite outgrowth following RA-treatment of the three cell lines (Figure 3B to K). Indeed, as expected, disabling the expression of TrkB completely blocked the RA-induced neurite outgrowth in SH-SY5Y (Figure 3D) and LAN-5 (Figure 3G) cells but had no effect on SK-N-BE cells morphology (Figure 3J). Taken together, the results demonstrate that disabling the expression of either Ret or TrkB causes a failure in RA-dependent cell differentiation, thus raising the question of the existence of a cooperative cross-talk between TrkB and Ret in neuroblastoma cells. It does exist cooperation between TrkB and Ret to induce RA-dependent NB differentiation? Based on the recent findings that demonstrate that TrkA is able to transactivate Ret [8], we asked whether active TrkB could as well induce the transactivation of Ret in NB cells. To this aim, we decided to hamper the function of the TrkB receptor by siRNA and monitor the level of phosphorylation of Ret following RA-treatment of SH-SY5Y (Figure 4A, left panel ) and LAN-5 cells (Figure 4A, right panel ). As shown, transfecting both the cell lines with the TrkB-specific siRNA for 72 h inhibits RA-induced Ret activation, but not expression, at the same extent of treating the cells with Ret specific siRNA (compare lanes 4 to 5, left and right panels). As a negative control, a non-related siRNA was also transfected into the cells that had no visible effects on phosphorylation (lane 3, left and right panels). In contrast, TrkB phosphorylation was not affected by inhibition of Ret by siRNA (data not shown). Thus, these results are compatible with the existence of a transactivation mechanism triggered by TrkB activity and acting on Ret in a ligand-independent manner. 10.1371/journal.pone.0001643.g004Figure 4 Existence of a cross-talk between TrkB and Ret in SH-SY5Y and LAN-5 cells. (A) RA-treated SH-SY5Y (left panels) and LAN-5 (right panels) cells were transfected with siRNATrkB (lane 4), siRNARet (lane 5) or negative control siRNAns (lane 3). C is referred to untreated cells (lane 1). Cell lysates were immunoblotted with anti-TrkB, anti-pRet, anti-Ret, or anti-GAP-43 antibodies, as indicated. The equal protein loading has been confirmed by immunoblotting with anti-atubulin? antibodies. (B) RA-treated LAN5, SH-SY5Y and SK-N-BE cells were left un-treated (lanes 2, 5 and 8, respectively) or treated with BDNF (lanes 3, 6 and 9, respectively). Lanes 1, 4 and 7 are mock-treated cells. Cell lysates were immunoblotted with anti-pRet or anti-Ret antibodies. To confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies. In (A) and (B), quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to Figure 1A). Blots shown in (A) and (B) are representative of at least three independent experiments. As predicted by these results, blocking TrkB activation and in turn Ret phosphorylation caused a failure in RA-dependent cell differentiation, monitored as inhibition of the RA-dependent induction of GAP-43 protein (Figure 4A, left and right panels ). These results strongly indicate that RA-induced overexpression and activation of TrkB in SH-SY5Y and LAN-5 cells is mediated by Ret activation and leads to neuroblastoma cells differentiation, indeed blocking TrkB expression prevents Ret activation and cell differentiation at the same extent reached by silencing the Ret receptor. Exogenous BDNF stimulation of LAN-5 and SH-SY5Y cells induces Ret activation To further confirm that Ret activity in LAN-5 and SH-SY5Y cells is TrkB-dependent, we asked whether the Ret receptor could be transactivated by exogenous BDNF addition. To increase TrkB and Ret expression, LAN-5 and SH-SY5Y were treated with RA for 72 h and cells were then stimulated with BDNF (50 ng/ml) for 10 min. As shown in Figure 4B, the BDNF acute stimulation of the cells led to an increase of Ret phosphorylation with respect to the un-stimulated cells of 2.75 and 4.8 folds for LAN-5 and SH-SY5Y cells, respectively (compare lane 3 to 2 and lane 6 to 5). As expected the BDNF stimulation of the cells does not modify the expression levels of Ret in RA-treated as well in mock treated cells. Consistently, the stimulation with BDNF of SK-N-BE does not affect the RA-induced phosphorylation of Ret (Figure 4B, compare lane 9 to 8) thus reflecting the absence of a functional TrkB expression in this cell line (see Figure 2C). This supports our above finding that the activation of Ret in RA-treated SH-SY5Y and LAN-5 cells is dependent on TrkB activity. The cross-talk mechanism stimulating Ret tyrosine kinase activity is GDNF-independent We have previously demonstrated that in SK-N-BE cells (lacking of a functional TrkB receptor) RA-induced differentiation is preceded by the accumulation of Ret and GDNF, and that Ret activation depends on the synthesis and the extracellular secretion of GDNF [18]. For what it concerns LAN-5 and SH-SY5Y cells, our results indicate that Ret phosphorylation following RA-treatment is due exclusively or at least in large part to a GDNF-independent mechanism that requires TrkB activity. To further confirm these findings we blocked GDNF-dependent activation of Ret by two different inhibitors: 1) the D4 aptamer, that inhibits Ret activity by directly binding its extracellular domain [27], [18]; 2) a truncated Ret protein, the EC-Retwt peptide, that competes for binding to GDNF, thus depleting the culture medium of the functional ligand [28], [29], [18]. Indeed, if in LAN-5 and SH-SY5Y cells Ret is activated by a GDNF-independent mechanism, using these molecules should have not to affect RA-induced Ret phosphorylation and differentiation. As shown in Figure 5, the treatment of SK-N-BE cells with D4 aptamer (Figure 5A, left panel, lane 3) or the soluble EC-Retwt protein (Figure 5B, left panel, lane 3) strongly inhibited Ret activation, while, in SH-SY5Y and in LAN-5 cells, any attempt to interfere with Ret stimulation either by using D4 (Figure 5A, middle panel, lane 3 and right panel, lane 3, respectively) or EC-Retwt (Figure 5B, middle panel, lane 3 and right panel, lane 3, respectively) failed and the levels of tyrosine phosphorylated Ret remained high. In addition, no inhibiting effect was observed by increasing neither the concentrations nor the incubation time of the aptamer or of the protein on the cells (not shown). As negative controls, we used a scrambled sequence of the D4 aptamer (D4Sc in the Figure 5A) and the recombinant EC-Ret protein lacking of the domains responsible of the functional interaction between Ret and the GFRα1/GDNF complex (EC-Ret1-387 in Figure 5B). 10.1371/journal.pone.0001643.g005Figure 5 The RA-induced stimulation of Ret activity is GDNF-independent in SH-SY5Y and LAN-5 cells. (A) SK-N-BE (left panels), SH-SY5Y (middle panels) and LAN-5 (right panels) cells were left untreated (lane 1) or treated for the indicated incubation times with RA either in the absence (lane 2) or in the presence of D4 (lane 3) or D4sc (lane 4). Cell lysates were immunoblotted with anti-pRet, anti-Ret, anti-tTG or anti-VGF antibodies. Equal loading was confirmed by immunoblotting with anti-αtubulin antibodies. (B) SK-N-BE (left panels), SH-SY5Y (middle panels) and LAN-5 (right panels) cells were incubated either in the absence (lane 2) or in the presence of ECRetwt (lane 3) or EC-Ret1-387 (lane 4) as indicated. Lanes 2 to 4, treatment with RA. Cell lysates were immunoblotted with anti-pRet or anti-Ret and and to confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies. In (A) and (B), quantitations were done as reported in legend to Figure 1A and relative abundances are expressed relative to controls, arbitrarily set to 1. Blots shown in (A) and (B) are representative of at least three independent experiments. Further, in SH-SY5Y and LAN-5, the cell differentiation was unaffected by D4 treatment, thus mirroring the activation of Ret. Indeed, as shown in Figure 5A, in both SH-SY5Y and in LAN-5 cells, VGF and tTG levels were induced by RA and remained high in presence of D4 (compare lane 3 to 2 in middle and right panels, respectively). As expected, in SK-N-BE cells D4 inhibited VGF as well tTG RA-dependent increase (compare lane 3 to 2 in left panel, and 18). On the basis of these results it is possible to conclude that in SH-SY5Y and LAN-5 cells, Ret is activated by a GDNF-independent mechanism. Furthermore, given that both the aptamer and the recombinant EC-Retwt interfere with Ret dimerization acting by the outside of the cell membrane, their inability to interfere with Ret suggests that in these cells, a cross-talk mechanism acting on Ret, stimulates its tyrosine kinase activity by an intracellular pathway. Discussion Receptor tyrosine kinases stimulation is a tightly regulated process that involves high affinity binding by external ligand polypeptide growth factors. Even tough this is recognised as the main mechanism that regulates RTK activity, recent reports indicate that alternative ways of activation exist that implicate transactivation triggered by other unrelated membrane receptors in the cytoplasm. Here we report for the first time that a physiological process, as the RA-induced differentiation, requires a novel transactivation mechanism which places Ret tyrosine kinase receptor downstream of TrkB receptor. As previously reported [15], [17], [18] retinoic acid treatment of several human neuroblastoma cells is rapidly followed by a strong increase in the expression of both these transmembrane receptors that become activated in the absence of exogenously added BDNF and GDNF ligands. Here we show that at difference of SH-SY5Y and LAN-5 cells, upon RA-treatment, in SK-N-BE the TrkB protein even increased in expression levels is neither tyrosine phosphorylated nor competent for BDNF acute stimulation. On the other hand, Ret is induced and phosphorylated at comparable levels in the three cell lines. Furthermore, we demonstrate that a functional TrkB receptor is needed for RA-mediated cell differentiation, indeed silencing TrkB causes a failure in morphological and biochemical cell differentiation in SH-SY5Y and LAN-5 whereas no effects were observed in SK-N-BE cells that express a non-functional TrkB receptor. On the other hand, we show that blocking Ret expression and activation by siRNA hampers as well the process of differentiation thus indicating that in LAN-5 and SH-SY5Y cells the concurrent activity of both receptors is needed for differentiation. In this scenario, the fact that, in SK-N-BE, cell differentiation exclusively depends on Ret activation [18] raises the obvious question of by which mechanism TrkB and Ret cooperate in order to shift the NB cells toward a differentiated phenotype. Given that silencing of Ret impairs NB differentiation in the three cell lines this indicates that Ret signalling is a common critical step for differentiation. For what it concerns LAN-5 and SH-SY5Y cells, our results establish that the RA-mediated Ret activation and cell differentiation are, in turn, dependent on TrkB activity, since inhibition of TrkB by siRNA significantly impairs Ret phosphorylation. Furthermore, we can exclude the presence of a GDNF-dependent mechanism of Ret activation since neither the D4 aptamer nor the EC-Retwt soluble protein reveal effective in inhibiting Ret phosphorylation following RA treatment of LAN-5 and SH-SY5Y cells. Indeed, the D4 aptamer interferes with the GDNF-mediated Ret dimerization and the EC-Retwt molecule competes for binding to GDNF thus depleting the culture medium of the functional ligand [27], [28], [29]. Taken together, our findings strongly indicate that at least two mechanisms can lead to stimulation of Ret activity by RA: the first, characteristic of SK-N-BE cells, lacking of a functional TrkB receptor, implicates the expression and secretion in the medium of GDNF thus providing maintenance of an autocrine loop that mediates cells differentiation (18 and current results); the second, in LAN-5 and SH-SY5Y cells, implicates that Ret activation is mediated by TrkB activity and occurs in the absence of GDNF stimulation. This indicates that a cross-talk mechanism between TrkB and Ret is functional in human NB cells. Furthermore, we show that the BDNF acute stimulation of SH-SY5Y and LAN-5 cells leads to Ret phosphorylation and transactivation in the absence of GDNF, thus confirming the existence of such inter-RTK signalling. In conclusion here we demonstrate that in the three NB cell lines, the RA-dependent Ret activation, both if GDNF-mediated (SK-N-BE) or TrkB-mediated (SH-SY5Y and LAN-5) is needed to induce cell differentiation since the outcome of blocking Ret phosphorylation is a failure in NB cell differentiation. Two major mechanisms could explain the TrkB-dependent Ret activation. First, TrkB may heterodimerizes with Ret following BDNF stimulation. Second, TrkB, once phosphorylated by BDNF could in turn activate Ret by an intracellular way. The heterodimerization of the platelet-derived growth factor receptor with EGFR [30], [31] or fibroblast growth factor receptor [32] and heterodimerization of insulin-like growth factor receptor and EGFR [33] are examples showing that cross-talk of different receptors may be mediated, at least in some cases, by their heterodimerization. This is unlikely to happen between TrkB and Ret on the basis of the following observations. The D4 aptamer inhibiting properties base on the capability of the RNA molecule to fold in a specific three-dimensional shape that binds at high affinity to Ret monomer and interferes with homodimerization and activation of the receptor [27]. Therefore, it is plausible that this molecule once bound to Ret monomer could obstacle as well Ret/TrkB dimerization. Our result showing that the D4 is unable to hamper RA-dependent Ret activation and differentiation of LAN-5 and SH-SY5Y cells, lends support to the second mechanism. Furthermore, it has been demonstrated the existence of cross-talk between the signal pathways mediated by Ret and by another member of the Trk-family, the TrkA receptor, in developing sympathetic neurons, whereby increased Ret expression and phosphorylation takes place as a consequence of TrkA activation by NGF by intracellular mechanisms [8]. These can include down-regulation of protein tyrosine phosphatases or up-regulation of tyrosine kinases acting on Ret and/or up-regulation of adaptor protein that activates Ret in the absence of GDNF. Work is in progress to clearly understand the mechanism and molecules involved in Ret transactivation. Here we demonstrate that the Ret receptor can be recruited by the TrkB receptor to function in the NB cells differentiation thus revealing a novel mechanism of functional cooperation between receptor tyrosine kinases. The understanding of the functional interplays between receptor is of major importance for the rationale design of new therapeutics in cancer. Materials and Methods Cell Culture and siRNA transfection Human NB cell lines SK-N-BE (2), SH-SY5Y and LAN-5 (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle medium supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Hyclone, Celbio, South Logan, UT), and 50 µg/ml gentamycin. All-trans RA (Sigma, St. Louis, MO) was dissolved (10 mM) in DMSO and diluted at 10 µM-final concentration in serum-containing culture medium. For BDNF acute stimulations, SH-SY5Y, LAN-5 and SK-N-BE (900.000 cells/10 cm-plate) were RA-treated for 72 h, serum starved for 4 h and then incubated for 10 min in the absence or in the presence of BDNF (Alomone Labs, Jerusalem, Israel) at a final concentration of 50 ng/ml. Ret or TrkB gene silencing was established by transfection of high performance validated Ret siRNA [34] specifically targeting Ret exon-2 (5′-GGGAUGCUUACUGGGAGAAtt3′) or TrkB stealth siRNA (5′-AACUUAAGCAGAAACAGCAUUACCA) custom-designed by Invitrogen, into SK-N-BE(2), SH-SY5Y and LAN-5 NB cell lines. Cells (35.000 cells per 6 cm plate) were grown and overlaid with the transfection mixtures containing 100 nM Ret siRNA or 150 nM TrkB siRNA and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM I reduced serum medium (Invitrogen). After 5-h incubation, complete culture medium containing 10 µM RA was added to the cells and incubation was prolonged up to 72 h. Controls were performed using a non-related siRNA that do not lead to the specific degradation of Ret and TrkB mRNA. Immunoprecipitation and western blot analysis Cells were washed twice in ice-cold phospate-buffered saline (PBS), and lysed in buffer A (50 mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl, 1% Nonidet P-40, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 1 mM Na3VO4). Protein concentration was determined by the Bradford assay using bovine serum albumin as the standard. For immunoprecipitation analysis, cell extracts (500 µg) were incubated with anti-panTrk antibody (C-14) (Santa Cruz Biotechnology Inc, Santa Cruz CA) for 2 h at 4°C and then immunoprecipitated with protein A/G- agarose (Santa Cruz Biotechnology Inc, Santa Cruz CA) overnight at 4°C. Immunoprecipitates were washed three times with buffer A and denatured in Laemmli buffer for 5 min at 100°C. The cell lysates or immunoprecipitates were subjected to 8% SDS-PAGE. Gels were electroblotted into polyvinylidene difluoride membranes (Millipore Co., Bedford, MA), and filters were probed with the indicated primary antibodies: anti-Ret (H-300), anti-TrkB (H-181), anti-VGF (R-15) (Santa Cruz Biotechnology Inc, Santa Cruz CA); anti-(Tyr-phosphorylated) Ret (referred as pRet) (Cell Signaling, Beverly, MA); anti-phospho-tyrosine (referred as pTyr) (4G10, Upstate Biotechnology Incorporated, Charlottesville, VA); anti-GAP-43 (Zymed Laboratories Inc., South San Francisco, CA); anti-α-tubulin (DM 1A) (Sigma, St. Louis, MO); anti-tTG antibodies (kindly provided by G. Peluso CNR, Naples, Italy) [35]. Proteins were visualized with peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system (Amersham-Pharmacia Biosciences LTD, Uppsala, Sweden). Where indicated, filters have been stripped as described [28]. Reverse Transcription-PCR analysis 106 NB cells were plated on 10-cm dishes and treated with 10 µM RA for 72 h for analysis of full-length TrkB and BDNF mRNA. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control to normalize the levels of mRNA in all samples. Total RNA was isolated using a RNA extraction kit (Ambion Austin, TX). RNA (5 µg) was reverse transcribed for 60 min at 42°C in a 20 µl-volume reaction mixture containing 20 units of Moloney Murine Leukaemia Virus Reverse Transcriptase (M-MV-RT) (Roche, Basel, Switzerland) and random hexanucleotides (Amersham Pharmacia). The resulting cDNA fragments were used as PCR templates. The following pairs of forward and reverse primer sets were used for amplification: TrkB, 5′-GAGCATCATGTACAGGAAAT-3′ and 5′-CTTGATGTTCTTCCTCATGT-3′ (PCR product size, 235 bp); BDNF, 5′-GCAACGGCAACAAACCACAACATTATC-3′ and 5′-GTCCCTGTATCAAAAGGCCAACTGAAG-3′ (PCR product size, 492 bp); GAPDH, 5′-CATCAAGAAGGTGAAGC-3′ and 5′-TCTTACTCCTTGGAGGCCAT-3′ (PCR product size, 240 bp). Amplifications were performed by 30 cycles of PCR by using the following conditions: TrkB: 30 sec at 95°C; 30 sec at 57°C and 30 sec at 72°C; BDNF: 1 min at 94°C, 1 min at 60°C and 2 min at 72°C; GAPDH: 20 sec at 95°C, 30 sec at 58°C and 1 min at 72°C. Neurite Outgrowth Bioassay NB cells were plated at equal density on 10-cm dishes. To evaluate the effects of TrkB gene silencing on morphologic cell differentiation, cells were transfected with TrkB-specific or non-related siRNA as reported in Cell Culture and siRNA trasfection. At 72 h of RA treatment, at least 15 random fields were photographed by a phase-contrast light microscope and 50 cells per frame were counted and scored as having neurites or not. A neurite was operationally defined as a process outgrowth that was long more than twice the diameter of cell body. Inhibitors of GDNF-dependent Ret activation D4 is a 93-base 2′-fluoro-RNA-based aptamer that specifically bind the extracellular domain of Ret thus interfering with ligand-induced stimulation of its intrinsic tyrosine kinase activity [18]. A scrambled sequence of D4 (D4sc) is used as control. The entire Ret receptor extracellular portion (EC-Retwt) and the protein containing just the first three NH2-terminal cadherin-like domains of EC-Retwt (named EC-Ret1-387) were produced as reported previously [28], [29]. To assess the effects of RNA aptamers and of EC-Ret proteins on Ret activity, SK-N-BE, SH-SY5Y and LAN-5 cells (160.000 cells/3.5 cm-plate) were treated for 18 h with 1 ml-culture medium containing 10 µM RA plus 50 µg of indicated EC-Ret or 1600 nM of indicated aptamers after a short denaturation-renaturation step. To evaluate the effects of D4 aptamer on cell differentiation, cells were incubated in 12-well cell plate with 10 µM RA together with 3 µM aptamer for 72 h. The used RNA concentrations ensure the continuous presence of a concentration of at least 200 nM, which takes into account the 6 h-half life of the D4 aptamer in 10% serum. This concentration is effective in inhibiting Ret activity as previously reported [18], [27]. We thank M. Gottesman for helpful discussions. Competing Interests: The authors have declared that no competing interests exist. 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Cure of chronic viral infection and virus-induced type 1 diabetes by neutralizing antibodies METTE EJRNAES1 , MATTHIAS G. VON HERRATH1 , & URS CHRISTEN1,2 1 La Jolla Institute for Allergy and Immunology, San Diego, CA, USA, and 2 Johann Wolfgang Goethe University, Frankfurt am Main, Germany Abstract The use of neutralizing antibodies is one of the most successful methods to interfere with receptor-ligand interactions in vivo. In particular blockade of soluble inflammatory mediators or their corresponding cellular receptors was proven an effective way to regulate inflammation and/or prevent its negative consequences. However, one problem that comes along with an effective neutralization of inflammatory mediators is the general systemic immunomodulatory effect. It is, therefore, important to design a treatment regimen in a way to strike at the right place and at the right time in order to achieve maximal effects with minimal duration of immunosuppression or hyperactivation. In this review, we reflect on two examples of how short time administration of such neutralizing antibodies can block two distinct inflammatory consequences of viral infection. First, we review recent findings that blockade of IL-10/IL-10R interaction can resolve chronic viral infection and second, we reflect on how neutralization of the chemokine CXCL10 can abrogate virus-induced type 1 diabetes. Keywords: Autoimmune disease, chronic infection, CXCR3, dendritic cells, LCMV , molecular mimicry Introduction Lymphocytic choriomeningitis virus (LCMV) infection of mice has proven to be one of the most informative experimental systems for investigating various aspects of virology and immunology. The various LCMV models offer several acute and persistent infection systems that are well suited to reflect immune kinetics as they might occur in a variety of human chronic infections. In addition, transgenic expression of LCMV proteins as model target antigens in the pancreas or the CNS is being used in animal models for type 1 diabetes (Ohashi et al. 1991; Oldstone et al. 1991) and multiple sclerosis (Evans et al. 1996), respectively. LCMV belongs to the arena virus family and is a natural pathogen for both humans and mice (Zinkernagel and Doherty 1974). The viral genome is comprised of two single stranded RNA segments; both segments are antisense, each encoding two proteins. The larger (L) segment encodes the L protein (the viral polymerase) and the Z protein with an undefined function. The short (S) segment encodes the nucleoprotein (NP) and the glycoprotein (GP), which undergoes post-translational cleavage to generate the two mature glycoproteins, GP1 and GP2 (Borrow and Oldstone 1997). LCMV can cause acute or persistent infection in vivo depending on the strain, route of infection and dose. Whereas adult mice inoculated intravenously with LCMV strain Armstrong (LCMV-Arm) rapidly clear the infection and remain immune-competent with the establishment of a stable memory T cell pool (figure 1) (Marker and Volkert 1973; Moskophidis et al. 1987), inoculation with the LCMV variant Cl13, which differs from its parent (LCMV-Arm) virus at only one amino acid ISSN 1740-2522 print/ISSN 1740-2530 online q 2006 Taylor & Francis DOI: 10.1080/17402520600579028 Correspondence: M. Ejrnaes, Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr, San Diego, CA 92121, USA. Tel: 1 858 558 3581. Fax: 1 858 558 3579. E-mail: [email protected]. Urs Christen, Pharmacenter, Johann Wolfgang Goethe University, Theodor-Stern Kai 7, 60590 Frankfurt am Main, Germany. Tel: 49 69 6301 83105. Fax: 49 69 6301 7942. E-mail: [email protected]. Clinical & Developmental Immunology, March 2006; 13(1): 67-77 positions in the virus GP is associated establishment of a chronic infection. This chronic infection is associated with both the functional impairment and deletion of virus-specific CD8 T cells and general immunosuppression (Moskophidis et al. 1993; Lin and Welsh 1998). Chronic-persisting protracted LCMV variant Clone 13 The LCMV variant Clone 13 (Cl13) was originally isolated from the spleen of a 2-month-old mouse infected at birth with LCMV-Arm (Matloubian et al. 1990). The molecular basis of persistence and suppression of the anti-LCMV cytotoxic T lymphocyte (CTL) response has been mapped to a single amino acid change in LCMV-GP; LCMV-Arm has a phenylalanine at amino acid position 260, whereas the variant Cl13 has a leucine at this position (Salvato et al. 1991; Dockter et al. 1996). Infection of mice with high doses of Cl13 leads to a persistent infection that is associated with a lack of viral antigen specific CD8 effectors (figure 1) (Moskophidis et al. 1993; Lin and Welsh 1998). A peripheral membrane protein, alpha-dystroglycan (a-DG), was identified as the receptor for LCMV (Cao et al. 1998). Additionally, it was observed that Cl13 binds more strongly to a-DG as compared to the Armstrong strain (Smelt et al. 2001). The affinity of the viral binding to its receptor might be a crucial element in determining the outcome of infection with LCMV Armstrong (lower affinity to a-DG) versus Cl13 (high affinity to a-DG). It was shown that differences in binding affinities of LCMV strains to a-DG correlated with viral tropism and disease kinetics (Smelt et al. 2001). LCMV-Arm and Cl13 appear to exhibit differences in their tropism within the spleen, with Cl13 causing a higher level of infection of antigen presenting cells (APCs) in the white pulp, including periarterial interdigitating DCs. Cl13 could thereby render these cells targets for more effective destruction by the antiviral CD8 CTL response which is induced at an early time point following infection with Cl13 compared to LCMV-Arm. As a consequence this CD8-dependent destruction of dendritic cells (DCs) in the spleen of Cl13-infected mice leads to the ensuing immunosuppression (Borrow et al. 1995). In vitro studies showing a decline of DCs after infection with Cl13 but not LCMV-Arm support the notion that the loss was related to infection (Sevilla et al. 2004). Additionally, it was reported that DCs obtained from cultures infected with Cl13, but not LCMV-Arm were non-functional, as demonstrated by their ability to stimulate allogeneic T cells (Sevilla et al. 2004). Interestingly, upon closer look, this publication also shows that the loss of DCs was mostly restricted to the CD11c CD8a population and affected CD11c CD8aneg DCs to a much lesser extent. IL-10 as a modulator of APC function Interleukin-10 (IL-10) is an immunosuppressive cytokine with implications for various immune and inflammatory diseases. It inhibits a broad spectrum of cellular immune responses, acting on APCs by, i.e. preventing DC maturation and thereby keeps the cells in an immature state and T cells by inhibiting pro-inflammatory cytokine production. Investigations have revealed that IL-10 play important roles in blocking cytokines production (Fiorentino et al. 1991) costimulatory molecules expression, there under MHC class II, CD80 and CD86 (Ding et al. 1993; Willems et al. 1994) as well as chemokine secretion (Jinquan et al. 1993; Kasama et al. 1994) and modification of chemokine receptor expression (Sozzani et al. 1998; Takayama et al. 2001, for review see Pestka et al. 2004). Apart from the reported potent immune-modulatory effects on APCs, IL-10 also affects TH1- and TH2-type immune responses, as evidenced by findings in asthma, transplantation and autoimmunity models (Moore et al. 2001). IL-10 regulates the proliferation and differentiation of TH1-type T lymphocytes, which appear to control many effector immune responses (i.e. host defense, anti-tumor immunity, autoimmunity) in vivo (Fiorentino et al. 1991). In different cell types, the duration of stimulation affects IL-10 expression differentially. In T cells IL-10 expression occurs shortly after stimulation Figure 1. Mice infected with medium dose LCMV Armstrong develop an acute viral infection characterized by an initial expansion of anti-viral specific T CD8 cells (blue line) with the ability to efficiently eliminate virus infected target. During their expansion, viral antigen-specific CD8 T cells acquire effector functions, including the ability to rapidly produce cytokines. Viral clearance is dependent on the presence of CD8 virus-specific cytotoxic T lymphocytes (CTLs) (Zinkernagel and Welsh 1976; Oldstone et al. 1986; Jamieson et al. 1987; Moskophidis et al. 1987); and the infection is cleared by day 10-12 from blood and spleen (Blue shaded area). After expansion of effectors and their contraction, a stable memory CD8 T cell population is generated that can be maintained in the absence of antigen (blue line) (Lau et al. 1994; Murali-Krishna et al. 1999; Homann et al. 2001; Kaech et al. 2002). In contrast, infection with Cl13, results in a prolonged infection that persists (red shaded area). This chronic infection is associated with both the functional impairment, deletion of virus-specific CD8 T cells and due to lack of establishment of a memory T cells pool inability mount sufficient recall responses (red line). M. Ejrnaes et al. 68 and the levels of IL-10 increase with the duration of stimulation (Wolk et al. 2002). Elevated levels of IL-10 mRNA have been observed in immune-responsive vs. non-responsive metastatic melanoma lesions (Mocellin et al. 2001). Moreover, treatment with a combination of anti-IL-10 receptor (IL-10R) monoclonal antibody (mAb) and toll-like receptor 9 (TLR9) ligands has been shown to have potent therapeutic anti-tumor effects (Vicari et al. 2002; Vicari and Trinchieri 2004), pointing to the role of IL-10 in the development of cancer. Furthermore, IL-10 has also been shown to play a role in the establishment of certain chronic viral infections such as human immunodeficiency virus (HIV-1) (Granelli-Piperno et al. 2004), hepatitis C (HCV) and human cytomegalovirus (HCMV) infections (Rigopoulou et al. 2005). Interestingly, an increase in systemic IL-10 production has been demonstrated upon HCV infection (Accapezzato et al. 2004), and in some cases of infection with HIV (Akridge et al. 1994; Ameglio et al. 1994; Autran et al. 1995; Granelli-Piperno et al. 2004; Ji et al. 2005). Current immunotherapeutic approaches in persistent infections As the hallmark of chronic infection is an impaired virus specific effector cell response, numerous efforts have been undertaken to increase anti-LCMV immunity. Conventional immunotherapy attempting to augment anti-viral immunity directly in persistent infected individuals have failed to affect the outcome so far, but lowering the viral antigenic load (by interferons or anti-viral drugs) has clear beneficial effects. Additionally, specific immunization strategies have been combined with direct anti-viral drug treatments, for example protease inhibitors and highly active antiretroviral therapy (HAART) in HIV (Palella et al. 1998) and interferon administration, and ribavirin in hepatitis infections (Torriani et al. 2004; Kamar et al. 2005). In all the situations, where anti-viral drugs were employed, viral loads were significantly reduced and, for HIV, long-term deleterious consequences of the persistent infection were decreased. Interestingly, the outcome of ribavirin administration in chronic HCV was not quite as promising, although viral titers were lowered, since liver fibrosis was enhanced, possibly as a rather direct effect of the drug (Torriani et al. 2004; Kamar et al. 2005). However, complete elimination of the pathogen has remained elusive, despite the fact that anti-viral immunity was significantly increased in many instances. Much attention has been paid to the role if IL-10 as a potent anti-inflammatory immuno-suppressive cytokine with important potential clinical applications. So far it remains unclear, whether IL-10 affects the outcome of infection, amount of immunopathology and complications and could be the actual cause for persistence. One major concern in clinically manipulating the levels of IL-10 is its critical role in the immune homeostasis. Long-term application of IL-10 could cause immunodeficiency, whereas continuous use of anti-IL-10 may lead to hyper immune reactivation. However, blocking the cytokine receptor itself for a short period of time is a relative novel approach to control the signaling effects of the cytokine. To gain further insight into the function of IL-10 in the establishment and maintenance of persistent viral infections, we have investigated how blockade of the IL-10/IL-10 receptor signaling pathway affects LCMV chronic infection in its natural host, the mouse. Neutralization of IL-10 resolves chronic viral infection LCMV Cl13-infection results in a prolonged period of elevated IL-10 production, which is predominantly produced by CD4 T cells already early upon infection (Ejrnaes et al. 2006). This interestingly coincides with the loss of the systemic CTL response against the virus. Treatment with a neutralizing IL-10R antibody that was previously shown to block IL-10/IL-10R interaction in vitro (BD Biosciences) results in accelerated viral clearance (Ejrnaes et al. 2006). This is associated with a numeric increase of total spleen cells in comparison to non-treated mice indicating that development of lymphopenia is reversed upon anti-IL10R mAb treatment (Ejrnaes et al. 2006). Furthermore, this rapid resolution of viral infection is associated with diminished levels of endogenous IL-10 and enhanced anti-viral CD8 memory T cell responses (Ejrnaes et al. 2006). Importantly, overall clinical appearance is improved through such an intervention as reflected in an increase in bodyweight, healthy shiny coat, and increase in physical activity (Ejrnaes et al. 2006). Interestingly, this protection from chronic infection was achieved with only a few injections of neutralizing anti-IL-10R mAb immediately after virus infection. Thus, the duration of treatment could be minimized in order to avoid long-term systemic immunosuppressive effects. Previous reports suggest that different DC subsets vary in their ability to prime effector T cells (Liu 2001), and in particular, evidence suggests that DCs can be converted to APCs, which skew the immune response towards TH2-domination, when treated with anti-inflammatory cytokines such as IL-10 (Buelens et al. 1995; Liu 2001). In this context we found that activation of LCMV-specific T cells in chronically infected mice is preferentially achieved by CD8a2 DCs, which induced IL-10 production by virus-specific CD4 T cells (Ejrnaes et al. 2006). Subclasses of DCs have been shown to have the potential to differentially skew cytokine production towards Cure of chronic viral infection 69 TH1- or TH2-profiles (Mosmann and Coffman 1989). Notably, it has been suggested that CD8a2 DCs induce TH2-profiles whereas CD8a DCs preferentially stimulate IFN-g production and therefore induce TH1 profiles (Maldonado-Lopez et al. 1999), a result we could confirm when analyzing the ability of CD8a and CD8a2 DCs to polarize nai ve LCMV-reactive CD4 T cells (Ejrnaes et al. 2006). The generalized state of lymphopenia induced by Cl13 infection might be mediated by APCs inducing IL-10 production. However, the mechanisms by which IL-10 enables Cl13 to persist are unknown. IL-10 could either down-regulate pro-inflammatory responses in a general manner or, more specifically, inhibit the induction or expansion of anti-viral CTLs. In fact, IL-10 may directly decrease the viability of CD8a DCs as has been previously suggested (Maldonado-Lopez et al. 2001; Re and Strominger 2004). In the state of chronic Cl13 infection, prominent CD8a2 DCs with reduced ability to prime TH1/TC1 effectors "by default" become the modulators of the T cell response and thus derail anti-LCMV immunity through the production of IL-10 (figure 2). While the precise mode of action of IL-10 is unknown, LCMV-specific CD4 T-cells activated in this context may in turn acquire the ability to produce IL-10 and provide inappropriate or insufficient anti-viral help to other cell types, in particular CD8 T cells, thus leading to persistent infection (figure 2). Additionally, it is possible that the remaining CD8a2 DCs, which appear ill-equipped to propagate anti-viral effectors, will continue to support IL-10 production. The resulting high concentration of IL-10 in the milieu may thus lead to further modulation of DC function and in this way become a self-fulfilling phenomenon (figure 2). As a consequence, only disruption of IL-10 signaling will have the ability to break this vicious circle and enable the recovery of appropriate anti-viral immunity by the infected host. Since blockade of IL-10 signaling likely directly acts on DCs, a central switch in immunity could be implanted in this way directly at the core, where most immune responses are orchestrated. Virus-induced type 1 diabetes Another possible consequence of virus infection and its accompanying inflammatory cascades is the initiation or acceleration of autoimmune disease. One scenario that might be involved in the etiology of autoimmune disease is that virus infection could break self-tolerance due to an inherent structural similarity to self-components (Oldstone 1989; Cantor 2000; Miller et al. 2001; Olson et al. 2001; Christen et al. 2004b; Christen and Herrath 2004). This concept of molecular mimicry has been integrated into animal models for various human autoimmune diseases such as type 1 diabetes (T1D), multiple sclerosis (MS), or systemic lupus erythematosus (SLE) (Christen and von Herrath 2004a). The RIP-LCMV mouse model for type 1 diabetes that was established in 1991 in the laboratories of Michael Oldstone (Oldstone et al. 1991) and Rolf Zinkernagel (Ohashi et al. 1991). These mice express the GPor NPof LCMV-Arm in the insulin-producing b-cells of the islets of Langerhans in the pancreas. Such b-cells are the target of the auto-aggressive immune system in T1D. Since the transgenically expressed viral protein is considered a component of "self", RIP-LCMV mice are tolerant and do not mount an immune response to the LCMV protein. However, infection with LCMV-Arm itself breaks this self-tolerance, auto-aggressive Figure 2. LCMV Cl13 persistence is the result of an early CTL-mediated loss of CD8a DCs, which indirectly supports immune responses high in IL-10 production that might be predominantly driven by the remaining CD8aneg CD11c DCs. M. Ejrnaes et al. 70 LCMV-specific T cells are generated and expanded, the b-cells are being destroyed and ultimately clinical overt diabetes results (figure 3). In the context of this review article it is important to reiterate here that LCMV-Arm is one of the isolated LCMV strains that has no immunosuppressive properties and does not result in persistent infection. In the past, this model was used to investigate mechanisms of how autoimmune processes are involved in the pathogenesis of T1D and to evaluate possible treatments for human T1D in an animal model. Just as proposed for human T1D, the onset of diabetes in RIP-LCMV mice depends on the action of both, autoreactive CD4 and CD8 T-cells and correlates with the numbers of auto-aggressive lymphocytes generated. In accordance, the incidence of disease varied between the individual transgenic lines ranging from 2 weeks (RIP-GP lines) to 1-6 months (RIP-NP lines). Further studies revealed the mechanism involved in the rapid compared to the slow onset diabetes: Transgenic lines expressing the LCMV-GP transgene exclusively in the b-cells of the islets manifested rapid-onset T1D (10-14 days after viral challenge) (von Herrath et al. 1994). In these lines the high systemic numbers of auto-aggressive CD8 T-cells were sufficient to induce diabetes and did not require help from CD4 cells. In contrast, in lines expressing the LCMV-NP transgene in both the b-cells and in the thymus, T1D took longer to occur after subsequent LCMV challenge. Several lines of evidence indicated that in RIP-NP mice the anti-self (viral) CTL were of lower affinity and that CD4 T-cells were essential to generate anti-self (viral) CD8 lymphocyte-mediated T1D (von Herrath et al. 1994). In addition, mouse models in which transgene-encoded "target-antigens" are expressed in the Figure 3. Therapeutic intervention during the ongoing destruction of islet cells in the pancreas. After LCMV infection active virus is found in the pancreas but not necessarily in the islets. In an initial stage local resident macrophages or dendritic cells are activated and release pro-inflammatory cytokines, such as TNFa. Blocking of TNFa at this early stage after infection can abrogate T1D (Christen et al. 2001 Christen and von Herrath 2004c) (panel B). Chemokines, most prominently CXCL10, are released by endothelial cells and b-cells (Christen et al. 2003, Frigerio et al. 2002) (panel C) which in turn attract islet-specific auto-aggressive T lymphocytes as well as non-specific bystander cells into the islets of Langerhans (Christen et al. 2003, Rhode et al. 2005) (panel D). At that time neutralization of CXCL10 prevents the subsequent development of T1D by interfering with the migration of auto-aggressive T lymphocytes to the pancreas (Christen et al. 2003). When not prevented from doing so, the first islet-specific auto-aggressive CD8 cells destroy some b-cells by perforin dependent cytolysis resulting in release of b-cell antigens (panel E). These antigens include transgenic viral proteins and additional non-viral components and are processed and presented to infiltrated CD8 and CD4 cells by APC (panel E). In the terminal stage of immunopathogenesis the majority of b-cells are being destroyed by auto-aggressive CD8 cells in a IFNg dependent manner (Seewaldt et al. 2000). It is only in this final stage where clinically overt diabetes is apparent and can be assessed by blood glucose measurements. The destructive process at this end stage can however be reversed in the presence of high local concentrations of TNFa (islet-specific TNFa expression in a transgenic system) which can induce a status of hyper-activation resulting in apoptosis of auto-aggressive CD8 (Christen et al. 2001, Christen and Von Herrath 2002) (panels D and E). Similarly, expression of CXCL10 at an auxiliary site, such as the PDLN, can cause a recruitment of auto-aggressive CD8 cells away from the islets and a subsequent apoptosis in the PDLN by hyper-activation (Rhode et al. 2005). Cure of chronic viral infection 71 pancreatic b-cells, such as the RIP-LCMV and the INS-HA (Lo et al. 1992, 1993) mouse, have demonstrated that the presence of auto-aggressive T-cells alone is not enough to cause disease. For example, when RIP-LCMV mice were crossed with mice expressing an inactive mutated form of IFNgR the diabetes incidence was drastically reduced (Seewaldt et al. 2000). These results indicate that unspecific "bystander factors", such as cytokines and chemokines generated during the acute inflammation after LCMV infection, are important to drive the auto-aggressive response (b-cell destruction) in "antigen-specific" models for T1D. Hence, the RIP-LCMV model has become a very useful tool to study the etiology and the mechanisms of human autoimmune diabetes and to evaluate possible treatments, such as blockade of specific inflammatory factors, as discussed below (Christen et al. 2001, 2003), oral tolerance induction (Homann et al. 1999) or DNA-vaccination (Coon et al. 1999). Besides having a clearly defined initiation point (LCMV-infection), the advantage of the RIP-LCMV system over other established models for T1D, such as the NOD mouse, is the presence of extensively characterized target antigens (GP, NP). The immune response against these target antigens can be visualized using flow cytometry by stimulation of splenocytes with LCMV-peptides or direct staining of CD8 T-cells with MHC class (-peptide tetramers (Murali-Krishna et al. 1998). In addition, we recently demonstrated tracking of LCMV-specific CD8 T-cells by in situ MHC class-peptide staining of quick-frozen tissue sections (McGavern et al. 2002). CXCL10--A sentinel chemokine during inflammation Among the inflammatory mediators generated upon LCMV-infection of the pancreas, CXCL10 proved to play a key role in imprinting a pattern for the subsequent development of autoimmune disease in the RIP-LCMV model. In general, ligands of the CXCR3 chemokine receptor including CXCL10 (interferon-induced protein of 10 kDa, IP-10), CXCL9 (monokine induced by gamma-interferon, Mig) and CXCL11 (interferon-inducible T-cell a chemoattractant, I-TAC) have been suggested to play an essential role during inflammation and autoimmunity. CXCR3 chemokine ligands are mainly expressed by keratinocytes, macrophages, fibroblasts and endothelial cells upon stimulation with IFNg or TNFa (Luster et al. 1985; Luster and Ravetch 1987) but are also generated by activated T-cell hybridomas, normal T-cells, and thymocytes (Gattass et al. 1994). CXCL10 was suggested to function as a "sentinel molecule" in host defense against viruses (Liu et al. 2000) and is generated very early after infection with a wide variety of viruses such as HIV, adenovirus, LCMV, Theiler's virus and mouse hepatitis virus (Asensio and Campbell 1997; Lane et al. 1998; Charles et al. 1999; Hoffman et al. 1999; Kolb et al. 1999). Further, CXCR3 chemokine ligands are have been implicated in the host defense against foreign pathogens by promoting local inflammation. Blockade of CXCL10 disturbed the control of parasite propagation after Toxoplasma gondii infection by abrogating T-cell migration into tissues and impairing antigen-specific T-cell function resulting in amplified tissue parasite burden and an increased mortality (Khan et al. 2000). Further, transgenic (tg) CXCL10 expression in keratinocytes resulted in delayed wound healing and disorganized neo-vascularization due to a more intense inflammatory phase (Luster et al. 1998). CXCR3 is the only cellular receptor for CXCL9, CXCL10 and CXCL11 identified to date, and is predominantly found on activated Th1-type T-cells (Loetscher et al. 1996; Sallusto et al. 1998). Thus, CXCR3 chemokines direct the anti-viral defense towards the more aggressive type 1 T-cell-domination and act as "bystander effectors" that unspecifically activate T-cells (including autoreactive T-cells) and subsequently drive an auto-aggressive immune response that may result in autoimmune disease. Unique role of CXCL10 in imprinting a pattern for autoimmune disease Data from our lab suggest that among CXCR3 chemokines, CXCL10 plays a unique role in imprinting a pattern for the subsequent development of autoimmunity (Christen et al. 2003; Christen and von Herrath 2004b). As previously reported for infection of the CNS by various viruses (Asensio and Campbell 1997; Lane et al. 1998; Charles et al. 1999; Hoffman et al. 1999; Kolb et al. 1999), infection of mice with LCMV caused a very rapid and strong expression of CXCL10 in the pancreas (Christen et al. 2003). Interestingly, CXCL10 was the only chemokine whose expression was strongly induced as early as 1 day after infection. The other two CXCR3 chemokine ligands were either expressed later (CXCL9) or were only faintly upregulated (CXCL11). Further, chemokines such as CCL3 (MIP-1a) or CCL5 (RANTES) were not increased until day 7 post-infection, a time where most pro-inflammatory cytokines including TNFa, and IFNg are strongly expressed as well (Christen et al. 2003). Very recently, the important role of CXCL10 during virus-induced diabetes was underlined in RIP-CXCL10 transgenic mice, which showed a spontaneous infiltration of islets by a mixed leukocyte population (Rhode et al. 2005). These mice were not diabetic but had a reduced capacity to response to a high glucose challenge possibly due to "inflammatory stress" to the islets (Rhode et al. 2005). In addition, when crossed to the RIP-LCMV mouse line the RIP-CXCL10 mice had accelerated T1D after M. Ejrnaes et al. 72 infection with LCMV (Rhode et al. 2005). Thus, CXCL10 is a prime candidate for a neutralization attempt to rescue mice from autoimmune disease. Neutralization of CXCL10 abrogates type 1 diabetes We used a very characterized neutralizing monoclonal antibody to CXCL10 that was previously shown to block the biological activity of CXCL10 Toxoplasma gondii infection model (Khan et al. 2000). Briefly, neutralization of CXCL10 inhibited the influx of activated T-cells into tissue and a 1000-fold higher parasite burden resulting in a higher mortality of infected mice (Khan et al. 2000). In our T1D mouse model we found that indeed blockade of CXCL10 with this neutralizing antibody abrogated disease in .60% of all LCMV-infected mice (Christen et al. 2003). In contrast blockade of CXCL9 with a neutralizing anti-CXCL9 antibody neither reduced the incidence nor prolonged the onset of diabetes (Christen et al. 2003). Mechanistically, blockade of CXCL10 interfered with the expansion of LCMV-specific auto-aggressive CD8 T-cells and their migration to the pancreatic islets of Langerhans (Christen et al. 2003). Thus, neutralization of one critical inflammatory factor at the right time could prevent the subsequent development of autoimmune disease. It is important here to note that the treatment had to be administered at the precise time when CXCL10 was expressed at high density. Rescued mice received 5 intravenous injections of 100 mg neutralizing antibody at days 0, 1, 2, 4 and 6 post infection. Treatment of mice before LCMV-infection or at a later time (days 7-14) was not successful in preventing autoimmune disease (U.CH. unpublished observations and Ref. Christen et al. 2003). The same monoclonal anti-CXCL10 antibody was used to neutralize CXCL10 in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) (Fife et al. 2001). Similar to our experiences in the RIP-LCMV model, CXCL10 neutralization decreased the accumulation of antigen-specific lymphocytes to the site of autoimmune damage (Fife et al. 2001). A different approach to neutralize CXCL10 was recently published by the Narumi group who made use of the non-obese diabetic (NOD) mouse model for spontaneous T1D (Morimoto et al. 2004). They administrated young NOD mice with a DNA plasmid encoding CXCL10. This DNA vaccination approach resulted in the in vivo generation of neutralizing anti-CXCL10 antibodies and suppressed the development of diabetes (Morimoto et al. 2004). In contrast to the findings in the RIP-LCMV model, the treatment did neither reduce insulitis nor alter the islet-specific immune response. It rather enhanced the proliferation of the insulin-producing b-cells resulting in a higher b-cell mass (Morimoto et al. 2004). Another interpretation of these data would be that CXCL10 neutralization relieves the islets from "inflammatory stress". Thus, b-cells from DNA-vaccinated NOD mice might proliferate at a normal rate, whereas b-cells from untreated NOD mice show reduced proliferation rates and therefore insufficient insulin production. However, the experiments by Morimoto et al. demonstrate that neutralization of CXCL10 can be successful even in a spontaneous model such as the NOD mouse. It was previously demonstrated that CXCL10 is predominantly expressed by b-cells upon LCMV-infection of the pancreas (Frigerio et al. 2002; Rhode et al. 2005). However, recent observations in our group show that diabetogenic T-cell clones produce CXCL10 as well (Ejrnaes et al. 2005). In this study, CD8 T-cell clones specific for the islet antigens insulin, glutamic acid decarboxylase (GAD) and LCMV-GP were analyzed for expression of various chemokines and cytokines. Interestingly, there was a clear correlation between diabetogenicity and the expression of CXCL10 (Ejrnaes et al. 2005). Additional CXCL10-expression by auto-aggressive T-cells might lead to a perpetuated infiltration rate of the islets and subsequent acceleration of the destructive process. Auxiliary CXCL10 expression can abrogate autoimmune disease Whereas expression of CXCL10 at the site of autoimmune damage clearly accelerates the immunopathogenic process and exacerbates disease (Rhode et al. 2005), auxiliary expression of CXCL10 can, under certain circumstances, abrogate the ongoing autoimmune destruction. In the late 1980ies it was observed that infection of NOD mice with LCMV can be used as a therapeutic intervention to prevent T1D (Oldstone 1988, 1990). In the RIP-LCMV model T1D could be abrogated by a secondary infection with one particular strain of LCMV (strain Pasteur, LCMV-Past) but not with LCMV-Arm (strain Armstrong) that was used for the initial induction of disease (Christen et al. 2004a). The major difference between those two LCMV strains, that share all the immunologically relevant epitopes, is a striking discrepancy in viral growth in the pancreatic draining lymph node (PDLN). Secondary infection with LCMV-Past resulted in higher viral titers in the PDLN when compared to the pancreas and to infection with LCMV-Arm. Along with this augmented viral proliferation a differential expression of the inflammatory chemokine CXCL10 between PDLN and pancreas was observed (Christen et al. 2004a). Interestingly, a similar increase in PDLN-specific CXCL10 expression could be detected in NOD mice infected with LCMV-Past as well as LCMV-Arm (Christen et al. 2004a). In both LCMV-Past-infected Cure of chronic viral infection 73 NOD and RIP-LCMV mice a significant decrease in the infiltration of islets and an increase in cellular apoptosis in the PDLN was detected (Christen et al. 2004a). Apparently, auto-aggressive cells migrated away from the islets and got stuck in the PDLN due to the high CXCL10 concentration. Once arrived at the highly inflamed PDLN the cells were hyper-activated and died by apoptosis. Thus, in such a scenario, the LCMV-infected, highly inflamed auxiliary site may the act as a filter for auto-aggressive lymphocytes (Christen and von Herrath 2005). Summary Neutralizing antibodies have been widely used to interfere with receptor-ligand interactions in vivo. Blockade of soluble inflammatory mediators and/or their cellular receptors is a highly effective way to down-regulate inflammation or prevent its negative consequences. In contrast to non-specific chemical immunomodulators or systemic treatment with cytokines, such as IFN-a, which act on a relatively broad range and thus can cause either unwanted severe side effects or general immunosuppression or -activation, neutralizing monoclonal antibodies act in a more specific way by preventing the binding of key mediators of inflammation to their cellular receptor. Nevertheless, long-term systemic administration of neutralizing antibodies inactivates the function of such mediators in their natural role in defense against harmful pathogens, such as viruses. It is, therefore, important to design a therapeutic regimen in way to reach maximal efficacy with a minimal side effects. This can only be achieved by a detailed knowledge of the mechanisms of action and the expression kinetics of such inflammatory mediators. In this review, we highlighted two recent findings from our lab that demonstrate that short term neutralization of inflammatory mediators can abrogate or reverse adverse effects of chronic inflammation. First, blockade of IL-10/IL-10R interaction can resolve chronic viral infection. Mechanistically, IL-10 neutralization restores the anti-viral immune response that was derailed by viral infection of a specific subgroup of APCs, namely the CD8a- DCs. Second, short-term neutralization of CXCL10 after in virus infection prevented the progression from inflammation to autoimmune disease in a mouse model for type 1 diabetes. In both examples, the precise timing and duration of administration of neutralizing antibodies was critical for success. Similarly, treatment of LCMV-infected RIP-LCMV mice with TNFR55-IgG1 fusion protein to neutralize TNFa was only successfully, when given early after infection (Christen et al. 2001). 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1838268107-PONE-RA-03133R110.1371/journal.pone.0001908Research ArticleComputational BiologyOncologyComputational Biology/GenomicsGenetics and Genomics/Cancer GeneticsGenetics and Genomics/Gene ExpressionGenetics and Genomics/Genetics of DiseaseGenetics and Genomics/GenomicsOncology/Breast CancerOncology/Oncology AgentsPharmacology/Drug ResistanceWomen's Health/Breast CancerAn Integrated Approach to the Prediction of Chemotherapeutic Response in Patients with Breast Cancer Chemotherapeutic ResponseSalter Kelly H. 1 Acharya Chaitanya R. 1 Walters Kelli S. 1 Redman Richard 1 2 Anguiano Ariel 1 2 Garman Katherine S. 1 2 Anders Carey K. 1 2 Mukherjee Sayan 1 3 Dressman Holly K. 1 Barry William T. 1 3 Marcom Kelly P. 2 Olson John 1 4 Nevins Joseph R. 1 Potti Anil 1 2 * 1 Duke Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America 2 Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America 3 Institute for Statistics and Decision Sciences, Duke University, Durham, North Carolina, United States of America 4 Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America Ouchi Toru EditorNorthwestern University, United States of America* E-mail: [email protected] and designed the experiments: JN JO AP KM KS. Performed the experiments: CA KS. Analyzed the data: SM HD AP CA KS KW WB. Contributed reagents/materials/analysis tools: SM HD JN JO AP CA WB. Wrote the paper: JN AP KG KM KS KW RR AA. Other: Designed the study: AP. 2008 2 4 2008 3 4 e190821 12 2007 22 2 2008 Salter et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background A major challenge in oncology is the selection of the most effective chemotherapeutic agents for individual patients, while the administration of ineffective chemotherapy increases mortality and decreases quality of life in cancer patients. This emphasizes the need to evaluate every patient's probability of responding to each chemotherapeutic agent and limiting the agents used to those most likely to be effective. Methods and Results Using gene expression data on the NCI-60 and corresponding drug sensitivity, mRNA and microRNA profiles were developed representing sensitivity to individual chemotherapeutic agents. The mRNA signatures were tested in an independent cohort of 133 breast cancer patients treated with the TFAC (paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide) chemotherapy regimen. To further dissect the biology of resistance, we applied signatures of oncogenic pathway activation and performed hierarchical clustering. We then used mRNA signatures of chemotherapy sensitivity to identify alternative therapeutics for patients resistant to TFAC. Profiles from mRNA and microRNA expression data represent distinct biologic mechanisms of resistance to common cytotoxic agents. The individual mRNA signatures were validated in an independent dataset of breast tumors (P = 0.002, NPV = 82%). When the accuracy of the signatures was analyzed based on molecular variables, the predictive ability was found to be greater in basal-like than non basal-like patients (P = 0.03 and P = 0.06). Samples from patients with co-activated Myc and E2F represented the cohort with the lowest percentage (8%) of responders. Using mRNA signatures of sensitivity to other cytotoxic agents, we predict that TFAC non-responders are more likely to be sensitive to docetaxel (P = 0.04), representing a viable alternative therapy. Conclusions Our results suggest that the optimal strategy for chemotherapy sensitivity prediction integrates molecular variables such as ER and HER2 status with corresponding microRNA and mRNA expression profiles. Importantly, we also present evidence to support the concept that analysis of molecular variables can present a rational strategy to identifying alternative therapeutic opportunities. ==== Body Introduction One of the major challenges facing the field of oncology is the selection of the most effective chemotherapy agents for individual patients. While steps have been taken towards using biomarkers to select patients eligible to receive certain targeted therapies, selection of the more common cytotoxic agents remains largely arbitrary [1]. For example, patients with breast cancer may be given the “TFAC” (paclitaxel (T), 5-fluorouracil (F), adriamycin (A), and cyclophosphamide(C)) treatment regimen, DC (docetaxel and cyclophosphamide) treatment, or AC (adriamycin and cyclophosphamide) treatment in the neo-adjuvant setting, with little guidance as to which will actually be most effective for their particular disease. Furthermore, the administration of ineffective chemotherapy agents increases the probability of side effects and decreases the quality of life of many cancer patients[2], [3], which further emphasizes the need to develop strategies that evaluate each individual patient's probability of responding to commonly used chemotherapeutic agents and limiting the agents used to those most likely to be effective. Recent advances in our understanding of cancer biology have offered a potential approach to meeting this challenge by using gene expression signatures of sensitive or resistant cell lines to predict patient response to a panel of commonly used chemotherapy agents [4], [5], [6], [7]. These initial gene signatures, however, were created using U95 Av2 Affymetrix gene array chips, while in recent months the volume of usable data has shifted to include the more comprehensive U133 gene expression platform. Thus, U133A expression data from the NCI-60 panel of cell lines, chosen using identical approaches as previously described[4], was used to develop robust, refined gene expression signatures of chemosensitivity. Using an independent dataset (n = 133) of breast cancer tumor samples from patients treated neoadjuvantly with combination (TFAC) chemotherapy, we validated the performance of the individual predictors of sensitivity to paclitaxel (T), 5-fluorouracil (F), adriamycin (A), and cyclophosphamide (C), as well as a combined predictor of TFAC sensitivity. We also use microRNA data from the NCI-60 cell lines to develop microRNA gene expression profiles of chemosensitivity, in an attempt to understand the biologic interplay between relevant microRNA expression in conjunction with the corresponding messenger RNA data, which could potentially refine the predictive ability of gene signatures. Finally, we use signatures of deregulated oncogenic signaling pathways in breast tumors to develop a strategy that identifies opportunities for other novel therapeutic drugs in patients resistant to chemotherapy, using the cohort of patients treated with TFAC as an example. Results mRNA signatures of paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide sensitivity Using identical cell lines identified previously as representing the extremes of sensitivity, gene signatures representative of sensitivity to paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide were developed. The leave-one-out cross-validation probabilities for each chemotherapy sensitivity signature showed separation of sensitive and resistant cell lines with an accuracy always exceeding 95% (Figure S1). The heatmaps (Fig 1a) of the probability values of the top genes used for prediction showed a clear demarcation between sensitive and resistant cell lines and the genes that were upregulated or downregulated in each. 10.1371/journal.pone.0001908.g001Figure 1 mRNA (a) and miRNA (b) gene signatures of sensitivity to paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide. Gene number is displayed on the vertical axes, while sample number is listed horizontally. The gene ontology of the genes used in each predictor, as discovered by performing a Batch Query on the Affymetrix website (www.affymetrix.com), included a plethora of genes and pathways thought to be important in cancer pharmacology and biology (Table S1). Genes used in predictors included those key in cell cycle regulation, signal recognition, tumor necrosis factor pathways, and growth arrest. Of note, the paclitaxel sensitivity signature included the Jun proto-oncogene, while the adriamycin signature included multiple members of the Ras-associated domain family as well as the epidermal growth factor receptor (EGFR) gene. The 5-fluorouracil signature included genes for transcription regulation and a cyclin-dependent kinase inhibitor (CDKN2A) thought to play a role in cancer progression, while the cyclophosphamide signature included genes (e.g. SET) commonly involved in leukemogenesis. Together, the ontology of the genes that constitute the four signatures reaffirms our confidence in their predictive ability. MicroRNA profiles characterize biology underlying chemotherapeutic resistance As a further step to advance our understanding of gene expression phenotypes of chemotherapeutic sensitivity and to begin to understand the role of microRNAs in predicting chemosensitivity, we also made use of relevant microRNA data from the individual NCI-60 cell lines. The heatmaps representing the microRNA chemosensitivity profiles showed demarcations between chemosensitive and resistant cell lines similar to the mRNA predictors (Figure 1b). Of particular interest is the fact that many of the individual microRNAs implicated in the individual profiles of chemosensitivity have previously been shown to have altered expression in human tumors (Table S2). Interestingly, miR-34, which was recently reported to mimic TP53 activity[8], [9], [10], [11], [12], was downregulated in the sensitive cell lines in the 5-fluorouracil and paclitaxel microRNA signatures, indicating a possible connection between chemosensitivity to those drugs and the well-known TP53 tumor suppressor network. The fact that miR-34 was downregulated in cell lines sensitive to paclitaxel was surprising, as mutations in the TP53 pathway are thought to confer resistance to taxanes[13]. This finding suggests that, while the inhibitory pathways of TP53 include abnormal tubulin detection and subsequent cell cycle arrest, miR-34 follows a separate path toward tumor suppression. In addition, miR-17 was downregulated in cell lines sensitive to adriamycin and cyclophosphamide, drugs commonly used in the treatment of lymphoproliferative disorders. The downregulation of miR-17 may represent an explanation for the effectiveness of these drugs in specific hematologic malignancies that are dependent on Myc pathway deregulation for their pathogenesis (e.g. chronic lymphocytic leukemia and aggressive lymphomas), since miR-17 functions synergistically with Myc to promote aggressive tumor growth in lymphoma[14]. Another microRNA thought to be involved with the Myc pathway, let-7, was implicated in the cyclophosphamide sensitivity signature in our analysis. Further, miR-let-7a, which has been shown to repress expression of the Myc transcription factor[15], was found to be downregulated in the cell lines sensitive to cyclophosphamide, an alkylating agent. These findings would imply that, if let-7 is downregulated in sensitive cell lines, Myc is upregulated, but the resultant proliferation is tempered by the repressive action of cyclophosphamide on cell replication. Lastly, miR-200b, which when inhibited confers sensitivity to chemotherapy in malignant cholangiocytes[16], was also found in our signature to be downregulated in cells sensitive to adriamycin, the current standard of care in the treatment for hepatobiliary tumors. MiR-200b suppresses a tumor suppressor gene (PTPN12) which is thought to inactivate the common oncogenes Src and Ras[16], such that the downregulation of miR-200b in sensitive cell lines allows the resultant proliferating cells to be targeted by adriamycin, a Topoisomerase II inhibitor. These findings suggest that, when utilized in an integrated approach, microRNA expression profiles in conjunction with corresponding gene expression data may provide a critical link for understanding mechanisms involved in chemosensitivity and chemoresistance. Validation of mRNA predictors in independent patient samples The true clinical value of gene expression signatures lies in their ability to predict response in large patient populations. Using previously developed methods to identify cell lines from the NCI-60 that represent the extremes of drug sensitivity and the corresponding updated Affymetrix U133A data, we developed genomic predictors of TFAC sensitivity [4], [6]. This study thus represents a refined predictive ability due to the use of U133A as opposed to the U95 data previously used. An independent dataset of 133 breast cancer tumor samples was used to evaluate the predictive ability of the U133A T, F, A, and C classifiers. When the predictive probability values of non-responders and responders to each individual chemotherapy agent were plotted, the median probability of sensitivity of the responders was higher than that of the non-responders (Figure 2) in each case. The probability of 5-fluorouracil (p = 0.02), adriamycin (p = 0.01), and cyclophosphamide (p = 0.02) sensitivity were all significantly different between responders and non-responders in a Mann-Whitney U test, and the paclitaxel probability values showed a predictive trend, although not statistically significant (p = 0.07). Importantly, when the combined TFAC probability was plotted, the probability of sensitivity in the responders (n = 34, Figure 2) was significantly higher than the probability of sensitivity of the non-responders (n = 99, p = 0.002, Mann-Whitney). The sensitivity of the combined predictor was 59%, while the specificity was 63%. Importantly, the negative predictive value, which is arguably the most relevant in a clinical scenario, as it should implicate patients who will not benefit from the treatment in question and should therefore be considered for other therapeutic options, was found to be 82%. 10.1371/journal.pone.0001908.g002Figure 2 Development and validation of chemotherapeutic response predictors. (a) Strategy for generating the chemotherapeutic response predictors. (b) Prediction of single-agent chemotherapy response in patient breast samples. Probability values of non-responders (NR) are shown in red, while probability values of responders (R) are shown in blue. Response was defined as complete pathologic response upon completion of TFAC neoadjuvant therapy. (c) Combined prediction of sensitivity to the TFAC chemotherapy regimen separated by non-responders (n = 99, red) and responders (n = 34, blue). Effect of molecular variables on predicted chemosensitivity patterns Because molecular variables such as ER, PR, and HER2 status, as well as Topoisomerase IIA expression, have been shown to have an effect on chemotherapy sensitivity[17], [18], we tested the effect of these molecular variables on the ability of gene signatures to accurately identify patients sensitive to TFAC therapy. While the prediction was significantly more robust in ER and HER negative patients, PR status had no major effect on TFAC prediction (Figure 3). When we separated the patients based on ‘basal-like’ (ER, HER, and PR negative) or ‘non basal-like’ status, the predictive ability of the TFAC combined signature was significantly greater in the basal-like patients (Figure 3). It may be important to note that the basal-like patients also showed a much higher likelihood of response (48%) as opposed to the non basal-like patients (19%), as might be expected based on previous findings reported in the literature [18]. 10.1371/journal.pone.0001908.g003Figure 3 Effect of molecular variables on combined TFAC prediction. Left, TFAC probability values of basal-like (HER2, ER, and PR negative) and non basal-like patients as separated by non-responders (NR) and responders (R). Right, TFAC probability values of non-responders and responders separated by ER score less than or greater than 50, PR status, and HER2 status. Likewise, because adriamycin is known to be a Topoisomerase IIA inhibitor, and HER2 status has been linked to Topoisomerase IIA expression[19], we analyzed the effects of HER2 status and Topoisomerase IIA expression independently on the predictive ability of the adriamycin sensitivity signature in the 133 patients. HER2 status seemed to have no major effect on adriamycin prediction, as both HER2 positive and negative groups of patients showed a significant difference between the medians of the non-responders and responders (Figure 4). Topoisomerase IIA expression, however, had a major effect on adriamycin prediction. When samples were divided into those with Topoisomerase IIA expression above and below the median value for each probe, the predictor for adriamycin sensitivity was significantly more robust in the samples with high Topoisomerase IIA expression as compared to low Topoisomerase IIA expression (p values of 0.0021 and 0.0022 versus 0.49 and 0.46, respectively, Figure 4). Thus, it is likely that stratifying patients first by Topoisomerase IIA expression before the application of genomic predictors of chemotherapeutic response may improve the ability of the classifiers in predicting clinically significant response to adriamycin. 10.1371/journal.pone.0001908.g004Figure 4 Effect of HER2 status and Topoisomerase IIA expression levels on adriamycin prediction. Left, patients were divided depending on HER2 negative or positive status, and predictive probability values of sensitivity to adriamycin were plotted for non-responders (NR) and responders (R). Right, patients were divided on the basis of whether their expression of Topoisomerase IIA (obtained using two different probes, 201291_s_at and 201292_at, in the U133A platform) was above or below the median value. Non-responders and responders were separated and their predictive probability values plotted. More broadly, this indicates a potential strategy to refine the predictive ability of gene expression-based signatures of chemosensitivity in breast cancer by first identifying cohorts based on relevant tumor biology such as ER, HER2 and Topoisomerase IIA expression. Patterns of oncogenic pathway activation and alternative chemotherapeutic options in patients resistant to standard chemotherapy The ability to accurately identify patients resistant to standard chemotherapy also emphasizes the need to dissect cancer biology further and identify alternative therapeutic strategies for patients resistant to therapy. To this end, we employed gene signatures representative of E2F, PI3K, Myc, β-catenin, Src, and Ras pathway activation, as well as signatures of response to other commonly used cytotoxic agents in breast cancer (docetaxel, etoposide, vinorelbine and cisplatin), in the cohort of 133 patients. Interestingly, none of the oncogenic signaling pathways alone were associated with the likelihood of response to TFAC therapy (Figure S2). While the knowledge gained from investigating individual pathways is sometimes critical in elucidating clinically relevant biology, breast cancer represents a very heterogeneous disease, the complexity of which may only be dissected by evaluating the oncogenic cooperation that exists between different signaling pathways. An approach to evaluating patterns of oncogenic cooperation between pathways is to use clustering strategies to demonstrate meaningful interactions between biologically relevant mechanisms. As shown in Figure 5, unsupervised hierarchical clustering of the oncogenic pathways E2F, PI3K, Myc, β-catenin, Src, and Ras in the cohort of 133 patients demonstrated significant, biologically relevant patterns of pathway activation. Importantly, analysis comparing the percent of responders in each cluster showed that two clusters had significantly higher or lower responder percentages (Figure 5). Whereas the entire population of patients was comprised of 25% responders, Cluster 3 had a much higher percentage of responders at 54%, and Cluster 4 had a lower population of responders at only 8%. Clusters 1, 2, and 5 had numbers of responders not significantly different from the entire population. A t-test between Clusters 3 and 4 showed that they were significantly different (p = 0.003, Mann-Whitney). In the interest of examining the two extremes of sensitivity, Clusters 3 and 4 were compared for further analyses. While Cluster 3, with the higher percentage of responders, seemed to show predominant activation of the Myc and Ras pathways, Cluster 4 showed activation of the Myc and E2F pathways. Therefore, we can hypothesize that E2F activation, in combination with Myc as opposed to Ras activation, may confer resistance to TFAC chemotherapy in patients with breast cancer. 10.1371/journal.pone.0001908.g005Figure 5 Patterns of predicted oncogenic pathway activation and alternative chemotherapeutic options in human breast cancer tumors. Above, hierarchical clustering of a collection of breast tumors (n = 133) according to patterns of oncogenic pathway activation. Below, predictions of sensitivity to other commonly used chemotherapeutic agents in breast tumors. Predictions were plotted as heatmaps in which a high probability of sensitivity (or response) is indicated by red, and low probability (or resistance) is indicated in blue. The percentage of responders in each cluster is reported. Further, to identify alternative chemotherapeutic options in patients resistant to TFAC therapy (Cluster 4), predictive probabilities of sensitivity to cisplatin, docetaxel, etoposide, and vinorelbine (Figure 5), all agents commonly used to treat breast cancer, were generated using U133A data. Interestingly, the most distinctive characteristic of Cluster 4 was the high probability of sensitivity to docetaxel (Figure 5). Importantly, a predictor of docetaxel sensitivity was also validated (p = 0.02, Figure S3) in an independent dataset of 24 patients treated neoadjuvantly with the single agent docetaxel[20]. This provided us with the opportunity to compare a robust predictor of docetaxel sensitivity across the current dataset of 133 patients that were clustered based on their oncogenic pathway activation status. When compared to patients in other clusters, the non-responders in Cluster 4 had a significantly greater probability of responding to docetaxel (P<0.001, Figure S4). Furthermore, when the probability of sensitivity to docetaxel was plotted against E2F activation in non-responders to chemotherapy, a trend towards a positive correlation was observed (P = 0.07, Figure S4). This implies that patients with an activated E2F pathway that are resistant to TFAC may benefit from the alternative treatment of docetaxel. Previous studies[21] showed that head and neck squamous cell carcinoma samples that were resistant to cisplatin and sensitive to docetaxel (similar to our Cluster 4 tumor samples) showed decreased E2F activation after treatment with docetaxel, which further supports the argument that the subset of patients in Cluster 4 may benefit from alternative treatment with docetaxel. The finding of the relationship between E2F and docetaxel is interesting biologically and emphasizes the importance of a validated predictor of docetaxel sensitivity in patients with early stage breast cancer. To this extent, we have employed independent data from Chang J et al [20], a study involving the neoadjuvant use of single agent docetaxel in patients with breast cancer. As shown in Figure S3, a U133A predictor of docetaxel (Table S3) accurately separates responders from non-responders (P = 0.02, sensitivity: 90%, specificity: 57%, positive predictive value: 60%, negative predictive value: 89%, Figure S3). It is important to emphasize that the results shown here represent an example of the complexity involved in defining phenotype of chemosensitive or chemoresistant disease. Knowledge of clinico-pathologic variables likely to affect an individual patient's response to a particular drug or regimen will only lead to the application of gene signatures in a more effective and refined matter. Discussion The selection of the right chemotherapeutic agent for individual cancer patients remains a challenge. Chemotherapy-associated morbidity is a major concern, and strategies to limit therapies used to those that are most likely to be effective have the potential to change the current paradigm of cancer treatment. The results we present here provide further evidence that the use of gene expression data to predict chemotherapy response and oncogenic pathway activation may assist in selecting therapeutics on a patient-by-patient basis. While the cell lines used and methods described are identical to previously validated signatures of sensitivity, we believe that the development of robust U133A-platform gene expression signatures of chemosensitivity may be a very useful tool in further validation strategies and eventually in guiding patient treatment. Importantly, the paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide signatures developed showed clinically relevant predictive ability in a population of human breast tumor samples obtained from patients treated neoadjuvantly with TFAC therapy. Beyond the development of U133A mRNA signatures for chemosensitivity, we also present data to suggest that microRNA expression may be useful in further dissecting the phenomenon of chemotherapy resistance and in predicting patterns of sensitivity. This finding is not very surprising, given the fact that microRNAs can function as either tumor suppressors or as oncogenes, and that altered microRNA levels are found in various human cancers [22], [23]. Notably, although preliminary, these profiles are the first to use microRNA data to constitute signatures of chemosensitivity and resistance. Thus, pending further validation, our results present preliminary data supporting the hypothesis that microRNA signatures may be used to complement mRNA gene expression signatures of chemosensitivity. While the chemotherapy sensitivity signatures described are useful for predicting which patients will respond to a specific regimen (TFAC), used alone they do not address the important issue of alternative therapeutic options for patients who would not respond to TFAC. Here, we use TFAC as a representative example of the bigger challenge in oncology with respect to chemotherapy, the issue of viable options for non-responders to traditional chemotherapeutic regimens. For this reason, we used signatures of oncogenic pathway activation[24] as well as signatures for other commonly used chemotherapy agents in breast cancer (cisplatin, docetaxel, etoposide, and vinca alkaloids) to identify potentially activated pathways that might be future targets for therapy, while also suggesting alternative chemotherapeutic options. Our data suggest that, in the case of Cluster 4 (which had the lowest percentage of responders) the activated E2F pathway may be driving chemoresistance, and alternative treatment with a regimen including docetaxel may have resulted in an increased number of responders. Importantly, the oncogenic pathway activation signatures employed were unable alone to predict responder status of patients, but combined with alternative chemotherapeutic signatures, they suggest a cohort of patients to that may benefit from alternative treatment. While our body of data represents a total of 157 patients, additional studies will be needed to confirm the trends observed, especially the benefit of docetaxel treatment in E2F-activated breast cancer cell lines resistant to other commonly used agents. In conclusion, the reported chemotherapy sensitivity signatures can be used effectively in the prediction of response, while other predictors of oncogenic pathway activation [24] and even tumor microenvironment should add information that further improves our understanding of cancer biology while also leading to alternative therapeutics for predicted non-responders. Importantly, relevant molecular or pathologic information may aid our understanding of chemotherapy resistance and sensitivity in breast cancer. It is likely that the most optimal approach to prediction of response to cytotoxic therapy will involve a combination of gene expression data, microRNA profiles, and molecular variables such as ER, HER2, and PR status. The goal is to develop a strategy to determine a personalized treatment approach to breast cancer, so that each individual patient will have a better chance of a favorable outcome by matching specific treatment options to their molecular profiles. Methods Development of mRNA and microRNA signatures The NCI-60 cell lines that were most resistant or sensitive to each chemotherapy agent were identified as previously described[4], and the Affymetrix HG-U133A based NCI-60 cell line expression data was obtained from the National Institute of Health (courtesy John Weinstein). Mas5 expression values were log2 transformed, and class labels (zero or one) were assigned to sensitive and resistant cell lines. The predictors were optimized for performance by applying multiple t-tests with a cut-off probability value of 0.01 using statistical package R[25] (version 2.5.1). Using methods previously described[4], [24], top gene probe identifiers that best separate sensitive from resistant cell lines were identified. Leave-one-out cross-validation, in which each cell line is removed from the signature one-by-one and its predictive probability value generated using the remaining cell lines, was used to assess the accurate and robust nature of each individual predictor. As a proof-of-concept approach to combining probabilities, the TFAC predictor was generated by taking the mean of the mean-centered probability values generated by the four individual chemotherapy sensitivity signatures. The same NCI-60 cell lines selected for the Affymetrix HG-U133A-based mRNA signatures were used to develop microRNA signatures of chemotherapy sensitivity and resistance. MicroRNA pin-spotted expression data was downloaded from the Cell Miner database in .gpr format. Using GeneSpring (Agilent), the signal intensity of each spot was calculated by subtracting local background (based on the median intensity of the area surrounding each spot) from the median signal. As a means of nonspecific filtering, raw values used were limited to those between 8 and 65. Signal intensities were log2 transformed and duplicate spots were averaged. Quantile normalization was performed across the 3 microarray batches, all values <5 were replaced with the median of such values, and the value for each control cell line was set to the mean of its five replicates. All control and mouse probes were removed, and only the resulting 273 human microRNA probes were used in remaining analyses. Bayesian binary regression performed in MatLab identified the top gene probe identifiers used in separating resistant from sensitive cell lines, and those probes are reported in Table S2. Patients and Samples A validation set of 133 human breast cancer samples was obtained from the MD Anderson Cancer Center website (http://bioinformatics.mdanderson.org/pubdata.html). This dataset was selected due to its clinically annotated data (Affymetrix Human Genome U133A) for patients treated with neoadjuvant chemotherapy, an ideal setting to evaluate the efficacy of gene expression-based predictors of response. All patients were treated with 24 weeks of sequential paclitaxel and fluorouracil-adriamycin-cyclophosphamide neoadjuvant chemotherapy, except two patients who were treated with either only paclitaxel or a combination of paclitaxel, 5-fluorouracil, epirubicin, and cyclophosphamide[26]. The clinical data designated patients as either non-responders (n = 99) or responders (n = 34) on the basis of pathologic complete response; in other words, responders showed no signs of residual invasive cancer in the breast or lymph nodes at the time of surgery. HER2 status, ER status (based on ER score), and PR status were obtained from the clinical data provided on the M.D. Anderson website and used in the analysis of the effect of molecular variables on chemotherapy sensitivity prediction. ‘Basal-like’ patients were defined as patients with negative ER, HER2, and PR status. Topoisomerase IIA expression was obtained from the log2-transformed expression data from two representative probes, 201281_s_at and 201292_at, and divided into values above and below the median. For the docetaxel validation, an independent dataset of 24 samples treated neoadjuvantly with docetaxel was used[20]. Application of oncogenic pathway activation and alternative chemotherapeutic agent signatures Oncogenic pathway signatures described previously[24] were used to create predictive probability values for activation of the β-catenin, E2F, Myc, PI3K, Ras, and Src pathways. The pathway signatures, combined with the validation set of breast tumor samples, were standardized using MatLab (version 7.1). Bayesian probit binary regression analysis was then performed on the datasets, and the resultant probability values generated. Unsupervised hierarchical clustering was performed using complete linkage clustering with Pearson correlation coefficient in the Gene Pattern[27](Hierarchical Clustering module, version 3.0) software package, and a heatmap was then generated. Clusters were assigned by visualization of the corresponding dendrogram, and the percent of responders in each cluster were calculated. Genomic signatures for docetaxel, etoposide, cisplatin, and a vinca alkaloid (a surrogate for vinorelbine) were applied to the breast tumor validation set to identify alternative chemotherapeutic options for non-responders to TFAC. The signatures for docetaxel and etoposide were developed as described above for the individual TFAC predictors. Briefly, cell lines selected from the NCI-60 for the Affymetrix HG-U95 based Av2 predictors[4] were used in the HG-U133A-based docetaxel and etoposide predictors, multiple t-tests were applied on the dataset using statistical package R (version 2.5.1), and the binary regression parameters were adjusted to achieve optimal cross-validation accuracy. The signatures for cisplatin and vinca alkaloid were developed using a set of 30 human cancer cell lines[28]. Resultant predictive probability values were displayed as a heatmap using HeatmapViewer module of the GenePattern software based on patient sample order obtained earlier from the unsupervised hierarchical clustering of the oncogenic pathway activation probability values. Clusters previously defined based on the patterns of oncogenic pathway activation were analyzed to assess whether alternative chemotherapeutic options were likely to be effective for specific patient clusters predicted to be predominantly resistant to the original chemotherapy agent. Supporting Information Figure S1 mRNA gene signatures of sensitivity to paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide. Heatmaps, with gene number on the horizontal axis and sample number on the vertical axis, are shown above. Below, leave-one-out cross-validation of samples selected to represent resistance (blue) and sensitivity (red) to each chemotherapeutic agent. (4.86 MB TIF) Click here for additional data file. Figure S2 Individual oncogenic pathway signatures as applied to the cohort (n = 133) of breast cancer patients. Non-responders and responders were separated and their predictive probability values plotted. (2.05 MB TIF) Click here for additional data file. Figure S3 Validation of a U133A predictor of docetaxel sensitivity in an independent dataset of 24 samples from patients treated neoadjuvantly with single agent docetaxel (1.21 MB TIF) Click here for additional data file. Figure S4 Docetaxel sensitivity and E2F activation in TFAC non-responders. The predicted probability of sensitivity to docetaxel of the TFAC non-responders as divided by individual clusters (Panel A) and Cluster 4 compared with the other clusters (Panel B). Panel C shows a linear regression analysis of the probability of sensitivity to docetaxel plotted against the E2F oncogenic pathway activation. (2.37 MB TIF) Click here for additional data file. Table S1 Ontology of genes used in the TFAC chemotherapy sensitivity signatures. (0.57 MB DOC) Click here for additional data file. Table S2 Ontology of miRNA genes used in chemotherapy sensitivity signatures. (0.07 MB DOC) Click here for additional data file. Table S3 Ontology of genes used in the docetaxel sensitivity signature. (0.21 MB DOC) Click here for additional data file. The authors thank the research support from the Jimmy V foundation and the Emilene Brown Cancer Research Fund. Competing Interests: The authors have declared that no competing interests exist. Funding: The current work is supported by research grants from the Department of Defense (CDMRP) and the Jimmy V. Foundation. ==== Refs References 1 Staunton JE Slonim DK Coller HA Tamayo P Angelo MJ 2001 Chemosensitivity prediction by transcriptional profiling. Proc Natl Acad Sci U S A 98 10787 19792 11553813 2 Herbst RS Bajorin DF Bleiberg H Blum D Hao D 2006 Clinical Cancer Advances 2005: major research advances in cancer treatment, prevention, and screening–a report from the American Society of Clinical Oncology. 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==== Front Br J Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 6602837 10.1038/sj.bjc.6602837 16234815 Molecular Diagnostics DNA methylation-associated inactivation of TGFβ-related genes DRM/Gremlin, RUNX3, and HPP1 in human cancers Suzuki M 123* Shigematsu H 13 Shames D S 1 Sunaga N 1 Takahashi T 1 Shivapurkar N 1 Iizasa T 2 Frenkel E P 1 Minna J D 1 Fujisawa T 2 Gazdar A F 1 1 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Bld NB, Room 8206, 6000 Harry Hines Blvd., Dallas, TX 75390, USA 2 Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan * Author for correspondence: [email protected] 3 These two authors contributed equally to this work. 18 10 2005 25 10 2005 31 10 2005 93 9 10291037 10 05 2005 14 09 2005 20 09 2005 Copyright 2005, Cancer Research UK 2005 Cancer Research UK The transforming growth factor β (TGFβ)-signalling pathway is deregulated in many cancers. We examined the role of gene silencing via aberrant methylation of DRM/Gremlin and HPP1, which inhibit TGFβ signalling, and RUNX3, which facilitates TGFβ-signalling, of all genes that are thought to be tumour suppressors, are aberrantly expressed, and are thus thought to have important role in human cancers. We examined DRM/Gremlin mRNA expression in 44 cell lines and the promoter methylation status of DRM/Gremlin, HPP1, and RUNX3 in 44 cell lines and 511 primary tumours. The loss of DRM/Gremlin mRNA expression in human cancer cell lines is associated with DNA methylation, and treatment with the methylation inhibitor-reactivated mRNA expression (n=13). Methylation percentages of the three genes ranged from 0–83% in adult tumours and 0–50% in paediatric tumours. Methylation of DRM/Gremlin was more frequent in lung tumours in smokers, and methylation of all three genes was inversely correlated with prognosis in patients with bladder or prostate cancer. Our results provide strong evidence that these TGFβ-related genes are frequently deregulated through aberrant methylation in many human malignancies. DRM/Gremlin (CKTSF1B1) HPP1 RUNX3 TGFβ methylation ==== Body The transforming growth factor β (TGFβ) superfamily of paracrine and autocrine signalling molecules regulates many intra- and extracellular functions, including development, proliferation, differentiation, extracellular matrix and bone formation, angiogenesis, and immune responses (Balemans and Van Hul, 2002; Gumienny and Padgett, 2002). The TGFβ family includes TGFβs, bone morphogenetic proteins (BMPs), activins, and several other subfamilies. Transforming growth factor β family members and their receptors are expressed by many types of normal and malignant cells. Both upregulation and downregulation of TGFβ family member signalling may occur in cancer cells during different stages of pathogenesis (de Caestecker et al, 2000; Teicher, 2001; Roberts and Wakefield, 2003). Downregulation frequently occurs early in tumour development and is associated with increased epithelial growth and inhibition of apoptosis, while upregulation is more frequent during later stages and is associated with increased angiogenesis, stromal remodelling, suppression of immune responses, and metastatic spread. These cellular changes may occur via deregulation of interaction with Smad proteins at the nuclear level, via increased secretion of ligands by tumour cells, and/or by inactivation of ligands by soluble or intracellular inhibitors (de Caestecker et al, 2000; Teicher, 2001; Roberts and Wakefield, 2003). A large number of soluble inhibitors of TGFβ family members have been identified (Gumienny and Padgett, 2002). The DRM/Gremlin (CKTSF1B1) gene, a member of the Cerberus/Dan family of BMP-soluble antagonists (Pearce et al, 1999), was independently isolated by two groups (Topol et al, 1997; Hsu et al, 1998). Topol et al (1997) isolated Drm from a rat model in which they demonstrated that transfection of Drm induced apoptosis and inhibited growth in rat fibroblasts. Hsu et al (1998) isolated Gremlin in Xenopus and demonstrated that it was a secreted protein that functioned as a BMP antagonist. DRM/Gremlin has been reported to influence BMP2-associated signalling pathways stimulated by fibroblast growth factor (Zuniga et al, 1999) and platelet-derived growth factor (PDGF) (Ghosh Choudhury et al, 1999), and it also negatively modulates embryonic lung morphogenesis (Shi et al, 2001). Although the importance of DRM/Gremlin has been demonstrated during development and in the pathogenesis of nephropathy (Zuniga et al, 1999; McMahon et al, 2000; Khokha et al, 2003), its role in cancer pathogenesis is poorly understood. Topol et al (2000) recently mapped the human homologue of DRM/Gremlin to chromosome 15q13–15 and demonstrated that DRM/Gremlin mRNA expression is downregulated in several human tumour types. These researchers also found that the DRM/Gremlin transcript is normally expressed only in healthy breast epithelium. While these findings suggest that DRM/Gremlin is a tumour suppressor gene (TSG), how it is silenced in cancer cells is not known. The HPP1 (TMEFF2) gene belongs to another, possibly unique, class of TGFβ antagonists. HPP1 is a transmembrane receptor containing two follistatin modules and a single epidermal growth factor (EGF)-like domain (Uchida et al, 1999). Follistatin, a secreted soluble inhibitor, binds and neutralises the activity of many TGFβ family members, including BMPs and activins, as well as PDGF and vascular endothelial growth factor (Patel, 1998; Lin et al, 2003). The EGF-like domain in HPP1 appears to be a ligand for c-erbB-4 (Uchida et al, 1999). Recently, Gery et al (2002) demonstrated that HPP1 exhibits antiproliferative effects in prostate cancer cell lines . These researchers also demonstrated an inverse correlation between HPP1 activity in prostate cancer xenografts and c-Myc expression (Gery and Koeffler, 2003). Two soluble forms of HPP1 protein that differ in the presence/absence of the EGF-like domain arise by proteolytic cleavage (Uchida et al, 1999). Currently, it is not known which isoforms of HPP1 are responsible for its tumour suppressor function. HPP1 maps to chromosome 2q32.3, where loss of heterozygosity (LOH) frequently occurs in a number of tumours types, including lung cancer and breast cancer (Otsuka et al, 1996; Huiping et al, 1999). RUNX3 is a Runt domain transcription factor that interacts with Smad proteins, suggesting that it may play an important role in TGFβ signalling. This gene is a candidate TSG localised to 1p36, a region commonly deleted in a wide variety of human cancers, including lung cancer and breast cancer (Ragnarsson et al, 1999; Girard et al, 2000). DNA methylation in the 5′ region is emerging as the primary mechanism of TSG inactivation (Jones and Baylin, 2002; Suzuki et al, 2004). Aberrant methylation of the HPP1 and RUNX3 genes has been demonstrated in gastrointestinal and other human tumours (Liang et al, 2000; Young et al, 2001; Guo et al, 2002; Li et al, 2002; Sato et al, 2002; Shibata et al, 2002; Kato et al, 2003; Xiao and Liu, 2004). Using a microarray strategy, we recently identified DRM/Gremlin as a gene that was differentially expressed in a non-small-cell lung carcinoma (NSCLC) cell line after treatment with a demethylating agent (5-aza-2′-deoxycytidine (5-Aza-CdR)). Interestingly, lung cancer cell lines frequently demonstrate LOH at this gene location (Girard et al, 2000). In this study, we examined mRNA expression and methylation status of DRM/Gremlin in lung cancer, breast cancer, and malignant mesothelioma (MM) cell lines, as well as the methylation status of DRM/Gremlin, HPP1, and RUNX3 in several primary malignant tumours. MATERIALS AND METHODS Cell lines and tumour samples In all, 28 lung cancer cell lines (15 NSCLC cell lines and 13 small-cell lung cancer (SCLC)] cell lines), 10 breast cancer cell lines, and six MM cell lines that were established by our group (Phelps et al, 1996; Gazdar et al, 1998) and deposited in the American Type Culture Collection (Manassas, VA, USA), were used in this study. Cell cultures were grown in RPMI-1640 medium (Life Technologies Inc., Rockville, MD, USA) supplemented with 5% fetal bovine serum and incubated in 5% CO2 at 37°C. Cell lines established at the National Cancer Institute have the prefix NCI while those established at UT Southwestern Medical Center have the prefix HCC. Normal bronchial epithelial cells (NHBEC), normal mammary epithelial cells (NHMEC), and normal mesothelial cells (NMC) were cultured as reported previously (Suzuki et al, 2005), and normal trachea RNA was obtained from Clontech (Palo Alto, CA, USA). In all, 13 tumour cell lines with DRM/Gremlin methylation and lack of DRM/Gremlin gene expression were incubated in culture medium with 4 μM (5-Aza-CdR) for 6 days, with medium changes on days 1, 3, and 5. Cells were harvested and RNA was extracted on day 6. Primary lung tumours were obtained from the Chiba University Hospital, Japan, and other tumours were obtained from the hospital system of the University of Texas Southwestern Medical Center, after obtaining Institutional Review Board approval and signed informed consent. Samples were immediately frozen and stored at −80°C until use. Reverse transcriptase–PCR (RT–PCR) assay for DRM/Gremlin An RT–PCR assay was used to examine DRM/Gremlin mRNA expression. Total RNA was extracted from samples with Trizol (Life Technologies, Rockville, MD, USA) following the manufacturer's instructions. The RT reaction was performed on 4 μg total RNA with Deoxyribonuclease I and the SuperScript II First-Strand Synthesis using the oligo(dT) primer System (Life Technologies), and aliquots of the reaction mixture were used for subsequent PCR amplification. Primer sequences for DRM/Gremlin amplification were: forward, 5′-ACTCAGCGCCACGCGTCGAAA-3′; reverse, 5′-ACTGAGTCTGCTCTGAGTCATT-3′ (GenBank accession number AC090877; forward, nucleotides 52619–52639; reverse, nucleotides 65324–65345), and we confirmed that genomic DNA was not amplified with these primers which cross an intron. The amplification programme for the DRM/Gremlin transcript was 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C for 40 cycles. The housekeeping gene GAPDH was used as an internal control to confirm the success of the RT reaction. Primer sequences for GAPDH amplification were: forward, 5′-CACTGGCGTCTTCACCACCATG-3′; reverse, 5′-GCTTCACCACCTTCTTGATGTCA-3′ (GenBank accession number NM_002046). These primer sequences were identical to the endogenous human target genes as confirmed by a BLAST search. PCR products were analysed on 2% agarose gels. Normal bronchial epithelial cells, NHMEC, NMC, and normal trachea were used as normal controls for RT–PCR. Map of the 5′ flanking region of DRM/Gremlin and bisulphite DNA sequencing The locations of CpG dinucleotides, the MSP amplicon (region of MSP (RMSP)), and the area that underwent bisulphite DNA sequencing (region of bisulphite sequencing (RBSSQ)) in the 5′ region of DRM/Gremlin are shown in Figure 3. Bisulphite-treated DNA was PCR-amplified using the following primers: forward, 5′-TGTGATTTGTTGTGTATTTTAGG-3′; reverse, 5′-ATAATTCTTCACAATTCACCCC-3′ (GenBank accession number AC090877, 52248–52821, 574 bp). These primers were designed to exclude binding to any CpG dinucleotides to ensure amplification of both methylated and unmethylated sequences. PCR products were cloned into plasmid vectors using the Topo TA cloning kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Five positive clones were purified from each test cell line using the Wizard Plus miniprep kit (Promega), and were then sequenced by the Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp., Foster City, CA). This region included the MSP primer sites and amplicon and encompassed 77 CpG dinucleotides. DNA extraction and methylation-specific PCR Genomic DNA was obtained from cell lines, primary tumours, and normal cells by digestion with proteinase K (Life Technologies), followed by phenol/chloroform (1 : 1) extraction (Suzuki et al, 2003). DNA methylation patterns in the CpG island of DRM/Gremlin were determined by the methylation-specific PCR (MSP) method as reported as described previously (Herman et al, 1996). Primer sequences of DRM/Gremlin for methylated reaction were as follows: forward, 5′-ATTTAAACGGGAGACGGCGCG-3′; reverse, 5′-GACCAAAACCGCCGAAACTCG-3′; those for the unmethylated reaction were: forward, 5′-ATTTAAATGGGAGATGGTGTG-3′; reverse, 5′-AACCAAAACCACCAAAACTCA-3′. Primer sequences for amplification of HPP1 and RUNX3 for MSP have been previously described (Li et al, 2002; Sato et al, 2002). Briefly, 1 μg genomic DNA was denatured by NaOH and modified by bisulphite. The modified DNA was purified using Wizard DNA purification kit (Promega), treated with NaOH to desulfonate, precipitated with ethanol and resuspended in water. PCR amplification was performed with bisulphite-treated DNA as a template using specific primer sequences for the methylated and unmethylated forms of the genes. DNA from peripheral blood lymphocytes (n=10) from healthy subjects (non-smoking) was used as negative controls for MSP assays. DNA from lymphocytes of a healthy volunteer treated with Sss1 methyltransferase (New England BioLabs, Beverly, MA) and then subjected to bisulphite treatment was used as a positive control for methylated alleles. Water blanks were included with each assay. Results were confirmed by repeating bisulphite treatment and MSP for all samples. Data analysis Statistical differences between groups were examined using Fisher's exact test, the chi-square test, and the Mann-Whitney test. Survival was calculated from the date of initial diagnosis until death or the date of the last follow-up (censored). Survival was analysed according to the Kaplan-Meier method, and differences in distribution were evaluated by means of the log-rank test. A P-value of less than 0.05 was defined as being statistically significant. RESULTS DRM/Gremlin mRNA expression in normal and malignant breast, lung, and mesothelial cells We used RT–PCR to examine the expression of DRM/Gremlin (Table 1); representative examples are shown in Figure 1A. DRM/Gremlin mRNA was detected in NHBEC, normal trachea, NHMEC, and NMC, indicating that this gene is normally expressed in respiratory cells, breast epithelial cells, and mesothelial cells. However, loss of DRM/Gremlin expression was observed in 12/15 (80%) of NSCLC cell lines, 5/13 (38%) of SCLC cell lines, 5/10 (50%) of breast cancer cell lines, and 4/6 (67%) of MM cell lines (Table 1 and Figure 2). Bisulphite genomic DNA sequencing of the 5′ region of DRM/Gremlin We sequenced bisulphite-treated DNA in the 5′ region of DRM/Gremlin to clarify any correlation between DNA methylation and gene silencing in various cancer cell lines. Using methylation-independent primers, we amplified and sequenced the 5′ region and exon 1 of the DRM/Gremlin gene (Figure 3). The 574-bp amplicon contains 77 CpG dinucleotides as well as exon 1. The translation start site is in exon 2. The G+C percentage is 74%, with a CpG ratio of 1. This region therefore satisfied the criteria for a CpG island (Gardiner-Garden and Frommer, 1987). Eight cell lines showed an excellent concordance between MSP and RT–PCR assay results and clonal sequencing results. Two normal tissues (normal lung and peripheral blood lymphocytes) also showed concordance between MSP assay and sequencing results. We developed MSP primers based on these sequencing results. DNA methylation of DRM/Gremlin in cell lines and tissues The DNA methylation status of DRM/Gremlin in cell lines as assessed by MSP assays are detailed in Table 1 and Figure 3, and representative examples are illustrated in Figure 4. Methylation of DRM/Gremlin was absent in DNA from peripheral blood lymphocytes from healthy volunteers and in respiratory cells, breast epithelial cells, and mesothelial cells. In contrast, DRM/Gremlin methylation was detected in 11/15 (73%) of NSCLC cell lines, 4/13 (31%) of SCLC cell lines, 5/10 (50%) of breast cancer cell lines, and 4/6 (67%) of MM cell lines. Either the methylated or the unmethylated forms of the gene were present in most cell lines (37/44 (84%)), while both forms were present in the remaining seven (16%) cell lines. Overall concordance between DRM/Gremlin expression and methylation was 42/44 (95%). Restoration of DRM/Gremlin expression in 5-Aza-CdR-treated cancer cells A total of 13 tumour cell lines (five NSCLC, one SCLC, three breast cancer, and four MM) that showed loss of expression and methylation of DRM/Gremlin were cultured with 5-Aza-CdR. DRM/Gremlin expression was restored after treatment in all 13 cell lines (Figure 1B). DNA methylation of DRM/Gremlin, HPP1, and RUNX3 in primary tumours and tissues and its correlation to clinicopathologic features DRM/Gremlin is aberrantly methylated and downregulated in thoracic cancer cell lines. HPP1 and RUNX3 have also been shown to be aberrantly methylated in certain cancer types. These three genes are TGFβ related and are all thought to be tumour suppressors. We next examined the methylation status of these three genes in primary tumours and tissues by MSP assay (Tables 2 and 3, Figure 4). Methylation of DRM/Gremlin was observed in 50% of lung cancer tissues (n=140), 54% of breast cancer tissues (n=37), and 60% of MM tissues (n=63), while methylation of HPP1 in these tissues occurred at a frequency of 28, 35, and 35%, respectively, and methylation of RUNX3 occurred at a frequency of 18, 22, and 33%, respectively. The methylation frequency for all three genes was significantly higher in adult tumours compared to paediatric tumours (DRM/Gremlin, P<0.0001; HPP1, P<0.0001; RUNX3, P<0.0001). Methylation of all three genes appeared to be tumour specific in all adult tumours, when compared to corresponding adjacent normal tissues (P<0.0001). Methylation of these genes was not detected in bronchial carcinoids. In lung cancer tissue, DRM/Gremlin methylation was not associated with age, postsurgical stage, or prognosis, but it was associated with gender (male, 68/88 (77%); female, 18/35 (51%); P=0.008) and smoking history (smoker, 74/97 (76%); nonsmoker, 12/26 (46%); P=0.007). In breast cancer samples, methylation of DRM/Gremlin gene was associated with older age (P=0.009). In addition, the frequency of methylation for RUNX3 was higher in estrogen receptor (ER)-positive cases than in ER-negative cases (ER positive, 8/22 (36%); ER negative, 0/8 (0%); P=0.02). In bladder cancer tissues, methylation of DRM/Gremlin was associated with poorer prognosis (P=0.026, log-rank test) (Figure 5). The frequencies of methylation for DRM/Gremlin and HPP1 were higher in advanced-stage bladder cancer cases (stages III–IV) than in early-stage cases (stages 0–II, P=0.03 and 0.003, respectively). The frequency of methylation for RUNX3 was higher in the presence of muscle invasion cases (20/39 (51%)) than in the absence of muscle invasion cases (4/18 (22%); P=0.048). In prostate cancer tissues, methylation of these three genes did not appear to be correlated with age, stage, Gleason score, or serum prostate-specific antigen level. However, RUNX3 and HPP1 methylation-positive status were associated with poorer disease-free prognosis (P=0.007 and 0.014, respectively; log-rank test). DISCUSSION DRM/Gremlin encodes a 184-amino-acid protein that is a member of the cysteine knot superfamily (Hsu et al, 1998). This protein has been highly conserved during evolution and it belongs to a novel family of BMP antagonists that includes the tumour suppressor DAN. The BMPs play a major role in bone formation and may facilitate bone metastases derived from prostate tumours (Masuda et al, 2003) as well as other cancers. DRM/Gremlin protein blocks the activity of BMP2, BMP4, and BMP7 with high affinity (Hsu et al, 1998; Merino et al, 1999) and possibly that of other growth factors in the TGFβ superfamily. BMP2 is overexpressed in NSCLC tissues and has been shown to stimulate growth of A549 lung cancer cells (Langenfeld et al, 2003). DRM/Gremlin is also known to affect lung development (Shi et al, 2001). BMP2 exposure has been shown to increase phosphatase and tensin homolog (PTEN) protein levels in the breast cancer cell line MCF-7 (Waite and Eng, 2003). Blocking BMP signalling by overexpression of a dominant-negative type II BMP receptor inhibits the growth of human breast cancer cells (Pouliot et al, 2003). Recently, Chen et al demonstrated that overexpression of Drm in the tumour-derived cell lines Daoy (primitive neuroectodermal, HTB186) and Saos-2 (osteoblastic, HTB-85) transcriptionally activates p21Cip1 via a novel mechanism, independent of p53 and both p38 and p42/44 MAP kinases, and inhibits neoplastic transformation (Chen et al, 2002). Thus, silencing of the DRM/Gremlin gene by DNA methylation may play a role in carcinogenesis both by affecting the cell cycle as well as by upregulation of BMP signalling. We observed frequent methylation of DRM/Gremlin in many human adult cancer tissues and cell lines, and methylation appeared to be correlated with reduced DRM/Gremlin mRNA expression, suggesting that epigenetic phenomena (i.e., methylation and the related mechanism of histone deacetylation) were the major causes of gene silencing. Expression of these genes was reactivated following treatment with the demethylating agent 5-Aza-CdR, providing further evidence that methylation is indeed the silencing mechanism involved. Methylation of DRM/Gremlin, HPP1, and RUNX3 appeared to be tumour specific in these cancer types when compared to corresponding adjacent normal tissues. Methylation of these genes in paediatric tumours was relatively rare, which is consistent with our previous reports (Harada et al, 2002). Methylation of these genes was not detected in bronchial carcinoids, which are lung tumours with relatively low invasive and metastatic potential. In a previous study, we found that the methylation profile of carcinoids was similar to that of SCLC, although the methylation frequencies of most genes were lower in carcinoids (Toyooka et al, 2001). In our previous studies, we observed that the methylation frequencies of MGMT and GSTP1 in lung cancers were significantly higher in US and Australian cases than in those from Japan and Taiwan (Toyooka et al, 2003). In addition, methylation frequencies were either similar, or slightly higher (seldom significantly) in lung tumour cell lines than in primary tumours (Toyooka et al, 2001). In our present series, the primary lung tumours were from Japan while all of the other primary tumours as well as the cell lines were from the US. Although the methylation frequencies of DRM/Gremlin between primary tumours and cell lines for lung cancer, breast cancer, and MM were similar, further interethnic studies need to be performed to clarify this matter. Although only a small number of breast cancer tissues were examined, tissues from older women showed a higher frequency of DRM/Gremlin methylation than did those from young women. Age-related methylation of TSGs has also been reported in colonic epithelium and cancer (Waki et al, 2003). Methylation of DRM/Gremlin was significantly more frequent in lung cancers arising in smokers compared to nonsmokers. We and others have noted a relationship between the methylation of certain genes, including p16 and APC, as well as an increased overall methylation status in smoking-related lung cancers (Kim et al, 2001; Toyooka et al, 2003). In bladder cancers, DRM/Gremlin methylation-positive status was associated with poorer prognosis, while RUNX3 and HPP1 methylation-positive status was associated with poorer disease-free prognosis in prostate cancers. Although the number of samples tested in this study is too small to draw definitive conclusions, deregulation of TGFβ signalling through hypermethylation of these genes may affect tumour progression as well as patients' prognosis, resulting in more aggressive local and distant metastatic spread, including the bone. Of interest, bone metastases are frequent in the cancers examined in this study, particularly in SCLC, breast cancer, and prostate cancer. In conclusion, we found that two inhibitors and one modulator of TGFβ signalling, DRM/Gremlin, HPP1, and RUNX3, respectively, are often methylated and thereby silenced in human cancers. Correlation between methylation of any of these genes with various clinicopathological features, including smoking status and survival, indicates that our findings may be of both biological and clinical relevance. Figure 1 RT–PCR for DRM/Gremlin (CKTSF1B1) expression in lung and breast cancer cell lines. (A) Representative examples of RT–PCR results for DRM/Gremlin expression. (B) Effect of 5-Aza-CdR treatment on DRM/Gremlin-negative cell lines with DRM/Gremlin methylation. Treatment with 5-Aza-CdR restored expression of DRM/Gremlin in all 13 methylated cell lines tested. Expression of the housekeeping gene GAPDH was measured as a control for RNA integrity. M, molecular size marker; LC, lung cancer; BC, breast cancer; MM, malignant mesothelioma; NEG, negative control (genomic DNA). Before (B) and after (A) treatment with 5-Aza-CdR. Figure 2 DRM/Gremlin (CKTSF1B1) expression and methylation in tumour cell lines. Closed box, positive (POS) band detected; open box, negative (NEG) band detected; N, NSCLC cell line; S, SCLC cell line; B, breast cancer cell line; M, MM cell line; ND, not done. Figure 3 The location and methylation status of methylated CpG dinucleotides in the region of DRM/Gremlin that underwent bisulphite genomic DNA sequencing (RBSSQ). Positive numbers indicate the nucleotide position from the transcription start site (TSS; indicated with an arrow). Thin vertical lines and the numbers indicate the positions of CpG dinucleotides in the RBSSQ. The horizontal closed bars between numbers indicate the positions of CpG dinucleotides included in the MSP primers. Open circles indicate unmethylated CpG sites and filled circles indicate methylated CpG sites. Figure 4 Representative examples of MSP results for DRM/Gremlin in cell lines and primary tumours. (A) Representative examples of MSP results for DRM/Gremlin in lung cancer cell lines, breast cancer cell lines, and MM cell lines. DRM/Gremlin M, DRM/Gremlin-methylated form; DRM/Gremlin U, DRM/Gremlin-unmethylated; POS, positive control, that is, artificially methylated DNA; NEG, negative control (water blank). (B) Lung cancer (T), matched normal lung tissue (N), breast cancer (T), matched normal breast tissue (N). (C) MM, malignant pleural mesothelioma; BC, bladder cancer; PCa, prostate cancer; ML, malignant lymphoma. A visible band indicates amplification of a methylated form of a gene. Owing to contamination by normal tissues, either the unmethylated band only or both the methylated and unmethylated bands were present in several samples. Figure 5 Survival curves of bladder and prostate cancer cases. Survival was analysed according to the Kaplan–Meier method, and differences in distribution were evaluated using the log-rank test. (A) Overall survival curve according to DRM/Gremlin methylation status in bladder cancer cases (n=57). (B) Disease-free survival curve of prostate cancer cases (n=28) according to RUNX3 and HPP1 methylation status. Poorer overall and disease-free survival were observed in bladder cancer cases with DRM/Gremlin methylation and in prostate cancer cases with RUNX3 and HPP1 methylation. Table 1 Methylation and expression of DRM/Gremlin in cell lines DRM/Gremlin Samples Expression (%) Methylation (%) Lung cancer cell lines (n=28) NSCLC (n=15) 3 (20) 11 (73) SCLC (n=13) 8 (62) 4 (31) Breast cancer cell lines (n=10) 5 (50) 5 (50) MM cell lines (n=6) 2 (33) 4 (67) NHBEC (n=1) 1 (100) 0 (0) NHMEC (n=2) 2 (100) 0 (0) NMC (n=3) 3 (100) 0 (0) MM=malignant mesothelioma; NHBEC=normal human bronchial epithelial cells; NHMEC=normal human mammary epithelial cells; NMC=normal human mesothelial cells. Table 2 Methylation of DRM/Gremlin, HPP1, and RUNX3 in human cancers No. methylated (%) Samples Total no. DRM/Gremlin HPP1 RUNX3 Adult tumours 367 Primary NSCLC Adenocarcinoma 60 22 (37) 18 (30) 13 (22) Squamous cell carcinoma 51 40 (78) 15 (29) 8 (16) Large cell carcinoma 6 5 (83) 4 (67) 4 (67) Primary SCLC 5 3 (60) 2 (40) 0 Bronchial carcinoid 18 0 0 0 Malignant mesothelioma 63 38 (60) 22 (35) 21 (33) Breast cancer 37 20 (54) 13 (35) 8 (22) Prostate cancer 50 10 (20) 19 (38) 5 (10) Bladder cancer 57 29 (51) 20 35) 24 (42) Lymphoma 20 10 (50) 6 (30) 1 (5) Paediatric tumours 150 Osteosarcoma 10 1 (10) 0 5 (50) Wilm's tumour 25 0 0 0 Neuroblastoma 27 0 0 0 Rhabdomyosarcoma 17 1 (6) 0 0 Medulloblastoma 12 0 0 0 Hepatoblastoma 22 0 0 0 Ewing sarcoma 7 0 0 0 Retinoblastoma 30 0 0 0 Normal tissues 138 Lung tissuesa and NHBEC 51 1 (2) 0 0 Mesothelial cells 3 0 0 0 Breast tissuesa 23 0 0 0 Protate tissuesa 4 0 0 0 Bladder tissuesa 5 0 0 0 Peripheral blood lymphocytesb 14 0 0 0 a Adjacent to resected tumours. b From healthy volunteers. Table 3 Clinicopathologic correlation with the methylation of DRM/Gremlin, HPP1 and RUNX3 in adult solid tumours Clinicopathologic features DRM/Gremlin methylation (%) Pa HPP1 methylation (%) P RUNX3 methylation (%) P Lung cancers Gender Male (n=88b) 68 (77) 0.008 25 (28) 0.3 18 (20) 0.9 Female (n=35) 18 (51) 14 (40) 7 (20) Agec ⩽65 (n=60) 42 (70) 0.9 19 (32) 0.9 13 (22) 0.8 >65 (n=63) 44 (70) 20 (32) 12 (19) Smoking history Nonsmoker (n=26) 12 (46) 0.007 8 (31) 0.9 5 (19) Smoker (n=97) 74 (76) 31 (32) 20 (21) 0.9 Histology Adenocarcinoma (n=60) 38 (63) 18 (30) 13 (22) Squamous cell ca. (n=51) 40 (78) 15 (29) 8 (16) Large cell ca. (n=6) 5 (83) 4 (67) 4 (67) Small cell ca. (n=5) 3 (60) 2 (40) 0 (0) Carcinoid (n=18) 0 (0) 0 (0) 0 (0) Postsurgical stage Stages I and II (n=52) 42 (81) 0.03 19 (36) 0.3 8 (15) 0.3 Stages III and IV (n=71) 44 (62) 20 (28) 17 (24) Breast cancers Age ⩽53 (n=17) 5 (29) 0.009 3 (18) 0.08 2 (12) 0.2 >53 (n=20) 15 (75) 10 (50) 6 (30) Postsurgical stage Stages I and IIA (n=21) 9 (43) 0.1 5 (24) 0.2 5 (24) 0.9 Stages IIB and IIIA (n=16) 11 (69) 8 (50) 3 (19) Estrogen receptor status Positive (n=22) 14 (64) 0.4 11 (50) 0.08 8 (36) 0.02 Negative (n=11) 6 (55) 2 (18) 0 (0) Progesterone receptor status Positive (n=20) 13 (65) 0.4 10 (50) 0.1 7 (35) 0.08 Negative (n=13) 7 (54) 3 (23) 1 (8) Bladder cancers Gender Male (n=36) 19 (53) 0.8 12 (33) 0.8 14 (39) 0.6 Female (n=21) 10 (48) 8 (38) 10 (48) Age ⩽67 (n=29) 14 (48) 0.8 10 (34) 0.9 12 (41) 0.9 >67 (n=28) 15 (54) 10 (36) 12 (43) Grade Grades 1 and 2 (n=13) 5 (38) 0.5 2 (15) 0.2 2 (15) 0.05 Grade 3 (n=43) 23 (53) 17 (40) 21 (49) Growth pattern Nonpapillary (n=26) 15 (58) 0.4 10 (38) 0.8 13 (50) 0.3 Papillary (n=31) 14 (45) 10 (32) 11 (35) Muscle invasion Noninvasion (n=18) 7 (39) 0.3 3 (17) 0.07 4 (22) 0.048 Invasion (n=39) 22 (56) 17 (44) 20 (51) Stage Stages 0–II (n=16) 4 (25) 0.03 1 (6) 0.003 3 (19) 0.1 Stages III and IV (n=30) 18 (60) 15 (50) 14 (47) Prostate cancers Age ⩽64 (n=27) 6 (22) 0.7 7 (26) 0.2 1 (4) 0.2 >64 (n=26) 4 (15) 12 (46) 4 (15) Gleason score ⩽6 (n=22) 6 (27) 0.3 11 (50) 0.2 1 (5) 0.4 ⩾7 (n=28) 4 (14) 8 (29) 4 (14) Preoperative serum PSAd ⩽7.5 ng/ml (n=25) 5 (20) 0.7 8 (32) 0.9 2 (8) 0.9 ⩾7.5 ng/ml (n=22) 3 (14) 7 (32) 2 (9) a Fisher's exact probability test. b Detailed data were available on the number in parentheses. c Devided by median age. d Prostate-specific antigen. ==== Refs Balemans W Van Hul W Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators Dev Biol 2002 250 231 250 12376100 Chen B Athanasiou M Gu Q Blair DG Drm/Gremlin transcriptionally activates p21(Cip1) via a novel mechanism and inhibits neoplastic transformation Biochem Biophys Res Commun 2002 295 1135 1141 12135612 de Caestecker MP Piek E Roberts AB Role of transforming growth factor-beta signaling in cancer J Natl Cancer Inst 2000 92 1388 1402 10974075 Gardiner-Garden M Frommer M CpG islands in vertebrate genomes J Mol Biol 1987 196 261 282 3656447 Gazdar AF Kurvari V Virmani A Gollahon L Sakaguchi M Westerfield M Kodagoda D Stasny V Cunningham HT Wistuba II Tomlinson G Tonk V Ashfaq R Leitch AM Minna JD Shay JW Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer Int J Cancer 1998 78 766 774 9833771 Gery S Koeffler HP Repression of the TMEFF2 Promoter by c-Myc J Mol Biol 2003 328 977 983 12729735 Gery S Sawyers CL Agus DB Said JW Koeffler HP TMEFF2 is an androgen-regulated gene exhibiting antiproliferative effects in prostate cancer cells Oncogene 2002 21 4739 4746 12101412 Ghosh Choudhury G Kim YS Simon M Wozney J Harris S Ghosh-Choudhury N Abboud HE Ghosh Choundhury G Ghosh-Choundhury N Bone morphogenetic protein 2 inhibits platelet-derived growth factor-induced c-fos gene transcription and DNA synthesis in mesangial cells. 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==== Front Br J Cancer Br J Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 6602595 10.1038/sj.bjc.6602595 15870708 Molecular Diagnostics The Akt inhibitor KP372-1 suppresses Akt activity and cell proliferation and induces apoptosis in thyroid cancer cells Akt inhibitor KP372-1 induces cancer cell apoptosis Mandal M 1 Kim S 1 Younes M N 1 Jasser S A 1 El-Naggar A K 2 Mills G B 3 Myers J N 1* 1 Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Molecular Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA * Department of Head and Neck Surgery, Unit 441, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail: [email protected] 23 05 2005 03 05 2005 92 10 18991905 10 11 2004 17 02 2005 24 03 2005 Copyright © 2005 Cancer Research UK 2005 Cancer Research UK https://creativecommons.org/licenses/by/4.0/ This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. The phosphatidylinositol 3′ kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway, which is a critical regulator of cell proliferation and survival, is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens, and hypothesised that KP372-1, an Akt inhibitor, would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells, leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling, further preclinical evaluation of this compound for treatment of thyroid cancer is warranted. molecular therapy growth factors anaplastic thyroid cancer KP372-1 ==== Body pmcThe incidence of thyroid cancer in the United States is expected to be approximately 23 600 in 2004 (Jemal et al, 2004). Thyroid carcinomas can be classified into papillary thyroid carcinoma, follicular thyroid carcinoma, and anaplastic thyroid carcinoma (Jemal et al, 2004). The papillary and follicular thyroid carcinomas constitute the majority of thyroid carcinomas and are grouped together as well-differentiated thyroid carcinomas. This group of thyroid carcinomas can often be cured with surgical resection and with radioactive iodide therapy. However, there are no effective alternative therapies for patients with metastatic well-differentiated thyroid cancer who do not respond to radioactive iodine therapy, suggesting an urgent need for development of novel therapies. The pathogenesis of thyroid cancer is characterised by the alterations of multiple signalling pathways and by abnormalities in a variety of tumour-suppressor genes and cell-cycle proteins (Fagin, 2002). The activation of the Akt protein kinase B (Akt/PKB) signalling pathway appears to play an important role in the development and progression of thyroid tumours. Interestingly, Akt has been found to be activated by a genetic loss of expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN), a tumour-suppressor gene, in Cowden's syndrome, an autosomal dominant multi-organ hamartoma syndrome characterised by benign and malignant thyroid tumours and breast and colon cancers (Dahia et al, 1997; Liaw et al, 1997). Akt activation, probably through a variety of mechanisms including aberrant stimulation of upstream cancers, occurs in most sporadic thyroid cancers (Ringel et al, 2001). In benign thyroid cell models, Akt signalling is important for cell growth in response to insulin, insulin-like growth factor-1, and serum (Kimura et al, 1999, 2001; Coulonval et al, 2000; Saito et al, 2001) and is activated by several oncogenes involved in thyroid cancer, including activated p21ras and chimeric rearrangements involving the ret gene (RET/PTC oncogenes) (Borrello et al, 1994; Rodriguez-Viciana et al, 1994). Despite the central role for Akt activation in thyroid tumorigenesis, little is known about the biological effect of inhibition of the Akt kinase in the progression of thyroid carcinoma. Based on the putative central role of the Akt kinase in thyroid oncogenesis, we hypothesised that KP372-1, a specific Akt kinase inhibitor (molecular weight, 224.20; QLT Inc., Vancouver, BC, Canada), would inhibit the proliferation and induce apoptosis of thyroid cancer cells in vitro. KP372-1 was identified in a screen of kinase-inhibiting compounds tested on more than 100 different cellular kinases, and was selected for its high specificity for the Akt kinase (unpublished data from QLT). In this study, we demonstrated the key role of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway in thyroid cancer and explored the effect of KP372-1 using thyroid cancer cells as model systems. We assessed the effects of KP372-1 on the inhibition of the PI3K/Akt pathway biochemically and on cell proliferation and apoptosis. MATERIALS AND METHODS Cell lines A papillary thyroid carcinoma cell line, NPA187, a follicular thyroid cancer cell line, WRO, and anaplastic thyroid cancer cell lines KAT4, C643, K18, HTH74, ARO, and DRO were used. NPA187 and WRO were obtained from Dr Yan Oh, The University of Texas MD Anderson Cancer Center, Houston, TX, USA, and KAT4, C643, K18, HTH74, and DRO were obtained from Dr Sai-Ching Jim Yeung, Department of Endocrine Neoplasia and Hormonal Disorders, MD Anderson Cancer Center, Houston, TX, USA. All the cell lines were grown in RPMI medium supplemented with 10% foetal bovine serum, L-glutamine, penicillin, sodium pyruvate, nonessential amino acids, and vitamin solution (Life Technologies, Inc., Grand Island, NY, USA). Adherent monolayer cultures were maintained on plastic and incubated at 37°C in 5% carbon dioxide and 95% air. The cultures were free of Mycoplasma species. The cultures were maintained no longer than 12 weeks after recovery from frozen stocks. Compounds KP372-1 (Figure 1) was synthesised by QLT Inc., Vancouver, BC, Canada. KP372-1 is a mixture of two isomers present in approximately equal amounts. A stock solution of KP372-1 for enzyme or cellular assays was prepared in dimethyl sulphoxide (DMSO) and then diluted in the medium. The final concentration of DMSO in the incubation mixture did not exceed 0.1% v v−1. Tissue samples and Western blotting Fresh frozen human thyroid tissue specimens were obtained from the thyroid tissue bank (The University of Texas MD Anderson Cancer Center) with the approval of the Institutional Review Board at the MD Anderson Cancer Center. Thyroid specimens from patients who had undergone surgery were carefully harvested by an experienced pathologist (AKE) and were snap frozen in liquid nitrogen and stored at −80°C. Thawed tissue samples were homogenised in Triton X-100 lysis buffer (20 mM HEPES, 50 mM NaCl, 1% Triton X-100, 0.1% deoxycholate, 2 mM EDTA, 2 mM sodium vanadate, and protease inhibitor cocktail), and equal amounts of protein were analysed by Western blotting. The following antibodies were used for Western blotting: rabbit anti-pAkt (S473), rabbit anti-pAkt (T308), and rabbit anti-Akt (Cell Signaling, Beverly, MA, USA), rabbit anti-p85 and rabbit anti-PTEN (Santa Cruz, Santa Cruz, CA, USA), and rabbit anti-β-actin (Sigma, St Louis, MO, USA). β-Actin was used as a loading control. Cell proliferation For MTT assays involving treatment with KP372-1, the cells were diluted to 1000 cells per 100 μl of complete medium, from which 100 μl was added to each well of a 96-well plate (Falcon; Becton-Dickinson, Franklin Lakes, NJ, USA). On the following day, 100 μl of medium supplemented with two times the desired concentration of KP372-1 was added to the appropriate wells. The cells were then kept at 37°C in 5% CO2 for 72 h. At this point, 10 μl of a 5 mg ml−1 stock solution of MTT (Sigma) dissolved in water was added to each well, and the plates were returned to the 37°C incubator for 2 h. The supernatant was aspirated out of each well, and 200 μl of DMSO was added to each well. The plates were then shaken for 5 min and the optical density measured at 570 nm using a spectrophotometer. To measure the cell proliferation, we plated the NPA187 and WRO cells at a concentration of 1 × 104 cells well−1 in six-well plates. The cells were then treated with KP372-1 at a concentration of 30 and 60 nM for NPA187 and WRO cell lines, respectively, and were counted using a haemocytometer on days 1, 2, and 3. 3H-thymidine incorporation DNA synthesis in the control and KP372-1-treated cells was assessed by the incorporation of 3H-thymidine into newly replicated DNA. NPA187 and WRO cells were plated at a concentration of 5000 cells well−1 in 96-well plates. After 24 h, the cells were treated with different concentrations of KP372-1 for 48 h and treated with 5-μCi ml−1 3H-thymidine during the last 2 h (NEN Life Science Products, Inc., Boston, MA). Cells were washed with PBS and then extracted with 0.1 N KOH and counted by liquid scintillation. DNA fragmentation assay For the DNA fragmentation assay, low-molecular-weight DNA was prepared (Mandal et al, 1996). Briefly, NPA187 and WRO cells (3 × 106 per plate) were seeded in 100 mm plates and treated with KP372-1 (30 nM for NPA187 and 60 nM for WRO) for 1, 2, or 3 days. Both floating and attached cells were scraped and collected in medium, washed three times with PBS, and resuspended in 1 ml of lysis buffer (20 mM Tris-HCl (pH 8), 10 mM EDTA (pH 8), and 0.5% Triton X-100). After incubation on ice for 30 min, the lysates were spun at 12 000 rpm in a microcentrifuge for 10 min. Low-molecular-weight DNA in the supernatant was extracted with equal volumes of phenol and chloroform for 1 h at 4°C. Ammonium acetate (2 M) was added to the aqueous phase, and the DNA was precipitated with two volumes of ethanol at −20°C overnight. The DNA was treated with RNAse A (1 mg ml−1) at 37°C for 1 h, and total DNA was analysed using 1.5% agarose gel and visualised with ethidium bromide staining. Western blot analysis of thyroid carcinoma cell lines after treatment with KP372-1 In order to show the induction of apoptosis-related proteins by KP372-1, NPA187 and WRO cells (3 × 106 per plate) were seeded in 100 mm plates and treated with KP372-1 (30 nM for NPA and 60 nM for WRO) for 1, 2, or 3 days. Both floating and attached cells were scraped and collected in medium, washed three times with PBS, and the cells were lysed in Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 50 mM NaF, 10 mM β-glycerol phosphate) containing 1 mM sodium vanadate, 1 mM phenylmethylsulphonyl fluoride, 10 μg ml−1 apoptinin, and lysis buffer (20 mM Tris-HCl (pH 8), 10 mM EDTA (pH 8), and 0.5% Triton X-100). After incubation on ice for 30 min, the lysates were spun at 12 000 rpm in a microcentrifuge for 10 min. Equal amounts of protein were then analysed by Western blotting using the following antibodies: mouse anti-poly(ADP-ribose)polymerase (PARP) antibody (Trevigen, Gaithersburg, MD, USA), rabbit anti-caspase-3 antibody (Cell Signaling), and rabbit anti-β actin antibody (Sigma). β-Actin was used as a loading control. In order to show the effect of KP372-1 on various signal transduction pathways in thyroid carcinoma cell lines, we performed Western blot analysis on NPA187 and WRO cells after treating the cells with KP372-1. The cells were plated as described above. After treating the cells with KP372-1 (30 nM for NPA187 and 60 nM for WRO) for 4 h, both floating and attached cells were scraped and collected in medium, washed three times with PBS, and lysed with a lysis buffer as described above. After incubation on ice for 30 min, the lysates were spun at 12 000 rpm in a microcentrifuge for 10 min. Equal amounts of protein were then analysed by Western blotting using the following antibodies: rabbit anti-pAkt (S473), rabbit anti-Akt, rabbit anti-p-mitogen-activated protein kinase (MAPK), rabbit anti-pmTOR, rabbit anti-pS6R, and rabbit anti-S6R (Cell Signaling). Akt enzyme assay to detect in vitro kinase activity Cells were lysed using the lysis buffer provided in the Akt enzyme assay kit (Cell Signaling). The cells were scraped and placed in an Eppendorf centrifuge tube incubated on ice for 15 min and spun in a centrifuge at 4°C for 15 min at full speed. The lysates were then transferred to a new tube and stored at −80°C until assayed. Immunoprecipitation was carried out as follows: 500 μg of protein was added to 5 μl of anti-Akt antibody (Cell Signaling) and rotated at 4°C overnight. Protein A sepharose beads (50 μl) were then added and rotated for 3 h at 4°C. The protein A sepharose beads were then washed three times with lysis buffer and three times with the 1 × kinase buffer provided in the kit. Then the beads were aspirated, and 40 μl of kinase buffer was supplemented with 200 μM ATP and a mixture (1 μg per 40 μl) of fusion protein (provided in the kit). The tubes were then incubated at 30°C for 30 min, after which 20 μl of 3 × sample buffer consisting of 187.5 mM Tris-HCl (pH 6.8), 6% (w v−1) sodium dodecyl sulphate (SDS), 30% glycerol, 150 mM DTT, and 0.03% (w v−1) bromophenol blue was added to each tube. The tubes were then boiled for 5 min at 95°C, and glycogen synthetase kinase-3 (GSK-3) phophorylation was measured using phospho antibodies (Cell Signaling). RESULTS Akt is phosphorylated in many thyroid cancer cell lines In an attempt to delineate the role of Akt signalling in thyroid cancer cells, we first profiled the expression of pAkt, total Akt, and the p85 subunit of PI3K in a panel of thyroid cancer cell lines. As seen in Figure 2, most thyroid cancer cell lines expressed readily detectable levels of pAkt-Ser473, pAkt-Thr308, total Akt, and subunits of the PI3K p85. PTEN was present in all the cell lines. The low levels of pAkt in some cell lines was likely due to the relative levels of pAkt rather than complete absence of this molecule. Three cell lines were selected for further characterisation: NPA187, which expressed relatively high levels of pAkt, and total Akt, K18, which expressed high levels of pAkt and low levels of total Akt, and WRO, which expressed lower levels of pAkt with high levels of total Akt. The presence of similar amounts of PTEN (most mutant PTEN molecules are unstable) in these cell lines suggests that the difference in pAKT levels was likely not due to defective PTEN function. Akt expression in human thyroid cancer tissues After profiling the expression of Akt and pAkt in thyroid cancer cell lines, we focused our attention on the role of Akt in well-differentiated thyroid carcinoma in subsequent experiments. To determine whether our in vitro findings with cell lines reflected the biology of human thyroid cancer in vivo, we evaluated the expression of Akt and pAkt in fresh papillary thyroid tumour specimens using Western blotting. The status of Akt activation was examined using a phosphorylation-specific antibody against pAkt-Ser473 and antibody against total Akt in thyroid tumours and adjacent normal-appearing tissues. As shown in Figure 3, six of eight tumours had higher levels of phosphorylated Akt-Ser473 than did normal tissues despite similar levels of total Akt. Akt phosphorylation was higher in the thyroid tumours than in the neighbouring normal tissues, suggesting a potential role for Akt phosphorylation in the carcinogenesis of thyroid cancer. The high levels of Akt phosphorylation in neighbouring tissue samples from some patients may reflect a ‘field effect’ due to genetic aberrations or, alternatively, the production and action of paracrine growth factors by the tumours. KP372-1 inhibits proliferation and induces the apoptosis of thyroid cancer cells in vitro The effect of KP372-1 on the growth of NPA187 and WRO cells was evaluated using an MTT assay, cell counting, and 3H-thymidine incorporation. The proliferation of these cell lines was inhibited by KP372-1 with an IC50 (concentration at which 50% inhibition occurs) of 30 and 60 nM for NPA187 and WRO, respectively (Figure 4). The proliferation of the cell lines was also inhibited by KP372-1, as evidenced by cell counting (Figure 5A and B) and the 3H-thymidine incorporation assay (Figure 5C and D). As shown in Figure 2, different levels of pAkt and total Akt were seen in the three cell lines. As shown in Figure 4, the NPA187 cell line, which had high basal pAkt levels, was more sensitive to KP372-1 than was WRO, which had low pAkt levels, suggesting that high pAkt could indicate cell dependence on this pathway and thus higher sensitivity to the inhibition of Akt. This decreased MTT incorporation can be due to a decreased rate of cell cycle transit or increased cell death. To assess the latter possibility, we treated the NPA187 and WRO cells with KP372-1 for different lengths of time and determined the extent of apoptosis by DNA fragmentation (Figure 6A) and the accumulation of a sub-G0/G1 cell population by flow cytometry (data not shown). The effect of KP372-1 on the status of PARP and caspase-3 was also examined (Figure 6B). The induction of activated caspase-3 and cleavage of PARP by KP372-1 treatment were observed in both cell lines, although with different kinetics and different magnitudes. Consistent with the MTT data, NPA187 demonstrated greater degrees of PARP cleavage and DNA degradation at 72 hours than WRO. To determine the duration of exposure to KP372-1 required to commit cells to apoptosis, NPA187 and WRO cells were incubated with 30 and 60 nM of KP372-1, respectively, for 6 h in serum-free medium. The cells were then washed with PBS and grown in medium containing 10% FBS without the inhibitor for another 24 or 48 h. The cells were then assayed for the percentage of apoptotic cell death. Apoptosis was not induced under these conditions (data not shown). Thus, we concluded that KP372-1 must be present continuously in order to induce apoptosis at least at these doses and for these cell lines. KP372-1 inhibits Akt kinase activity, phosphorylation of Akt, and downstream targets of Akt in thyroid cancer cells We next determined the effect of KP372-1 on the phosphorylation of AKT (Ser473) and on downstream targets of Akt, including p-mTOR and p-S6 ribosomal protein (Ser240/244), and MAPK. We treated NPA187 and WRO cells with KP372-1 at their respective IC50 for 4 h and analysed the cell lysates with the specific antibodies indicated in Figure 7A. In the case of NPA187 and WRO, phosphorylation of Akt and S6 ribosomal protein was downregulated by treatment with KP372-1. However, the phosphorylation of mTOR and MAPK was not changed by treatment with KP372-1. Akt kinase activity was also downregulated by KP372-1 in multiple thyroid cancer cell lines, as tested by an in vitro kinase assay using GSK-β as substrate (Figure 7B). Our results indicate that KP372-1 blocks Akt kinase activity, thereby decreasing phosphorylation of the S6 ribosomal protein. The mechanism resulting in the decrease in Akt phosphorylation is under exploration, but may represent an allosteric change in the molecule, decreasing access to upstream kinases or increasing access to downstream phosphatases. DISCUSSION Our study shows that thyroid cancer cells expressed detectable levels of Akt Ser473, Akt-Thr308, total Akt, PTEN, and the p85 subunits of the PI3K and Akt kinase activity. Most of the tumours showed a higher level of Akt-Ser473 phosphorylation than matching normal tissues, suggesting an association between a high level of Akt phosphorylation and thyroid carcinogenesis. This association was further supported by evidence that blockade of Akt signalling with the selective inhibitor KP372-1 induced apoptosis and inhibited cell proliferation in human thyroid cancer cell lines in culture. Furthermore, KP372-1 was found to inhibit the phosphorylation and kinase activities of Akt in addition to the phosphorylation of downstream substrates. However, the mechanism responsible for decreased Akt phosphorylation is not clear. It is possible that the binding of KP372-1 to Akt may alter its conformation so that the relevant amino-acid residues are not available for phosphorylation. A similar effect has been seen with other inhibitors such as those for MEK1 and JNK where they decrease phosphorylation of their target in cells with an activated pathway. In our study, we found that the papillary thyroid cancer cell line NPA187 was more sensitive to the effects of KP372-1 compared with the follicular cell line WRO. However, Ringel et al (2001) found that the cell line NPA187 was more sensitive than WRO to the effects of LY294002, a phosphatidylinositol 3′ kinase (PI3K) inhibitor. This difference in sensitivities to two different agents that target the same pathway may be due to the fact that these agents show affinity for kinases other than the intended primary target kinase. It is also known that KP372-1 inhibits kinases other than Akt, such as CDK1, CK2, CSK, DNAPK, ERK1, GSK3b, LCK, MEK1, PIM, PKA, PKC, and S6K, albeit at relatively high concentrations (unpublished work from QLT). We have also found that the NPA187 cell line showed higher levels of Akt phosphorylation than WRO. This observation suggests that NPA187 may be more dependent than WRO on the activation of Akt for survival and proliferation. Inhibition of Akt might be of great benefit to patients with aggressive thyroid cancers, and support for the concept of targeting Akt comes from many observations. First, more than 54% of human cancers have active Akt that is detectable in situ (Bellacosa et al, 1991). Akt activation was identified in 10 of 10 follicular cancers, 26 of 26 papillary cancers, and two of 10 follicular variants of papillary cancers, but in only four of 66 normal tissue samples and two of 10 typical benign follicular adenomas (Vasko et al, 2004). Second, pAkt expression was found to be greatest in regions of capsular invasion and was localised to the nucleus in follicular cancers and to the cytoplasm in papillary cancers, except for invasive regions of papillary cancers, where it localised to both compartments (Vasko et al, 2004). Thus, small-molecule Akt inhibitors could have wide applicability as anticancer drugs. Third, inhibition of the PI3K/Akt pathway by biochemical or genetic means increases the efficacy of chemotherapy, radiotherapy, or both, in vitro and in vivo (Hu et al, 2000; Brognard et al, 2001; Bondar et al, 2002). Finally, several standard chemotherapeutic and chemopreventive agents inhibit the PI3K/Akt pathway when administered in vitro, and, in some cases, inhibition of Akt is directly responsible for these agents' cytotoxicity (West et al, 2002). Despite the acknowledged need for Akt inhibitors, none is widely available and none that inhibits the kinase activity of Akt is in clinical evaluation. The current studies indicate that KP372-1 acts to inhibit Akt and has activity in cells with high levels of pAkt. This is similar to other inhibitors of the PI3K/Akt pathway, such as Wortmannin and LY294002. Wortmannin and LY294002 may have limited clinical utility because they lack specificity and have potential adverse side effects, poor pharmacological properties, low stability, and poor solubility (West et al, 2002). Wortmannin inhibits myosin light-chain kinase; phospholipases C, D, and A2; and DNA-dependent protein kinase (West et al, 2002). LY294002 also inhibits the aryl hydrocarbon receptor, a ligand-activated transcription factor (Guo et al, 2000). In vivo use of LY294002 in mice has been associated with many adverse effects, including death (Hu et al, 2002). Similarly, Wortmannin has demonstrated hepatic and haematopoietic toxicity. Therefore, although Wortmannin and LY294002 inhibit the PI3K/Akt pathway, their drawbacks raise doubts about their suitability as leading candidates for additional development. The major advantage of KP372-1 over Wortmannin and LY294002 as PI3K inhibitors is its greater efficacy and the marked induction of apoptosis in cancer cell lines. This may be due to its targeting a central downstream molecule and also due to the potential for a number of processes to bypass effects at the level of PI3K. However, the final determination will be in terms of therapeutic index, which will need to be evaluated in mice and eventually humans. Indeed, a potential downside of Akt inhibitors is toxicity because of the importance of Akt signalling in many normal cellular processes such as insulin signalling, and the lack of selectivity of the current Akt inhibitors including KP372-1 to different Akt isoforms. Identifying kinase inhibitors that target the ATP-binding site of a kinase can be fraught with specificity problems because all kinases and many other molecules possess ATP-binding sites. This was perhaps best observed with STI-571 (Gleevec, imatinib mesylate, Novartis Pharma, Basel, Switzerland), a competitive inhibitor of the ATP-binding site of many kinases (Klejman et al, 2002). The wide clinical application of STI-571 is partially due to its ability to inhibit many kinases, including bcr–abl, platelet-derived growth factor receptors, and c-Kit (Heinrich et al, 2000; McGary et al, 2002; von Bubnoff et al, 2002). The relatively nonspecific activity of STI-571 results in activity against Kit and the PDGFR in gastrointestinal stromal tumours (GIST) and against the PDGFR in hypereosinophilic syndrome. It is somewhat surprising and fortuitous that the relative broad activity of STI-571 was not associated with toxicity. In conclusion, thyroid cancer cell lines and well-differentiated human tumour specimens showed high levels of Akt phosphorylation on Ser473 and high Akt activity levels, which supported the findings of several other studies (Dahia et al, 1997; Liaw et al, 1997; Ringel et al, 2001), indicating that the Akt signalling pathway plays a role in thyroid cancer progression. In addition, specific inhibition of Akt kinase activity by KP372-1 resulted in decreased cell proliferation and induction of apoptosis of thyroid cancer cells in vitro. Although anaplastic thyroid cell lines were included in some of our experiments, our data lend support to the use of Akt kinase inhibitor in well-differentiated thyroid carcinoma rather than in anaplastic or poorly differentiated thyroid carcinomas. These findings indicate that further preclinical evaluation of this and other compounds targeting the PI3K/Akt pathway in well-differentiated thyroid cancer is warranted. Figure 1 Molecular structure of KP372-1. Figure 2 Expression of phosphorylated (p) Akt-Ser473, pAkt-Thr308, p85, subunits of PI3K, and PTEN in thyroid cancer cell lines. Cell lysates from exponentially growing cells were analysed by immunoblotting with antibodies against the indicated proteins. Results shown are representative of three independent experiments. Figure 3 Akt expressions in human thyroid cancer. Expression of pAkt (Ser473) and total Akt in thyroid tumours (T) and adjacent normal tissues (N) were detected with immunoblotting. Figure 4 Effects of KP372-1 on the proliferation of thyroid carcinoma cell lines in vitro. Thyroid carcinoma cell lines NPA187 and WRO were plated in a 96-well plate and treated with different concentrations of KP372-1 for 48 h. Cell growth was measured by MTT assay. Results shown are representative of three experiments. Figure 5 Effects of KP372-1 on the proliferation of thyroid carcinoma cell lines in vitro. (A, B) Thyroid carcinoma cell lines NPA187 and WRO were plated in six-well plates and treated with 30 and 60 nM KP372-1 for NPA187 and WRO cell lines, respectively, for 1, 2, or 3 days. Cell proliferation was then measured by cell counting using a haemacytometer. (C, D) Thyroid carcinoma cell lines NPA187 and WRO were plated in a 96-well plate and treated with various concentrations (0–120 nM) of KP372-1 for 48 h. Cell proliferation was then measured by 3H-thymidine incorporation. Results shown are representative of three experiments. Figure 6 KP372-1 induces apoptosis in thyroid cancer cells in vitro. (A) Cells were treated with KP372-1 as indicated for various periods. DNA fragmentation was measured by ethidium bromide staining after the DNA was resolved on an agarose gel. (B) Cells were treated with KP372-1 for different time periods, and cell extracts were immunoblotted with the indicated antibodies. Results shown are representative of three experiments with similar results. Figure 7 KP372-1 inhibits Akt phosphorylation and some of the downstream signalling molecules as well as Akt kinase activity. (A) NPA187 and WRO cells were treated with the IC50 concentrations of KP372-1 (30–60 nM, respectively) for 4 h in RPMI medium without serum. Equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and immunoblotted with different antibodies as indicated. (B) KP372-1 inhibits Akt kinase activity. 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==== Front Br J Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 6602376 10.1038/sj.bjc.6602376 15756262 Molecular Diagnostics Aberrant methylation of SPARC in human lung cancers Suzuki M 12* Hao C 2 Takahashi T 1 Shigematsu H 1 Shivapurkar N 1 Sathyanarayana U G 1 Iizasa T 2 Fujisawa T 2 Hiroshima K 3 Gazdar A F 1 1 Hamon Center for Therapeutic Oncology Research, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 2 Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba 260-8607, Japan 3 Department of Basic Pathology, Graduate School of Medicine, Chiba University, Chiba 260-8607, Japan * Author for correspondence: [email protected] 01 03 2005 08 03 2005 14 03 2005 92 5 942948 16 08 2004 24 11 2004 09 12 2004 Copyright 2005, Cancer Research UK 2005 Cancer Research UK SPARC (secreted protein acidic and rich in cysteine) is an extracellular Ca2+-binding matricellular glycoprotein associated with the regulation of cell adhesion and growth. We investigated loss of expression of SPARC gene and promoter methylation in lung cancers and correlated the data with clinicopathological features. We observed loss of SPARC expression in 12 of 20 (60%) lung cancer cell lines. Treatment of expression-negative cell lines with a demethylating agent restored expression in all cases. Methylation frequencies of SPARC gene were 55% in 20 lung cancer cell lines. Primary tumours had methylation at a rate of 69% (119 of 173), while nonmalignant lung tissues (n=60) had very low rates (3%). In lung adenocarcinomas, SPARC methylation correlated with a negative prognosis (P=0.0021; relative risk 4.65, 95% confidence interval 1.75–12.35, multivariate Cox's proportional-hazard model). Immunostaining revealed protein expression in bronchial epithelium (weak intensity) and in juxtatumoral stromal tissues (strong intensity) accompanied by frequent loss in cancer cells that correlated with the presence of methylation (P<0.001). Our findings are of biological interest and potentially of clinical importance in human lung cancers. methylation SPARC lung cancer immunostaining ==== Body The matricellular protein SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin/BM-40, is a calcium-binding glycoprotein that shows a high degree of interspecies sequence conservation and plays an important role in cell–matrix interactions during tissue remodelling, wound repair, morphogenesis, cellular differentiation, cell migration, and angiogenesis (Sage et al, 1989; Jendraschak and Sage, 1996; Yan and Sage, 1999; Bradshaw and Sage, 2001; Brekken and Sage, 2001). Its role in tumorigenesis is complex, and expression is often being downregulated in tumour cells accompanied by upregulation in juxtatumoral stromal cells. SPARC is highly expressed in reactive fibroblasts, and expression is deregulated in many types of human malignant tumours (Porte et al, 1995; Le Bail et al, 1999; Rempel et al, 1999; Paley et al, 2000). SPARC may promote vascularisation of tumours, tumour progression, or invasiveness by modulating the activity of cytokines and stimulating secretion of tissue remodelling metalloproteases (Lane and Sage, 1994; Motamed and Sage, 1997; Sage, 1997). Owing to the correlation between expression and invasion, SPARC was thought to be a proinvasive protein (Everitt and Sage, 1992a, 1992b; Lane et al, 1994; Ledda et al, 1997). However, ovarian cancer cells treated with SPARC showed inhibition of cell proliferation and underwent apoptosis (Yiu et al, 2001). SPARC potently inhibited angiogenesis and significantly impaired neuroblastoma tumour growth in vivo (Chlenski et al, 2002). Implanted Lewis lung carcinoma cells grew more rapidly in SPARC null mice (Brekken et al, 2003). Thus, the exact role of SPARC in tumour growth and progression is unclear. DNA methylation is a major mechanism associated with the inactivation of tumour suppressor genes in cancer (Esteller et al, 2001; Jones and Baylin, 2002; Suzuki et al, 2004). It has been reported that SPARC is silenced through DNA methylation in pancreatic cancer cells (Sato et al, 2003). SPARC protein by immunostaining was overexpressed in stromal fibroblasts immediately adjacent to the neoplastic epithelium in pancreatic cancers, suggesting SPARC expression in juxtatumoral tissues is regulated through tumour–stromal interactions (Sato et al, 2003). As exogenous SPARC inhibits the growth of pancreatic cancer cells in vitro, tumour–stromal interactions of SPARC may act to facilitate or retard tumour progression (Sato et al, 2003). In non-small-cell lung cancer (NSCLC), SPARC expression by immunostaining was also found strongly in stromal fibroblasts, persistently in chondrocytes of bronchial cartilage, weakly in bronchial epithelium, but not in alveolar cells nor in tumour cells (Koukourakis et al, 2003). SPARC, is located at 5q33.1, a region that demonstrates frequent loss of heterozygosity in idiopathic pulmonary fibrosis and small-cell lung cancer (SCLC) (Girard et al, 2000; Demopoulos et al, 2002). In this study, we examined the mRNA expression and methylation of SPARC in lung cancer cell lines, and examined the methylation and protein by immunostaining in primary tumours. We correlated these findings with clinicopathologic features. MATERIALS AND METHODS Cell lines and tumour samples We studied 12 NSCLC cell lines (NCI-H460, NCI-H1437, NCI-H1770, NCI-H2087, NCI-H2122, NCI-H2126, HCC15, HCC95, HCC193, HCC366, HCC515, HCC1171) and eight SCLC cell lines (NCI-H69, NCI-H146, NCI-H209, NCI-H211, NCI-H524, NCI-H526, NCI-H1672, NCI-H2171) that were established by us (Phelps et al, 1996), several of which are deposited in the American Type Culture Collection (Manassas, VA, USA). Cell cultures were grown in RPMI-1640 medium (Life Technologies Inc., Rockville, MD, USA) supplemented with 5% fetal bovine serum and incubated in 5% CO2 at 37°C. Cell lines established at the National Cancer Institute have the prefix NCI, while those established at UT Southwestern Medical Center have the prefix HCC. Nonmalignant human bronchial epithelial cells (NHBEC) were cultured as reported previously (Toyooka et al, 2002), and normal trachea RNA was obtained from Clontech (Palo Alto, CA, USA). Surgically resected samples were obtained from patients with lung cancer who had not received treatment prior to resection at the Chiba University Hospital, Japan, after obtaining Institutional Review Board approval and informed consent. Samples were immediately frozen and stored at −80°C until used. Reverse transcription–polymerase chain reaction (RT–PCR) assay An RT–PCR assay was used to examine SPARC mRNA expression. Total RNA was extracted from the samples with Trizol (Life Technologies, Rockville, MD, USA) following the manufacturer's instructions. RT reaction was performed on 4 μg total RNA with deoxyribonuclease I and the SuperScript II First-Strand Synthesis using oligo-(dT) primer System (Life Technologies), and aliquots of the reaction mixture were used for the subsequent PCR amplification. The forward PCR amplification primer of SPARC was 5′-AAGATCCATGAGAATGAGAAG-3′ (Ex8-S), and the reverse primer 5′-AAAAGCGGGTGGTGCAATG-3′ (Ex9-AS) (Accession number NM_003118; forward, nucleotides 649–669; reverse, nucleotides 847–865). These sequences are separated by an intron, and we confirmed that genomic DNA was not amplified with these primers. Polymerase chain reaction amplification was carried out for 12 min at 95°C for initial denaturation, followed by 33 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. The housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control to confirm the success of the RT reaction. The primers for GAPDH amplification were as follows: forward primer, 5′-CACTGGCGTCTTCACCACCATG-3′; and reverse primer, 5′-GCTTCACCACCTTCTTGATGTCA-3′. Polymerase chain reaction amplification was carried out for 12 min at 95°C for initial denaturation, followed by 25 cycles of 94°C for 30 s, 65°C for 45 s, and 72°C for 30 s. These primer sequences were identical to the human target genes as confirmed by a BLAST search. Polymerase chain reaction products were analysed on 2% agarose gels. Nonmalignant human bronchial and normal trachea were used as normal controls for RT–PCR. 5-Aza-2′-deoxycytidine (5-Aza-CdR) treatment In all, 11 tumour cell lines with SPARC hypermethylation and absent gene expression were incubated in culture medium with 4 μM 5-Aza-CdR for 6 days, with medium changes on days 1, 3, and 5. The cells were harvested and RNA was extracted at day 6. DNA extraction and methylation-specific PCR (MSP) Genomic DNA was obtained from cell lines, cultured nonmalignant cells, primary tumours, and nonmalignant tissues by digestion with proteinase K (Life Technologies), followed by phenol/chloroform (1 : 1) extraction (Herrmann and Frischauf, 1987). DNA methylation patterns in the CpG island of SPARC were determined by the method of MSP as reported by Herman et al (1996). Primer sequences of SPARC for unmethyalted reaction were 5′-TTTTTTAGATTGTTTGGAGAGTG-3′ (sense) and 5′-AACTAACAACATAAACAAAAATATC-3′ (antisense), and for the methylated reaction, 5′-GAGAGCGCGTTTTGTTTGTC-3′ (sense) and 5′-AACGACGTAAACGAAAATATCG-3′ (antisense) (Sato et al, 2003). Briefly, 1 μg of genomic DNA was denatured by NaOH and modified by bisulfite. The modified DNA was purified using Wizard DNA purification kit (Promega, Madison, WI, USA), treated with NaOH to desulphonate, precipitated with ethanol, and resuspended in water. Polymerase chain reaction amplification was carried out for 12 min at 95°C for initial denaturation, followed by 38 cycles of 94°C for 20 s, 62°C for 20 s, and 72°C for 30 s. DNA from peripheral blood lymphocytes (n=14) from healthy nonsmoking subjects was used as negative controls for methylation-specific assays. DNA from lymphocytes of a healthy volunteer treated with Sss1 methyltransferase (New England BioLabs, Beverly, MA, USA) and then subjected to bisulfite treatment was used as a positive control for methylated alleles. Water blanks were included with each assay. Polymerase chain reaction products were visualised on 2% agarose gels stained with ethidium bromide. Immunostaining SPARC was detected on paraffin-embedded tissue sections by the avidin–biotin peroxidase complex method using a mouse monoclonal antibody generated against the N-terminal region of SPARC (AON-5031; Hematologic Technologies Inc., Essex Junction, VT, USA). After immunostaining, the sections were counterstained with haematoxylin, dehydrated, and mounted. Immunostaining was graded by two independent observers unaware of clinical or laboratory findings. Staining was categorised from 0 to 4+ based on the percentage of cells stained. Score 0 was assigned if less than 5% of cells were positive. A weak staining (+1) was assigned if 6–25% of cells were positive, a moderate staining (2+) was assigned if 26–50% of cells were positive, high sore (3+) was assigned if 51–75% of cells were positive, and a very high score (+4) was assigned if more than 75% of cells stained positive. The degree of staining intensity was also noted. Statistical analysis Statistical differences between groups were examined using Fisher's exact test, χ2 test, and Mann–Whitney test. Survival was calculated from the date of initial diagnosis until death or the date of the last follow-up (censored). Survival was analysed according to the Kaplan–Meier method, and differences in their distribution were evaluated by means of the log-rank test. A multivariate Cox's proportional-hazard model was developed to evaluate the covariates' joint effects. All P's are two-sided, and P of less than 0.05 was defined as being statistically significant. RESULTS Aberrant methylation and expression of SPARC in cell lines Expression of SPARC was examined by RT–PCR, and representative examples are shown in Figure 1A. SPARC expression was present in NHBEC and normal trachea. However, loss of SPARC expression was observed in 10 of 12 (83%) NSCLC cell lines, and two of eight (25%) SCLC cell lines (Table 1 and Figure 1B). Aberrant methylation was absent in DNA from peripheral blood lymphocytes from healthy nonsmoking volunteers (n=14) and NHBEC. Aberrant methylation was found in nine of 12 (75%) NSCLC cell lines, and two of eight (25%) SCLC cell lines. Both loss of expression and lack of methylation of SPARC were found in NSCLC cell line NCI-H2126, suggesting an alterative method of gene silencing in this cell line. The concordance between loss of gene expression and aberrant methylation of SPARC was 92% in NSCLC cell lines, and 100% in SCLC cell lines (overall concordance 95%). 5-Aza-2′-deoxycytidine treatment Nine NSCLC cell lines (NCI-H460, NCI-H1437, NCI-H2087, NCI-H2122, HCC15, HCC95, HCC366, HCC515, HCC1171) and two SCLC cell lines (NCI-H146, NCI-H211) that showed loss of expression and methylation of SPARC were cultured with the demethylating agent 5-Aza-CdR. SPARC expression was restored after treatment in all 11 methylated cell lines tested (Figure 1C). Aberrant methylation of SPARC in primary tumours Results of aberrant methylation of SPARC in primary tumours and nonmalignant tissues are detailed in Table 1 and Figure 1D. SPARC methylation was a tumour-specific event in lung cancers (P=0.0001) when compared with corresponding adjacent nonmalignant tissues. Of 173 lung cancers, 119 (69%) were found to be methylated. There was a significant relationship between the major histologic types of lung cancer, NSCLC (115 of 161, 71%) and SCLC (four of 12, 33%) (P=0.01). When compared with clinicopathologic data, there was no significant association with gender, age, smoking history, or tumour stage. However, the frequency of methylation was significantly higher in squamous cell carcinomas (52 of 61, 85%) compared to adenocarcinomas (57 of 84, 68%) (P=0.01). Overall survival of adenocarcinomas was poorer in patients with SPARC methylation than in those without methylation (P=0.008; log-rank test; Figure 2) and for patients with advanced stages (P=0.0005). In a multivariate Cox's proportional-hazard model, promoter methylation of SPARC was an independent adverse prognostic factor (P=0.0021; relative risk (RR) 4.65, 95% confidence interval (CI) 1.75–12.35) next to stage (P=0.0003; RR 4.44, 95% CI 1.98–9.90) in adenocarcinoma cases (Table 2). Immunostaining of SPARC protein in lung cancer The expression of SPARC protein was examined in 162 primary lung cancers by immunostaining (Figure 3). In nonmalignant lung tissues, 73 of 128 cases (57%), negative (0) to weak (1+) SPARC expression was found in bronchial epithelial cell, while moderate (2+) to very strong (4+) expression was found in 55 cases. Alveolar cells were negative except for one case, which showed weak expression. Stromal fibroblasts distant from tumours showed negative to weak expression. Inflammatory cells including lymphocytes were negative expression, while chondrocytes of bronchial cartilage were moderate (2+) to strong (4+) SPARC expression. In 132 of 162 cases, negative (0) to weak (1+) SPARC expression was found in the cancer cells, while moderate (2+) to strong (4+) SPARC expression was found in 30 cases. Moderate (2+) to very strong (4+) SPARC expression was found in 154 of 162 cases in stromal cells near to or surrounding cancer cells. Thus, the predominant immunostaining pattern was negative in tumour cells while being positive in corresponding juxtatumoural stromal cells (125 of 162, 77%). However, there was no correlation of immunostaining pattern between tumour cells and stromal cells (Table 3A). Immunostaining pattern of neither tumour cells nor stromal cells correlated with patients' survival (data not shown). Of 162 cases, 105 showed loss (0) or weak (1+) expression of SPARC protein and methylation of SPARC gene, and 19 cases showed moderate (2+) to very strong (4+) expression without methylation (Table 3B). Thus, the concordance between methylation and loss of gene expression in tumour cells was 77% (P<0.0001). There was no correlation between tumour methylation and stromal cell immunostaining. DISCUSSION Sato et al demonstrated downregulation of SPARC mRNA in pancreatic cancer cells through DNA methylation (Sato et al, 2003). In our study, we observed that decreased SPARC expression in lung cancer cell lines is associated with DNA methylation of the gene promoter, and it is re-expressed by treatment with the demethylating agent 5-Aza-CdR in lung cancer cell lines. Although there are other possible mechanisms for downregulation of SPARC expression, the excellent concordance between mRNA expression by RT–PCR or protein expression by immunostaining and DNA methylation of SPARC indicates that the gene is downregulated mainly through DNA methylation in lung cancer. As with other tumours, SPARC is downregulated in lung cancer cells while being upregulated in juxtatumoural stromal cells. Upregulation was unusual in stromal cells distant from the tumour. These findings result in a complex pattern of simultaneous selective downregulation in a specific cell type (tumour cells) accompanied by selective upregulation in adjacent stromal cells. A similar pattern has been previously described in ovarian, pancreatic, and lung cancers (Brown et al, 1999; Koukourakis et al, 2003; Sato et al, 2003). Our findings demonstrate that the mechanism of downregulation of SPARC in lung cancer cells is due to methylation. The dual up- and downregulation of SPARC in tumours makes interpretation difficult, while its role in tumorigenesis remains controversial. SPARC may function similar to TGFβ, where signaling is downregulated in cancer cells early in tumorigenesis, but is overexpressed at later stages, thus acting either as tumour suppressor or oncogene, depending on the tumour stage (Wakefield and Roberts, 2002). Our data indicate that tumour–host interactions between lung cancer cells and stromal cells through SPARC protein play an important role in the pathogenesis of lung cancers. Our data demonstrated that methylation of SPARC gene in tumour cells had poorer prognosis in lung adenocarcinomas, whereas reactivity of stromal fibroblasts had been reported to correlate worse prognosis in NSCLC (Koukourakis et al, 2003). The expression of SPARC in cancer tissues or functional analyses of SPARC gene in tumour cell lines have been widely studied (Table 4). Expression, as analysed by RT–PCR or immunostaining, was downregulated in lung, pancreatic, and ovarian cancers, whereas it was upregulated in oesophageal, bladder, metastatic prostate, hepatocellular cancers, and invasive meningiomas and malignant melanomas, and some of those findings were supported by functional analyses. The expression of SPARC by both normal and tumour cells is highly dependent on tumour type and culture conditions. Expression of SPARC in cancer tissues correlated with poor prognosis in malignant melanoma, bladder, and oesophageal carcinoma as also reported by others, although some of these reports was analysed by RT–PCR using whole specimens (Massi et al, 1999; Yamanaka et al, 2001; Yamashita et al, 2003). Aberrant expression of SPARC in primary tumours may result in negative prognosis. In conclusion, SPARC was downregulated in cancer cells through DNA methylation and overexpressed in its stromal cells of lung cancer, and DNA methylation correlated with prognosis in adenocarcinomas. DNA methylation of SPARC may play a role in the pathogenesis of lung cancers. This work was supported by a grant from the UT Specialized Program of Research Excellence in Lung Cancer (P50CA70907) and Early Detection Research Network, National Cancer Institute, Bethesda, MD, USA. Figure 1 (A) Representative examples of RT–PCR for SPARC in lung cancer cell lines, NHBEC, and normal trachea. GAPDH was used as a control for the RNA integrity and RT reactions. NC=negative control. (B) Representative examples of MSP assay in cell lines. Polymerase chain reaction products were visualised on 2% agarose gels stained with ethidium bromide. M=methylated band; U=unmethylated band. (C) Representative examples of RT–PCR for SPARC mRNA in lung cancer cell lines before (−) and after (+) treatment with 5-Aza-CdR. (D) Representative examples of MSP assay in primary tumours and nonmalignant tissues. All PCR products were visualised on 2% agarose gels stained with ethidium bromide. M=methylated band; U=unmethylated band; T=lung cancer tissues; N=nonmalignant lung tissue; PC=positive control. Figure 2 Overall survival for 84 lung adenocarcinoma patients with early stages (n=34) or advanced stages (n=50) (A), or with (n=57) or without (n=27) the methylation of SPARC (B). Probability of survival curves was calculated using the Kaplan–Meier product-limit method and compared via the log-rank test between groups. Figure 3 Immunostaining for SPARC in normal and malignant lung tissues. (A) Weak to moderate expression (1–2+) of SPARC by bronchial epithelium. Most of the staining is present in the apical surface of ciliated surface cells. (B) Bronchioli showed negative (0) expression. (C) Strong (3+) reactivity for SPARC in adenocarcinoma cells that lacked DNA methylation of SPARC gene, while alveolar cells and stromal cells are negative. (D) Negative immunostaining for SPARC in an adenocarcinoma demonstrating DNA methylation, while the adjacent stromal cells demonstrate very strong (4+) reactivity for SPARC; × 200 magnification. Table 1 SPARC methylation in lung cancers Samples Total no. No. methylated (%) Tumours 193 Cell lines NSCLC cell lines 12 9 (75) SCLC cell lines 8 2 (25) Primary NSCLC Adenocarcinoma 84 57 (68) Squamous cell carcinoma 61 52 (85) Large-cell carcinoma 11 6 (55) Others 5 0 (0) Primary SCLC 12 4 (33) Nonmalignant 74 Lung tissues and NHBEC 60 2 (3) Peripheral blood mononuclear cellsa 14 0 (0) NSCLC=non-small-cell lung cancer; SCLC=small-cell lung cancer; NHBEC=nonmalignant human bronchial epithelial cells. a From healthy nonsmoking volunteers. Table 2 Univariate and multivariate statistics of the prognostic value of gender, age, smoking, stage, and methylation status of SPARC for survival in lung adenocarcinomas Number Univariate P-value Multivariate Risk ratio 95% CI P-value Gender Female 40 0.76 0.71 0.30–1.65 0.42 Male 44 Age (years) <64a 42 0.58 1.73 0.87–3.42 0.12 >64 42 Smoking Nonsmoker 39 0.43 1.52 0.65–3.57 0.34 Smoker 45 Stage I, II 34 0.0005 4.44 1.98–9.90 0.0003 III, IV 50 SPARC methylation − 27 0.008 4.65 1.75–12.35 0.0021 + 57 Results of univariate analyses using the log-rank test and multivariate analyses using Cox's proportional-hazard model of prognostic factors for overall survival. Stage and SPARC methylation are significantly associated with poor survival. CI=confidence interval; SPARC=secreted protein acidic and rich in cysteine. a Divided by median age of adenocarcinoma cases. Table 3 (A) SPARC immunostaining in tumour and stromal cell (n=162) and (B) methylation and immunostaining of SPARC in tumours (n=162) Tumour cell Stromal cell No. (%) P-valuea (A) Positive Positiveb 29 (18) >0.9 Positive Negativec 1 (1) Negative Positive 125 (77) Negative Negative 7 (4) Tumour methylation Tumour immunostaining No. (%) P-value (B) Methylated Positive 11 (7) <0.0001 Methylated Negative 105 (65) Unmethylated Positive 19 (12) Unmethylated Negative 27 (17) Tumour methylation Stromal immunostaining No. (%) P-value Methylated Positive 110 (68) >0.9 Methylated Negative 6 (4) Unmethylated Positive 44 (27) Unmethylated Negative 2 (1) SPARC=secreted protein acidic and rich in cysteine. a Fisher's exact probability. b Positive reveals moderate (2+) to very strong (4+) immunostaining. c Negative reveals negative (0) to weak (1+) immunostaining. Table 4 (A) Expression of SPARC in various tumours and (B) functional analyses of SPARC for tumorigenesis in tumour cell lines Tumour type Type of material Method for detecting expression Frequency or level of expression compared to nonmalignant tissues Reference (A) Lung cancer Tissue RT–PCR 18% Schneider et al (2004) Lung cancer Tissue Immunostaining 5% Koukourakis et al (2003) Barrett's oesophagus Tissue RT–PCR High (P=0.004) Brabender et al (2003) Pancreatic adenocarcinoma Cell lines/tissue RT–PCR/immunostaining 12%/32% Sato et al (2003) Oesopahgeal carcinoma Tissue Immunostaining 100% Yamashita et al (2003) Bladder cancer Tissue RT–PCR High (P<0.0001)a Yamanaka et al (2001) Ovarian cancer Tissue Immunostaining 14% Paley et al (2000) Metastatic prostate cancer Tissue Immunostaining High (most)b Thomas et al (2000) Invasive meningioma Tissue Immunostaining 100% Rempel et al (1999) Malignant melanoma Tissue Immunostaining 63.8% Massi et al (1999) Hepatocellular carcinoma Tissue Immunostaining 91%c Le Bail et al (1999) Tumour type Expressiond Effect for tumorigenesis Reference (B) Pancreatic adenocarcinoma High Growth suppression Sato et al (2003) Prostate cancer High Promotion of cell migration to bone De et al (2003) Lewis lung adenocarcinoma Null More rapid growth Brekken et al (2003) Neuroblastoma High Impairing of tumour growth Chlenski et al (2002) Breast cancer High with c-Jun Increase of motility and invasion Briggs et al (2002) Glioma High Promotion of invasion Schultz et al (2002) Ovarian cancer High Inhibition of the proliferation Yiu et al (2001) SPARC=secreted protein acidic and rich in cysteine; RT–PCR=reverse transcription–polymerase chain reaction. a Stage T2 or greater invasive tumours compared to stages T1 or less tumours. b High levels of SPARC protein were observed in most of the metastatic foci. c High in the stromal myofibroblasts of the tumour tissues. d Forced expression by endogenous or exogenous SPARC. ==== Refs Brabender J Lord RV 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==== Front Br J Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 6602254 10.1038/sj.bjc.6602254 15599381 Molecular and Cellular Pathology Id-1 stimulates cell proliferation through activation of EGFR in ovarian cancer cells Id-1 induces cell proliferation in ovarian cancer cells Zhang X 1 Ling M-T 1 Feng H 1 Wong Y C 1 Tsao S W 1 Wang X 1* 1 Cancer Biology Group, Department of Anatomy, The University of Hong Kong, 1/F, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong * Cancer Biology Group, Department of Anatomy, The University of Hong Kong, 1/F, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong. E-mail: [email protected] 13 12 2004 14 12 2004 91 12 20422047 15 07 2004 12 10 2004 18 10 2004 Copyright © 2004 Cancer Research UK 2004 Cancer Research UK Increased EGFR (epidermal growth factor receptor) expression has been reported in many types of human cancer and its levels are positively associated with advanced cancers. Recently, upregulation of Id-1 (inhibitor of differentiation or DNA binding) protein was found in over 70% of ovarian cancer samples and correlated with poor survival of ovarian cancer patients. However, the molecular mechanisms responsible for the role of Id-1 in ovarian cancer are not clear. The aim of this study was to investigate the effect of Id-1 on ovarian cancer proliferation and its association with the EGFR pathway. To achieve this, we transfected an Id-1 expression vector into three ovarian cancer cell lines and examined cell proliferation rate by flow cytometry and bromodeoxyuridine staining. We found that ectopic Id-1 expression led to increased cell proliferation demonstrated by increased BrdU incorporation rate and S-phase fraction. The Id-1-induced cell growth was associated with upregulation of EGFR at both transcriptional and protein levels. In contrast, inactivation of Id-1 through transfection of an Id-1 antisense vector resulted in downregulation of EGFR. Our results indicate that increased Id-1 in ovarian cancer cells may promote cancer cell proliferation through upregulation of EGFR. Our findings also implicate that Id-1 may be a potential target for the development of novel strategies in the treatment of ovarian cancer. Id-1 EGFR cell proliferation ovarian cancer ==== Body Ovarian cancer is the second leading cause of death from gynaecologic malignancy worldwide, with a mortality rate of 114.2 per 100 000 women (Parkin et al, 2001). Approximately 75% of women present with ovarian cancer at advanced stage; therefore, prognosis for this disease is poor, with a 5-year survival rate of less than 40% (Jemal et al, 2004). Although the molecular basis for the development of ovarian cancer is not clear, upregulation of the epidermal growth factor receptor (EGFR) is reported to be a frequent event in ovarian cancer and associated with tumour progression, invasion and poor survival rate (Kohler et al, 1989; Simpson et al, 1995; Skirnisdottir et al, 2001; Cloven et al, 2004). For example, expression of EGFR is significantly higher in malignant ovarian cancer cells compared to borderline and benign tumours (Simpson et al, 1995). A study on 226 patients with different stages of ovarian cancer found that EGFR-positive staining was evident in approximately 50% of the patients, and among them 59.8% had recurrent or died of this disease (Skirnisdottir et al, 2004). Furthermore, in a separate study, the survival time of patients (n=111) with EGFR-positive tumour was much shorter than patients with EGFR-negative tumours (Kohler et al, 1989). These results indicate that EGFR may be a key factor in promoting ovarian cancer growth as well as progression. Recently, using antisense technology, suppression of EGFR led to inhibition of cellular proliferation, cell adhesion and tumorigenicity in ovarian cancer cells (Alper et al, 2000). In addition, targeting the EGFR using an active EGFR-specific tyrosine kinase inhibitor ZD1839 also resulted in the reduction of ovarian cancer cell growth (Sewell et al, 2002). These results further suggest that inactivation of EGFR pathway may provide a therapeutic target for the treatment of ovarian cancer. However, the molecular basis of the EGFR-induced ovarian cancer cell proliferation is still not clear. Recently, Id-1 (inhibitor of differentiation or DNA binding) has been suggested as one of the upstream regulators of the EGFR pathway (Ling et al, 2004). Id-1 is a member of the helix–loop–helix (HLH) transcription factor family. It lacks the basic domain for DNA binding and acts as a dominant inhibitor of the basic HLH transcription factors by forming heterodimers (Benezra et al, 1990). Like EGFR, upregulation of Id-1 is frequently found in many types of human cancer such as breast (Lin et al, 2000), pancreas (Maruyama et al, 1999), cervical (Schindl et al, 2001), head and neck (Langlands et al, 2000) and prostate cancer (Ouyang et al, 2002a), and increased Id-1 expression levels are associated with advanced tumour stage and poor prognosis (Maruyama et al, 1999; Schindl et al, 2001; Ouyang et al, 2002a). Recently, it is reported that over 70% of ovarian cancer samples (among a total of 101 cases) are found to express Id-1 protein, while none of the nonmalignant cystadenomas shows positive Id-1 staining examined by immunohistochemistry as well as Western blotting (Schindl et al, 2003). In addition, the cancer samples with poor or moderate histological differentiation show stronger Id-1 expression than the well-differentiated tumours. Furthermore, the overall survival is much shorter in the patients with higher Id-1 expression than the patient with relatively lower Id-1 expression (Schindl et al, 2003). These results indicate that Id-1 may play an important part not only in tumorigenesis but also progression of ovarian cancer. The fact that both Id-1 and EGFR protein expression levels increase with progression and poor prognosis as well as chemoresistance of ovarian cancer (Alper et al, 2001; Maihle et al, 2002; Schindl et al, 2003; Cloven et al, 2004) raises a hypothesis that these two proteins may either share similar functions or regulate through same pathways. In addition, evidence from our previous studies has shown that Id-1 promotes prostate cancer cell proliferation through activation of EGFR (Ling et al, 2004), indicating that Id-1 may be an upstream regulator of EGFR. To study the role of Id-1 on ovarian cancer cell growth and its association with EGFR pathway, in this study, we first transfected an Id-1 expression vector into three ovarian cancer cell lines and then examined the effect of ectopic Id-1 expression on ovarian cancer cell proliferation using bromodeoxyuridine (BrdU) staining and flow cytometric analysis. The effect of Id-1 on EGFR expression at both transcriptional and protein levels was also determined by luciferase assay and Western blotting. These results were further verified through transfection of an antisense Id-1 vector in two ovarian cancer cell lines with high levels of Id-1. Our results suggest that ectopic Id-1 expression stimulates ovarian cancer cell proliferation and this process is mediated through upregulation of EGFR. Our results provide novel evidence to suggest Id-1 as an upstream regulator of the EGFR pathway in promoting ovarian cancer cell growth. MATERIALS AND METHODS Cell lines and cell culture conditions Five ovarian cancer cell lines, Skov3 (obtained from ATCC, Manassas, VA, USA), Ovca420, Ovca432, Ovca433, Ovca429, were maintained in RPMI 1640 (Life Technologies Inc., Carlsbad, CA, USA) supplemented with 2 m-glutamine and 5% (v v−1) foetal calf serum (FCS) at 37°C. Ovca420, Ovca432, Ovca433 and Ovca429 were established from freshly isolated ascites or tumour explants from patients with late-stage ovarian adenocarcinomas with distinct characteristics (Rauh-Adelmann et al, 2000). Generation of stable Id-1-expressing transfectants The pBabe–Id-1 retroviral expression vector and its corresponding vector control were used for generation of stable transfectants. Details on the vectors as well as transfection procedures have been described previously (Ouyang et al, 2002b). All the transfectants were selected and maintained in puromycin (1–2 μg ml−1). The pool of more than 20 individual clones transfected with either Id-1 or pBabe was generated. Cell culture medium was changed to serum-free medium (SFM) before performing additional experiments. BrdU staining Detailed experimental procedures have been described previously (Wang et al, 2002a). Briefly, monolayer cells were grown on 4-mm Chamber slides (ICN, Biomedicals, Aurora, OH, USA) and the culture medium was changed to SFM for 48 h. Then, the cells were treated with BrdU (10 m) for 1 h and then washed once with PBS. The cells were then fixed in cold methanol for 5 min at room temperature and washed in PBS. The cells were incubated with mouse monoclonal antibody against BrdU (1 : 10, Roche Diagnostics, Indianapolis, IN, USA) for 1 h at 37°C and then with anti-mouse IgG-FITC for 1 h at 37°C after washing with PBS. The percentage of FITC-positive cells was evaluated and at least 500 cells were evaluated in each experiment. The percentage of BrdU-positive cells in the control vector (pBabe) was considered as 100. The error bars represent the standard deviation generated from three independent experiments. Cell cycle analysis Cells (5 × 105) were plated in 5% FCS culture medium. After 24 h, the culture medium was replaced by SFM for 48 h. The cells were harvested by trypsinisation and then fixed in ice-cold 70% ethanol. The cells were then washed with PBS and incubated with propidium iodide (50 μg ml−1) and RNase (1 μg ml−1) for 30 min. Flow cytometric analysis was performed on an EPICS profile analyzer and analysed using the ModFit LT2.0 software (Coulter) as described previously (Wang et al, 2002a). Western blotting Detailed experimental procedures were described previously (Ouyang et al, 2002b). Briefly, whole-cell lysate was prepared by resuspending cell pellet in lysis buffer (50 m Tris-HCl (pH 8.0), 150 m NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS) including protease inhibitors (1 μg ml−1 aprotinin, 1 μg ml−1 leupeptin, 1 m PMSF), and protein concentrations were measured using the protein assay kit (Bio-Rad, Hercules, CA, USA). Protein suspension from the whole-cell lysate (20 μg) was loaded onto a sodium dodecylsulphate–polyacrylamide gel (SDS–PAGE) for electrophoresis and then transferred to a PVDF membrane (Amersham, Piscataway, NJ, USA). The membrane was then incubated with primary antibody for 1 h at room temperature against Id-1, EGFR, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS-T, the membrane was incubated with secondary antibody against mouse or rabbit IgG and the signals were visualised using ECL plus Western blotting system (Amersham, Piscataway, NJ, USA). Luciferase assay Cells were plated into a 12-well plate at a density of 1 × 105 cells well−1. After 24 h, the medium was changed to SFM. pER-1 (luciferase reporter containing the EGFR promoter, kindly provided by Dr A Johnson, NCI, MD, USA) and pRL-CMV-Luc (internal control) were cotransfected with either the pcDNA, pcDNA-Id-1 or pcDNA-Id-1-AS, respectively (Ling et al, 2004), into the cells using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN, USA). Cells were lysed 48 h after transfection and were assayed for luciferase activity using the Dual-luciferase reporter assay system (Promega, WI, USA). Each data point represented the mean of three experiments and error bars indicated the standard deviation. RESULTS Generation of stable Id-1 transfectants in ovarian cancer cells Under in vitro culture conditions, Id-1 expression is usually dependent on FCS stimulation and this characteristic is more evident in cell lines exhibiting less aggressive phenotype. For example, the androgen- or oestrogen-dependent cell lines (i.e. LNCaP (prostate cancer) and MCF7 (breast cancer)), which represent less aggressive tumours, show serum-dependent Id-1 expression. In contrast, the androgen- or oestrogen-independent cell lines (i.e. PC3 (prostate cancer) and MDA-MB-231 (breast cancer)), which represent aggressive tumours, express the Id-1 protein constitutively regardless of serum concentrations (Lin et al, 2000; Ouyang et al, 2002b). This phenotype provides a tool for studying the direct role of Id-1 on human cancer cells through either ectopic introduction or inactivation of the Id-1 gene. In this study, we first examined Id-1 protein expression in five ovarian cancer cell lines in a range of FCS concentrations (5, 2.5, 1 and 0%). As shown in Figure 1AFigure 1 Effect of FCS on Id-1 expression in ovarian cancer cell lines and Id-1 expression in stable transfectants. (A) Western blotting analysis of Id-1 protein expression in five ovarian cancer cell lines, Ovca420, Ovca432, Ovca433, Ovca429 and Skov3, in culture medium containing different serum concentrations. Note that the expression of Id-1 is dependent on FCS in Ovca420, Ovca432 and Ovca433 cell lines but independent in Ovca429 and Skov3 cell lines. (B) Three cell lines Ovca420, Ovca432 and Ovca433 were transfected with an Id-1 expression vector (pBabe–Id-1) or the empty control (pBabe) and stable transfectants were generated. Id-1 protein expression was examined by Western blotting after culturing in SFM for 48 h. Expression of actin was examined as an internal loading control. Note that Id-1 protein levels are much higher after ectopic expression of the pBabe–Id-1 vector in Ovca420, Ovca432 and Ovca433 cell lines. Results represent three experiments. , ovarian cancer cell lines Ovca420, Ovca432 and Ovca433 showed a serum-dependent Id-1 expression, while the expression of Id-1 was much less serum dependent in the Ovca429 and Skov3 cells. The Id-1 expression pattern in these ovarian cancer cell lines also seemed to correlate with their malignant phenotype. For example, Ovca429 cell line has been reported to be much more aggressive demonstrated by its highly invasive nature and increased expression of metastasis promoting proteins such as uPA and MMP compared to Ovca432 and Ovca432 cells (Ellerbroek et al, 1998; Ahmed et al, 2002). We then transfected an Id-1 expression vector into Ovca420, Ovca432 and Ovca433 cell lines and generated stable transfectants. As shown in Figure 1B, after selection in puromycin, the Id-1 stable transfectants showed constitutively high levels of Id-1 protein expression compared to the vector controls when cultured in serum-free conditions. Effect of Id-1 expression on serum-independent cell proliferation in ovarian cancer cells To investigate the effect of Id-1 expression on ovarian cancer cell proliferation, the DNA synthesis rate and the cell cycle S-phase fraction were determined using BrdU staining and cell cycle analysis, respectively, in the cells cultured in SFM. As shown in Figure 2Figure 2 Effect of ectopic Id-1 expression on ovarian cancer cell proliferation. (A) BrdU incorporation rate between the Id-1 transfectants and the vector controls. Cells were cultured in SFM for 48 and 72 h, respectively, and stained with an antibody against BrdU. At least 500 cells were counted in each experiment and the percentage of BrdU-positive cells was calculated and compared with the vector controls at 48 h time point, which was assigned as 1. Results presented as the mean and standard deviation from three experiments. (B) Representative results of BrdU staining. Strong nuclear FITC-positive signal was considered as BrdU positive. The background was enhanced to facilitate the counting of total cell numbers. Note that the percentage of BrdU-positive cells is higher in Id-1 transfectants than the pBabe control transfectants. , after culturing in SFM for 48 and 72 h, the percentage of BrdU-positive cells in Id-1 transfectants (solid columns) was higher than the vector controls (open columns) in all three ovarian cancer cell lines, especially in Ovca432 (up to 10-fold increase). The small increase of BrdU incorporation in Ovca433 Id-1 transfectant (approximately 1.7–2-fold) may be due to the relatively high basal levels of Id-1 in the parental cells, which may facilitate cell proliferation in SFM conditions. Nevertheless, these results indicate that ectopic expression of Id-1 in ovarian cancer cells led to increased DNA synthesis rate. To confirm these results, we performed flow cytometric analysis and studied the S-phase fraction. As shown in Figure 3Figure 3 Effect of ectopic Id-1 expression on cell cycle S-phase fraction. Flow cytometric analysis was performed on the Id-1 and pBabe transfectants after culturing in SFM for 48 and 72 h, respectively. Note that the percentage of S cells is higher in Id-1 transfectants than in the vector control. , the Id-1 transfectants from all three cell lines showed higher percentage of S-phase cells after culturing in SFM for 48 and 72 h, respectively. For example, the S-phase fraction was 25.17% in Ovca432–Id-1 transfectant, while the vector control only had 9.72% of S-phase cells after culturing in SFM for 48 h. Taken together, these results suggest that upregulation of Id-1 promotes cell proliferation in ovarian cancer cells. Effect of ectopic Id-1 expression on EGFR in ovarian cancer cells As previously suggested, Id-1 promotes cell proliferation through EGFR pathway in prostate cancer cells (Ling et al, 2004). Next, we studied if the Id-1-induced cell proliferation in ovarian cancer cells was mediated through EGFR pathway. As shown in Figure 4AFigure 4 Effect of ectopic Id-1 expression on EGFR. (A) EGFR protein expression in the Id-1 transfectants and the vector control cells were cultured in SFM for 48 h and EGFR expression was examined by Western blotting. (B) EGFR promoter activity in Ovca420, Ovca432 and Ovca433 cells transiently transfected with pcDNAId-1 and the vector control pcDNA. pER-1 (luciferase reporter containing the EGFR promoter) and pRL-CMV-Luc (internal control) was cotransfected with pcDNA or pcDNA-Id-1, respectively. Cells were lysed 48 h after transfection and assayed for luciferase activity using the Dual-luciferase reporter assay system (Promega, WI, USA). The luciferase activity of the cells transfected with pER-1 and pcDNA was considered as 100%. Each experiment was repeated three times and the error bars represent the standard deviation from three independent experiments. Note that ectopic expression of Id-1 in Ovca420, Ovca432 and Ovca433 cells induces upregulation of EGFR at both transcriptional and protein levels. , the vector-transfected control Ovca420 and Ovca433 cells showed detectable basal levels of EGFR protein, while EGFR was absent in Ovca432 cells. After exogenous Id-1 expression, the EGFR protein level was increased remarkably in all of the Id-1 transfectants compared with the vector control, suggesting that ectopic Id-1 expression resulted in upregulation of EGFR protein expression. To confirm these results, luciferase assay was performed to examine if ectopic Id-1 expression could lead to EGFR activation at transcriptional level. After cotransfection of pcDNA-Id-1 or pcDNA vectors with pER-1 (luciferase reporter containing the EGFR promoter) and pRL-CMV-Luc (internal control) in the parental cell lines, we found that the EGFR promoter activity was increased in all three cell lines transfected with the Id-1 expression vector (solid columns, 250–350% increase) than the cells transfected with the pcDNA vector control (open columns) (Figure 4B). Taken together, these results indicate that upregulation of Id-1 in Ovca420, Ovca432 and Ovca433 cells has led to the upregulation of EGFR expression at both transcriptional and protein levels. Effect of antisense Id-1 on the expression of EGFR in ovarian cancer cells To further confirm the association between Id-1 and EGFR expression, we next studied whether blockage of Id-1 expression could lead to the downregulation of EGFR. As shown in Figure 1A, the Skov3 and Ovca429 cell lines had constitutively high levels of Id-1 expression regardless of serum concentrations; we then transfected a vector containing the antisense sequence to the Id-1 gene (Ling et al, 2003) into these two cell lines and tested the expression of EGFR. As shown in Figure 5AFigure 5 Effect of Id-1 inactivation on EGFR. (A) Id-1 and EGFR protein expression in ovarian cancer cells transiently transfected with an antisense Id-1 expression vector (pcDNA-Id-1-AS) and the control vector (pcDNA). Cells were cultured in SFM for 48 h after transfection and Id-1 and EGFR expression was examined by Western blotting. (B) EGFR promoter activity in ovarian cancer cells transiently transfected with Id1-AS and the vector control. pER-1 (luciferase reporter containing the EGFR promoter) and pRL-CMV-Luc (internal control) were cotransfected with pcDNA or pcDNA-Id-1-AS, respectively, to Skov3 and Ovca429 cells. Cells were lysed for luciferase assays 48 h after transfection and the luciferase activity was tested using the Dual-luciferase reporter assay system (Promega, WI, USA). Samples transfected with pER-1 and pcDNA was considered as 100%. The error bars represent standard deviation from three independent experiments. Note that inhibition of Id-1 expression in Skov3 and Ovca429 cells reduces EGFR expression at both transcriptional and protein levels. , 48 h after transfection, the Id-1 protein level was decreased in both cell lines compared to the pcDNA vector control (up to 70% decrease), indicating a successful inhibition of Id-1 expression in these cells. In addition, the expression of the EGFR protein was also downregulated at similar levels in these two cell lines. For example, Id-1 expression was decreased by 70% in Skov3 cells and the EGFR protein level was also reduced by approximately 70% compared to the vector-transfected cells. These results were further confirmed by luciferase assay, which showed that the EGFR promoter activity was inhibited in the cells transfected with the antisense Id-1 vector (solid columns) compared to the vector control (open columns) (Figure 5B). These results further support the hypothesis that Id-1 may be an upstream regulator of the EGFR signalling pathway. DISCUSSION In this study, we have demonstrated the positive effect of Id-1 expression on cell proliferation in three ovarian cancer cell lines (Figures 1, 2, and 3). In addition, the evidence provided in this study that ectopic expression (or downregulation) of Id-1 led to upregulation (or downregulation) of EGFR at both transcriptional and protein levels indicates that Id-1 may be an upstream positive regulator of the EGFR signalling pathway (Figures 4 and 5). Since upregulation of EGFR is a common event in ovarian cancer, our evidence implicates a novel mechanism responsible for EGFR activation in this cancer. As EGFR is one of the key factors in promoting ovarian cancer cell growth (Alper et al, 2000, 2001; Pack et al, 2004), our results also suggest an alternative target to suppress EGFR signalling pathway through inactivation of Id-1, thus inhibiting cancer cell growth. Although the Id-1-induced cell proliferation in normal as well as in cancer cells has been reported in several previous studies (Lin et al, 2000; Ouyang et al, 2002b; Wang et al, 2002b), this study is the first to demonstrate its positive role on ovarian cancer cell growth, especially its relation with EGFR pathway in ovarian cancer cells. Several mechanisms have been suggested for the role of Id-1 in cell proliferation. For example, Id-1 inhibits p16INK4a/RB pathway leading to the bypass of replicative senescence in primary cells (Alani et al, 2001; Ohtani et al, 2001) and induces serum-independent proliferation in human prostate cancer cells (Ouyang et al, 2002b). Recently, the Id-1-induced cell proliferation has been linked to the Raf-MEK and NF-κB pathways (Ohtani et al, 2001; Ling et al, 2002, 2003). Since Id-1 is a regulator of transcription, it is not surprising that it may regulate gene expression involving multiple signalling pathways. In this study, we found that ectopic Id-1 expression led to transcriptional activation of EGFR (Figure 4), while inactivation of Id-1 resulted in suppression of EGFR expression (Figure 5). Our results indicate a novel downstream effector of Id-1 in ovarian cancer cells. It is possible that increased Id-1 expression in ovarian cancer cells may provide autocrine signals to stimulate EGFR activity, resulting in promotion of cell proliferation. Since inactivation of EGFR has shown promising results in inhibition of ovarian cancer cell growth as well as suppression of metastatic phenotypes, our results suggest a new target in inhibition of EGFR signalling. Previously, it was also reported that Id-1 protected against anticancer drug taxol-induced cell death in nasopharyngeal carcinoma cells (Cheung et al, 2004) and suppression of Id-1 resulted in sensitisation to TNFα-induced apoptosis in prostate cancer cells (Ling et al, 2003). Since the front-line treatment strategy for advanced ovarian cancers is chemotherapy, downregulation of Id-1 may provide a novel strategy in improving the efficiency of chemotherapeutic drugs through suppression of Id-1-induced protection against apoptosis. Activation of EGFR as well as upregulation of Id-1 have been associated with aggressive behaviour and poor clinical outcome in ovarian cancer patients, respectively (Schindl et al, 2003; Skirnisdottir et al, 2004). In this study, we also found that the cell lines (i.e. Ovca432, Ovca433) that showed serum-dependent Id-1 expression had lower basal levels of EGFR and exhibited less invasive characteristics as reported in previous studies than the ones with serum-independent Id-1 expression (i.e. Ovca429) (Ellerbroek et al, 1998; Ahmed et al, 2002). These results further suggest a strong link between Id-1 and EGFR in promoting ovarian cancer progression. Recently, the positive role of Id-1 in metastasis has been reported through promoting tumour angiogenesis in a breast cancer animal model (Fong et al, 2003). In ovarian cancer, similar effects have been reported in the cells expressing high levels of EGFR. For example, Ovca8 cells expressing high levels of EGFR were much more invasive and contained high levels of integrins and MMP activity and these malignant phenotypes were suppressed when EGFR was inactivated through antisense technology (Alper et al, 2001). It is possible that increased expression of Id-1 and EGFR proteins may provide growth advantage for ovarian cancer cells to progress to more advanced malignant phenotype leading to poor prognosis in patients. However, how Id-1 and EGFR interact with each other and what signalling pathways are responsible for mediating ovarian cancer proliferation and progression remain to be elucidated. In summary, we have provided first evidence that Id-1 plays an important part in the proliferation of ovarian cancer cells and this function is mediated through upregulation of EGFR. Although further investigations are needed to elucidate the precise molecular mechanisms responsible for the role of Id-1 in ovarian cancer, our results suggest a novel upstream regulator of the EGFR pathway. Since inhibition of EGFR is effective in the suppression of ovarian cancer cell growth, inactivation of Id-1 may provide an alternative strategy for the treatment of this cancer. This work was supported by RGC Grants to XH Wang (HKU7478/03M) and YC Wong (HKU 7314/01M and HKU7490/03M). ==== Refs Alani RM Young AZ Shifflett CB 2001 Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a Proc Natl Acad Sci USA 98 7812 7816 Alper O Bergmann-Leitner ES Bennett TA Hacker NF Stromberg K Stetler-Stevenson WG 2001 Epidermal growth factor receptor signaling and the invasive phenotype of ovarian carcinoma cells J Natl Cancer Inst 93 1375 1384 11562388 Alper O De Santis ML Stromberg K Hacker NF Cho-Chung YS Salomon DS 2000 Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumourigenicity in ovarian cancer cells Int J Cancer 88 566 574 11058872 Ahmed N Pansino F Baker M Rice G Quinn M. 2002 Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer J Cell Biochem 84 675 686 11835393 Benezra R 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==== Front J Transl MedJournal of Translational Medicine1479-5876BioMed Central 1479-5876-6-271849863910.1186/1479-5876-6-27ResearchMolecular analysis of the effects of Piroxicam and Cisplatin on mesothelioma cells growth and viability Verdina Alessandra [email protected] Irene [email protected] Angela [email protected] Rossella [email protected] Simona [email protected] Lucia [email protected] Ada [email protected] Alfonso [email protected] Laboratory D, Dept. for the Development of Therapeutic Programs, CRS, Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158, Rome, Italy2 Department of General Pathology and Oncology, "Centro Sperimentale S. Andrea delle Dame", Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy3 Department of Biochemistry and Biophysics, Section of Pathology, Second University of Naples, Via L. Armanni 5, 80138 Naples, Italy4 Campania Regional Operating Center (COR) of the National Mesothelioma Registry (ReNaM) and Department of Experimental Medicine, Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy2008 22 5 2008 6 27 27 23 2 2008 22 5 2008 Copyright © 2008 Verdina et al; licensee BioMed Central Ltd.2008Verdina et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed for prevention and treatment of a variety of human cancers. Piroxicam, in particular, has been recently shown to exert significant anti-tumoral activity in combination with cisplatin (CDDP) on mesothelioma cells. However, the mechanisms through which NSAIDs regulate the cell cycle as well as the signal pathways involved in the growth inhibition, remain unclear. In the present study, using two mesothelioma cell lines, MSTO-211H and NCI-H2452, we have investigated the influence of piroxicam alone and in association with CDDP on proliferation, cell cycle regulation and apoptosis. In both cell lines a significant effect on cell growth inhibition, respect to the control, was observed with all the drugs tested. Moreover, treatment with piroxicam or CDDP alone altered the cell cycle phase distribution as well as the expression of some cell cycle regulatory proteins in both cell lines. These effects were increased, even if in a not completely overlapping manner, after treatment with the association of piroxicam and CDDP. In particular, the two drugs in NCI cell line had a synergistic effect on apoptosis, probably through activation of caspase 8 and caspase 9, while the most evident targets among the cell cycle regulators were cyclin D1 and p21waf1. These results suggest that the association of piroxicam and CDDP specifically triggers cell cycle regulation and apoptosis in different mesothelioma cell lines and may hold promise in the treatment of mesothelioma. ==== Body Background Nonsteroideal anti-inflammatory drugs (NSAIDs) are commonly used as anti-inflammatory and analgesic drugs. However, several epidemiological studies have found that treatment with NSAIDs is associated with a reduced risk for cancer [1,2]. Since then, the antineoplastic effects of NSAIDs have been evaluated in many randomized clinical trials [3-8]. NSAIDs inhibit cyclooxygenases (COX-1 and COX-2), key enzymes in arachidonic acid metabolism, which catalyze an intermediate step in the production of prostaglandins, prostacyclins and thromboxanes [9]. Although COX-1 is constitutively expressed in many tissues, COX-2 is detected negligibly in most tissues but can be induced by cytokines and stress in various cell types. In several cancers COX-2 is over-expressed and this over-expression appears to be involved in the development of cancer by promoting cell division [10,11], inhibiting apoptosis [12,13], altering cell adhesion and enhancing neovascularization [14-16]. The inhibition of COX-2 by NSAIDs blocks these activities and, thus, may account for the anti-carcinogenic effects of these drugs. However, NSAIDs can also act through COX-independent mechanisms and each NSAID appears to have its own, more or less specific, COX-independent target [17,18]. Recently, an overexpression of COX-2 has been demonstrated in malignant mesothelioma [19-21] and this has provided the rationale to explore the use of COX inhibitors for the prevention and/or treatment of this tumour. Malignant mesothelioma (MM) is one of the most lethal human tumours, which incidence is expected to increase in Europe within the next 20 years [22]. Prognosis is poor and patients have a median survival of few months in either treated or untreated cases [23,24]. Mesothelioma represents a therapeutic problem since it is resistant to radiation, chemotherapy or surgical resection. Recent randomized studies on treatment of mesothelioma with combined chemotherapy demonstrate a survival benefit when a combination of cisplatin and antifolate drugs has been used [25,26]. Moreover, the combination of chemotherapy followed by surgery supplemented by postoperative radiotherapy in cases of incomplete resection, seems to be a promising treatment [27]. Unfortunately, none of these forms of treatment has significant impact on the progression and the outcome of mesothelioma and new therapeutic approaches must be investigated for a more successful treatment of this disease. Recently, the anti-tumour effects of NSAIDs have been studied on in vitro and in vivo experimental MM models. In particular, NS398 has produced a significant reduction of proliferation level in MM cell lines established and derived from previously untreated patients [28] and celecoxib has proved to be efficient in inhibiting mesothelioma cell growth [29] In a previous work we have demonstrated a significant anti-proliferative effect of piroxicam in two mesothelioma cell lines (MSTO-211H and NCI-H2452), not expressing COX-2, treated with piroxicam alone or in combination with CDDP [30]. The combination of the two drugs resulted in a synergistic effect, suggesting that piroxicam sensitizes mesothelioma cells to CDDP cytotoxicity. This result was confirmed also in vivo, by using a mesothelioma flank tumour model and a mesothelioma orthotopic tumour model [30]. In this work we have investigated the molecular mechanisms of cell cycle perturbation caused by piroxicam, CDDP and their association in two mesothelioma cell lines MSTO-211H and NCI-H2452. The resulting knowledge of the biological events elicited by these drugs in exerting their anti-tumour effects, could represent the basis for identifying specific molecular target of mesothelioma cells and for leading to advances in therapy. Methods Reagents Piroxicam (FELDENE; Pfizer, New York, NY) was supplied as a 60 mmol/L injectable solution and CDDP (Pharmacia-Italia, Nerviano, MI, Italy) as a 50 mmol/L injectable solution. Primary mouse monoclonal antibody against human p27Kip1 (p27) and primary rabbit polyclonal antibody against human p21waf1 (p21) were supplied by S. Cruz Biotechnology, Inc. Santa Cruz, CA, U.S.A.. Anti cyclin D1 (Cyc D1) monoclonal antibody was supplied by Cell signalling Technology, Inc. Danvers, MA, U.S.A. and anti cyclin A (Cyc A) monoclonal antibody by Calbiochem, EMD Chemicals, Inc. La Jolla CA, U.S.A. Anti actin monoclonal antibody was supplied by SIGMA, Saint Louis, Missouri, U.S.A. and anti COX-2 monoclonal antibody by Cayman Chemical, Ann Arbor, MI, U.S.A. Horseradish peroxidase-conjugated secondary antibodies were supplied from Santa Cruz Biotechnology, Inc. Santa Cruz, CA, U.S.A., and ECL and Super ECL Western blotting detection reagents from Amersham-Pharmacia, Uppsala, Sweden. Cell lines The human mesothelioma cell lines MSTO-211H (MSTO) and NCI-H2452 (NCI) were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured as monolayers in flasks using American Type Culture Collection complete growth medium in a humidified atmosphere containing 5% CO2 at 37°C. Cell treatment with piroxicam and CDDP Cells were seeded in complete growth medium and 16 hours later were treated with piroxicam and CDDP alone or in combination (administered at the same time) for 3 h, 6 h, 24 h, 48 h. MSTO were treated with piroxicam 0.76 mM (IP 50 [30]) and CDDP 4.5 μg/ml (IP 50 [30]); NCI were treated with piroxicam 0.68 mM (IP 50 [30]) and CDDP 10 μg/ml (IP 50). Controls were untreated. Cell growth Cells were treated as mentioned above and were counted 3, 6, 24 and 48 hours after beginning of treatment. Experiments were repeated in triplicate and media values were calculated. Cell growth was expressed as percent of control (untreated cells) and was compared between different treatment groups by Bonferroni test. P values < 0.05 was regarded as statistically significant. SPSS software (version 14.00, SPSS, Chicago) was used for statistical analysis. Cell cycle analysis on cancer cells Unsynchronized cells in the mid log phase were seeded at a density of 106 in T25 flasks. After 16 hours, cells were treated with piroxicam and/or CDDP, as described in the previous section. At 24 and 48 hours, adherent and floating cells were harvested, resuspended in staining solution containing propidium iodide (50 μg/ml), RNAse A, sodium citrate (0.1%), NP40 (0.1%) in PBS 1×, and incubated for 30 minutes in the dark. Cell cycle distribution of 20.000 cells was analyzed with a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) by ModFit version 3 Technology (Verity) as previously reported [31,32]. Pre-G1 picks were analysed as indicative of sub-G1 apoptotic population. All the experiments were performed at least 3 times and values were expressed in mean ± SD. Caspase 3, 8 and 9 assays Caspase activity was detected within whole living cells using BIOMOL and B-BRIDGE Kits supplied with cell-permeable fluorescent substrates. The fluorescent substrates for caspase 3, 8 and 9 were respectively FAM-DEVD-FMK, FAM-LETD-FMK, FAM-LEHD-FMK. Cells were washed twice in cold PBS and incubated for 1 h in ice with the corresponding substrates as recommended by suppliers. Cells were analysed after washing using the CellQuest software applied to a FACScalibur (BD). Experiments were performed in triplicate and values were expressed in mean ± SD. Protein analysis by western blotting Cell lysates were prepared by treating cells with ice-cold lysis buffer (Roche Applied Science, Mannheim, Germany) for 20 minutes followed by centrifugation at 4°C for 15 minutes. 40 μg of proteins were separated on 10% SDS-PAGE gels and then transferred on polyvinylidene fluoride (PVDF) membrane. For p21 and Cyc D1 detection in NCI were used 80 μg of proteins. Membranes were incubated with specific antibodies diluted 1:250 (p21, p27 and Cyc A), 1:500 (anti Cyc D1) and 1:1,000 (anti COX-2). Probing with anti-actin antibody diluted 1:10,000 was used to normalize the sample loading. Horseradish peroxidase-conjugated secondary antibodies were used at 1:3,000 dilution. Antibody reaction was visualized using ECL and Super ECL Western blotting detection reagents. The experiments were done in triplicate with comparable results and electrophoretic bands were analyzed by Scion Image program. Prostaglandin E2 assay Prostaglandin E2 levels were detected in medium from cell culture by using the Correlate-EIA High Sensitivity Prostaglandin E2 Enzyme Immunoassay kit from Assay Designs (Ann Arbor, MI). Results Effects of piroxicam alone and in combination with CDDP on mesothelioma cells growth To determine the effects of piroxicam alone or in combination with CDDP on cellular growth, MSTO and NCI cells were treated with the two drugs (as described in Methods) for different times. Cell growth was assessed by cell counts using as control the untreated cells (Fig 1). In both cell lines a significant effect on cell growth inhibition, respect to the control, was observed at 24 and 48 hours with all the drugs tested. Interestingly, in MSTO the combination of piroxicam and CDDP resulted in a stronger growth inhibition, respect to the other treatments, at 3 and 6 hours. Figure 1 Inhibition of MSTO (left panel) and NCI (right panel) by piroxicam and/or CDDP. MSTO and NCI were treated for the indicated time periods as previously specified. Cell growth was assessed by cell counts and was expressed as percent of control (untreated cells). Experiments were repeated in triplicate and media values were calculated and indicated in the upper table. P values at the different points of the treatments respect to the control and of the combined treatment (P+C) respect to the single drugs treatment were indicated in the lower tables. CTRL = control; P = piroxicam; C = CDDP. COX-2 and prostaglandin E2 protein expression levels in the MSTO and NCI cell line In order to determine if some of the anti-proliferative effects of piroxicam were due to its role as COX inhibitor, COX-2 protein levels in MSTO and NCI cells were assessed by western blot. Both mesothelioma cell lines expressed not detectable level of COX-2 (fig. 2). As positive controls, a human prostate cancer cell line (PC3) lysate expressing high levels of COX-2 [30], a human osteosarcoma cell line (U-2 OS) lysate expressing low levels [unpublished results], and ovine COX-2 standard were used. The not detectable expression of COX-2 was further confirmed by the lack of detectable levels of prostaglandin E2 in cell medium analyzed (detection limit for the used kit was 8 to 10 pg/ml) (data not shown). Figure 2 COX-2 expression level in MSTO and NCI cell lines at two different times. Ovine COX-2 standard, PC-3 (human prostate cancer cell line) lysate were used as positive controls and U-2 OS lysate as negative control. Normalization with actin level. The experiments were done in triplicate with comparable results. Effects of piroxicam alone and in combination of CDDP on Cell Cycle Phase Distribution To dissect the effects on cell cycle distribution of the treatment with piroxicam and/or CDDP, we performed FACS analysis (fig. 3). Cells were treated with piroxicam and/or CDDP for 24 and 48 hours. Cell cycle analysis on MSTO (fig. 3 left panel) showed that piroxicam was able to induce only a mild alteration, in particular a decrease in the S and an increase in the G1 phase of the cell cycle. On the other hand, CDDP treatment induced a significant block of the cells in S phase at 24 hours that, subsequently, evolves in part in apoptosis and in part into G2/M phase. Cell cycle analysis on NCI (fig. 3 right panel), on the other hand, showed that piroxicam was not able to induce a significant modification in the cell cycle distribution, except for a slight increase in the apoptosis fraction. CDDP, on the contrary, caused, as in MSTO, an increase in the S and apoptotic fractions, while it determined a complete disappearance of cells in G2/M phase. Figure 3 Effects of piroxicam and/or CDDP on cell cycle regulation in MSTO (left panel) and NCI cells (right panel). Cells were treated with piroxicam and/or CDDP for 24 and 48 hours and analyzed to determine cell cycle phase distribution. All the experiments were performed at least 3 times. Media ± Standard deviation of experiments is expressed as percentage of total cells. CTRL = control; P = piroxicam; C = CDDP. The results obtained with the combination of the two drugs showed a stronger and sinergic induction of apoptosis respect to single treatment in both cell lines. Piroxicam and CDDP treatment induces caspase activation In order to deeply investigate the apoptotic pathways activated by the two drugs, we monitored the enzymatic activity of the initiator caspases 8 and 9 and of the effector caspase 3 using flow cytometry technology (fig. 4). When apoptosis was analysed by caspase 9 and 8 activity in MSTO and NCI, we observed that, in both cell lines, caspase 9 was activated more in presence of the double treatment, which thereby showed at least an additive effect in induction of cell death. On the other hand, caspase 8 was significantly activated in MSTO by both the single drugs and their combination in a similar manner, whereas in NCI all treatments only produced a slight increase. Aiming to understand the effects of these initiatior caspase activations, we tested the activity of the effector caspase 3 in these conditions. As shown in fig. 4, we detected in NCI an increased activation by the combined treatment, whereas MSTO seems more directly sensitive to the CDDP treatment alone. The effects of treatments in NCI is in agreement with the hypothesis that piroxicam and CDDP cooperates for the induction of apoptosis via caspase 8, 9 and 3. Figure 4 Determination of enzymatic activity of the caspase (CASP) 3, 8 and 9. Following 16 h treatments with piroxicam and/or CDDP as indicated, the activity of caspase 3, 8 and 9 was measured and expressed as percentage ± Standard deviation of total cells. Experiments were performed in triplicate. CTRL = control; P = piroxicam; C = CDDP. Effects of piroxicam alone and in combination with CDDP on cell cycle regulatory proteins To identify the molecular pathways targeted by the two drugs, the expression levels of several cell cycle regulatory proteins were determined by western blotting in MSTO and NCI cells treated with piroxicam, CDDP and a combination of piroxicam and CDDP. In both cell lines we observed a decrease of Cyc D1 (fig. 5), and in NCI an evident increase of p21 expression (fig. 6), after treatment with piroxicam or CDDP. Interestingly, in NCI cell line the effect on p21 was more evident when a combination of CDDP and piroxicam was used. Figure 5 Effects of piroxicam alone and in combination with CDDP on cell cycle regulatory proteins. CycD1 was analysed by western blotting in MSTO (left side) and NCI (right side) treated with piroxicam and/or CDDP for different times. Electrophoretic bands were analyzed using Scion Image program. Experiments were repeated in triplicate and media values and standard deviations were calculated. CTRL = control; P = piroxicam; C = CDDP. Figure 6 Effects of piroxicam alone and in combination with CDDP on cell cycle regulatory proteins. p21 was analysed by western blotting in MSTO (left side) and NCI (right side) treated with piroxicam and/or CDDP for different times. Electrophoretic bands were analyzed using Scion Image program. Experiments were repeated in triplicate and media values and standard deviations were calculated. CTRL = control; P = piroxicam; C = CDDP. No appreciable changes were observed in the levels of Cyc A and p27 in both mesothelioma cell lines with all the different drug treatments (data not shown). Discussion MM is an insidious tumor with a dismal prognosis. Due to the low incidence of the disease, only few randomized studies have been performed to date. The reported response rates to the different therapeutic protocols ranged from 10 to 45% with no clear advantage in terms of survival that is between 4 and 12 months [25,33]. Various drugs have been tested in different combinations so far; among the most commonly employed are doxorubicin, cyclophosphamide, CDDP, carboplatin, gemcitabine, and pemetrexed. Recently, a benefit in response rate was observed with a combination of premetrexed and cisplatin and, similarly, by adding raltitrexed to cisplatin alone [25,26]. However, new and more effective chemotherapic drugs are urgently required for a more successful treatment of this deadly disease. Cancer, indeed, is viewed now not only as being the consequence of uncontrolled proliferation, but is also considered to be the result of an altered balance between cell proliferation and cell apoptosis. Therefore, therapies combining abrogation of cell cycle checkpoints and enhancement of the cell death mechanisms should be investigated in MM. Our previous studies demonstrated that piroxicam induced a significant inhibition of proliferation in two mesothelioma cell lines (MSTO and NCI). Moreover, we demonstrated a marked tumour growth inhibition and an extended survival of mice treated with a combination of piroxicam and CDDP in peritoneal mesotheliomas induced by MSTO intra-peritoneal injection [30]. Intrigued by the possible convergent activities exerted by CDDP and piroxicam, we studied the effects of those treatments in single dosage or in combination on cell growth in NCI and MSTO cells. Our data suggest that piroxicam has anti-proliferative effects in both cell lines, a finding that is consistent with data from the literature showing that piroxicam may target multiple component of the molecular machinery regulating cell cycle. Moreover, in MSTO, piroxicam in association with CDDP caused a stronger growth inhibition at 3 and 6 hours respect to the single drug treatments. Based on the fact that in both cell lines the level of COX-2 is very low and PGE2 is undetectable, we assume that piroxicam in these cells exerts its anti-proliferative activity via COX-2/prostaglandin E2-independent mechanisms. These data confirm recent reports that some of the anti-proliferative and anti-neoplastic effects of NSAIDs are independent of the inhibition of COX enzymes [34-36]. For example, in colon carcinoma the regulation by NSAIDs of the molecular pathways of cellular proliferation includes modulation of Ras and MAP Kinase signal transduction pathways, nuclear factor kB protein activation and cyclin expression [37-40]. Moreover, the treatment of human colon carcinoma cells either with indomethacin or aspirin results in a decrease in β-catenin/TCF transcriptional activity and cyclin D1 expression [41]. To dissect the effects on cell cycle distribution and apoptosis of the treatment with piroxicam and/or CDDP, we performed FACS analysis. This analysis demonstrated that the combination of the two drugs is able to perturb the cell cycle regulation of the mesothelioma cells in a not completely overlapping manner in the two cell lines. In particular, in MSTO cells the combination of the two drugs was very effective in causing an important increase of apoptotic fraction essentially due to CDDP action. Probably, the slight increase of apoptotic index between CDDP alone and the combined treatment is not a consequence of a direct action of piroxicam on cell cycle distribution but is the result of a sensibilization of cells to CDDP action, as we previously demonstrated [30]. On the other hand, in NCI cells there is an important synergic effect on apoptosis. In this last case the better efficacy of the combined treatment could be correlated with the increase of the three analyzed caspases. This is in agreement with the hypothesis that piroxicam and CDDP cooperate for the induction of apoptosis via caspase 8, 9 and 3 activation in NCI cells. Nevertheless, the greater sensitivity of the MSTO cell line to apoptosis induced by the single CDDP is in line with the higher caspase 8 and 9 activation. Our data support previous observations [30,42,43] of a synergistic effect of piroxicam, when used in combination with CDDP on cell cycle regulation and apoptosis. Interestingly, the specific check-points affected by this treatment are not overlapping in different cell lines, this demonstrating that the effects of piroxicam could be on multiple targets. In our experimental model, when we looked at the molecular regulators of cell cycle, we detected in MSTO and NCI a significant down-regulation of Cyc D1 and in NCI an up-regulation of p21 expression level. These effects are consistent with the results of growth inhibition described above. Interestingly, our research group has recently demonstrated that p21 expression is correlated with prognosis in mesothelioma patients, thus further confirming the key role played by this molecule in mesothelioma progression [44,46]. Nevertheless, genomic and proteomic technologies should be used to confirm and better analyze the molecular effects demonstrated by our biochemical approach. Conclusion Piroxicam is a widely used, well tolerated, easily administrable medication that could be readily associated not only to CDDP but also to a broad spectrum of chemotherapy and immunotherapy agents to improve efficacy of therapeutic protocols for mesothelioma. Our data support the hypothesis that piroxicam could sensitize mesothelioma cells to cisplatin treatment by acting on several molecular pathways. Indeed, careful molecular dissection of the molecular pathways elicited or turned off by piroxicam treatment should be better carried on by genomic and proteomic experimental approaches in order to more clearly define the most suitable targets of this drug and, eventually, propose the use of piroxicam in clinical trial setting, even if the cardiac risks associated with COX-inhibitors should be considered. Competing interests The authors declare that they have no competing interests. Authors' contributions All authors read and approved the final manuscript. AV set up the protocols and treated the Cells; IC, AN, RG, SM, and LA contributed in the experimental procedures and in the interpretation of the data, AS gave advise on the work and helped in the interpretation of the data, AB supervised all the work and wrote the paper together with AV Acknowledgements This work was supported by grants from Regina Elena Cancer Institute to Lab. 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==== Front PPAR ResPPAR ResPPARPPAR Research1687-47571687-4765Hindawi Publishing Corporation 1856668710.1155/2008/961753Review ArticleA Role for PPARγ in the Regulation of Cytokines in Immune Cells and Cancer Yang Xiao Yi 1 Wang Li Hua 1 Farrar William L. 2 1Basic Research Program, SAIC-Frederick, National Cancer Institute-Frederick, Frederick, MD 21702, USA2Cancer Stem Cell Section, Laboratory of Cancer Prevention, Division of Basic Sciences, National Cancer Institute-Frederick, Frederick, MD 21702, USAWilliam L. Farrar: [email protected] by Dipak Panigrahy 2008 12 6 2008 2008 96175327 3 2008 22 5 2008 Copyright © 2008 Xiao Yi Yang et al.2008This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. PPARγ and its ligands appear to serve diverse biological functions. In addition to the well-studied effects of PPARγ on metabolism and cellular differentiation, abundant evidence suggests that PPARγ is an important regulator of the immune system and cancers. Since cytokines are not only key modulators of inflammation with pro- and anti-inflammatory functions but they also can either stimulate or inhibit tumor growth and progression, this review summarizes the role for PPARγ in the regulation of cytokine production and cytokine-mediated signal transduction pathways in immune cells and cancer. ==== Body 1. INTRODUCTION Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily [1–6]. PPARs exist in three isoforms, PPARα, PPARβ/δ, and PPARγ, which are encoded by different genes and harbour isotype-specific expression patterns and functions. PPARs were initially identified as mediators of peroxisome proliferation in rodent liver, where PPARα plays the major role. However, none of the PPARs could be attributed to peroxisome proliferation in humans [7–10]. Among the various subtypes of PPARs, PPARγ is the best characterized receptor in humans. There are at least two PPARγ isoforms derived from the alternative promoters, PPARγ1 and PPARγ2. PPARγ2 isoform is longer than PPARγ1 by additional 30 N-terminal amino acids [11, 12]. Synthetic ligands including the thiazolidinedione (TZD) class of drugs, L-tyrosine-based compounds, and diindolymethanes as well as natural ligands including a broad range of polyunsaturated fatty acids 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE) and the eicosanoids 15-deoxy-Δ12, 14-prostaglandin J2 (15-d-PGJ2) function as efficacious PPARγ activators [13–15]. PPARγ is expressed at high levels in adipose tissue and is an important regulator of adipocyte differentiation, which functions as a ligand-dependent, sequence-specific activator of transcription. Expression of PPARγ in immune system was initially documented in 1994. Kliewer et al. reported that PPARγ is expressed at high levels in mouse spleen [8]. Greene et al. detected the expression of PPARγ2 in normal neutrophils and peripheral blood lymphocytes by Northern blot analysis in 1995 [9]. Monocytes and macrophages were the first cells of the immune system in which the physical presence and anti-inflammatory properties of PPARs were first described [16, 17]. Subsequently, PPARγ has been reported to exist in other immune cell types of hematopoietic origin, including T lymphocytes [18–22], B lymphocytes [23], NK cells [24], dendritic cells [25–28], eosinophils [29], and mast cells [30–32]. Multiple lines of evidence suggest that PPARs, especially PPARγ, are known to be expressed or overexpressed in several cancers such as epithelial tumor cells, renal cell carcinoma cells, myeloid and lymphoid malignancies, and multiple myeloma cells [33–37]. Ligands of PPARγ have been shown to promote differentiation and to inhibit cell growth and induce apoptosis in several types of human cancer, including colon cancer [38–40], breast cancer [41, 42], lung cancer [43], prostate cancer [44, 45], gastric cancer [46], liposarcoma [47, 48], and leukaemia [49], supporting a role for PPARγ ligands as potential tumor suppressors in PPARγ-dependent or -independent manner [50, 51], although several murine models suggest that, under certain circumstances, PPARγ ligands may stimulate cancer formation [36]. The cytokines are a large family of secreted molecules consisting of more than 100 peptides or glycoproteins. Each cytokine is secreted by particular cell types in response to a variety of stimuli and produces a characteristic constellation of effects on the growth, motility, differentiation, or function of its target cells. Cytokines can act in an autocrine manner to affect the behavior of the cell that releases the cytokine and/or in a paracrine manner to affect the behavior of adjacent cells. Moreover, some cytokines are stable enough to act in an endocrine manner to affect the behavior of distant cells, although this depends on their abilities to enter the circulation and their half-life in the blood. Cytokines are especially important for regulating immune and inflammatory responses with pro- and anti-inflammatory functions, and have crucial functions in controlling both the innate and adaptive arms of the immune response. Not only do cytokines govern the development and homeostasis of lymphocytes, but they also direct the differentiation of helper T cells and promote the generation of memory cells [52]. During formation and development of tumor, the mixture of cytokines that is produced in the tumor microenvironment has an important role in cancer pathogenesis. Cytokines can either stimulate or inhibit tumor growth and progression [53–57]. Specific polymorphisms in cytokine genes are associated with an increased risk of cancer [58]. Cytokines are produced by immune cells as a host response to cellular stress caused by either exogenous or endogenous agents to control and minimize cellular damage. However, an uncontrolled and sustained generation of cytokines can lead to altered cell growth, differentiation, and apoptosis. Therefore, cytokines are a linker among immunity, inflammation, and cancer [59]. In addition to their antiproliferative and proapoptotic activities on immune cells and cancer cells, effects of PPARs and their ligands in immune system and cancer cells may be mediated through influencing cytokine production or cytokine-mediated signal transduction pathways. Conversely, the expression of PPARs is also modulated by cytokines. In this review, we recapitulate molecular mechanisms on PPARs regulating cytokine production or cytokine-mediated signal transduction and cell responses, and enumerate their physiological and pathological consequences in immune responses, inflammation, and cytokine-responsive tumors. 2. MECHANISM(S) OF CYTOKINE GENE REGULATION BY PPARγ Like other nuclear receptors, the structure of PPARs is comprised of: an amino-terminal activation function, AF-1 (A/B domain), which can activate transcription in a ligand-independent fashion, the DNA-binding domain (DBD), a hinge region, and a carboxy-terminal ligand-binding domain (LBD) [1–3, 60, 61]. The DBD allows them to bind to and activate target genes, thus defining them as transcription factors. The LBD also contains a second activation function (AF-2) that maps to a surface-exposed hydrophobic pocket, proving a docking site for coregulatory proteins, and modulates their activities, making them hormone-dependent transcription factors. Upon ligand binding, PPARs heterodimerize with retinoid X receptors (RXRs) and form a complex that translocates to the nucleus and regulates gene expression. This heterodimeric complex binds to peroxisome proliferator response elements (PPREs) located within the promoter regions of target genes that consist of a direct repetition of the consensus AGGTCA half-site spaced by one or two nucleotides (DR1 or DR2). In addition to the heterodimer complex, it has been reported that a host of accessory proteins, named “coactivators” or “corepressors,” bind to the nuclear receptors PPAR/RXR in a ligand-dependent manner and impact the transcriptional process by either remodeling chromatin structure and/or acting as adapter molecules that link the nuclear receptor complex to key transcriptional machinery. Ligand binding to PPARs appears to trigger conformational changes that permit their dissociation from corepressors and favor their association with coactivators. The coactivators possess or recruit histone acetyltransferase activity to the transcription site. Subsequently, acetylation of histone proteins alters chromatin structure, thereby facilitating the binding of RNA polymerase and the initiation of transcription. In the absence of ligand, PPARγ has the potential to silence genes to which it is bound by recruiting transcriptional corepressor complexes and repress gene expression [1–5, 62, 63, 64]. Surprisingly, most of the effects of PPARs on cytokine expression result from crosstalk with other transcriptional factors through nongenomic transrepressive mechanisms. It is well known that some key transcriptional factors such as nuclear factor of activated T cells (NFAT), nuclear factor-kappa B (NF-κB), GATA-3, T-bet, AP-1, or signal transducers and activators of transcription (STAT) regulate the expression of cytokine genes. Transrepression by PPARs can occur either by inhibiting the binding of transcriptional factors to DNA through direct protein to protein interactions or by sequestrating cofactors necessary to their activity. A protein-to-protein interaction between PPARs and other transcription factors completely prevents these transcription factors from binding to their own response elements and therefore blocks their transcriptional activation of cytokine genes [63, 64]. Activation of PPARγ negatively influences the production of inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), Interleukin (IL)-6, and IL-1β by macrophages. A well-established example is PPARγ coassociation with NFAT, a T-cell specific transcription factor, in regulation of IL-2 gene expression [18]. The transcription factor NFAT plays an essential role in gene expression of IL-2 by T lymphocytes and is also involved in the proliferation of peripheral T lymphocytes. Therefore, we evaluated transcriptional activity and DNA binding of NFAT to determine whether NFAT might be a target for negative regulation of T-cell activation by PPARγ ligands. Utilizing the gel-shift experiment, we found that PPARγ ligands significantly inhibited the specific binding of NFAT probe corresponding to the human IL-2 promoter. The transcriptional activation of the reporter construct directed by the NFAT distal site of the IL-2 promoter was abrogated by 15-d-PGJ2 or ciglitazone in the presence of PPARγ over expression. We further tested for complex formation between PPARγ and NFAT in a coimmunoprecipitation experiment. The NFAT can be coprecipitated with PPARγ in T cells induced by PMA/PHA and 15-d-PGJ2 or ciglitazone. Furthermore, the addition of anti-PPARγ antibody induced high-affinity binding of extracts to the NFAT probes as determined by using an electronic mobility shift assay, demonstrating that removal of PPARγ with this antiserum increases the target specificity of NFAT. This data indicated that a direct physical protein-protein interaction occurs between nuclear receptor PPARγ and transcription factors NFAT, in turn inhibiting transcription of IL-2 in T lymphocytes. 3. CROSSTALK OF PPARγ WITH CYTOKINE-MEDIATED SIGNAL TRANSDUCTION PATHWAYS Cytokines induce a variety of biological responses by binding to specific cell surface receptors and activating cytoplasmic signal transduction pathways, such as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, which transmits information received from extracellular polypeptide signals, through transmembrane receptors, directly to target gene promoters in the nucleus, providing a mechanism for transcriptional regulation without second messengers [65–74]. JAKs bind specifically to intracellular domains of cytokine receptor signaling chains and catalyze ligand-induced phosphorylation of themselves and of intracellular tyrosine residues on the receptor, creating STAT docking sites. Phosphorylation of STATs on activating tyrosine residues leads to STAT homo- and heterodimerization. STAT dimers are rapidly transported from the cytoplasm to the nucleus and are competent for DNA binding. Binding of the activated STAT dimer to a target promoter initials formation of a primary transcription complex and dramatically increases the transcription rate from this promoter of target gene. Transcription of target genes induced by the STAT dimers reflects an intrinsic ability of STAT transcriptional activation domains to recruit nuclear coactivators that mediate chromatin modifications and communication with the core promoters [73]. Several lines of evidence indicated that activated PPARγ crosstalks with cytokine-mediated signal transduction pathways in modulation of immune responses and tumor cell growth and apoptosis [75–82]. Interestingly, in the case of interactions between PPARγ and STAT3 [83–87], two structurally distinct PPARγ ligands suppress IL-6 activated-STAT3 through the divergent types of crosstalk including direct or a corepressor SMRT-mediated association (see Figure 1). The 15-d-PGJ2 is a naturally occurring ligand with low affinity of PPARγ, whereas a class of antidiabetic drugs known as thiazolidinediones is a type of high-affinity synthetic ligands of PPARγ. Because the ligand-binding pocket is not static, each PPARγ ligand has the potential to induce a different conformation of the receptor. Additionally, a non-PPARγ-dependent mechanism may be involved in the difference between the effects of 15-d-PGJ2 and the thiazolidinediones on STAT3. Therefore, it is reasonable that these two structurally distinct PPARγ agonists suppress IL-6 activated STAT3 through diverse molecular mechanisms. The multiplicity of crosstalk between nuclear receptors and other transcriptional factors is an important factor that contributes to both signal diversification and specification. Direct protein-protein interaction between transcription factors and ligand-activated nuclear receptors has been shown involved in the regulation of some transcription factors. In multiple myeloma cells, we demonstrated that upon 15-d-PGJ2 binding, PPARγ indeed interacted with phosphorylated STAT3 and represses IL-6 signaling by inhibiting the binding of STAT3 to target genes [84]. Ligand-induced activation of PPARγ induces growth arrest by antagonizing the prosurvival signaling cascade induced by IL-6. PPARγ impedes IL-6 signaling by inhibiting the transcription of a number of STAT3-regulated genes such as mcl-1 and c-myc that are important in cell growth and survival. The exact mechanism through which PPARγ represses STAT3 has not been fully elucidated. PPARγ has been shown to physically associate with STAT3, which may inhibit STAT3 from binding DNA or possibly facilitate the export of STAT3 out of the nucleus. However, certain agonists that induced growth arrests of these cells did not induce SMRT to dissociate from PPARγ, suggesting that this nuclear hormone receptor may use numerous mechanisms to inhibit multiple myeloma cell growth. An alternative mechanism for PPARγ-mediated STAT3 repression has also been suggested, in which PPARγ agonist treatment of multiple myeloma cells induces the corepressor protein SMRT to dissociate from PPARγ; SMRT could then complex with and inhibit the transcriptional activities of STAT3. The corepressor SMRT has also to be demonstrated to mediate PPARγ downregulation of STAT3 in multiple myeloma cells. PPARγ can form weak interactions with the corepressor NCoR/SMRT complex. PPARγ cannot bind to DNA while it is associated with the corepressor complex. After ligand binding, PPARγ disassociates from the corepressor complex, and then binds to DNA through a peroxisome proliferator response element. We first clarified that treatment of MM cells with troglitazone decreased association of SMRT with PPARγ, which results in redistribution of corepressor SMRT from PPARγ to activated STAT3. Furthermore, this interaction between SMRT and IL-6-activated STAT3 can be attenuated by a PPARγ antagonist GW9662, confirming the specificity of the exchange of corepressor SMRT induced by the liganded PPARγ. Recruitment of SMRT, which is associated with histone deacetylase, by STAT3 leads to transcriptionally inactivating STAT3 and consequently downregulating IL-6 mediated MM cell growth and gene expression. These observations support that coactivators or corepressors function is not only for regulation of the ligand-dependent DNA binding and transcriptional activities of nuclear receptors themselves but also acts as a bridge protein to modulate nuclear receptors crosstalk with other transcription factors. 4. PPAR REGULATION OF CYTOKINE IN IMMUNE CELLS The immune response can be classified into two fundamental types: innate and adaptive immunity. The innate immune response functions as the first line of defense against infection. It consists of soluble factors, such as complement proteins, and diverse cellular components including granulocytes (basophils, eosinophils, and neutrophils), mast cells, macrophages, dendritic cells, and natural killer cells. The adaptive immune response is slower to develop but manifests as increased antigenic specificity and memory. It consists of antibodies, B cells, and CD4+ and CD8+ T lymphocytes. Natural killer T cells and γ δT cells are cytotoxic lymphocytes that straddle the interface of innate and adaptive immunity [57]. In immune responses innate and adaptive immunity are interlocked and complement each other. Signaling in the immune system can be either a direct interaction of cells or be mediated by cytokines and antibodies that are carrying signals to all cells with the appropriate receptors. Although PPARγ involvement in the regulation of innate immunere sponses has been studied since the late 1990s [16, 17], only recently it has the role of PPARγ in adaptive immunity been investigated [18–32]. Here, we focus on PPARγ regulation of cytokine-mediated immune responses in immune cells. 4.1. PPAR and IL-2 IL-2 is an autocrine and paracrine growth factor that is secreted by activated T lymphocytes and is essential for clonal T cell proliferation. Although originally described as a potent T cell growth factor in vitro, the main nonredundant role of IL-2 in vivo is now known to be the maintenance of peripheral T cell tolerance. As well as promoting the proliferation and survival of recently activated effector T cells, IL-2 also plays a critical role in regulatory T cell (Treg) homeostasis and has been variously described as promoting the thymic development, peripheral homeostasis and suppressive function of Tregs. These observations, stemming largely from studies on various murine models of IL-2 and IL-2 receptor deficiency, have prompted a greater understanding of the protolerogenic nature of IL-2 dependent signaling. Greene et al. detected the expression of PPARγ2 in normal neutrophils and peripheral blood lymphocytes in 1995 [9]. In human peripheral blood T cells, we detected inhibition of PHA-induced proliferation and IL-2 production by 15-d-PGJ2 and TZD troglitazone in a dose-dependent manner [18]. When PPARγ2 wild type expression vector was transfected into Jurkat cells, we found that troglitazone and 15-d-PGJ2 inhibited transcription and production of IL-2 in Jurkat cells in a PPARγ-dependent manner. Cotransfection assays with PPARγ and PPRE-driven/IL-2 promoter luciferase reporter constructs revealed that the inhibitory effects of troglitazone and 15-d-PGJ2 on IL-2 promoter activity are dependent on the expression and activation of PPARγ. Finally, we demonstrated that activated PPARγ inhibited the DNA-binding and activity of transcription factor NFAT regulating the IL-2 promoter in T cells. Clark et al. described the expression and function of PPARγ in mouse T-lymphocytes [20]. They demonstrated that murine SJL-derived Th1 clones and freshly isolated T cell-enriched splenocytes from SJL mice express PPARγ1 mRNA but not PPARγ2. To test its functional significance, they used two PPARγ ligands, 15-d-PGJ2 and a TZD, ciglitazone. Both ligands could inhibit antigen-induced and anti-CD3 antibody-induced T cell proliferative responses of T cell clones, and the freshly isolated T cell enriched splenocytes. In these studies, it was also demonstrated that the two PPARγ ligands mediated inhibition of IL-2 secretion by the T cell clones, whereas inhibition of IL-2 induced proliferation was not detected. 4.2. PPAR and IL-4 IL-4 is a pleiotropic and multifunctional cytokine produced by activated T cells, mast cells, and basophils [88]. IL-4 plays a critical role in regulating the outcome of an immunere sponse by facilitating the differentiation of CD4+ T cells into IL-4-producing T helper (Th) type 2 cells and suppressing the differentiation of interferon-γ producing Th1 cells, thereby favoring humoral immune responses [89]. Regulation of IL-4 gene expression, therefore, is critically important for the differentiation of Th2 cells and Th2-dependent immune responses [90]. Dysregulated expression of IL-4-producing cells has been linked with autoimmune and allergic diseases [91]. In T cells, IL-4 gene expression is regulated at the transcriptional level by both ubiquitous and cell type-restricted factors, including NF-AT, c-Maf, GATA-3, STAT6, JunB, and other transcription factors [90]. These factors interact with a proximal promoter region composed of multiple regulatory elements that can both positively and negatively affect transcriptional activation. IL-4 gene transcription is mediated by subset-specific transcription factors such as GATA-3 and c-Maf during the differentiation of naive T cells into Th2 cells. A phase of short-term gene transcription, elicited by the interaction of differentiated T cells with antigen, requires the antigen-induced transcription factor NFAT. Treatment of CD4+ T cells with ciglitazone or 15-d-PGJ2 triggered the physical association between PPARγ and NFATc1, resulting in IL-4 promoter inhibition and decreased IL-4 production [92]. Huang et al. [93] reported that IL-4 induces expression of PPARγ and 12/15-lipoxygenase in macrophages, suggesting the potential of coordinated induction of both receptor and activating ligands. Therefore, it appears likely that PPARγ is a key factor in regulating at least some aspects of macrophage lipid metabolism and primarily as a repressor of inflammatory responses. The ways how these two processes are connected, and the contribution of macrophage specific PPARγ-induced gene expression and transrepression to inflammatory responses in vivo remains to be explored. We reported an interesting PPARγ ligand-mediated immunoregulatory circuit between monocyte/macrophages and T cells [19]. Traditionally, T helper cells can be divided into two functional subsets consisting of Th1 and Th2 cells on the basis of the immunoregulatory cytokines that these T cells produce. Some of these immunoregulatory cytokines possess cross-regulatory properties and can enhance or suppress cytokine production by Th1 or Th2 subset. Thp cells are the pluripotent precursors of Th1 and Th2 cells. Moreover, the development of either Th1 or Th2 helper cells is believed to be determined by the effects of cytokines directly on helper Thp cells. IL-4 is principally produced by helper T cells of theTh2 phenotype. IL-4 has been shown to induce 12/15 lipoxygenase in monocytes/macrophages, which converts arachidonic acid into several metabolic products, including the potential PPARγ ligand 13-HODE [93]. Based on this finding, we tested the relevance of the regulation of soluble mediators (PPARγ ligands) released by IL-4 treated monocytes/macrophages on T cell activation. The medium of macrophages cultured with or without IL-4 was added to T cells stimulated with anti-CD3 or PHA/PMA. We found that T cells with the conditioned medium from IL-4-treated macrophages produced significantly less IL-2. The medium of IL-4-treated macrophages contained a sufficient amount of 13-HODE and anti-13-HODE antibody could neutralize the inhibitory effects of the IL-4-conditional medium on T cell IL-2 production. Using human T lymphocytes and the PPARγ-transfected Jurkat T cells, we demonstrated the specific inhibition by 13-HODE of the transcription factors NFAT and NF-κB, the IL-2 promoter reporter, and IL-2 production. These observations led us to hypothesize that IL-4, produced by Th2 cells, may indirectly affect the production of IL-2 by Thp or Th1 helper cells by inducing the production of these potential PPARγ ligands by macrophage 12/15-lipoxygenase, which in turn interferes with the subsequent development of T helper cells (see Figure 2) [19]. Since many complicated pathological situations cannot be simply explained by the Th1 cell and Th2 cell paradigm, efforts to resolve these issues in recent years have resulted in the discovery of many new T helper cell subsets such as Treg cell and Th17 cell subsets. Therefore, it is interesting to explore further how PPARγ regulates these new Th subsets (see Section 5). 4.3. PPAR and IFNγ IFNγ plays a central role in inflammatory reactions and is predominately produced by CD4, CD8, and NK cells. IFNγ drives inflammatory reactions by stimulating the release of NO, TNF-α, and IL-1β bymonocytes/macrophages. IFNγ is also a major effector cytokine, responsible for driving cell-mediated immunity and mediating organ-specific autoimmunity. Recent studies have shown that PPARγ ligands inhibit IFNγ production by T lymphocytes; however, the mechanism underlying this observation has not been clarified [94]. Based on previous studies, PPARγ ligands could indirectly decrease IFNγ by inhibiting activation of T cells, production of IL-2, or induction of apoptosis, or inhibiting IL-12 production by antigen-presenting cells [95–98]. Cunard et al. demonstrated that PPARγ is expressed in both murine CD4 and CD8 cells and that PPARγ ligands directly decrease IFNγ expression by murine and transformed T cell lines. In contrast, GW9662, a PPARγ antagonist, increases IFNγ expression. Transient transfection studies reveal that PPARγ ligands, in a PPARγ-dependent manner, potently repress an IFNγ promoter construct. Repression localizes to the distal conserved sequence of the minimal IFNγ promoter. They also demonstrate that PPARγ acts on the minimal IFNγ promoter by interfering with c-Jun activation. These studies suggest that many of the observed anti-inflammatory effects of PPARγ ligands may be related to direct inhibition of IFNγ by PPARγ [94]. 5. PPAR REGULATION OF CYTOKINES IN TH17 AND REGULATORY T CELLS Recently, Th17 cells and CD4+ CD25+ regulatory T (Treg) cells have been described as two distinct T helper cell subsets from Th1 and Th2 cells. Th17 cells play critical roles in the development of autoimmunity and allergic reactions by producing IL-17 and, to a lesser extent, TNF-α and IL-6 [99, 100], while Treg cells expressing the forkhead/winged helix transcription factor (Foxp3) have an anti-inflammatory role and maintain tolerance to self components by contact-dependent suppression or releasing anti-inflammatory cytokines [transforming growth factor (TGF)-β1 and IL-10], therefore, the balance between Th17 and Treg may be important in the development/prevention of inflammatory and autoimmune diseases [101, 102]. 5.1. PPAR, IL-17 and Th17 cells Production of IL-17 is a defining feature of a recently identified class of effector T cells termed Th17 cells [99, 100]. Th17 cells act as a distinct effector subset and secrete the signature cytokine IL-17, a proinflammatory cytokine that recruits and activates neutrophils, enhances T cell priming, and promotes the release of inflammatory mediators. Th17 cells provide defense against extracellular bacteria, mediate inflammation, and are critical for many types of autoinflammatory disorders (i.e., experimental autoimmune encephalomyelitis, type II collagen-induced arthritis, inflammatory bowel disease, and psoriasis). The discovery and initial characterization of these Th17 cells have provided a potential explanation for various chronic disease pathologies that were unclear with an understanding of only the Th1 and Th2 cell subsets. IL-10-deficient (IL-10−/−) mice spontaneously develop inflammatory bowel disease with a Th1-polarized cytokine pattern. In addition to showing high colonic expression of the Th1-derived cytokine IFNγ, IL-10−/− mice also show high expression of IL-17. Lytle et al. observed that rosiglitazone, a high-affinity ligand for PPARγ, had its greatest effect in suppressing IL-17 production in IL-10 knockout mice [103]. Interestingly, the PPARα ligand fenofibrate has been shown to repress IL-17 expression in cultured splenocytes activated by PMA plus ionomycin and by Th17 cells in a pathogenic CD4+ T cell line cultured from C3H Bir mice treated with cecal bacterial antigens [104]. 5.2. PPAR, TGFβ and Treg cells At least two subtypes of CD4+ CD25+ regulatory T cells (Tregs) have been described: thymically derived natural Tregs (nTregs) and inducible Tregs (iTregs) generated peripherally from CD4+ CD25− T effector cells (Teff) [100, 101]. Induced Treg are more functionally and phenotypically heterogeneous in comparison to natural Treg and can be subdivided into: induced Foxp3+ Tregs, Th3, and Tr1. Which signals drive Treg cell proliferation in the tumor setting? TGFβ is the cytokine that is thought to foster Treg-cell amplification [101]. Both tumor cells directly or “tumor educated” immune cells can locally produce large amounts of TGFβ [102]. Some mouse and rat tumors actively induce myeloid immature dentritic cells to secrete TGFβ and this promotes Treg cell proliferation. There is also substantial evidence that indicates the involvement of TGFβ in Treg cell conversion. Wohlfert et al. have used ciglitazone, a synthetic PPARγ ligands, to characterize the relationship between PPARγ ligands and both iTregs and nTregs. They reported that ciglitazone-activated PPARγ enhances the TGFβ-dependent conversion of naive T cells into Foxp3+-induced Tregs in vitro, although the mechanism by which PPARγ enhances Treg activity remains unknown [105]. Hontecillas and Bassaganya-Riera have used PPARγ deficient CD4+ cells obtained from tissue-specific PPARγ null mice to investigate the role of endogenous PPARγ on Treg and effector CD4+ T cell function. They demonstrated that only PPARγ-expressing Treg was able to completely prevent inflammation induced by effector cells of either genotype, suggesting that PPARγ expression and/or activation by endogenous agonists is required for optimal Treg function [106]. 6. PPAR REGULATION OF CYTOKINES IN CANCER CELLS Cytokines that are released in response to infection, inflammation, and immunity can function to inhibit tumor development and progression. Alternatively, cancer cells can respond to host-derived cytokines that promote growth, attenuate apotosis, and facilitate invasion and metastasis. Proinflammatory cytokines implicated in carcinogenesis include IL-1, IL-6, IL-15, colony stimulating factors, TNF-α, and the macrophage migration inhibitory factor. A unique immune response signature, consisting predominantly of humoral cytokines, promotes metastasis in hepatocellular carcinoma. Likewise, a signature consisting of 11 cytokine genes in the lung environment predicted lymph node metastasis and prognosis of lung adenocarcinoma with IL-8 and TNF-α as the top 2 genes for predicting prognosis. IL-8 was originally described as a monocyte-derived neutrophil chemotactic factor that specifically attracted neutrophils and was renamed due to its multiple function. IL-8 can have angiogenic activities in several cancers including nonsmall cell lung cancer and can function as a positive autocrine growth factor. Both TNF-α and IL-6 contributed to the chemically induced skin tumors and lymphomas in mice. Collectively, cytokines are considered as a linker between inflammation and cancer [55–57]. A considerable amount of research has shown that PPARγ ligands suppress the proliferation rates of many types of cancer cells, particularly those derived from liposarcoma, colon cancer, breast cancer, prostate cancer, myeloid leukemia, glioblastoma, and many others. Various in vitro studies have shown that treatment of many types of cancer cells with TZD resulted in the induction of cell differentiation or apoptosis as well as improvement in levels of various markers for invasion and metastasis. Furthermore, activation of PPARγ by glitazones inhibits angiogenesis and neovascularization both in vitro and in vivo and blocks the release of vascular endothelial growth factor from smooth muscle cells [107, 108]. In addition to the above direct antiproliferative and proapoptotic activities on cancer cells, effects of PPARs and their ligands in cancer cells may function through influencing cytokine production or cytokine-mediated signal transduction pathways. The mechanisms are probably linked to: (1) PPAR ligands may sensitize cancer cells to the antitumor effects of cytokines such as TNFα, (2) PPAR ligands may suppress production of cytokines for tumor cell growth, and (3) PPAR ligands may affect tumor microenvironment by regulation of Treg through influencing associated cytokines. A good example is that PPARγ ligands suppress multiple myeloma through inhibiting IL-6 and IL-6 activated signal pathway in both PPARγ-dependent and -independent manner. 6.1. PPAR and IL-6 Interleukin-6 (IL-6) is a cytokine with multiple biologic activities on a variety of cells. IL-6 plays a major role in the response to injury or infection and is involved in the immune response, inflammation, and hematopoiesis. Its deregulation impacts numerous disease states, including many types of cancer. Consequently, modulating IL-6 may be an innovative therapeutic strategy in several diseases. IL-6 is a pleiotropic cytokine that is involved in the physiology of virtually every organ system. Aberrant expression of this cytokine has been implicated in diverse human illnesses, most notably inflammatory and autoimmune disorders, coronary artery and neurologic disease, gestational problems, and neoplasms. In cancer, high levels of circulating IL-6 are observed in almost every type of tumor studied and predict a poor outcome. Furthermore, elevated IL-6 levels are associated strongly with several of the striking phenotypic features of cancer. Several molecules have been developed recently that target the biologic function of IL-6. Early results in the clinic suggest that this strategy may have a significant salutary impact on diverse tumors. The field of cytokine research has yielded a deep understanding of the fundamental role of IL-6 and its receptor in health and disease. Therapeutic targeting of IL-6 and its receptor in cancer has strong biologic rationale, and there is preliminary evidence suggesting that targeting of the IL-6 system may be beneficial in the treatment of cancer [109]. One of the most studied tumor types in relation to IL-6 is multiple myeloma, a malignancy of differentiated B-lymphocytes. Multiple myeloma is characterized by accumulation of clonal plasma cells in the bone marrow, accounts for 10% of all hematologic cancers, and remains an incurable hematological malignancy [110–112]. Recently, we investigated how PPARγ ligands suppress IL-6 gene expression through crosstalk between PPARγ and NF-κB or between PPARγ and C/EBPβ [86]. C/EBPβ and NF-κB bind to the promoter region of the IL-6 gene, and their cooperation is needed to activate IL-6 transcription. The nuclear receptor PPARγ can be activated by troglitazone. Predominately, the complex between C/EBPβ and troglitazone-bound PPARγ leads to decreased DNA binding and transactivation of C/EBPβ, inhibiting gene expression of IL-6. In addition, PGC-1, a coactivator, is shared by both PPARγ and NF-κB. After activation by ligands, ligand-bound PPARγ competes for the limited amounts of PGC-1. Therefore, NF-κB dissociates with PGC-1 and decreases NF-κB DNA-binding and transactivation, leading to blocked IL-6 transcription. In the case of 15-d-PGJ2 inhibition of IL-6 transcription, although 15-d-PGJ2 also shares the above ligand-bound PPARγ downregulation mechanisms on C/EBPβ and NF-κB, 15-d-PGJ2, compared with troglitazone, prefers to use PGC-1 as a bridging protein to associate with NF-κB. In addition, 15-d-PGJ2 inactivates NF-κB through decreasing phosphorylation of IKK and IκB in PPARγ-independent manner. The molecular mechanisms of PPARγ ligands on the regulation of multiple transcription factors have proven, not surprisingly, complex. Given that IL-6 is the key growth and survival factor of multiple myeloma cells, and is particularly involved in the origin of all benign and malignant plasma cell expansions as well as MM cell resistance, the effects and targets of the PPARγ ligands on aspects of multiple myeloma biology and bone marrow stromal cells may be clinically relevant. 7. CONCLUSIONS Most proinflammatory cytokines produced by either host immune cells or tumor cells themselves promote tumor development. By contrast, proapoptotic and anti-inflammatory cytokines usually interfere with tumor development [55]. There is emerging evidence that the nuclear receptor PPARγ interacts with transcriptional factors to modulate cytokine production and action in immunity, inflammation, autoimmune diseases, and tumors. PPARγ regulation may occur at the levels of gene expression of cytokines themselves and their receptors or cytokine-mediated signaling transduction pathways in immune cells and cancer. The crosstalk between PPARs and cytokine signaling pathways mediating inflammatory effects at the cellular level is also effective to induce the expression of PPAR genes. The molecular basis of this interaction has remained elusive, despite the proposal of several distinct mechanisms. One of the most important mechanistic aspects is protein-protein interaction through a direct or cofactor-mediated indirect manner. On the basis of insights into the mechanisms on interaction between these two distinct families of transcriptional factors activated by different signaling pathways, new targeting drug design and/or therapeutic strategies will be discovered and developed for treatment of cytokine-related diseases ranging from inflammation to cancer. ACKNOWLEDGMENT This project has been funded in whole or in part with Federal funds from the NCI/NIH under Contract NO1-CO-12400. ABBREVIATIONS AF:Activation function AP-1:Activation protein 1 C/EBP:CCAT/enhancer-binding protein DBD:DNA binding domain ER:Estrogen receptor IFN:Interferon IL:Interleukin Jaks:Janus kinases LBD:Ligand binding domain MAPK:Mitogen-activated protein kinase MM:Multiple myeloma NcoR:Nuclear receptor corepressor NFAT:Nuclear factor of activated T cells NF-κB:Nuclear factor-kappa B PPAR:Peroxisome proliferator-activated receptor PPRE:PPAR response element RAR:Retinoic acid receptor RXR:Retinoid-X receptor STAT:Signal transducer and activator of transcription SMRT:Silencing mediator of retinoid and thyroid receptors SRC:Steroid receptor coactivator TGF:Transforming growth factor Th:T helper cell TNF:Tumor necrosis factor Treg:Regulatory T cell TZD:Thiazolidinedione. Figure 1 PPARγ crosstalk with IL-6-activated STAT3 signaling pathway. Upon IL-6 binding, the IL-6R/gp130 dimer induces phosphorylation of JAK1,3, which in turn phosphorylates STAT3. The phosphorylated STAT3 dimerizes and translocates to the nucleues, where they bind to the STAT3 binding element (SBE) in the responsive gene to initiate transcription. Two structurally distinct PPARγ agonists suppress IL-6-activated STAT3 through diverse molecular mechanisms. 15-d-PGJ2 enhances direct physical protein-protein interaction between PPARγ and phosphorylated STAT3 and represses IL-6 signaling by inhibiting the binding of STAT3 to target promoters; Troglitazone inhibits the interaction between PPARγ and the corepressor SMRT, thereby inducing the redistribution of SMRT from PPARγ to activated STAT3, in turn transcriptionally inactivating STAT3 signaling. Figure 2 PPARγ regulation of cytokine-mediated immunoregulatory circuit between monocytes/macrophages and T lymphocytes. T helper (Th) lymphocytes can be traditionally divided into two functional subsets consisting of Th1 and Th2 cells on the basis of the immunoregulatory cytokines that these T cells produce. Thp cells are the pluripotent precursors of Th1 and Th2 cells. IL-4 is principally produced by helper T cells of theTh2 phenotype. IL-4 can induce the upregulation of expression of the enzyme 12/15 lipoxygenase in monocytes/macrophages, providing a potential PPARγ-specific ligands 13-HODE. The mediator secreted by monocytes can be taken up by neighboring Thp or Th1 cells and activate PPARγ in these cells. 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Experimental Diab. Res., 5:237-244, 2004 Copyright c Taylor and Francis Inc. ISSN: 1543-8600 print / 1543-8619 online DOI: 10.1080/154386090506148 Fructose Diet-Induced Skin Collagen Abnormalities Are Prevented by Lipoic Acid V. Thirunavukkarasu, A. T. Anitha Nandhini, and C. V. Anuradha Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar, India Nonenzymatic glycation of proteins, leading to chemical modification and cross-linking are of importance in the pathology of diabetic complications. We studied the effect of -lipoic acid (LA) on the content and characteristics of the protein collagen from skin of high-fructose fed rats. The rats were divided into 4 groups of 6 each. Two groups of rats were fed with a high fructose diet (60 g/100 g diet) and administered either LA (35 mg/kg b.w., i.p) (FRU+LA) or 0.2 ml vehicle (saline) (FRU) for 45 days. The other 2 groups were fed with control diet containing starch (60 g/100 g diet) and administered either saline (CON) or lipoic acid (CON+LA). The rats were maintained for 45 days and then sacrificed. Plasma glucose, insulin, fructosamine, protein glycation, and blood glycated hemoglobin (HbA1C) were measured. Collagen was isolated from skin and the physicochemical properties of collagen were studied. Fructose administration caused accumulation of collagen in skin. Extensive cross-linking was evidenced by enhanced glycation and AGE-linked fluorescence. Increased peroxidation and changes in physicochemical properties such as shrinkage temperature, aldehyde content, solubililty pattern, susceptibility to denaturing agents were observed in fructose-fed rats. SDS gel pattern of collagen from these rats showed elevated component of type I collagen. These changes were alleviated by the simultaneous administration of LA. Administration of LA to fructose-fed rats had a positive influence on both quantitative and qualitative properties Received 10 May 2004; accepted 9 July 2004. The authors thank Dr. L. Suguna, Project Fellow, Bioproducts Laboratory, Central Leather Research Institute, Chennai, for her help in carrying out the electrophoresis of collagen samples. Address correspondence to C. V. Anuradha, Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu-608 002, India. E-mail: [email protected] of collagen. The results suggest a mechanism for the ability of LA to delay diabetic complications. Keywords Fructose Diet; Glycation; Collagen; Cross Linkage; Lipoic Acid INTRODUCTION Collagens form the major structural proteins of all connective tissues and the interstitial tissue of all parenchymal organs and contribute to the stability and structural integrity of tissues and organs. Collagens are centrally involved in the formation of the fibrillar and microfibrillar network of the extracellular matrix and basement membranes [1]. Among the 20 different collagens so far identified, type I collagen is the most abundant collagen which forms more than 90% of the organic mass of bone and is the major collagen of tendons, skin, ligaments, cornea, and many interstitial connective tissues [2]. Glycation (Maillard reaction) is a nonenzymatic binding reaction between proteins and glucose that leads to the formation of advanced glycation end products (AGE). Glycation could cause changes in the structural and functional properties of proteins and are of importance in the etiology of secondary complications in diabetes [3]. Collagen is a protein with slow turnover rate that contains several basic amino acids with free amino groups and is a strong candidate for extensive modification by glycation [4]. AGEs have been shown to be associated with structural alterations in collagen by forming cross-links between collagen fibers and this creates an increase in mechanical stiffness [5] and a decrease in susceptibility to enzymatic digestion [6]. The diabetes-associated changes in collagen function have been documented to be a biochemical link between persistent hyperglycemia and diabetic microvascular disease [7]. 237 238 V. THIRUNAVUKKARASU ET AL. -Lipoic acid (LA) (1,2, dithiolane-3-pentanoic acid), a natural cofactor of mitochondrial dehydrogenases complexes, is a physiological constituent of mitochondrial membranes and an endogenous antioxidant. Four distinct antioxidant actions of LA have been observed including reactive oxygen species scavenging activity, capacity to regenerate endogenous antioxidants such as glutathione, vitamins C and E, metal chelating activity, and repair of oxidized proteins [8, 9]. Dietary fructose has adverse effects on carbohydrate metabolism [10]. Fructose-rich diet induces insulin resistance, hyperinsulinemia, glucose intolerance, hypertriglyceridemia and hypertension in rats. Fructose feeding is frequently used as an animal model for insulin resistance. Fructose feeding has been shown to cause glycation and crosslinking of skin collagen in rats [10]. Positive influence of LA has been observed in studies involving high carbohydrate feeding. LA was found to prevent the rise in blood pressure and to improve insulin sensitivity in chronic glucose-fed [11] and fructose-fed rats [12]. However, no experimental data are available on the effect of LA on collagen structure and properties. The present study explores whether LA administration would prevent collagen accumulation in skin and would improve variables such as solubility, shrinkage temperature, glycation and fluorescence in high fructose-fed rats. MATERIALS AND METHODS MaleWistarratsofbodyweightsof150-170gwereobtained from the Central Animal House of Rajah Muthiah Medical College, Annamalai University. They were housed two per cage under 12 hr- light and 12 hr- dark cycle. The rats were fed during the acclimatization period of 10 days with a standard pellet diet (Karnataka State Agro Corporation Private Ltd., Agro, feeds division, Bangalore, India). The animals used in the present study were cared for in accordance with the principles and guidelines of the Institutional Animal Ethics Committee, Rajah Muthiah Medical College, Annamalai University, Annamalai Nagar. Animals and Treatment The animals were divided into 4 groups of 6 rats each. Group 1: (CON): received control diet and water ad libitum. 0.2 ml of vehicle (saline) was given intraperitoneally daily. Group 2: (FRU): received fructose-enriched diet and water ad libitum. 0.2 ml of vehicle (saline) was given intraperitoneally daily. Group 3: (FRU+LA): received fructose diet and administered with lipoic acid (35 mg/kg b.w) dissolved in saline by intraperitoneal injection once daily. Group 4: (CON+LA): received the control diet and were given lipoic acid (35 mg/kg b.w) in saline by intraperitoneal injection once daily. The compositions of control and fructose diets are given in Table 1. The diets were prepared fresh daily. The animals were maintained in their respective groups for 45 days. Body weight changes were measured weekly. At the end of the experimental period the rats were sacrificed by cervical decapitation and blood was collected and processed for plasma separation. Skin was dissected from the dorsal side of the abdomen and removed of hairs. Glucose [13], fructosamine [14], and glycated protein levels [15] were measured in plasma, while glycated hemoglobin (HbA1C) was measured in blood [15]. Plasma insulin was estimated by the microparticle enzyme immuno assay method, using a reagent kit (Boehringer Manheim, Germany). Aweighedamountoffreshskintissue(5g)wasdefattedwith a chloroform:methanol (CM, 2:1) mixture and lyophilized. The lyophilized sample was hydrolysed with 6 N HCl for 18 h at 110 C. After hydrolysis the digested sample was evaporated to dryness, dissolved in water and hydroxyproline content was measured [16]. Total collagen content was determined by multiplying the hydroxyproline content determined by a factor 7.46. The extent of glycation was determined by the method of Rao and Pattabiraman [15] using concentrated sulphuric acid and 80% phenol. TABLE 1 Composition of diet (g/100g) Ingredients Control diet High fructose diet Corn starch 60 -- Fructose -- 60 Casein (fat free) 20 20 Methionine 0.7 0.7 Groundnut oil 5 5 Wheat bran 10.6 10.6 Salt mixture 3.5 3.5 Vitamin mixture 0.2 0.2 The composition of mineral mix (g/Kg) - MgSO4.7H2O-30.5; NaCl-65.2; KCl-105.7; KH2PO4-200.2; 3MgCO3, Mg (OH)2. 3H2O-38.8; FeC6H5O7.5H2O-40.0; CaCO3-512.4; KI-0.8; NaF-0.9; CuSO4.5H2O-1.4; MnSO4-0.4 and CONH3-0.05. 1 kg of vitamin mix contained thiamine mono nitrate, 3 g; riboflavin, 3 g; pyridoxine HCl, 3.5 g; nicotinamide, 15 g; d-calcium pantothenate, 8 g; folic acid, 1 g; d-biotin, 0.1 g; cyanocobalamin, 5 mg; vitamin A acetate, 0.6 g; -tocopherol acetate, 25 g and choline chloride, 10 g. LIPOIC ACID PREVENTS FRUCTOSE-INDUCED COLLAGEN ABNORMALITIES 239 Collagen-linked fluorescence was measured by the method of Monnier et al. [17]. About 5 g of fresh skin tissue was minced in phosphate buffered saline (PBS, 10 mM, pH 7.4) and washed successively with CM mixture and N-2-hydroxyethylpiperazine N-2-ethane sulphonic acid (HEPES) buffer (0.02 M, pH7.5). The pellet was suspended in HEPES buffer containing 120 units of type VII collagenase and digested at 37 C for 48 h and centrifuged. The fluorescence of supernatant was measured at 440 nm excitation and 370 nm emission. The solubility pattern was determined by the method of Miller and Rhodes [18]. Skin tissue (5 g) was extracted with neutral salt solvent containing 20 mM ethylene diamine tetra acetic acid and 2 mM N-ethyl maleimide. Extraction was re- peatedoncemoreandthesupernatantswerepooled.Theresidue obtained was again extracted twice with 0.5 M acetic acid. The resultingsupernatantswerepooledandtheresidueobtainedwas extracted with pepsin (100 mg/g wet tissue) and supernatants were collected. Hydroxyproline assay was done in the pooled supernatants [16]. The procedure of Adam et al. [19] was followed to assess the susceptibility of insoluble collagen to denaturing agents such as urea and potassium thiocyanate (KCNS). Insoluble collagen was suspended in 6 M urea and 2 M KCNS separately for 24 hours and then centrifuged. The supernatants were analyzed for hydroxyproline content. Aldehyde content in collagen samples were analysed according to the method of Paz et al. [20] and expressed as mol acetaldehyde/100 mg collagen. The level of thiobarbituric acid reactive substances was also measured by the method of Iqbal et al. [21]. The shrinkage temperature of collagen was determined as described by Nutting and Borasky [22]. For performing gel electrophoresis, collagen was extracted from skin by the method of Chandrakasan et al. [23]. The skin was dissected free, removed of hairs and washed in ice-cold TABLE 2 Levels of glucose, insulin, fructosamine, glycated protein in plasma, glycated hemoglobin in blood and hydroxyproline and collagen content in skin of control and experimental animals CON FRU FRU+LA CON+LA Glucose (mmol/L) 4.7 0.5 5.4 0.1 4.9 0.1# 4.8 0.1 Insulin (U/ml) 43.9 3.3 80.9 5.2 46.6 4.2# 43.1 1.5 Fructosamine (mmol/L) 0.84 0.06 1.36 0.28 0.93 0.06# 0.86 0.02 Glycated protein (mol/L) 2.26 0.21 3.44 0.25 2.37 0.36# 2.27 0.15 Glycated hemoglobin (% total Hb) 1.24 0.07 3.63 0.09 2.01 0.03# 1.25 0.04 Hydroxyproline (mg/100mg tissue) 6.04 0.49 7.62 0.58 6.47 0.58# 6.41 0.55 Total collagen (mg/100mg tissue) 48.61 3.58 59.20 2.62 48.69 2.96# 45.72 2.75 Values are mean SD of 6 rats from each group. Compared with CON (P < 0.05); # Compared with FRU (P < 0.05). ANOVA followed by DMRT. CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA- control+lipoic acid. PBS. The following procedure was carried out at 0 C. About 150-200 g of skin tissue was defatted with CM mixture and then extracted with 0.5 M acetic acid overnight and centrifuged. The supernatant was precipitated with the addition of solid sodium chloride (NaCl) to reach a concentration of 5% (W/V and kept overnight). The pellet obtained was suspended in 0.5 M acetic acid containing 100 mg pepsin/g wet tissue, centrifuged and the supernatant was precipitated with NaCl. The solution was then centrifuged and the precipitate obtained was dissolved in 0.5 M acetic acid and dialyzed overnight against 0.02 M disodium hydrogen orthophosphate for at least 5-6 changes. The purified collagen was lyophilized and subjected to sodium dodecyl sulphate gel (SDS) electrophoresis for quantification of and components using a 3% stacking gel and a 5% running gel. Coomassie brilliant blue was used for staining. The gels were scanned with a densitometer and / ratio of acid and pepsin-soluble collagen was calculated. STATISTICAL ANALYSES Values are expressed as means S.D. Data within the groups were analysed using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). A value of P < 0.05 was considered statistically significant. RESULTS Daily food consumption was similar in the experimental groups and no significant alterations in the weight gain of animals were observed between the groups. (Final body weight: CON-174.0 g 5.62; FRU-178.0 g 5.43; FRU+LA-185.0 g 3.76; CON+LA-185.0 g 4.47). Table 2 gives the levels of plasma glucose, insulin, fructosamine and glycated protein, HbA1C and the contents of hydroxyproline and total collagen in skin of control and 240 V. THIRUNAVUKKARASU ET AL. TABLE 3 Extent of glycation and AGE formation, levels of aldehyde, lipid peroxidation, and shrinkage temperature in skin collagen of control and experimental animals CON FRU FRU+LA CON+LA Glycation (g glucose/mg collagen) 7.91 1.06 14.60 0.54 8.67 0.87# 7.96 0.98 Fluorescence (AU/mol hydroxyproline) 35.47 2.23 40.40 1.99 39.18 3.17# 35.37 10.09 Aldehyde content (mol acetaldehyde/100 mg collagen) 7.81 0.88 14.93 1.61 9.43 1.02# 8.39 0.89 Lipid peroxidation (nmol/mg protein) 7.65 0.58 14.77 0.48 8.98 0.52# 8.28 0.31 Shrinkage temperature ( C) 60.69 1.14 66.44 1.60 61.67 0.69# 60.99 1.36 Values are means SD from 6 animals in each group. compared with CON, P < 0.05; # compared with FRU, P < 0.05. ANOVA followed by DMRT. CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA- control+lipoic acid. experimental animals. The levels were significantly elevated in fructose-fed rats as compared to control rats. Fructose-fed rats treated with LA showed near-normal levels of these parameters. Table 3 summarizes the effect of LA on the extent of glycation, AGE linked fluorescence, aldehyde content, peroxidation and shrinkage temperature in collagen obtained from skin of control and experimental rats. The rats fed fructose diet showed increased levels of these parameters as compared to rats fed control diet. LA treatment to fructose-treated rats restored the levels to normal control values. Table 4 shows the subunit ratio (/) of acid and pepsin soluble skin collagen in control and experimental rats. Rats fed fructose showed a significantly decreased ratio as compared to control rats. But fructose-fed rats treated with LA showed near normal levels. The ratio was unaltered in control rats treated with LA. Figures 1 and 2 show the solubility pattern of collagen in control and experimental animals. Rats fed fructose showed decreased solubility of collagen in neutral salt and acid and an increased solubility with pepsin. LA-treated fructose rats TABLE 4 Effect of LA on the / ratio of acid- and pepsin-soluble collagen in skin of control and experimental animals Acid soluble collagen Pepsin soluble collagen (Percentage of total collagen) (Percentage of total collagen) / ratio / ratio CON 60.5 38.2 1.58 69.8 27.9 2.50 FRU 52.7 47.2 1.11 62.7 37.2 1.68 FRU+LA 59.6 40.2 1.48# 68.9 31.8 2.17# CON+LA 59.6 37.6 1.58 70.6 29.4 2.41 Values are means SD from 6 animals in each group. compared with CON, P < 0.05; # compared with FRU, P < 0.05. ANOVA followed by DMRT. CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA-control+lipoic acid. showed improved solubility with neutral salt and acid as compared to fructose-fed rats. Fructose rats showed poor solubility of collagen in the presence of denaturing agents (Figure 2). Figures 3 shows the SDS-gel pattern of acid soluble and pepsin soluble collagen in skin of control and experimental rats. Fructose treated rat showed increased band width of different subunits of type I collagen and type III collagen, while fructose-treated LA supplemented rat showed a pattern similar to that of control rat. The lowest / ratio 1.11 (acid) and 1.68 (pepsin) was observed in the collagen from fructose-fed rat while collagen from the control rat showed the highest ratio of 1.58 and 2.50, respectively. LA treated fructose rat showed significantly higher values as compared to fructose-treated rat. DISCUSSION Fructose feeding induced a significant increase in total collagen content and AGE-related fluorescence of skin collagen. Excessive collagen can result from an imbalance between its synthesis and degradation by interstitial collagenases. Long-term fructosefeedingforayearinratsincreasedplasmafructosamine LIPOIC ACID PREVENTS FRUCTOSE-INDUCED COLLAGEN ABNORMALITIES 241 FIGURE 1 a) Neutral salt soluble collagen in skin. Compared with CON (P < 0.05); # Compared with FRU (P < 0.05) (one way ANOVA followed by DMRT). CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA- control+lipoic acid. b) Acid soluble collagen in skin. Compared with CON (P < 0.05); # Compared with FRU (P < 0.05) (one way ANOVA followed by DMRT). CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA- control+lipoic acid. c) Pepsin soluble collagen in skin. Compared with CON (P < 0.05); # Compared with FRU (P < 0.05) (one way ANOVA followed by DMRT). CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA-control+lipoic acid. and HbA1C, induced skin collagen crosslinking, altered the solubility of collagen and increased bone collagen fluorescence [10]. Prolonged fructose feeding thus accelerates aging by causing changes in age-related markers in collagen of skin and bones. In the present study the rats fed fructose for 45 days showed increased levels of glucose, insulin, fructosamine and glycated protein in plasma, HbA1C in blood and collagen glycation in skin. FIGURE 2 Susceptibility of skin collagen to denaturing agents. Compared with CON (P < 0.05); # Compared with FRU (P < 0.05) (one way ANOVA followed by DMRT). CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA-control+lipoic acid. Alterations in physicochemical properties indicate extensive crosslinking and maturation of collagen in fructose-fed rats. Excessive covalent cross links in collagen fibres and changes in the content of imino acids such as proline and hydroxyproline can increase the shrinkage temperature [24] and alter the solubility pattern [25]. The percentage solubility of collagen in neutral salt and acid were significantly reduced, while there was an increase in pepsin solubility of collagen from fructose-fed rats. The structure and properties of collagen are modified due to extensive crosslinking and have been reported in aging and in various pathological conditions including diabetes [26]. A significant increase in peroxidation in collagen samples were observed in skin of fructose-fed rats. Malondialdehyde, an end product of lipid peroxidation can react with the free amino groups of collagen and stimulate cross-linking [27]. Previous reports from our laboratory [28, 29] and by others [30] have shown increased lipid peroxidation products in blood and tissues indicating oxidative stress in fructose-treated rats. The SDS gel pattern of skin collagen in our present study confirms the increase in cross-linking of collagen in fructose-fed rats. The band size of -components of collagen in fructose-fed rats was higher than that of control rats. The relative abundance of high molecular weight collagen chains is demonstrated by the ratio of to chains. chains are dimers in which the inter chain cross links are not disulfide bridges [10]. Type III (1) fraction of collagen, which is specific for skin tissues are found to be significantly increased in fructose-fed rats as compared to that of control rats [10]. LA treatment prevented fructose-induced hyperglycemia and hyperinsulinemia and also abolished the alterations in 242 V. THIRUNAVUKKARASU ET AL. FIGURE 3 a) SDS gel pattern of acid soluble collagen from skin in normal and experimental rats. Compared with FRU (P < 0.05) (one way ANOVA followed by DMRT). CON-control, FRU-fructose, FRU+LA-fructose+lipoic acid, CON+LA-control+lipoic acid. b) SDS gel pattern of pepsin soluble collagen from skin in normal and experimental rats. Compared with FRU (P < 0.05) (one way ANOVA followed by DMRT). CON-control, FRU-fructose, FRU+LA- fructose+lipoic acid, CON+LA-control+lipoic acid. collagen properties. Collagen from LA-administered fructose-fed rats displayed decreased glycation, AGE formation, aldehyde and peroxide content in skin collagen, together with a decline in total collagen content as compared to untreated fructose-fed rats. The solubility pattern was improved with a relative increase in neutral salt and acid soluble collagen. These changes indicate the reduction in cross-linking of collagen in LA-treated rats. TheeffectsofLAcouldbeduetothepositiveinfluenceofLA on glycemia and glucose metabolism. LA treatment improves glucose utilisation in rat diaphragm [31]. LA has been reported to increase glucose uptake and disposal in muscle isolated from Zucker diabetic rats [32]. The antioxidant function of LA could also contribute to inhibition of protein glycation, AGE formation and cross-linking. The activity of prolyl hydroxylase an ascorbic acid-dependent enzyme has been reported to be altered in diabetic rats [33]. This enzyme is required to maintain the normal properties of collagen. This alteration is mainly due to the reduction in ascorbic acid concentrations. In a previous study, we reported a significant decrease in ascorbic acid concentration in plasma and tissues of fructose-fed rats and its reversal by LA at this dosage (35 mg/kg b.w) [28, 29]. LA effectively recycles ascorbic acid, -tocopherol and glutathione there by elevating their tissue levels. These endogenous antioxidants inhibit protein glycation and advanced glycation end product formation [34]. LA inhibits tissue lipid peroxidation in rats fed fructose [29] and protein glycation in RBCs exposed to high concentrations of glucose [35]. Crosslinking of corneal collagen by glucose in vitro is dependent on transition metal-catalyzed oxidation of glucose or Amadori products on collagen, requires oxygen, and involves the formation of superoxide, hydrogen peroxide, and hydroxyl radicals [36]. LA scavenges reactive oxygen species, chelates metal ions and participates in the repair of oxidized proteins [9]. We suggest that LA could prevent collagen abnormalities by a combination of its effect on glucose utilization and antioxidation. Consumption of fructose in diet has increased in the general population in recent years. Crystalline fructose is used extensively as a sweetener in pharmaceuticals and in food industry. High fructose sweeteners in soft drinks is estimated to account for almost half of the total added sugars in the U.S. diet [37]. Long-term fructose consumption may provoke glycation and collagen crosslinking [10] and hence may contribute to diabetic complications. 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==== Front J CarcinogJournal of Carcinogenesis1477-3163Medknow Publications 1477-3163-7-31866239710.1186/1477-3163-7-3ReviewPathogenesis of malignant pleural mesothelioma and the role of environmental and genetic factors Weiner Shoshana J [email protected] Siyamek [email protected] Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, 9500 Euclid Avenue, Cleveland, OH, USA2 University Hospitals, Case Western Reserve University School of Medicine, 11100 Euclid Avenue LKS Building 7th floor, Cleveland, OH, USA2008 28 7 2008 7 3 3 13 9 2007 28 7 2008 Copyright © 2008 Weiner and Neragi-Miandoab; licensee BioMed Central Ltd.2008Weiner and Neragi-Miandoab; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Malignant pleural mesothelioma (MPM) is a rare, aggressive tumor for which no effective therapy exists despite the discovery of many possible molecular and genetic targets. Many risk factors for MPM development have been recognized including environmental exposures, genetic susceptibility, viral contamination, and radiation. However, the late stage of MPM diagnosis and the long latency that exists between some exposures and diagnosis have made it difficult to comprehensively evaluate the role of risk factors and their downstream molecular effects. In this review, we discuss the current molecular and genetic contributors in MPM pathogenesis and the risk factors associated with these carcinogenic processes. ==== Body Introduction Malignant pleural mesothelioma (MPM) is a solid, locally aggressive tumor of the pleura that encases and invades the lung parenchyma in late stages of the disease (see figure 1) causing clinically significant morbidities such as dyspnea and chest pain [1,2]. Without treatment, MPM is associated with a poor median survival, ranging from 4 to 12 months [2]. Figure 1 Gross specimen of malignant pleural mesothelioma. Presented here is an advanced case of malignant pleural mesothelioma encasing the lung and limiting lung compliance. Diffuse growth into the lung parenchyma is also present. (Personal photo of Neragi-Mianoab). The single term MPM can be misleading in that these tumors present with substantial phenotypic variability and are, therefore, classified according to the relative proportions of epithelial and spindle cells. The three major histological types include epithelial (see figure 2), sarcomatous (see figure 3), and mixed types. In all of these types of malignant mesothelioma, the cells are more frequently bi- or multi-nucleated, arranged in clumps, and the nuclear and nucleolar sizes are proportionally larger [3]. Figure 2 Malignant pleural mesothelioma of epithelial type. Epithelial MPM derives from the mesothelial cells and consists of glands and tubules that resemble adenocarcinoma. Large spherical cells are arranged in solid masses and columns mostly within lymphatics. There may also be glandular formation as demonstrated in this photograph. (Personal photo of Neragi-Mianoab). Figure 3 Malignant pleural mesothelioma of sarcomatous type. Sarcomatous MPM originates from the deep connective tissue of the mesothelial surface and resembles fibrosarcoma. (Personal photo of Neragi-Mianoab). Epithelium-derived mesothelioma consists of large spherical cells arranged in solid masses and columns that form mainly within the lymphatics. These cells may also form glandular structures that resemble adenocarcinoma (see figure 2). The epithelial cells contain large numbers of desmosomes, tonofilaments, and long, slender branching microvilli that may have contact with extracellular collagen because the basement membranes are incomplete [4,5]. The sarcomatoid type originates from the deep connective tissue of the mesothelium. These tumors are characterized by ovoid-to-spindle-shaped cells similar to cells seen in fibrosarcomas (see figure 3) [6]. Epithelial mesothelioma is the most prevalent type, followed by mixed/biphasic, sarcomatous, and, rarely, desmoplastic types [7]. In a large study, epithelial cell type was observed in 61.5% of specimens (n = 930), followed by biphasic in 22% (n = 334) and sarcomatous in 16.4% (n = 247) [8]. Additional subtypes of epithelia histology include: tubular, papillary, solid, large/giant cell, small cell, clear cell, signet cell, glandular, microcystic, myxoid, and adenoid cystic [9,10]. Unlike some other tumors, there is no evidence for the existence of a premalignant, non-invasive phase for MPM [11]. This has made it difficult to determine which risk factors and molecular changes are responsible for the initiation and further progression of MPM development. However, through the use of cell culture, animal models, and epidemiology, an active field of research and debate has uncovered many players in MPM pathogenesis. The first part of this review considers the genetic and molecular changes that have been identified in the development of MPM. The second section will provide a more detailed review of the known risk factors that may cause these molecular changes and the controversies that surround these risk factors. Genetic and molecular processes in MPM In the development of cancer, a cell acquires alterations in gene expression and protein function that allow the cell to surpass its normal growth limits. One way of acquiring these alterations is through changes to the genome itself. Abnormal karyotypes, often with extensive aneuploidy and structural rearrangements, have been described for a number of genetic loci in MPM [12,13]. The effects of these mutations and additional effects from environmental risk factors have begun to explain how malignant mesothelioma cells form malignant tumors. Additional details about many of these pathways can be found in earlier reviews [14-16]. Tumor Suppressors Tumor suppressor genes (TSGs) play vital roles in regulating the cell cycle in response to DNA damage and other stressors. The loss of TGS function is one of the fundamental events in tumorigenesis [17]. Loss of heterozygosity (LOH) seems to be a consistent feature of MPMs, which commonly lead to the loss and/or inactivation of multiple TSGs. Pylkkanen et al. [18] demonstrated frequent deletions at specific sites within chromosomal arms 1p, 3p, 6q, 9p, 13q, 15q, and 22q. p16/CDKN2A, p15/CDKB2B and p14ARF at 9p21, FHIT gene at 3p and neurofibromatosis 2 (NF2) at 22q12 are frequently altered TSGs that account for some of these deleted sites in MPMs [18-22]. Epigenetic methods may also contribute to TSG inactivation. Promoter hypermethylation has been demonstrated for p16/CDKN2A in MPM tumor samples and cell lines, and for another TSG candidate RASSF1A located at 3p21 in cell lines [23,24]. Wilms tumor suppressor gene (WT1), another TSG associated with MPM, will be discussed in the section on pediatric mesothelioma. A surprising finding in mesothelioma research is the lack of frequent mutations in the two most notorious TSGs: p53 and pRb. Although p53 mutations have been found in MPM cell lines [21,25], there is a general sense that the contributions of p53 mutations in MPM pathogenesis are minor [21,26-28]. However, the importance of p53 deregulation is well-recognized. p53 is essential for cell cycle arrest in response to DNA damage and in genomic instability. When Vaslet et al. [29] induced mesothelioma growth in heterozygous p53+/- mice with crocidolite asbestos fibers, the mice that had lost the functioning allele of p53 had shorter latent periods and more aggressive tumors than the mice that still maintained one copy. Outside of this model, p53 function is more commonly affected by mutations in upstream and downstream members of the p53 pathways [16,25]. The most well-known mechanism is the inactivation of p53's upstream regulator p14ARF [14]. With the loss of p14ARF, the cell loses its ability to inhibit MDM2. This allows MDM2 to inhibit p53, which can no longer induce cell cycle arrest and apoptosis. The loss of pRb function occurs in a similar manner to p53. This time p16/CDKN2A is mutated. In brief, p16/CDKN2A can no longer inhibit cyclin-dependent kinases 4 and 6, which are responsible for the G1-S phase transition of the cell cycle. Left unchecked, these kinases can phosphorylate and thereby inactivate pRb, which allows an uncontrolled entry into S phase [14]. Homozygous deletion of p16/CDKN2A has been reported in >70% of malignant mesotheliomas, and has been associated with poor prognosis [30,31]. In addition to proteins within the p53 and pRb pathways, the role of a viral protein has also been shown to inactivate these TSGs. The large tumor antigen protein (SV40Tag) of the SV40 virus can bind to and inactivate both p53 and pRb [32-37]. However, the role of SV40 virus in mesothelioma is still a matter of debate, as discussed below. Not only must malignant tumors continue their growth, but they must also be able to move and invade. A recent paper by Poulikakos et al. [38] proposes a mechanism through which merlin, the product of NF2, may help mesothelioma cells acquire this ability. Through the use of in vitro assays, Poulikakos et al. [38] found that the re-expression of merlin in two human malignant mesothelioma cell lines significantly decreased cell motility and invasion. Furthermore, merlin may mediate its effects through the phosphorylation of focal adhesion kinase. Further evidence is needed in order to determine if merlin functions in this particular way in vivo. Oncogenes While the loss of TSGs allows the cell to grow in light of aberrant changes in cellular DNA and function, it is the activation of oncogenes that inspires cell growth and proliferation. AP-1 and β-catenin transcription factors are implicated in MPM pathogenesis. The AP-1 family of transcription factors is known for mediating a wide range of processes including proliferation, apoptosis and transformation in response to a variety of stimuli [39]. The activation and expression of AP-1 proteins are regulated by different mitogen-activated protein kinases (MAPK) [40]. Fra-1, a member of the Fos family of AP-1 transcription factors, has been implicated in MPM pathogenesis. Ramos-Nino et al. [41] found a high level of AP-1 DNA binding activity and an increased expression of Fra-1 in mesothelioma cell lines. In addition, rat mesothelial cells exposed to either asbestos or epidermal growth factor (EGF) showed an increase in Fra-1 expression. This expression was abrogated when cells were pretreated with an inhibitor to the MAPK, extracellular-signal-regulated kinase (ERK). When mesothelioma cell lines were treated with either the ERK inhibitor or were transfected with a vector carrying a dominant negative fra-1, there was a reversal of the transformed phenotype of the cells [41]. Previous work also examined the role of asbestos and the EGF receptor in ERK activation [42]. Zanella et al. [42,43] hypothesized that asbestos may activate the EGF receptor itself or may also induce the ERK pathway through the formation of reactive oxygen species. Hepatocyte growth factor (HGF) and signaling through its receptor, c-Met, may also activate ERKs in addition to the Akt pathway [44]. Furthermore, the small tumor antigen (SV40tag) protein of the SV40 virus may activate ERK. SV40tag is known to inhibit protein phosphatase 2A (PP2A). Due to this inhibition, PP2A can no longer dephosphorylate and inactivate members of the MAPK family [32,41]. The transcription factor β-catenin is regulated by an ubiquitin ligase pathway. In the absence of Wnt signaling, glycogen synthase kinase 3B (GSK3β) phosphorylates adenomatous polyposis coli (APC) and axin, which increases their affinity for β-catenin. GSK3β can then phosphorylate β-catenin, marking it for destruction. When Wnt is present, GSK3β phosphorylation is inhibited, allowing β-catenin to escape ubiquination, and enter the nucleus where it can bind to T-cell factor/lymphoid-enhancer factor (Tcf/Lef) transcription factor proteins thereby activating transcription of downstream effectors [45]. The role of this oncogenic pathway has been previously reviewed by Lee et al. [14] Here we briefly discuss the concerns surrounding the activation of β-catenin in MPM. It is interesting that activating mutations in the GSK3β phosphorylation sites of β-catenin have not been detected [46] since mutations in the p53 and pRb tumor suppressors also do not appear to play an important role in MPM pathogenesis as discussed above. Just as with these TSGs, we may need to look to upstream regulators in order to find our answer as to how β-catenin is activated. For example, an increase in disheveled expression has been observed in patient samples and mesothelioma cell lines, [47] and a lack of staining in some mesotheliomas for the C-terminus of APC has led to the hypothesis that inactivating mutations of APC may be involved [46]. A group of upstream negative-regulators of the Wnt pathway have also been implicated. Wnt inhibitory factor 1 (WIF-1) [48], Dickkopf-1 [49], and secreted frizzle-related proteins (sFRP) [50,51] have the ability to inhibit Wnt signaling at the level of cell membrane receptor activation. Thus far, data has demonstrated a possible role for promoter methylation in the inactivation of WIF-1 and sFRPs [48,50], a mechanism also described for TSG inactivation. Lee et al. [49] has also hypothesized that some of these mediators may have a role outside of the canonical Wnt-β-catenin pathway. Lastly, SV40tag may also be involved in the activation of the Wnt pathway, in addition to its role of ERK activation [32]. In this case, the inhibited PP2A cannot inactivate the transcription factor β-catenin, making entrance to the nucleus possible. Landscapers The role of growth factors in carcinogenesis has already been suggested above in the case of MAPK pathway activation by EGF. Growth factors can stimulate proliferative pathways through their contact with membrane receptors. They may also play a role in the process of tumor invasion and metastasis, which has been demonstrated by their ability to stimulate chemotactic and/or chemokinetic motility in mesothelioma cell lines [52]. Moreover, they can act on stromal cells in order to provide an environment favorable to tumor growth. For example, endothelial cells proliferate during the process of angiogenesis, which supplies the growing tumor with necessary oxygen and nutrients. Another important family of landscaper genes is the matrix metalloproteases (MMPs), which help to degrade the extracellular matrix that surrounds the tumor [53-57]. This process is also important in angiogenesis as well as in tumor migration and invasion. Here we also mention that the expression of cyclooxygenase 2 (COX-2) has been recognized as a prognostic factor in MPM as reviewed elsewhere [15,16]. It is possible that COX-2 may play a role in angiogenesis and/or resistance to apoptosis [58]. Many growth factors, such as insulin-like growth factor-1 (IGF-1) [16,59], hepatocyte growth factor (HGF) [44,60], basic fibroblast growth factor (b-FGF) [61], TNF-α [62], EGF [14,63], VEGF [14,64-68] and platelet-derived growth factor (PDGF) A and B [69], have been implicated in the development and progression of malignant mesothelioma. Growth factors may come from a variety of sources during the course of MPM pathogenesis such as the surrounding lung parenchyma [70], macrophages [62], and from the mesothelial cells themselves [44,62] in response to a number of stimuli including inflammatory cytokines [62,70], asbestos [62], and SV40 infection [64,65]. Some of these stimuli may also induce ectopic signaling through growth factor receptors without the need for ligand stimulation [42] and/or activating mutations of these receptors may result in signaling [44]. The role of growth factors in oncogenesis has become a provocative subject in cancer therapy since the development of cytotoxic drugs that target these growth factors offer fresh potential for the treatment of mesothelioma [67,71]. Here we discuss the role of VEGF, HGF, and TNF-α in more detail. Additional growth factors are reviewed elsewhere [14-16]. VEGF is an angiogenic peptide, which is an independent prognostic factor in MPM [66]. Higher VEGF levels were found in the pleural effusions of patients with MPM compared to its level in the effusions of patients with non-malignant pleural disease [68]. While VEGF is a fairly specific angiogenic factor, it has also been shown to increase the growth of mesothelioma cells in vitro [68]. The use of antisense oligonucleotides (ODN) to inhibit the production of both VEGF and VEGF-C slowed mesothelioma cell growth. In addition, antibodies to VEGF receptor (VEGFR-2) and VEGF-C receptor (VEGFR-3) had a synergistic effect in inhibiting mesothelioma cell growth [67]. One mechanism that may increase the production of VEGF in MPM is SV40 infection. Production and release of VEGF was greater in SV40Tag-positive MPM cells than in MPM cells that did not show evidence of SV40 infection [65]. It appears that this effect is mediated through more than just SV40Tag, since mesothelial cells transfected with the full length SV40 genome produced higher levels of VEGF as compared to cells transfected with SV40Tag only [65]. HGF has many possible roles in MPM pathogenesis. It may stimulate morphological changes [64], promote cell growth and migration [44,64] and induce angiogenesis by itself or through an increase in the production of VEGF [72]. Some of these effects may be mediated by downstream signaling via Akt and ERK pathways [44]. The mechanisms by which HGF and its receptor are activated are not fully understood. Cacciotti et al. proposes an SV40-mediated activation that is dependent on SV40Tag binding to pRb. The HGF that is produced in response to SV40 infection signals through autocrine and paracrine mechanisms [64]. HGF may also come from neighboring lung tissue that has been damaged by asbestos exposure and inflammation [70]. Lastly, signaling through c-Met may be caused through activating mutations [44]. TNF-α may help to explain the survival of mesothelial cells after exposure to asbestos [62]. Human mesothelial cells are sensitive and often die after phagocytosis of asbestos fibers [73]. The dilemma caused by this finding is well stated by Yang et al. [62]: "How can asbestos cause MM [malignant mesothelioma] if HM [human mesothelial cells] exposed to asbestos die?" They show in vitro that asbestos induces the expression of TNF-α and its receptor in human mesothelial cells, and that cell survival may be mediated by the NF-κB pathway downstream of receptor activation. They also propose that other cells, macrophages in particular, may be important contributors of TNF-α in vivo [62]. More research is needed in order to test this hypothesis. Apoptosis Genes Apoptosis frequently occurs in response to signals from outside the cell. For example, TNF-related apoptosis-inducing ligand (TRAIL), Fas ligand and a lack of growth factor stimulation may result in programmed cell death [74]. However, it is a couple of intracellular mediators of apoptosis that have thus far been implicated in MPM. The over-expression of BCL-2 helps to protect the cell from apoptosis as reviewed elsewhere [14-16]. MPM cells may also be protected from apoptosis by the ectopic expression of telomerase [75], and SV40 infection may be one way mesothelial cells activate this protein [76]. In the absence of telomerase, telomeres located at the terminal segments of chromosomes shorten with each cell division. At first, these segments protect the coding regions of chromosomal DNA from degradation. When the telomeres become too short, apoptosis ensues. The activation of telomerase allows MPM cells to escape this mechanism of cell death and to perpetuate mutations that might have otherwise been discarded in the normal process of "cell aging." Risk factors of MPM: Their contributions and controversies Many risk factors have been identified as contributors to MPM pathogenesis. Above, we began the discussion surrounding the contribution of genetics, asbestos, and SV40 infection. Here we will complete our discussion of these factors and further introduce concerns surrounding the relationship of MPM to radiation and to the pediatric population. Asbestos Exposure Asbestos' contribution to the pathogenesis of MPM is multifaceted with effects ranging from direct to indirect, genetic to molecular. Asbestos induces mutations in mesothelial cells. The more direct mechanism of injury includes deposition of asbestos fiber in the pleura. Longer fibers can penetrate deeply into parietal pleura and have a high likelihood of causing cancer [77]. Asbestos fibers may also damage the mitotic spindle of cells and thereby disrupt mitosis, resulting in aneuploidy and DNA damage [78,79]. In a less direct fashion, asbestos can lead to the formation of reactive oxygen species (ROS). The production of ROS can be catalyzed by the iron content of the fibers or can occur through additional reactions on the fiber surface [80,81]. Macrophages that have phagocytosed asbestos fibers release ROS and lymphokines, which can damage DNA and possibly suppress the immune system [82,83]. Asbestos and/or the resulting ROS may also directly activate cell-signaling pathways [42,43]. The response of mesothelial cells to asbestos and ROS is an important factor in MPM pathogenesis. Above, we described the possible role of TNF-α in protecting mesothelial cells from death after asbestos exposure [62]. The ability to manage oxidative damage may be another mechanism mesothelial cells use to protect themselves [84]. Ferritin heavy chain (FHC) is a subunit of ferritin involved in iron sequestration. Aung et al. [84] found that when two mesothelioma cell lines were treated with asbestos, the cells that expressed higher levels of FHC had a smaller percentage increase in hydrogen peroxide generation and experienced less apoptosis as compared to cells that expressed lower levels. In addition, polymorphisms in some genes of important free radical scavenging enzymes such as mitochondrial manganese superoxide dismutase (MnSOD), glutathione-S-transferase M1 and mEH have been associated with MPM [85,86]. The Asbestos Controversy: Which Fiber is Responsible? The use of the single term "asbestos" to describe at least five unique fibrous silicate minerals (see table 1) hides the underlying controversy as to which fibers truly carry carcinogenic potential. The asbestos fibers, with regard to their bio-persistence and dimensional properties, have been stratified into two main groups: serpentine fibers (mainly chrysotile), and amphibole fibers (consisting of crocidolite, tremolite, anthrophylite and amosite). Nearly 95% of asbestos used internationally is chrysotile, and only 5% is amosite and crocidolite [87], although these groups of fibers are commonly found mixed together. New members of the asbestos family and new fiber species have been found to have carcinogenetic potential [88,89]. Exposure to these fibers may help to explain why some communities experience a high incidence of MPM without any known evidence of asbestos exposure [89]. Table 1 Classification of asbestos fibers Type Subtype Known Chemical Formula Serpentine Chrysotile Mg6Si4O10(OH)8 Amphibole Crocidolite Na2(Fe3+)2(Fe2+)3Si8O22(OH)2 Tremolite Ca2Mg5Si8O22 (OH)2 Anthophyllite (Mg,Fe)7Si8O22(OH)2 Amosite Actinolite Ca2(Mg, Fe)5Si8O22(OH)2 Fluoro-edenite Erionite Zeolite The two different theories dealing with carcinogenicity of asbestos fibers are the Amphibole Hypothesis and Stanton's Theory. The Amphibole Hypothesis claims that only amphibole fibers can cause cancer, since chrysotile fibers are broken down and cleared too quickly to provoke carcinogenesis [90]. This is in contrast to amphibole fibers, which persist in the body for a longer period of time as a result of their durability and biopersistance [91]. The Stanton Theory suggests that long and thin fibers (≥ 8 micrometer in length and ≤ 0.25 microm in width) are strongly carcinogenic regardless of their physicochemical nature [92], since they can penetrate further into the pleura [77]. There is data that both supports and opposes these theories. In his recent reviews [93,94], Yarborough concludes that the epidemiological evidence for the role of chrysotile fibers in MPM pathogenesis is weak and that chrysotile may not play a significant role in this disease process. However, he does admit that a threshold for chrysotile most likely exists though it has not yet been adequately detected by epidemiological research [93]. Although Yarborough's conclusions mainly support the Amphibole Hypothesis, they do not completely rule out a role for chrysotile fibers in MPM pathogenesis. Conclusions concerning the Stanton Hypothesis are also mixed. A recent expert panel concluded that longer fibers have more carcinogenic potential [95] although an earlier review of animal and in vitro studies by Jaurand [96] did not find this correlation. The role of the different fiber types and the quantity of exposure necessary to cause MPM are still controversial. The data are complicated by the variability in study designs and definitions as well as by the role of other risk factors such as genetics, industrial hygiene, and concomitant smoking [96-99]. However, the general consensus holds that longer fibers and amphibole fibers have more carcinogenic potential than their shorter and chrysotile counterparts in regards to MPM. Radiation Recent advances in cancer treatment involve multimodality approaches that include surgery, chemotherapy, and irradiation. Although these combined therapies increase survival in certain types of cancer, they can also cause the development of new malignancies [2,100,101]. Ionizing radiation, in particular, has been shown to play an important role in secondary benign and malignant tumors [102,103]. Post-radiation malignant mesothelioma has been reported after radiation therapy for breast cancer [102,104,105], Hodgkin's disease [106], cervical cancer [107]. Wilm's tumor [108-110], and seminoma [111]. In order to rule out previous asbestos exposure as a risk factor in these cases, Wissman [112] measured the levels of ferruginous bodies in the lung of a patient who developed MPM after radiation for Hodgkin's disease. The lung tissue showed 250 ferruginous bodies per gram of lung tissue, which is consistent with no significant prior asbestos exposure. In addition, Cavazza et al. [113] reviewed the National Cancer Institute's SEER data for 30 patients who developed malignant mesothelioma after radiation therapy. According to Cahan's criteria, these cases of mesothelioma may be considered as treatment-related post-radiation mesothelioma or sarcoma [114]. Radiation seems to contribute to MPM development in a small percentage of patients after radiation therapy. This low incidence may be explained by a multifactorial cause of secondary malignancies. In addition to radiation, exposure to chemotherapy, genetic predisposition, environmental cocarcinogens and other factors may be needed in their development [115]. As the frequent use of radiation therapy has raised concerns about future increases in the incidence of these secondary malignancies [116-118], future research to recognize these additional risk factors may be useful in identifying and modifying the treatment of patients who are likely to develop these secondary cancers. SV40 virus SV40 is a poliomavirus with double-stranded circular DNA [33,119]. The virus has two regions, early and late. The early region encodes SV40Tag, SV40tag, and 17 KT. The late region encodes structural proteins of the virus [120]. Important roles of the early proteins in TSG, oncogene and growth factor regulation were described above. Briefly, SV40Tag can bind to and inhibit p53 and pRb TSGs, and SV40tag has been shown to inhibit PP2A, which may lead to the activation of Wnt and ERK signaling pathways. SV40 infection may also increase autocrine and paracrine signaling through a variety of growth factor pathways and induce the expression of telomerase [32]. Another possible role of SV40 in MPM that was not stated above is that SV40 infection may increase the transcription and activation of Notch-1, which may have an important role in mesothelial cell transformation and proliferation [121]. Lastly, SV40 has been hypothesized to work as a cocarcinogen with asbestos [73,122-125]. SV40 may provide another mechanism by which mesothelial cells escape asbestos' cytotoxic effects [122]. Cacciotti et al. [122] found that mesothelial and mesothelioma cells infected with SV40 were more resistant to asbestos treatment. They concluded that this effect may be mediated by the activation of the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway. This pathway may become activated downstream of SV40's ability to induce growth factor signaling pathways or through other mechanisms. They provided in vivo evidence from mesothelioma samples. 10 out of 11 tumors with detectable SV40Tag expression also stained positively for activated Akt. After helping mesothelial cells survive asbestos exposure, SV40 may also work with asbestos to cause DNA damage [125] and to transform cells [73,122,124]. When Bocchetta et al. [73] expressed SV40Tag and SV40tag in human mesothelial and fibroblast cells, cells that had been treated with asbestos showed a larger number of transformed foci as compared to cells that only expressed SV40. Controversy about SV40: Does SV40 really play a role? Not only does the above data supply strong mechanistic support for the role of SV40 infection in MPM pathogenesis, but in vitro experiments have also demonstrated a high susceptibility of mesothelial cells to develop stable infections by SV40 as compared to human fibroblasts, which quickly lyse after only semipermissive infection [73]. Despite this collection of mainly in vitro data, a role of SV40 infection in MPM pathogenesis has not been established due to the conflicting results of epidemiological studies. From 1955 to 1963, the polio vaccine supplied to the United States, Canada, Europe, Asia and Africa was contaminated with SV40. Furthermore, the possibility of horizontal transmission may have enlarged this exposure [32]. Many studies [123,126-130] have found evidence of SV40 infection. A meta-analysis conducted by Vilchez [131] reviewed molecular, pathological, and clinical data from 1,793 cancer patients. He concluded that there is significant data to support a role for SV40 infection in human brain cancers, bone cancers, malignant mesothelioma, and non-Hodgkin's lymphoma. However, whether these results can be attributed to polio virus contamination is not completely clear [128,129,132,133]. Populations in Finland and Turkey were not exposed to the contaminated vaccine. In these cases, SV40 contamination was not observed in MPM as would be expected if the source of infection was primarily vaccination. On the other hand, Engels et al. [134] reported that although a contaminated poliovirus vaccine was administered to most children in Denmark from 1955 to 1961, there was no increase in malignant mesothelioma incidence [134]. Additional studies argue against the significance of SV40 infection [135,136]. In a study by Manfredi et al. [135], SV40Tag DNA was not detectable in tumor tissue of 69 mesothelioma patients. SV40Tag protein was also undetectable in tumor samples and mesothelioma cell lines by immunohistochemistry. Perhaps the standardization of SV40-detection techniques and more comprehensive studies will determine if SV40 will be a worthwhile target for preventative measures in the future [32]. Pediatric mesothelioma, familial cases and genetic predisposition Mesothelioma is very rare in childhood with an estimated 2%–5% of all cases occurring in the first two decades of life. The diagnosis may be challenging because of its rarity and its pathologic similarities to other papillary or spindle cell neoplasms in the pediatric age group [137,138]. To date, little is known about the pathogenesis of MPM in children. Frair et al. [139] reviewed the risk factors in a series of 80 pediatric patients with malignant mesothelioma. Only four of the 80 children had exposure to known risk factors (two had history of exposure to asbestos, one had received radiation therapy, and one was exposed to isoniazid in utero). A causal relationship between malignant mesothelioma and asbestos exposure, radiation, and/or isoniazid could not be established. As far as asbestos is concerned, the long latency of asbestos-related mesothelioma makes a role for this risk factor in pediatric MPM unlikely [140,141] unless the natural history of asbestos-related cancer in children is very aggressive and rapidly progresses [140]. Pediatric and familial cases may open an avenue for the study of genetic contributions to MPM. Small case studies have found that pediatric patients with Wilm's tumor who were treated with radiation may be at an increased risk for mesothelioma [110,142]. This risk may represent a role of WT1 as a TSG in MPM pathogenesis. However, this picture is complicated by data that reports a lack of inactivating mutations in MPM [143-145]. Also, one study has reported a de novo activating-mutation in WT1 in a 45-year old woman with a perotineal mesothelioma [145], and many studies have demonstrated the expression of WT1 in mesothelioma as reviewed by Whitson et al. [16]. It may be possible that the WT1 plays different roles in pediatric versus adult mesotheliomas, or that the association of Wilm's tumor with mesothelioma is confounded by the exposure to radiation and other risk factors. The study of pediatric patients who have not been exposed to known risk factors may provide a key opportunity to evaluate WT1 involvement in MPM in addition to the role of other genetic contributors. Familial cases also support the role of genetic predisposition to MPM [146,147]. However, the interpretation of these studies may be complicated by the presence of common environmental exposures. Ascoli et al. [146] found that most of the familial cases in their study had been exposed to asbestos. One might conclude that this common exposure outweighs any contribution of genetics. On the other hand, these cases provide a resource to examine genetics-asbestos interactions in MPM pathogenesis, and may help to explain why less than 10% of people exposed to asbestos develop mesothelioma [130]. Already, various studies have begun to apply modern genetic association techniques to the study of mesothelioma [31,85,86,148,149]. However more rigorous studies with larger sample sizes will be needed in order to make the most out of these techniques. Ohar et al. [150] has also offered other demographic information that may be taken into consideration when planning and analyzing genetic studies. Conclusion MPM has a complex etiology in which asbestos, ionizing radiation, viruses, genetic factors, and even diet [151] may act alone or in concert to activate the molecular processes necessary for carcinogenesis. A multi-step process is supported by the observation that numerous chromosomal deletions accumulate in most malignant mesotheliomas, many of which result in the loss and/or inactivation of TSGs [18,22]. However, uncovering the temporality of these steps has been difficult. Although many experimental techniques have been employed, the study of MPM is complicated by its late stage at diagnosis and its rarity. The long latency between asbestos exposure and MPM diagnosis exemplifies these problems. It is unclear if the time lag between asbestos exposure and diagnosis reflects a slow-growing tumor after early genetic mutations, or if the accumulation of genetic changes reaches a threshold of malignant transformation [11] since the late stage of diagnosis makes it difficult to determine the temporality of various genetic and molecular events. In addition, the long latency and the rarity make asbestos-induced MPM a poor candidate for comprehensive cohort studies. Through the application of innovative animal models [21,124,152], in vitro studies, and epidemiology, we may be able to gain a better understanding of which risk factors and molecular targets are the most important for future preventative and therapeutic measures. Competing interests The authors declare that they have no competing interests. Authors' contributions SJW and SNM both contributed to the research and writing. 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==== Front Mol VisMVMolecular Vision1090-0535Molecular Vision 1762007MOLVIS0329 ReviewEstrogens and neuroprotection in retinal diseases Kumar D. Maneesh 1Simpkins James W. 2Agarwal Neeraj 11 Departments of Cell Biology and Genetics, UNT Health Science Center, Fort Worth, TX2 Pharmacology and Neuroscience, UNT Health Science Center, Fort Worth, TXCorrespondence to: Neeraj Agarwal, Departments of Cell Biology and Genetics, UNT Health Science Center, Fort Worth, TX, 76107; Phone: (817) 735-2000; FAX: (817) 735-2610 email: [email protected] Dr. Agarwal is presently at Division of Extramural Research, National Eye Institute, NIH Suite 1300, 5635 Fishers Lane, MSC 9300, Bethesda, MD, 20892-93002008 11 8 2008 14 1480 1486 22 10 2007 18 7 2008 2008Molecular VisionThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Estrogens play a critical role in the normal growth and development of humans and in recent years our understanding of their effects in the central nervous system (CNS) have been advancing rapidly. It is now known that estrogens influence synaptic plasticity, brain development, and memory. In addition, estrogens have been shown to be neuroprotective in degenerative disorders. The understanding of the influences of estrogens in the retina, as a component of the CNS, has not kept pace with the advances in understanding of the brain. Studies that have addressed the effects of estrogens on the retina, specifically those focusing on glaucoma, are examined here in the hope that estrogen therapy may be a viable option for treating retinal dystrophies and optic neuropathies. GalleyStatusExport to XML ==== Body Introduction Glaucomatous visual field deterioration is a neurodegenerative process commonly associated with an insidious increase in intraocular pressure (IOP), with characteristic optic disc and retinal ganglion cell (RGC) nerve fiber layer damage [1,2]. World Health Organization statistics indicate that glaucoma accounts for blindness in at least 5 million people, causing 13.5% of worldwide blindness [3]. In the United States, glaucoma is the second leading cause of blindness and the second most frequent reason for ambulatory visits to physicians by U.S. Medicare beneficiaries [4]. Extrapolating to the entire Medicare population, blindness and vision loss are associated with greater than $2 billion in non-ophthalmologic related costs. The staggering healthcare and economic burdens make vision loss a medical imperative [5]. In recent years, there has been compelling evidence to support the use of estrogens as neuroprotectants [6-13]. However, the Women’s Health Initiative Study concluded that conjugated equine estrogens and estrogen plus medroxyprogesterone acetate increase the risk for ischemic stroke in generally healthy postmenopausal women [14,15]. Additional findings indicate that low doses of 17β-estradiol may protect against ischemia and reperfusion injury [16,17]. A correlation between glaucoma and ophthalmic artery blood flow changes with hormone therapy (HT), female reproductive factors, as well as HT and IOP clearly illustrate that estrogens influence the health of the retina [18-22]. Nonetheless, the use of estrogens as neuroprotectants is complicated by the natural feminizing sequelae that would be undesired in male patients, and therefore it becomes important to investigate use of synthetic non-feminizing estrogen analogs. With an emphasis on glaucoma as the primary retinal neurodegenerative disease, this review examines whether estrogens can be effective neuroprotectants in the retina. Estrogen receptor expression in the retina Estrogen receptors α and β (ERα and ERβ) are involved in classic receptor-mediated genomic pathways as well as alternative mechanisms of orchestrating neuroprotection [12,13,23]. Bovine and rat retinas have been shown to express both receptor isoforms (ERα predominates in rat) throughout the retinal thickness, seen in greatest density within the ganglion cell layer, the inner nuclear layer, and the outer portion of the outer nuclear layer [24,25]. Human donor eyes from premenopausal women have been found to express ERα in the neurosensory retina and retinal pigment epithelium, but receptors have not been detected in tissues dissected from men and postmenopausal women [26]. Another study of ER expression in humans showed that ERβ protein was localized to the ganglion cell layer and choroid. However, at the transcriptional level both ERα and ERβ were observed with local differences in expression, suggesting variability in the ratio of ERα:ERβ [27]. ERs have also been detected in the neuroendocrine secretory and metabolic ciliary epithelium. 17β-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation for sex steroids, were shown to be under direct paracrine influence of 17β-estradiol, evidence of estrogen modulating its own fate within the eye [28]. The presence of estrogen receptors and metabolic machinery within the eye suggest that estrogens play more than a passive role in the retina. Retinal estrogenic effects Given the intimate association between estrogens, their receptors, and their metabolism in the eye, is it possible that estrogens could have deleterious effects similar to those seen with HT in the Women’s Health Initiative Study? Rather than producing hypercoagulability, it appears that HT in older women does not have a long-term vasodilatory effect on retinal arterioles. These studies were based on the coronary vessel and incidence of myocardial infarction studies in fertile women, and does not support the hypothesis that exogenous estrogen exposure accounts for greater observed retinal arteriolar diameters in women [29]. Curiously, blood viscosity is decreased and ocular vascularity is improved in women with glaucoma who receive HT [19]. Neither of these studies clearly indicate what role, if any, HT plays in glaucoma. Is it possible then that gender differences or exposure to endogenous feminizing hormones could affect IOP and the development of glaucoma? This question was the basis for investigations that examined the effect of HT on IOP in a single case study and three large international studies examining female reproductive factors as risks for the development of open angle glaucoma (OAG). An early case study examined IOP changes with initiation of HT through 12 weeks of treatment. After the initiation of HT treatment of one glaucoma patient who had menopausal symptoms, it was found IOP was reduced by an average of 4.5 mmHg at four weeks and by an average of 4 mmHg at 12 weeks [20]. In a larger cross-sectional controlled study, 107 women receiving HT and 107 women serving as controls underwent IOP assessment and cup-to-disc ratio assessment. In addition, they completed comprehensive medical and family history questionnaires. The two groups were found to not differ in mean IOP, cup-to-disc ratios, prevalence of elevated IOP, or prevalence of glaucoma. A personal history of ischemic heart disease was the only clear risk factor for increased IOP [30]. The Rotterdam Study published findings on the association between early menopause and the development of OAG. This population-based study concluded that early menopause (≤45 years of age), age-adjusted and controlled for HT use, was a significant risk factor for the development of OAG [18]. The Blue Mountain Eye Study, which examined the association between endogenous estrogen exposure and OAG, determined that early menarche (age ≤ 12 years) and increasing parity significantly increased the risk for OAG [22]. A similar study conducted in rural southern India found no connection between female reproductive factors and OAG [21]. This disparate finding may be explained by significantly greater phytoestrogen content in the predominantly vegetarian south Indian diet, possibly masking what may otherwise have been an association between female reproductive factors and OAG. This theory was supported by a study that examined retinal thickness in male and female rats whose diet was supplemented with soy phytoestrogens. Male rats fed a phytoestrogen-fortified diet showed an increase in retinal thickness, while female rats showed a decrease in retinal thickness compared to control diet-fed animals [33]. These studies equivocally suggest that HT and length of endogenous estrogen exposure may influence the development of OAG. Although not directly relevant to retina, the risk of cardiovascular disease (CVD) in postmenopausal women receiving estrogen and progesterone has been proposed to be due to increased levels of pro-inflammatory cytokine TNF-α [31]. However, estrogen can also lower markers of vascular inflammation (IL-6 secretion), and thus render protective effects of lower doses of 17β-estradiol in combination with trivalent chromium therapy in diabetic monocytes [32]. Estrogens as neuroprotectants Estrogen receptor independent and dependent neuroprotective mechanisms have been well established in the brain [7,12,13,34,35], but similar studies in the retina either are limited or have not been conducted. Those studies that demonstrate neuroprotective efficacy in models of retinal disease are examined here. One of the pioneering studies in estrogen-mediated retinal neuroprotection examined the efficacy of a synthetic estrogen analog in an in vivo model of retinitis pigmentosa (RP) and in an in vitro model of N-methyl-D-aspartate (NMDA)-induced excitotoxic glaucomatous RGC death. In the RP model, treatment with an estrogen analog at postnatal day 9 yielded an outer nuclear layer, containing the photoreceptors cells lost in RP, that was nearly twice the thickness in untreated controls [36]. In the glaucoma model, primary RGCs treated with the same estrogen analog were protected from NMDA-induced excitotoxic death [36]. Furthermore, the in vitro study demonstrated maintenance of mitochondrial stability and inhibition of lipid peroxidation. These findings were in accord with previous reports from brain parenchymal studies, offering a novel approach to neuroprotection in degenerative diseases of the retina [36]. Another study examined protection of cultured retinal pigment epithelial cells (RPE), the pathologic target in age-related macular degeneration (AMD) vision loss, by 17β-estradiol against hydrogen peroxide-induced cell death. This study not only demonstrated significant protection of RPE cells, but also showed that 17β-estradiol quenched hydrogen peroxide-induced upregulation of apoptosis-related proteins [37]. The large cross-sectional Salisbury Eye Evaluation Project supported these in vitro findings. This study evaluated the effects of HT and female reproductive factors on AMD, showing that current HT was associated with lower odds of large drusen predictive of advanced AMD [38]. Together, these studies suggest that estrogen treatment may be effective in AMD, in addition to RP and glaucoma as described in the previous section. Oxidative stress, induced by ischemia-reperfusion or other perturbations, is a ubiquitous pathway of degenerative vision loss. The effects of 17β-estradiol on leukocyte accumulation have been evaluated during ischemia–reperfusion injury and retinal damage after transient retinal ischemia. Treatment with 17β-estradiol significantly inhibited leukocyte accumulation and subsequently improved retinal function as assessed by electroretinogram [39]. Another ischemic retinal study showed that 17β-estradiol protected RGCs from early changes in synaptic connections that are associated with ischemia preceding apoptosis and ischemia-induced global apoptosis [40]. These studies created the foundation for the field and led to the examination of estrogen-mediated neuroprotective mechanisms. Estrogens are classically known to effect transcriptional modulation via receptor mediated genomic pathways, but they may also alter intracellular signaling cascades [35]. One study approached this subject by examining estrogen-mediated retinal neuroprotection in an in vitro hydrogen peroxide-induced model of retinal neurodegeneration along with an in vivo model of light-induced photoreceptor degeneration. Both 17β-estradiol and 17α-estradiol protected retinal neurons in vitro, with 17β-estradiol activating the phosphoinositide 3-kinase (PI3K) pathway, transiently increasing phospho-Akt levels. The estrogen receptor antagonist tamoxifen did not reverse the protective effect of 17β-estradiol, but inhibition of the insulin receptor beta blocked the PI3K mediated protective effects. This finding suggested that neuroprotection with 17β-estradiol may be independent of its receptors, but dependent on the PI3K-signaling pathway that is known to promote neuronal survival. Systemic administration of 17β-estradiol, in the in vivo arm of the study, demonstrated activation of insulin receptor beta as well, with a transient increase in PI3K activity and phosphorylation of Akt, protecting rat photoreceptor cells [41]. Similarly, two recent studies addressed the neuroprotective effect of 17β-estradiol via extracellular signal-regulated kinase (ERK) pathway induction. In the first, retinas were harvested from female rats, both those that had received oophorectomy and those that had not, to determine the effect of optic nerve transection on RGC survival. It was observed that RGC survival was significantly decreased in rats that had received oophorectomy, but RGC loss was reduced with intravitreal 17β-estradiol injection [42]. Protection was mediated via the ERK signal transduction pathway. Interestingly, ERK inhibitor U0126 inhibited the neuroprotective effect observed [42]. In the second study, the neuroprotective effect of 17β-estradiol against NMDA-induced retinal neurotoxicity was examined. In that study, retinal pretreatment with 17β-estradiol silastic implants attenuated RGC death due to intravitreal injection of NMDA [43]. However, co-administration of U0126 or estrogen receptor antagonist 13-methyl-7[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]=7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol (ICI) 182,780 with NMDA completely abolished the protective effect of 17β-estradiol. Moreover, NMDA treatment alone significantly increased the levels of phosphorylated ERK (p-ERK) that increased further with 17β-estradiol pretreatment. 17β-estradiol-induced increases in p-ERK levels were attenuated with administration of U0126 and ICI 182,780 [43]. Our own work has shown that 17β-estradiol and the synthetic estrone-derived non-feminizing estrogen analog 2-(1-adamantyl)-3-hydroxyxyestra-1,3,5(10)-triene-17-one (ZYC)-3 are effective neuroprotectants against glutamate-induced cytotoxicity of RGC-5 rat RGCs [25]. 17β-estradiol and ZYC-3 afforded complete protection against glutamate-induced RGC-5 cytotoxicity. As opposed to 17β-estradiol, ZYC-3 had no appreciable affinity for either estrogen receptor isoform, but was nearly 20-fold more effective at inhibiting lipid peroxidation [25]. This study also demonstrated that ZYC-3 pretreatment of glutamate-insulted RGC-5 cells elicited activation of a host of anti-apoptotic signal transduction pathways. Signal transduction pathways that are known to affect the survival of stressed neurons were examined in RGC-5 cells treated with glutamate. Phosphorylated-Akt (P-Akt), also known as PKB or Rac, plays a critical role in the balance between survival and apoptosis and is activated by a host of survival factors [44-46]. The p42 (Erk1) and p44 (Erk2) MAP kinase proteins (activated by phosphorylation) also play critical roles in growth and differentiation regulated by a host of factors including neurotransmitters and neurotrophins [47-50]. Activation of phosphorylation cascades in these pathways has been shown to promote survival of RGCs in a variety of models of RGC injury and protection [51-54]. The p90 S6 ribosomal kinase family of proteins responds to numerous growth factors and is activated by phosphorylation downstream of Erk1 and Erk2 [55,56]. Additionally, phosphorylation of p53 was increased with ZYC-3 pretreatment. Phosphorylated activation of tumor suppressor protein p53 plays a major role in response to DNA damage by arresting the cell cycle and initiating DNA repair or apoptosis [57-59]. This may indicate cell cycle arrest for DNA repair to promote cell survival. The pro-apoptotic p38 MAP kinase pathway serves as a gateway to cytokine and stress responses, with activation via phosphorylation leading to cell death [60-63]. In models of axotomy-induced apoptosis, phosphorylation of p38 peaked at one day postaxotomy, inhibition of p38 phosphorylation attenuated RGC loss, and selective inhibition of NMDA receptors showed dose-dependent attenuation of p38 activation, resulting in protection of RGCs [64,65]. These studies demonstrate that natural estrogens and synthetic analogs are effective neuroprotectants, an outcome classically attributed to receptor-mediated gene expression. These effects may also be due to receptor-independent activation of signal transduction pathways. While estrogens may hold promise in treating degenerative retinal disorders including AMD, RP, and glaucoma, most of the studies discussed were conducted with natural estrogens. As a consequence, these studies are subject to criticisms that have led to a fear of HT within the general population. An alternative approach would be to exploit the neuroprotective virtues of estrogens while attenuating their classic receptor-mediated feminizing effects. One of the key receptor-independent mechanisms by which estrogens afford neuroprotection occurs through scavenging and resonance stabilization of free radicals. The future neuroprotection Oxidative stress is a fundamental pathogenic mechanism of RGC death in glaucoma [66-68]. Conversely, estrogens have been identified as potent antioxidants [35,69-73]. Simple principles of organic chemistry suggest that synthetic estrogens have a future as designer neuroprotective drugs. Key to their receptor-independent neuroprotective effects is their ability to function as direct antioxidants by quenching free radicals and terminating their propagation [74-76]. This mechanism involves donation of a hydrogen atom from the phenolic group found on the A ring of natural estrogens (Figure 1B), to the free-radical (Figure 1C), resonance stabilizing the free radical and terminating chain reactions (Figure 1D). This process leaves behind a phenoxyl radical that can be regenerated in vivo [70]. Regeneration of phenolic antioxidants occurs via three known pathways using ascorbic acid, glutathione-dependent free-radical reductase, and a recently discovered NADPH-mediated reductive aromatization (Symbol X in Figure 1) [69,77,78]. This mechanism is key to their function as direct antioxidants. Figure 1 Termination, stabilization, and recycling of free-radical by phenolic estrogen derived drugs. Structure A represents the quinolic ring that can spontaneously convert to a phenolic ring found in the structure of estrogens. The phenol (B) can then scavenge free radicals (C) and resonance-stabilize them (C-D) until reduced (X). Reduction of phenoxy radicals (C) is an enzyme-mediated process that uses ascorbic acid, glutathione-dependent free radical reductase, and a newly discovered NADPH-mediated reductive aromatization. This illustration was loosely based on work of Prokai et al. [79]. Based on these principles, the efficacy of synthetic estrogen derivatives designed to harness the antioxidant capabilities of estrogens without their receptor-mediated feminizing effects will be the choice of future neuroprotective drugs in this class of compounds (Figure 2). Prodrug quinolic variants of some compounds have also been shown to attenuate estrogen receptor binding and are believed to enhance the ability of the compound to traverse the lipid bilayer (Figure 1A) [69]. These studies were conducted based on previously established protocols for treatment of RGC-5 with glutamate to induce an increased oxidative burden to examine the neuroprotective efficacy of novel compounds [25]. The drug ZYC-3 was identified as the most effective neuroprotectant and its mechanisms of action are the subject of a portion of our work in this field. Figure 2 Examination of the neuroprotective efficacy of estrogens and analogs. Maximal efficacy, with dose of drug, was examined in a glutamate-induced cytotoxicity model of in vitro retinal ganglion cell death using the retinal ganglion cell-5 cell line. This screening process was used to guide drug selection and design, and to identify highly efficacious compounds for detailed studies of mechanism of action. Abbreviations: 17β-estradiol (E2) is 1,3,5(10)estratriene-3,17 beta-diol; E2Q is 3,5(10)estratriene-3,17 beta-diol-quinol; Estrone (E1) is 3-hydroxyestra-1,3,5(10)-triene-3,17 beta-diol; E1Q is 3-hydroxyestra-1,3,5(10)-triene-3,17 beta-diol-quinol; 17α-estradiol is 1,3,5(10)estratriene-3,17 alpha-diol; 17α-estradiol-quinol is 1,3,5(10)estratriene-3,17 alpha-diol-quinol; ADAEI is 2-adamantyl-3-hydroxyestra-1,3,5(10)-triene-17-one; ADAEIQ is 2-adamantyl-3-hydroxyestra-1,3,5(10)-triene-17-one-quinol; THN is 1,3,6,8-tetrahydroxynapthalene; THNQ is 1,3,6,8-tetrahydroxynapthalene-quinol; ZYC1 is 17β estra-1,3,5(10), 9(11)-tetratriene-3,17-diol; ZYC-3 is 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-triene-17-one; ZYC10 is Enantiomer of ZYC1. Conclusions Estrogens clearly have an impact on the health of the retina. Current evidence indicates that estrogen receptors are found throughout the retinal thickness, concentrated prominently in the RGC and nerve fiber layers. At least in part, estrogen receptors mediate the influence of estrogens on blood viscosity and ocular vascularity. Estrogens and female reproductive factors may also have an impact on IOP and the development of OAG. The observation that estrogens may protect against degenerative vision loss in RP, AMD, and glaucoma is suggestive of their retinal neuroprotective capabilities, but also paves the way for the development of non-feminizing estrogen-like drugs that can be used in patients regardless of sex or health predispositions. Although not all novel drugs examined produced complete protection, even partial protection shows that outcome-directed drug design can be an effective method of developing neuroprotective drugs. These results may then be used to refine the design of compounds to improve their efficacy. The capabilities of medicinal, quantitative, and computational chemistry can be used to model free radical scavenging and resonance stabilization to refine the design of synthetic estrogens. Reexamination of such compounds in selective high throughput in vitro experiments could rapidly produce a host of efficacious drugs. Acknowledgments This work was partially supported by the National Glaucoma Program of the American Health Assistance Foundation (N.A.), grants AG 10485 and AG 22550 from the National Institute of Aging (J.W.S.), and a training grant, AG020494, from National Institute on Aging (D.M.K.). ==== Refs References 1 Wygnanski T Desatnik H Quigley HA Glovinsky Y Comparison of ganglion cell loss and cone loss in experimental glaucoma. Am J Ophthalmol 1995 120 184 9 7639302 2 Van Buskirk EM Cioffi GA Glaucomatous optic neuropathy. Am J Ophthalmol 1992 113 447 52 1558122 3 Thylefors B Negrel AD Pararajasegaram R Dadzie KY Global data on blindness. 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==== Front J Med Case ReportsJournal of Medical Case Reports1752-1947BioMed Central 1752-1947-2-2551867184310.1186/1752-1947-2-255Case ReportA novel observation of pubic osteomyelitis due to Streptococcus viridans after dental extraction: a case report Naqvi Naseem [email protected] Rizwana [email protected] Christopher [email protected] Sushmita [email protected] Department of Acute Medicine, Dumfries & Galloway Royal Infirmary, UK2 Department of Medicine, Macclesfield District General Hospital, East Cheshire NHS Trust, UK3 Department of Renal Medicine, Aintree University Hospitals NHS Trust, Liverpool, UK4 Department of Medicine, Royal Albert Edward Infirmary, Wigan, UK2008 31 7 2008 2 255 255 23 8 2007 31 7 2008 Copyright © 2008 Naqvi et al; licensee BioMed Central Ltd.2008Naqvi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Pubic osteomyelitis should be suspected in athletic individuals with sudden groin pain, painful restriction of hip movements and fever. It is an infrequent and confusing disorder, which is often heralded by atypical gait disturbance and diffuse pain in the pelvic girdle. The most common pathogen is Staphylococcus aureus but, on occasions, efforts to identify infectious agents sometimes prove negative. Pubic osteomyelitis due to Streptococcus viridans has not been reported previously in the literature. Case presentation We describe the case of a fit 24-year-old athlete, who had a wisdom tooth extracted 2 weeks prior to the presentation, which could have served as a port of entry and predisposed the patient to transient bacteraemia. Conclusion S. viridans is well known for causing infective endocarditis of native damaged heart valves, but to the best of the authors' knowledge it has not been reported previously as a cause of pubic osteomyelitis. We believe that this case should alert physicians to the association between dental procedures and osteomyelitis of the pubis secondary to S. viridans. ==== Body Introduction Pubic osteomyelitis is an uncommon osseous infection. It accounts for 2% of all osteomyelitis of bone [1,2]. Groups at risk include intravenous drug users [3], people with diabetes and patients who have undergone urological and/or obstetrical procedures [4]. Another, less well-known predisposing factor is strenuous physical activity in athletes [1]. The most common pathogen causing pubic osteomyelitis is Staphylococcus aureus. We describe the case of a patient with pubic osteomyelitis due to Streptococcus viridans, which developed after dental extraction. Case presentation A 24-year-old, previously fit and well, male fitness instructor and football player presented to the emergency department complaining of pain in his groin and buttocks. Symptoms started 3 days before he presented to the hospital, and he had engaged in strenuous exercise and jogging for 7 hours the day before admission to the hospital. He thought initially that he had sprained his groin muscles while exercising and decided to defer coming to the hospital. There was no other significant past medical history of any illnesses, although the patient reported having had an extraction of a wisdom tooth 2 weeks prior to this presentation. On examination in the emergency department, he was a fit-looking man who appeared very anxious. He was brought into the emergency department in a wheelchair. On initial assessment in the emergency department, he was pyrexial with a temperature of 38.6°C, blood pressure 127/77 mmHg, and a regular pulse of 73 beats per minute. On examination of his musculoskeletal system, he was unable to stand and required the support of two people. Examination of the lower limbs revealed painful restriction of hip flexion and extension, with a power of 5/5 in both hips and knees. There was no overlying erythema or tenderness over the hip joints. On neurological examination, sensations were intact to all modalities (intact light touch, pinprick, joint position and vibration sense) with a flexor plantar response. There were no cranial nerve palsies and the cerebellar examination did not reveal any ataxias or nystagmus. No abnormalities were detected on cardiovascular, respiratory and abdominal examination, apart from mild tenderness in the lower abdomen without guarding or rebound. Bowel sounds were present and genital examination was unremarkable with good anal tone. No tenderness was elicited on examination of the spine. Initial investigations revealed a normal full blood count (haemoglobin 15.2 g/litre, white blood cell count 8.7 × 109/litre with a neutrophil count of 6.8 × 109/litre and platelets of 412 × 109/litre), normal urea, creatinine and electrolytes with the exception of raised C-reactive protein (CRP) at 142 mg/litre. He was admitted to the acute assessment ward with the possible differential diagnosis of acute myositis or pelvic and/or psoas abscess, and blood cultures were performed. An urgent computed tomography (CT) scan of his abdomen and pelvis did not reveal any evidence of psoas or pelvic abscess and serum creatinine kinase levels were 40 U/litre. Other enzymes including lactate dehydrogenase, aldolase, aspartate transaminase and alanine transaminase were all within normal limits. On the second day of admission, he started having swinging pyrexia (body temperature spiking to 39°C and touching the baseline of 36.6°C every 6 hours) with rigors. At that time, his weakness and pain were severe enough to limit even turning and sitting up in bed. Repeat examination revealed severe tenderness to palpation over the symphysis pubis, more marked on the left. Initial sets of blood cultures revealed heavy growth of S. viridans. He was commenced on high doses of ceftriaxone (2 g three times a day) and gentamicin (80 mg once a day). A magnetic resonance imaging (MRI) scan of the pelvis suggested marrow oedema of the left pubic bone extending all the way up to the left sacro-iliac joint and soft tissue swelling of the pubic symphysis, changes highly suggestive of osteomyelitis of the left pubic ramus (Figure 1). Figure 1 Magnetic resonance imaging scan of the pelvis showing extensive marrow oedema of the left pubic ramus. A bone scan showed increased uptake of contrast over the left pubic ramus and subsequent needle aspiration of the left pubic bone grew S. viridans on cultures. Further investigations to exclude any potential cause of immunodeficiency, including human immunodeficiency virus testing, were negative. The patient gradually improved on intravenous antibiotics, started ambulating and became apyrexial after a week's course of antibiotics. The level of CRP came down to 50 mg/litre from an initial value of 142 mg/litre. He was later discharged from the hospital on 4 weeks of oral clindamycin. He was reviewed in the clinic 4 weeks after his discharge and showed complete clinical recovery. The CRP level had returned to normal (less than 5 mg/litre) and subsequent blood cultures were sterile. Discussion Pubic osteomyelitis should be suspected in athletic individuals with sudden abdominal, pelvic or groin pain, painful restriction of hip movements and fever. The pathogenesis of this disease in athletes is thought to involve pre-existing trauma or sports injury and subsequent seeding of this area during transient bacteraemia following surgical procedures, for example, dental extraction [4]. The main differential diagnosis of pubic osteomyelitis is osteitis pubis. Osteitis pubis is a painful, noninfectious, self-limited inflammatory condition of the pubic bone associated mainly with genitourinary surgery, but it also occurs following minor trauma or as a manifestation of overuse in athletes [5]. Whereas the initial clinical symptoms of the two conditions may be similar, the presence of fever and progressive clinical deterioration favours an infectious process and emphasises the need for repeated cultures. It is still unclear why athletes are at risk of developing this rare condition. This condition commonly occurs in specific athletic endeavours, such as football or running, that involve strenuous physical exercise and may produce excessive stress to the pelvis. In addition, it has been suggested that the immune system in athletes may be compromised during strenuous exercise, which might increase their susceptibility to transient bacteraemia caused by minor skin or mucous membrane trauma; however, this issue is debatable. Finally, a pre-existing subclinical osteitis pubis may make athletes locally susceptible to osteomyelitis [5]. It is important to recognise that both conditions may occur simultaneously in one patient [6]. Osteomyelitis of the pubic bone is an infrequent and confusing disorder, which is often heralded by atypical gait disturbance and diffuse pain in the pelvic girdle [7]. Diagnosis of pubic osteomyelitis is often delayed in young patients as it occasionally mimics pelvic pathology resulting in unnecessary invasive procedures in the search for the cause of an acute onset of lower abdominal pain. Symptoms of fever, nausea, vomiting, anorexia and lower abdominal pain and tenderness in a young patient can easily be mistaken for those of acute appendicitis. The classic symptoms of pubic osteomyelitis include pain in the groin or adjacent areas with radiation to the thigh and limitation of motion. The classic signs include local tenderness and swelling, a high temperature, occasionally an elevated erythrocyte sedimentation rate and leucocytosis. The port of entry of infection is often unclear and any history of preceding injuries, infections or dental procedures should be specifically looked for when eliciting history. Any history of painful restriction of hip movements should be specifically explored as it is often wrongly diagnosed as true muscular weakness of the pelvic girdle muscles or septic arthritis of the hip joint. We found 19 reported cases of pubic osteomyelitis in athletes, including our patient, in a review of the literature [8]. All patients were active athletes who participated in strenuous physical activity. In most of the 18 other patients, diagnosis was delayed. The average time from the start of symptoms to diagnosis was 13 days (range 1 to 30 days). Changes in plain radiographs of the pubic bone usually appear only several weeks after the clinical presentation of osteomyelitis and, therefore, are not reliable in making the diagnosis. Typical changes include pubic rarefaction and osteolysis. Sclerosis may appear later. A technetium bone scan shows increased uptake and may facilitate an earlier diagnosis. In three patients, diagnosis was made only after aspiration and culture. In most of the cases reviewed, the infectious agent was identified. The most common pathogen was Staphylococcus aureus, which was identified in cultures of blood or local aspirate [9] To the authors' knowledge, S. viridans has not been previously reported as a cause of pubic osteomyelitis, although there are case reports of vertebral osteomyelitis caused by S. viridans in people with diabetes [10,11] and two cases of femoral osteomyelitis due to S. viridans [12,13]. S. viridans are aerobic, Gram-positive cocci most abundant in oral flora as commensals and are well known for causing infective endocarditis of native damaged heart valves although, in our patient, there was no clinical evidence of endocarditis as evidenced by a normal transoesophageal echocardiogram. Dental extraction in our patient could have served as a port of entry and predisposed the patient to transient bacteraemia. Conclusion Pubic osteomyelitis is a challenging diagnostic dilemma. We believe that this novel observation should alert physicians to the association between dental procedures and pubic osteomyelitis due to S. viridans. It is important to take a history of dental extraction in all patients who present with fever and pelvic pain. It is also important to investigate patients with MRI scans as X-rays are neither sensitive nor specific enough for detecting osteomyelitis. Changes in plain radiographs of the pubic bone usually appear only several weeks after the clinical presentation of osteomyelitis and therefore are not reliable in making the diagnosis. Early diagnosis and treatment can prevent subsequent deformities of the pelvic bones and morbidity due to chronic osteomyelitis and joint deformities. Competing interests The authors declare that they have no competing interests. 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==== Front Adv UrolAdv UrolAUAdvances in Urology1687-63691687-6377Hindawi Publishing Corporation 10.1155/2008/937231Methodology ReportTechnique of Intravesical Laparoscopy for Ureteric Reimplantation to Treat VUR Thakre Atul A. 1, 2 Yeung C. K. 3 1Minimally Invasive Pediatric Urology Centre and Children's Continence Care Centre, Sterling Hospital, Ahmedabad, Gujarat, 380052, India2Department of Pediatric Surgery, Civil Hospital, Ahmedabad, Gujarat, 380016, India3Division of Pediatric Surgery and Pediatric Urology, Department of Surgery, Prince of Wales Hospital, Chinese University of Hong Kong, Hong KongAtul A. Thakre: [email protected] by Walid A. Farhat 2008 18 8 2008 2008 93723125 3 2008 30 6 2008 Copyright © 2008 A. A. Thakre and C. K. Yeung.2008This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The prevalence of vesicoureteral reflux (VUR) has been estimated as 0.4 to 1.8% among the pediatric population. In children with urinary tract infection, the prevalence is typically from 30–50% with higher incidence occurring in infancy. When correction of VUR is determined to be necessary, traditionally open ureteral reimplantation by a variety of techniques has been the mainstay of treatment. This approach is justified because surgical correction affords a very high success rate of 99% in experienced hands and a low complication rate. In that context the purpose of presenting our surgical technique: laparoscopic intravesical ureteric reimplantation is to highlight the use of laparoscopy to perform ureteric reimplantation for the management of pediatric VUR. ==== Body 1. SURGICAL TECHNIQUE: LAPAROSCOPIC INTRAVESICAL URETERIC REIMPLANTATION The patient is positioned supine with the legs separated apart for cystoscopy and bladder catheterization intraoperatively. For small infants, the surgeon can stand and operate over the patient’s head whereas for older children, the surgeon usually stands on the patient’s left side. The video column is placed between the patient’s legs at the end of the table. The port placement is preceded by transurethral cystoscopy to allow placement of the first camera port under cystoscopic guidance. The bladder is first distended with saline and a 2-0 monofilament traction suture is passed percutaneously at the bladder dome under cystoscopic vision, through both the abdominal and bladder walls. This helps to keep the bladder wall from falling away when the first camera port site incision is made and during insertion of the cannula. A 5-mm Step port (Tyco Healthcare Group LP, Conn, USA) is then inserted under cystoscopic vision. A urethral catheter is then inserted to drain the bladder and start carbon dioxide insufflation to 10–12 mm Hg pressure. The urethral catheter is used to occlude the internal urethral meatus to secure CO2 pneumovesicum, and it could also serve as an additional suction irrigation device during subsequent dissection and ureteric reimplantation. A 5-mm 30-degree scope is used to provide intravesical vision. Two more 3–5 mm working ports are then inserted along the interspinous skin crease on either side of the lower lateral wall of the distended bladder under vesicoscopic guidance (see Figure 1). A 3-4 cm long segment of an Fr 4 or 6 catheter is then inserted into the respective ureter as a stent to facilitate subsequent ureteral mobilization and dissection, and secured with a 4-zero monofilament suture (see Figure 2). Intravesical mobilization of the ureter, dissection of submucosal tunnel, and a Cohen’s type of ureteral reimplantation is then performed under endoscopic guidance, in a similar manner to the open procedure. The ureter is mobilized by first circumscribing it around the ureteral orifice using hook electrocautery (see Figure 3). With traction on the ureteric stent using a blunt grasper, the fibrovascular tissue surrounding the lower ureter can be seen and divided using fine 3-mm endoscopic scissors and diathermy hook, while preserving the main ureteric blood supply (see Figure 4). Mobilization of the ureter is continued for 2.5 to 3 cm to the extravesical space. Once adequate ureteral length is obtained, the muscular defect in the ureteral hiatus is repaired using 5-zero absorbable sutures, usually with an extracorporeal knot-tying technique (see Figure 5). A submucosal tunnel is then created as in an open Cohen’s procedure. Using a diathermy hook, a small incision is made over the future site of the new ureteral orifice, usually chosen to be just above the contralateral ureteral orifice. Dissection of the submucosal tunnel is then started from the medial aspect of the ipsilateral ureteral hiatus towards the new ureteral orifice, using a combination of endoscopic scissor dissection and diathermy hook for haemostasis. Once the submucosal tunnel dissection is completed, a fine grasper is passed and the mobilized ureter is gently drawn through the tunnel. Ureteroneocystostomy is performed under endoscopic guidance with intracorporeal suturing using interrupted 5-0 or 6-0 poliglecaprone or polydioxanone sutures (see Figures 6, 7). A ureteral stent is not routinely used except for selected patients undergoing bilateral ureteral reimplantation or those with megaureters requiring tapering ureteroplasty. The working ports are removed under endoscopic vision with evacuation of the pneumovesicum. The bladder-holding stitches are then tied. Each port site entry wound is then closed with a subcuticular monocryl suture. 2. RESULTS The operative success for laparoscopic Cohen’s is encouraging [1–3] and endoscopic intravesical ureteric mobilization and cross-trigonal ureteral reimplantation can be safely performed with routine pediatric laparoscopic surgical techniques and instruments under carbon dioxide insufflation of the bladder [1]. We have not faced any major complications with this technique. In the early part of the series when the cannulas were not secured to the bladder wall, displacement of the port outside the bladder wall occurred. This resulted in gas leakage into the extravesical space, with compromise of the intravesical space and endoscopic vision. It is usually possible to reintroduce the ports but securing the ports perfectly is the key to the success of this technique [1]. We have experienced mild to moderate scrotal and suprapubic emphysema immediately postoperatively, which subsided spontaneously within 24 hours. Persistent mild haematuria up to 72 hours has also been observed, which too settles spontaneously. A recent series has reported complications of postoperative urinary leak in (12.5%) and ureteral stricture at the neoureterovesical anastomosis in (6.3%). This series also reported higher complications in patients 2 years or younger with bladder capacity less than 130 cc. [2]. 3. DISCUSSION Laparoscopic surgery has gradually made its place in surgically dealing vesicoureteral reflux. Laparoscopic extravesical and intravesical surgeries have shown good early results [1–3]. It also showed that children benefit from the improved cosmesis, more rapid recovery, and decreased postoperative analgesia requirements with the laparoscopic technique. Initial experience reported increased operative time and a steep learning curve [2], but these issues have been overcome with greater experience [1]. Greatest technical merit with high level of surgical precision is required to do this surgery. The operation desires extreme care, gentleness, and tissue respect while dissecting out the ureters. Great care needs to be taken to prevent damage to the ureteric vascularity which is an important cause which leads to developing ureteric necrosis and strictures. Laparoscopy aids fine dissection of the ureter and the submucosal tunnel with minimal trauma to the bladder wall and mucosa. The bladder can be quickly rehabilitated after surgery and normal voiding is ensured in the long term. To obtain the highest possible success with this operation, the decisive technical details described should be meticulously observed [1] supported by very good laparoscopic reconstruction skills to achieve these results. Figure 1 5-mm working ports inserted along the interspinous skin crease on either side of the lower lateral wall of the distended bladder under vesicoscopic guidance. Figure 2 A 3-4 cm long segment of an Fr 4 or 5 catheter is inserted into the ureter as a stent to facilitate subsequent ureteral mobilization and dissection, and secured with a 4-zero monofilament suture. Figure 3 The ureter is mobilized by first circumscribing it around the ureteral orifice using hook electrocautery. Figure 4 With traction on the ureteric stent using a blunt grasper, the fibrovascular tissue surrounding the lower ureter can be seen and divided using fine 3-mm endoscopic scissors and diathermy hook, while preserving the main ureteric blood supply. Figure 5 Once adequate ureteral length is obtained, the muscular defect in the ureteral hiatus is repaired using 5-zero absorbable sutures, usually with an extracorporeal knot-tying technique. Figure 6 Ureteroneocystostomy is performed under endoscopic guidance with intracorporeal suturing using interrupted 5-0 or 6-0 poliglecaprone or polydioxanone sutures. Figure 7 Completed ureteroneoctostomy. ==== Refs 1 Yeung CK Sihoe JDY Borzi PA Endoscopic cross-trigonal ureteral reimplantation under carbon dioxide bladder insufflation: a novel technique Journal of Endourology 2005 19 3 295 299 15865516 2 Kutikov A Guzzo TJ Canter DJ Casale P Initial experience with laparoscopic transvesical ureteral reimplantation at the Children's Hospital of Philadelphia The Journal of Urology 2006 176 5 2222 2226 17070297 3 Peters CA Woo R Intravesical robotically assisted bilateral ureteral reimplantation Journal of Endourology 2005 19 6 618 621 16053348	18725986	PMC2517844	CC BY	2021-01-05 09:55:20	yes	Adv Urol. 2008 Aug 18; 2008:937231
==== Front J Exp Clin Cancer ResJournal of Experimental & Clinical Cancer Research : CR0392-90781756-9966BioMed Central 1756-9966-27-271869452210.1186/1756-9966-27-27ResearchsiRNA directed against c-Myc inhibits proliferation and downregulates human telomerase reverse transcriptase in human colon cancer Colo 320 cells Hao Huang [email protected] Yu [email protected] Fu [email protected] Wei [email protected] Su [email protected] Huang [email protected] Wu [email protected] Huang [email protected] Liu [email protected] Xiao [email protected] Center of Experimental Medicine, Wuhan No.1 Hospital, Wuhan, 430022, PR China2 Department of Pathogentic Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China3 Brain Research Center, University of British Columbia, Vancouver, BC, Canada2008 12 8 2008 27 1 27 27 22 5 2008 12 8 2008 Copyright © 2008 Hao et al; licensee BioMed Central Ltd.2008Hao et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The c-Myc and human telomerase reverse transcriptase gene (hTERT) gene are frequently deregulated and overexpressed in malignancy. hTERT activity is induced by c-Myc and strategies designed to inhibit c-Myc expression in cancer cells may have considerable therapeutic value. We designed and used a short hairpin RNA to inhibit c-Myc expression in Colo 320 cells and validated its effect on cell proliferation. In this study, four c-Myc-shRNA expression vectors were constructed and introduced into Colo 320 cells. The effects of c-Myc silencing on tumor cell growth was assessed by soft agar assay and DNA synthesis experiments. The expressions of c-Myc and hTERT were also assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it was found that expression of c-Myc and hTERT decreased in shRNA-transfected cells. The downregulation of c-Myc and hTERT inhibited cell growth, shortened telomere lengths, and suppressed telomerase activity. In conclusion, our findings demonstrate that shRNA of c-Myc can inhibit the DNA replication in Colo 320 cells effectively and reduce telomere length and telomerase activity, therefore, it could be used as a new potential anticancer tool for therapy of human colon cancer. ==== Body Background Telomerase is a ribonucleoprotein enzyme that synthesizes telomeres, i.e., DNA repeats that cap and protect the ends of chromosomes[1]. The core of the mammalian telomerase holoenzyme is the catalytic subunit, telomerase reverse transcriptase, which adds hexameric DNA repeats (TTAGGG) that correspond to the telomerase RNA component known as TERC[2,3]. Telomerase activation has been regarded as a crucial step in cellular carcinogenesis, and it is one of the most common molecular markers in a broad spectrum of malignancies [4]. It has been reported that telomerase activity is significantly high in about 80% of cancers and correlates well with the degree of malignancy[5]. Some studies have shown that hTERT gene expression is more specific and sensitive than telomerase activity in the diagnosis of malignant neoplasms, as the hTERT gene is overexpressed in about 90% of malignant tumors [6]. The control of c-Myc gene expression is a complex process and occurs at various steps of transcription, such as initiation, elongation, and attenuation, as well as during the post-transcriptional stages[7,8]. The protein product of the c-Myc protooncogene plays a vital role in the process of cellular growth and differentiation[9]. Deregulation of c-Myc expression has been detected in many cancers and is believed to be an important step in carcinogenesis [10]. In addition, c-Myc has also been implicated in the regulation of telomerase through its ability to induce the transcriptional activation of hTERT[11]. RNA interference (RNAi) has been described. Recently, a post-transcriptional gene silencing pathway mediated by double-stranded RNA (dsRNA), also called RNA interference (RNAi) has been described[12,13]. RNAi is a natural mechanism of defence, which protects cells against exogenous dsRNA, such as viral or deriving from transposones[14]. When a dsRNA enters the cytoplasm, RNAse III Dicer can process it to produce several small interfering RNAs (siRNAs), 21–23 nucleotide long RNA molecules with 2 nucleotide long 3'overhangs. Small interfering RNAs may get incorporated into the RNA induced silencing complex (RISC), which identifies and silences complementary RNAs generally through a cleavage mechanism. In the last few years, it has been demonstrated that siRNAs represent an efficient tool to modulate the expression of a large number of cancer-related genes[15]. In the present study, we selectively downregulated c-Myc expression in human colon cancer Colo 320 cells with siRNA delivered via a plasmid-based polymerase III promoter system. This approach allowed us to explore a possible role for c-Myc in regulating telomerase activity. Methods Short-interfering RNA design shRNA directed against c-Myc mRNA were selected using the computer program (web site ), and it was verified, by BLAST search, that there was no homology with another human gene. shRNA #1–4 sequences directed against c-Myc mRNA were selected according to Blast search score and GC content (40–60%). The shRNA expression cassette contained 19 nucleotide of the target sequence followed by the loop sequence (TTCAAGACG), reverse complement to the 19 nucleotide, stop codon for U6 promoter and BamHI site (c-Myc -1: 5'-CTATGACCTCGACTACGACTTCAAGACCGTCCTAGTCGAGGTCATAG-3'; c-Myc -2: 5'-AAATTCGAGCTGCTGCCC TTCAAGACG GGGCAGCAGCTCGAATTTC-3'; c-Myc -3: 5'GCCCCCAAGC TAGTTATC TTCAAGACG GATAACTACCTTGGGGGCC-3'; c-Myc -4: 5'CCACAGCATACATCCTGT TTCAAGACG ACAGGATGTATGCTGTGGC-3'). The shRNA cassettes and their complementary strands were synthesized (Wuhan Genesil Biotechnology, Wuhan, China). Cell culture and transfection The colon cancer cell line Colo 320 was obtained from China Center for TypeCulture Collection (GDC298). The cells were grown in RPMI-1640 medium (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco BRL), 50 units/ml penicillin, and 50 μg/ml streptomycin. The Colo 320 cells were maintained in a humidified 37°C incubator with 5% CO2, fed every 3 days with complete medium, and subcultured when confluence was reached. The cells were routinely passaged every 1 or 2 days. For transfection, 2 × 105 cells were seeded into each well of a six well tissue culture plate (Costar). The next day (when the cells were 70–80% confluent), the culture medium was aspirated and the cell monolayer was washed with pre-warmed sterile phosphate-buffered saline (PBS). Cells were transfected with the pGensil-c-Myc -1, -2, 3, and -4 harboring green pEGFP-C1 green fluorescence protein reporter gene by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's protocol. Cells were continuously cultured until harvest for analysis. Cell counts Colo 320 cells were trypsinized on the indicated days and counted with a hemocytometer. We quantify cell proliferation by comparing the number of cells counted before and after transfection. Colo 320 Approximately 200 cells were evaluated in each sample using the hemocytometer. cell viability was assessed by trypan blue exclusion (Sigma-Aldrich China Inc, Shanghai, China). DNA synthesis Colo 320 cells were treated with 1 mCi of 3H-thymidine per milliliter (Amersham Biosciences, Piscataway, NJ) for the last 6 h of day 3 after shRNA treatment. Cells were then rinsed twice with PBS, ice-cold 5% trichloroacetic acid, and 80% ethanol; the incorporated radioactivity of cell lysates was measured in a liquid scintillation counter. Soft agar colony assay Two days after transfection, Colo 320 cells (300 cells per well) transfected with indicated plasmids were mixed with tissue culture medium containing 0.7% agar to result in a final agar concentration of 0.35%. Then 1 ml sample of this cell suspension was immediately plated in six-well plates coated with 0.6% agar in tissue culture medium (2 ml per well) and cultured at 37°C with 5% CO2. After 2 weeks, the top layer of the culture was stained with 0.2% piodonitrotetrazolium violet (Sigma). The culture was analyzed in triplicate, and colonies larger than 100 μm in diameter were counted. Quantification of c-Myc and hTERT mRNA For the sqRT-PCR analysis, total RNA was isolated using Trizol reagent (Sigma) and reversely transcribed using random hexamers with Superscript II (Invitrogen). For c-Myc and hTERT detection, cDNA was mixed with the PCR reaction mixture and each c-Myc and hTERT-specific primer (LT5 and LT6). The PCR cycle number was set up to permit the distinction between mRNA expression profiles among the samples. PCR products were visualized on 1.2% agarose gel. Actin was used as an internal control, and positive and negative controls were always included. PCR reagents were from the Master Taq kit (Eppendorf). Telomere length measurement Determination of telomere length was performed by Southern analysis of telomere restriction fragment (TRF) length. Genomic DNA was isolated from transfected cells using DNA isolation kit (Qiagen, Valencia, CA, USA) and quantified by UV spectrophotometry. Two micrograms of DNA were digested with restriction endonucleases RsaI and HinfI. The DNA digests were electrophoresed through 0.8% agarose and transferred to nylon membranes by capillary transfer in 20 × SSC as described. After UV crosslinking (1200 μJ), the membranes were hybridized with a 3'-digoxigenin oligonucleotide probe with the sequence (CCCTAA)3'. After washing to remove unbound probe, an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Applied Science, Indianapolis, IN, USA) was used for immunodetection of bound probe, followed by CDP Star chemiluminescence substrate (Roche Applied Science). Blots were exposed to X-ray film for 10–60 s. Mean TRF length and percentage photo-stimulated luminescence were determined from densitometric analysis of digital images of exposed films as described. Measurements of TRF length were performed in duplicate for each membrane. Detection of telomerase activity The telomerase activity was measured using a PCR-TRAP ELISA kit (Roche, USA) according to the manufacturer's description with some modifications. For the TRAP reaction, 2 μg total RNA was added to 25 μl of reaction mixture with the appropriate amount of sterile water to create a final volume of 50 μl. Hybridization and the ELISA reaction were carried out following the manufacturer's instructions. Western blotting Transfected cells were washed twice with PBS and suspended in IPH lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 100 mM phenylmethyl sulfonyl fluoride leupeptin 1 mg/mL, aprotinin 1 mg/mL, and 1 M dithiothreitol). Cells were extracted at 4°C for 30 min. After centrifugation at 12,000 rpm for 20 min, the supernatant was subject to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was allowed to react with anti-β-actin polyclonal, anti-c-Myc and hTERT antibody (Santa Cruz Biotechnology). Specific antibodies were detected with a chemiluminescence kit (Sigma Life Science) according to the supplier's manual. Chemiluminescence was detected by exposure to X-ray film. Statistical analysis All data in the text and figures is presented as means ± standard deviation (means ± SD). Statistical analysis was performed using Student's t-test. Results Effect of the c-Myc small interfering RNA on cell growth As the first step, transfection efficiency of the plasmid encoding shRNA for c-Myc in Colo 320 cells was examined by coexpressing pEGFP green fluorescence protein. When the cells were examined under a fluorescence microscope 24 h after transfection, more than 75–85% of them was transfected (Figure 1A). We then tested whether RNAi-mediated c-Myc could influence the ability of Colo 320 cells to form colonies in soft agar. Colo 320 cells were transfected with pGensil-c-Myc or empty vector. At 48 hours after transfection, the cells were placed into medium with soft agar, and colonies were counted after 2 weeks. RNAi directed against c-Myc resulted in a significant decrease (about 65%) in colony formation in Colo 320 cells (Figure 1B-1,2,3). These results showed that suppression of c-Myc by RNAi could decrease the ability of colon cancer cells to form colonies in soft agar. Since four constructs gave similar results, we describe the results with pGenesil – c-Myc -1 in the subsequent experiments. We also evaluated the effect of c-Myc-specific shRNA on Colo 320 cell proliferation. Cell proliferation was measured by counting the number of viable cells using trypan blue staining. Transfected c-Myc-shRNA (Figure 1C-1) resulted in a marked inhibition of cell proliferation over this 2-day period and the shRNAs induced anti-proliferative effect was dose dependent. Cell growth was not influenced significantly by treatment with control-shRNA and vector. The statistical analysis shows that Colo 320 cell proliferation was affected by silencing of c-Myc. This is further confirmed with DNA synthesis assay using 3H-thymidine. In Figure 1C-2, the cell control and vector showed the maximum counts of (3H) thymidine (around 1400 and 1350 cpm/μgDNA) when compared to the treatment groups. In addition, the anti-proliferative effect was dose dependent and maximum inhibition was achieved with 12.5 μM shRNA. Figure 1 Effect of shRNA on proliferation of Colo 320 cells. A. Plasmid pGenesil-c-Myc-1,-2, -3, and -4 with pEGFP encoding green fluorescence protein were transfected to the cells. B. c-Myc-depletion inhibits colony formation of Colo 320 cells. B-1) Control Colo 320 cells. B-2) Colo 320 cells treated with pGenesil-c-Myc-1 for 24 h B-3) The percentage of colonies of pGenesil-c-Myc-1 treated cells standardized against the corresponding control. C. Cells were treated with increasing concentrations of shRNA for 48 h. C-1)Cell viability was determined by cell counts. Results are expressed as the percentage of viable cells in the treatment groups with respect to that in the corresponding control. C-2) Cell proliferation following shRNA treatments for 48 h was quantified by 3H-thymidine incorporation studies. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (P < 0.01, vs control. ##P < 0.01, vs vector). Down-regulation of c-Myc and hTERT by expression of shRNA Compared with untreated cells, c-Myc and hTERT mRNA abundance was significantly decreased in cells incubated with 5, 7.5, 10, or 12.5 μM shRNA for 48 h (P < 0.01). Control and empty vector group markedly expressed c-Myc mRNA (Figure 2A) (P < 0.01). Figure 2 Effect of shRNA on expression of c-Myc and hTERT gene in Colo 320 cells. A. c-Myc and hTERT mRNA expression of Colo 320 cells was detected by RT-PCR with different treatments. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. c-Myc protein and hTERT expression of Colo 320 cells was detected by Western blotting with different treatments. Typical Western blot results for c-Myc and hTERT protein. Protein expression of c-Myc in Colo 320 cells was quantified by densitometric analysis. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (P < 0.01, vs control. ##P < 0.01, vs vector). As Western blot analysis indicated, the shRNA was able to reduce c-Myc and hTERT protein expression. As indicated in (Figure 2B), shRNA treatment inhibited the protein expression levels of c-Myc genes, representative downstream targets for c-Myc and hTERT protein in Colo 320 cells (P < 0.01). Transfection with shRNA at different concentrations (0, 5,7.5, 10, 12.5) resulted in the significant attenuation of expression of c-Myc and hTERT protein (P < 0.01). In contrast, β-actin protein expression was not affected by shRNA. These data indicated that vector-based RNAi could effectively suppress c-Myc and hTERT over-expression. Effects of shRNA on telomere length and telomerase activity We evaluated the effect of shRNA on telomerase activity. Our data suggested that shRNA could down-regulate telomerase activity in shRNA-transfected groups. Telomerase activity were shown in Figure 3A. It revealed that shRNA-transfected groups had lower telomerase activity than the control groups. shRNA at a variety of concentrations resulted in significant reduction of telomerase activity. Figure 3 Effects of shRNA on telomere length and telomerase activity in Colo 320 cells. A. Representative the concentration-course analysis of telomerase activity, each groups cells were mixed with 1 ml TBA solution for preparation of protein extract and 1 μg protein was subjected to TRAP assay. After hybridization and ELISA procedure, the absorbance of the samples at 450 nm was measured. The cells were subjected to no treatment (control), pGenesil-vector group, 5, 7.5, 10, 12.5 μM shRNA for 48 h. B. Mean telomere restriction fragment length was detected by RT-PCR with different treatments by Southern analysis as described under Materials and methods. Typical Southern blot results for telomere restriction fragments. Locations of the base pair markers on the DNA ladder are indicated along the left side. Significant difference was observed between the mean telomere lengths of the control and shRNA-transfected groups cells. All data were obtained from three independent experiments. Error bars represent means ± SEM. Significantly different from the corresponding control (**P < 0.01, vs control. ##P < 0.01, vs vector). TRF length then was determined using pulse gel electrophoresis followed by Southern blot hybridization with telomere-specific probes. Average telomere length is shown in Figure 3B-1. Telomere length in shRNA-transfected groups telomeres was shorter than the one in the control groups. Transfection of shRNA resulted in significant increment of mean telomere length (Figure 3B-2, P < 0.05). Discussion Some researchers have shown that c-Myc act as a transcription factor, which binds with E-box sites (CACGTG) of a gene's cis-element to regulate other gene transcription, such as hTERT gene[16]. C-Myc forms a dimer with Max, then binds to the specific E-box site sequence 5'-CACGTG-3' to transactivate target genes[17]. In contrast, c-Myc may also form a dimer with mad1 at the same binding site to suppress the transcription of target genes[18]. It has been shown that telomerase is highly related with malignant neoplasms [19]. When cells change from the normal growth status to abnormal growth status, there is persistent telomerase activity[20]. Previous studies showed that hTERT expression is related to many factors; in particular, that hTERT transcript regulation is affected by different transcription factors such as c-Myc[21,22]. Others report that activation or inhibition of c-Myc expression can change hTERT promoter activity[23,24]. The up-regulation, of c-Myc can trans-activate hTERT promoter and consequently activates telomerase. Xu et al suggest that c-Myc can activate hTERT transcription in a dose-dependent manner in leukemia cells. Several studies have shown that downregulating c-Myc activity induces tumor shrinkage[25]. In the present study, we used vector-based shRNA technique and constructed the recombinant plasmid expressing c-Myc-shRNA to transfect Colo 320 cells. Our results demonstrated that a transient reduction of c-Myc protein level by RNAi could significantly inhibit the growth rate of Colo 320 cells and its ability to form colonies in soft agar. Based on the results of western blotting, we also confirmed that the specific c-Myc-shRNA designed and used in this study successfully reduce the expression of the c-Myc and hTERT. We also found the We also found the attenuation of c-Myc and hTERT protein expression was dose-dependent. Telomerase activity is strongly associated with telomere length maintenance. In the present study, we analyzed telomere length and telomerase activity in shRNA-transfected Colo 320 cells. Fig. 3 shows that the shRNA-transfected group had a shorter telomere length and lower telomerase activity than control group. Therefore, shRNA can significantly reduce telomere length and telomerase activity in siRNA-transfected Colo 320 cells. In summary, we have identified the c-Myc shRNA that specifically inhibits activated c-Myc and suppresses cell proliferation. The c-Myc shRNA is able to block c-Myc DNA-binding activity and reduces the levels of c-Myc mRNA. Meanwhile, c-Myc shRNA can significantly reduce telomere length and telomerase activity in shRNA-transfected Colo 320 cells. Targeting c-Myc activation with RNAi may hold therapeutic promise for colon cancer with c-Myc and telomerase activation. Acknowledgements This work is supported by Foundation for Medicine Scientific Research Project of Wuhan from Wuhan Ministry of Health, PR China. ==== Refs Morin GB The human telomere terminal transferase enzyme is ribonucleoprotein that synthesizes TTAGGG repeats Cell 1989 59 521 529 2805070 10.1016/0092-8674(89)90035-4 Counter CM Meyerson M Eaton EN Weinberg RA The catalytic subunit of yeast telomerase Proc Natl Acad Sci USA 1997 94 9202 9207 9256460 10.1073/pnas.94.17.9202 Mitchell JR Wood E Collins K A telomerase component is defective in the human disease dyskeratosis congenita Nature 1999 402 551 555 10591218 10.1038/990141 Wright WE Piatyszek MA Rainey WE Byrd W Shay JW Telomerase activity in human germline and embryonic tissues and cells Dev Genet 1996 18 173 179 8934879 10.1002/(SICI)1520-6408(1996)18:2<173::AID-DVG10>3.0.CO;2-3 Janknecht R On the road to immortality: hTERT upregulation in cancer cells FEBS Lett 2004 564 9 13 15094035 10.1016/S0014-5793(04)00356-4 Kim NW Piatyszek MA Prowse KR Harley CB West MD Ho PL Coviello GM Wright WE Weinrich SL Shay JW Specific association of human telomerase activity with immortal cells and cancer Science 1994 266 2011 2015 7605428 10.1126/science.7605428 Pelengaris S Khan M Evan G c-Myc: more than just a matter of life and death Nat Rev Cancer 2002 2 a 764 76 12360279 10.1038/nrc904 Nilsson JA Cleveland JL Myc pathways provoking cell suicide and cancer Oncogene 2003 22 9007 9021 14663479 10.1038/sj.onc.1207261 Miliani de Marval PL Macias E Rounbehler R Sicinski P Kiyokawa H Johnson DG Conti CJ Rodriguez-Puebla ML Lack of cyclin-dependent kinase 4 inhibits c-myc tumorigenic activities in epithelial tissues Mol Cell Biol 2004 24 7538 7547 15314163 10.1128/MCB.24.17.7538-7547.2004 Murphy DJ Swigart LB Israel MA Evan GI Id2 is dispensable for Myc-induced epidermal neoplasia Mol Cell Biol 2004 24 2083 2090 14966287 10.1128/MCB.24.5.2083-2090.2004 Gunes C Lichtsteiner S Vasserot AP Englert C Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1 Cancer Res 2000 60 2116 2121 10786671 Fire A Xu S Montgomery MK Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 1998 391 806 11 9486653 10.1038/35888 Hannon GJ Rossi JJ Unlocking the potential of the human genome with RNA interference Nature 2004 431 371 78 15372045 10.1038/nature02870 Yu JY Deruiter SL Turner DL RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells Proc Natl Acad Sci USA 2002 99 6047 52 11972060 10.1073/pnas.092143499 Dasgupta R Perrimon N Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research Oncogene 2004 23 8359 65 15517017 10.1038/sj.onc.1208028 Casillas MA Brotherton SL Andrews LG Ruppert JM Tollefsbol TO Induction of endogenous telomerase (hTERT) by c-Myc in WI-38 fibroblasts transformed with specific genetic elements Gene 2003 316 57 65 14563552 10.1016/S0378-1119(03)00739-X James L Eisenman RN Myc and Mad bHLHZ domains possess identical DNA-binding specificities but only partially overlapping functions in vivo Proc Natl Acad Sci USA 2002 99 10429 10434 12149476 10.1073/pnas.162369299 D'Cruz CM Gunther EJ Boxer RB c-MYC induces mammary tumorigenesis by means of a preferredpathway involving spontaneous Kras2 mutations Nat Med 2001 7 235 239 11175856 10.1038/84691 Poole JC Andrews LG Tollefsbol TO Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT) Gene 2001 269 1 12 11376932 10.1016/S0378-1119(01)00440-1 Meeker AK de Marzo AM Recent advances in telomere biology. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1880687508-PONE-RA-0524810.1371/journal.pone.0003258Research ArticleBiochemistryCell BiologyMolecular BiologyBiochemistry/Chemical Biology of the CellBiochemistry/Protein ChemistryBiochemistry/Replication and RepairCell Biology/Cellular Death and Stress ResponsesBiochemistryBiochemistry/Chemical Biology of the CellBiochemistry/Protein ChemistryBiochemistry/Replication and RepairCell BiologyCell Biology/Cellular Death and Stress ResponsesMolecular BiologyProtein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation Protein Repair and ApoptosisCimmino Amelia 1 Capasso Rosanna 1 Muller Fabbri 2 Sambri Irene 1 Masella Lucia 1 Raimo Marianna 1 De Bonis Maria Luigia 1 D'Angelo Stefania 3 Zappia Vincenzo 1 Galletti Patrizia 1 * Ingrosso Diego 1 1 Department of Biochemistry and Biophysics, School of Medicine and Surgery, Second University of Naples, Naples, Italy 2 Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, United States of America 3 Faculty of Motor Science, Parthenope University, Naples, Italy Abraham Edathara EditorUniversity of Arkansas for Medical Sciences, United States of America* E-mail: [email protected] and designed the experiments: PG DI. Performed the experiments: AC RC FM IS LM. Analyzed the data: VZ. Contributed reagents/materials/analysis tools: MR MLDB SD. Wrote the paper: PG DI. 2008 22 9 2008 3 9 e325825 6 2008 22 8 2008 Cimmino et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). Methodology/Principal Findings Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H2O2; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H2O2 concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H2O2 treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. Conclusion/Significance Apoptosis can be modulated by “on/off” switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein. ==== Body Introduction Protein deamidation occurs spontaneously in proteins at level of unstable Asn residues, which are flanked, on the α-carboxyl side, by small non-bulky residues, such as Gly, Ala, Ser or Thr [1], [2]. The deamidation mechanism entails the nucleophilic attack of the peptidyl nitrogen of the Asn+1 residue onto the β-carbonyl carbon of the Asn, leading to the formation of an aspartyl succinimidyl (ASU) intermediate, with the elimination of the ammonia moiety. (Fig. 1). ASU itself is unstable and its ring can open on either side of the nitrogen atom, yielding either a normal peptide or an atypical isopeptide containing a β-linked isoaspartyl residue (isoAsp) [3], [4] the latter form being generally prevalent [4]. The occurrence of such an abnormal residue can significantly alter protein structure and function, as it has been demonstrated for epidermal growth factor, calmodulin, tubulin, synapsin, eye lens crystallins, Alzheimer's β-amyloid, tissue plasminogen activator [5] collagen type-I [6], Protein Kinase A [7] and others. 10.1371/journal.pone.0003258.g001Figure 1 Mechanism for deamidation of asparaginyl residues in peptides. (Step 1): the nitrogen of the Asn+1 residue (a Gly in the example) attacks the β-carbonyl carbon of the Asn, thus forming the succinimidyl derivative of the peptide (ASU) with the ammonia elimination. The ASU ring can open spontaneously on either side of the nitrogen atom. In one case the α-aspartyl peptide is formed (Step 2). In the other case the β-isoaspartyl peptide does occur (Step 3). Protein isoaspartyl carboxyl O-methyltransferase (PCMT; 2.1.1.77) is a S-adenosylmethionine (AdoMet)–dependent methyltransferase, which specifically recognizes and methyl esterifies the free α-carboxyl groups of the isoaspartyl residues, raising from asparaginyl deamidation. This enzyme, hence, promotes the conversion of the abnormal L-isoAsp residue into L-aspartyl, eliminating the isopeptide bond which alters protein conformation, thus preventing the accumulation of dysfunctional proteins. This methylation-dependent repair activity has been demonstrated in vitro, with synthetic isoAsp-containing peptides as well as with deamidated purified proteins. Various ex vivo studies confirmed that a PCMT activity is related to the processing of deamidated-isomerized proteins. For example in the erythrocytes from patients with spherocytosis [8] or with a G6PD deficiency [9] the increase in membrane protein isomerization, associated with the disease, could be monitored ex vivo by an increase in methyl ester formation in the intact erythrocytes. On the other hand, in chronic renal failure, where PCMT is inhibited by the intracellular accumulation of S-adenosylhomocysteine, membrane proteins tend to accumulate isomerized aspartyls within the red cell membrane [10]. Further in vivo studies provided evidence on the role of this enzyme in preventing the accumulation of potentially harmful damaged proteins. In PCMT knockout mice isomerized proteins increased 4–8 fold compared with the levels detected in the wild-type mice, and the knockout animals exhibited brain damage and fatal epileptic seizures [11]. A number of cell stress conditions have been linked to an increased propensity of proteins to undergo deamidation. Oxidative conditions have been considered as a way through which proteins become more susceptible to deamidation. The underlying mechanism is still unclear, although the evidence suggests that oxidative conditions may induce an increased flexibility of the polypeptide backbone or a transient unfolding of proteins, allowing Asn deamidation and enhancing the formation of L-isoAsp residues. In this respect it has been shown that erythrocytes from glucose-6-phosphate dehydrogenase (G6PD)-deficient patients display a higher propensity to deamidation at membrane protein level [9]. Moreover, UV irradiation, which causes an increased formation of reactive oxygen species, leads to an increased protein deamidation in cultured melanoma cells [12]. On the other hand, analysis of the PCMT gene provided interesting clues about the regulation of this enzyme. This gene contains several motifs, as potential regulation sites in response to different stress conditions [13]. It has been proposed that protein methyl esterification, catalyzed by PCMT, may be able to mediate protection from apoptosis induced by Bax in a neuronal cell line [14]. This interpretation relies upon the evidence that cotransfection with a PCMT carrying vector prevents apoptosis induced by Bax, in this system. More recently it has been shown that Bcl-xL, an antiapoptotic member of the Bcl2 protein family, contains two labile asparaginyl sites which are deamidation-prone (i.e. positions 52 and 66) [15]. The deamidated Bcl-xL is not more able to block the action of pro-apoptotic proteins thus leading to cell death. A transient intracellular alkalinization has been related to Bcl-xL isomerization during cell stress [16]. It is worth noting in this respect that general alkaline pH conditions increase the tendency of labile asparaginyl residues to form ASU. Consistent with these observations the rate of Bcl-xL deamidation was found to be significantly reduced in hepatocellular carcinomas compared to normal liver tissue and this has been linked to a resistance of the transformed cells to undergo apoptosis [17]. We report here that PCMT, when overexpressed in endothelial cells, is able to prevent apoptosis induced by an oxidative treatment. Experiments using PCMT negative dominants show that the enzymatic activity must be preserved in order to exert its antiaptotic effect. In order to identify molecular mediators of apoptotic cascade involved in this mechanism, and specifically recognized and modified by PCMT, we have employed the human recombinant enzyme as a specific ligand. We thus identify in our experimental system, a number of methyltransferase targets, also including Bcl-xL. We could therefore infer the role of protein methylation in apoptotic cell death and its underlying implications. Results Characterization of PCMT plasmid constructs Porcine aortic endothelial cells (PAEC) were transfected with plasmid constructs carrying the PCMT sense or the antisense genes or either negative dominant mutants (Asp83→Phe and Asp83→Val), as described under “Methods”. The site of mutagenesis, which affects a conserved residue in sequence motif I, involved in AdoMet binding [18], [19], was chosen on the basis of the information available on PCMT structure and on its catalytic mechanism. PAEC extracts were then used as a PCMT source to check whether transfection successfully produced the overexpression of the proper protein species (wild type or mutant) or, in the case of antisense, silenced the endogenous PCMT. Methyltransferase activity was assayed, according to the vapor diffusion assay procedure, in the presence of saturating concentrations of both the methyl donor AdoMet and ovalbumin, a common in vitro methyl accepting substrate. Results in Fig. 2 panel A show that PAEC transfected with the sense PCMT gene displayed an increased methyltransferase activity, compared either with the antisense PCMT transfectants or with both negative mutants. 10.1371/journal.pone.0003258.g002Figure 2 Characterization of PCMT plasmid constructs. Panel A) Plasmids were transfected into endothelial cells and 48 h later methyltransferase activity was assayed using a methanol diffusion assay in the presence of saturating concentrations of the methyl donor AdoMet and ovalbumin as a methyl accepting protein. Results are given as the mean of three experiments. Error bars indicate standard deviation; (*) refer to statistically significant differences (p<0.05), as evaluated by t-test. Panel B) Cells transfected were then processed for immunoblotting with PCMT antibody. The immunoblot was reprobed for actin as a loading control. The sample “PCMT” is authentic human recombinant enzyme as a positive control. PAEc = PAE transfected with void plasmid. The same cell extracts were then analyzed by SDS-PAGE and Western blot, using a PCMT antipeptide antibody. All transfectants carrying PCMT sense (wild type or mutants) over-expressed the relevant protein, while the protein signal was almost undetectable in those carrying the antisense counterpart (Fig. 2, panel B). These results demonstrated that PAEC, transfected with either PCMT Asp83→Phe or Asp83→Val plasmids effectively overexpress the relevant mutant proteins, where the conserved Asp83 residue in the consensus region I is substituted by a residue with a non polar side chain. As the result, the mutant proteins are devoid of catalytic activity. Transient transfection of PAEC with a plasmid carrying the antisense PCMT significantly lowers the activity of the endogenous methyltransferase, by critically reducing its expression. Consequently PAEC transiently transfected with plasmids carrying various PCMT constructs, can be used as a model system to study the role of PCMT on apoptosis induced by an oxidative stress. PCMT overexpression prevents apoptosis induced by H2O2 treatment in endothelial cells PAEC transfected with plasmids carrying PCMT wild type, mutants or antisense, were exposed to an oxidative stress brought by H2O2 treatment, as described in the experimental section. Apoptosis was monitored by the occurrence of DNA fragmentation, according to the typical DNA ladder pattern. In addition, caspase 3 activation was detected in cell extracts by SDS-PAGE Western blot analysis using an anti-caspase 3 antibody, which specifically recognizes the cleaved (activated) form of this enzyme. The activation of the apoptosis cascade was further confirmed by checking PARP cleavage, as a typical caspase 3 substrate. Fig. 3 column A shows the results of the DNA fragmentation (ladder) detection assay. Treatment with 0.1 mM H2O2 was fully effective inducing apoptosis in PAEC cells transfected with the antisense PCMT plasmid, compared with the cells overexpressing the catalytically active “sense” PCMT. The latter transfectants were in fact resistant to H2O2 treatment, up to a concentration of the oxidant in between 0.3 and 0.4 mM. As for the mutants, cells overexpressing either Asp83→Val or Asp83→Phe PCMT showed a sensitivity to H2O2 comparable with that observed in the antisense PCMT transfectants, since 0.1 mM H2O2 is sufficient to generate an evident DNA fragmentation. 10.1371/journal.pone.0003258.g003Figure 3 Effect of PCMT expression levels and mutants on apoptosis induced by oxidative stress on PAE cells. Column A: Apoptotic DNA ladder in cells overexpressing PCMT constructs and subject to oxidative treatment. Apoptotic DNA ladder patterns, of transfected endothelial cells stimulated with different concentrations of H2O2, were detected after transfection with plasmid void (control) or carrying PCMT wild type (sense); antisense PCMT; PCMT Asp83 →Phe83 Mut and PCMT Asp83 →Val83 Mut. Column B: Caspase-3 activation and PARP cleavage in cells overexpressing PCMT constructs and subject to oxidative treatment. Immunoblot developed with a polyclonal antibody against caspase-3 and PARP (a final effector of various apoptotic pathways) on transfected endothelial cells stimulated with different concentration of H2O2 after transfection with plasmid void (control) or carring PCMT wild type (sense), antisense PCMT, PCMT Asp83 →Phe83 Mut and PCMT Asp83 →Val83 Mut. CP is a positive control obtained by treating a parallel cell sample with cisplatin. Column C: Flow cytometry analysis of cells overexpressing PCMT constructs and subject to oxidative treatment. Cells stimulated with 0.3 mM of H2O2 after transfection with void plasmid (control), PCMT wild type (sense), Antisense PCMT, PCMT Asp83 →Phe83 Mut. PCMT Asp83 →Val83 Mut. PI: propidium iodide. The enhanced sensitivity to the proapoptotic stimulus revealed by the DNA ladder assay was further confirmed by SDS-PAGE Western blot experiments, aimed at the detection of caspase-3 activation and poly (ADP-ribose) polimerase (PARP) cleavage (Fig. 3 column B). In cells transfected with antisense PCMT, indeed, apoptosis was apparent even at 0.1 mM H2O2 concentration. A similar behavior was observed in cells transfected with either PCMT mutants, in which apoptosis markers appeared already at 0.1 mM H2O2 concentration. Conversely, in the sense PCMT transfectants both markers of apoptosis were detected only at a H2O2 concentration in between 0.3 and 0.4 mM. These results, as a whole, demonstrated that PCMT overexpression is able to raise the threshold of cell sensitivity to apoptosis induced by oxidative stress up to a H2O2 concentration approaching 0.4 mM. In order to quantitate the effects of the oxidative treatment on the induction of apoptosis, cells were also analyzed by flow cytometry. PAEC samples transfected with plasmids carrying wild type or antisense or negative dominant mutants of PCMT, were subject to oxidative, pro-apoptotic treatment using 0.3 mM H2O2. This value, indeed, represented a clear cut off concentration which was able to induce apoptosis only in the negative dominants (PCMT antisense or mutants), according to the markers so far employed. Results of the FACS analysis basically confirmed that overexpression of wild type PCMT prevents apoptosis induced by an oxidative stress, as judged by the appearance of a pre-G0 peak in both the antisense and mutant PCMT cell samples, upon H2O2 treatment (Fig. 3 column C). The evaluation of the area under the peaks allowed a quantitative assessment of the different cell subpopulations. Results showed that overexpression of wild type PCMT renders cells resistant to apoptosis, while a high percentage of cells transfected with antisense or either negative mutants undergo apoptosis upon H2O2 treatment. Accumulation of deamidated isoaspartyl-containing proteins in PCMT negative dominants It is conceivable that the reduced PCMT activity in PAEC transfected with antisense or PCMT mutants may determine a significant build up of isoaspartyl-containing damaged proteins. Therefore we attempted: a) to establish whether or not PCMT overexpression was able to prevent the accumulation of deamidated-isomerized proteins, in PAEC upon oxidative pro-apoptotic treatment; b) to quantitatively evaluate the amount of isoAsp sites in proteins extracted from these cells. To this end an in vitro assay, using recombinant PCMT, was employed. Results showed that transiently transfected PAEC were all characterized by a high content of deamidated proteins, except those overexpressing the wild type PCMT gene (Fig. 4). Therefore, the ability of PCMT to prevent apoptosis upon oxidative treatment was strictly dependent on the retention of its catalytic activity. In fact negative dominants, carrying a point mutation involving the catalytic site of the enzyme, accumulated isomerized-damaged proteins and, hence, were more sensitive to the pro-apoptotic stimulus. These results led us to exclude that PCMT may prevent apoptosis though a direct interaction of the enzymatic protein with members of the apoptotic cascade. Such a direct interaction mechanism is instead operative in E. Coli, where PCMT overexpression improved heat shock survival by a mechanism independent of its methyltransferase activity [20]. 10.1371/journal.pone.0003258.g004Figure 4 Quantitation of the extent of protein deamidation in cells lysates after oxidative stress. Cells transfected with PCMT wild type (sense), antisense PCMT (anti), PCMT mutants [Asp83 →Phe83 (Phe) and Asp83→Val83 (Val)] and endothelial cells transfected with void plasmid (PAE) were stressed with 0.3 mM of H2O2. Deamidated proteins were quantitated in each lysate preparation using recombinant PCMT. Standard deviation error bars are included for each analysis. More remarkably, our results demonstrate that some molecular mediators, involved in apoptosis induced by pro-oxidant conditions, are deamidated proteins, which can be methylated by PCMT, since they contains the unique feature recognized by this enzyme: the isoaspartyl residue. We were then prompted to search for substrates of PCMT whose deamidation could be favored under oxidative pro-apoptotic conditions and which could no longer be “repaired” in the negative dominants. Identification of PCMT substrates In order to identify specific methyl accepting substrates involved in apoptosis, the purified human recombinant PCMT was cross-linked with sulfo-SBED, a trifunctional reagent containing a biotin, a sulfonated N-hydroxysuccinimide (Sulfo-NHS)active ester and a photoactivatable aryl azide. The moiety containing the active ester also exhibits a cleavable disulfide bond. A PCMT-interacting substrate is then captured by the photoreactive aryl azide moiety. The interacting complex is then isolated and the disulfide bond subsequently reduced. Upon reduction of the disulfide bond, PCMT is released and biotin is “transferred” onto the methyltransferase substrate (Label Transfer Method) [21] (Fig. 5 panel A). The biotinylated PCMT substrates can then be purified exploiting streptavidin-biotin interactions as described under “Methods” and subject to proteomic analysis. As an expression system, we chose human umbilical vein endothelial cells (HUVEC) infected with retrovirus carrying antisense-PCMT at a moi of 100. The retroviral transgene was expressed in nearly 100% of the cells, as assessed by confocal microscopy of GFP fluorescence. We used real time-PCR to analyze the silencing of PCMT gene in the cells infected with antisense-PCMT carrying virus. PCMT was effectively hypoexpressed in the antisense PCMT-transduced cells Relative expression of PCMT in the HUVEC infected with the vector carrying the antisense PCMT was 0.012 fold (±0.0035; standard deviation) compared to what detected in the uninfected HUVEC. HUVEC infected with retro-PCMT antisense were then treated with 0.3 mM H2O2 for 24 h. In order to identify PCMT substrates, protein extracts from these cells were incubated with biotinylated human recombinant PCMT, as above described. In order to discriminate the specifically PCMT-interacting proteins from a background of non-interacting proteins, a parallel assay was run, as a negative control, by incubating cell extracts with sulfo-SBED not cross-linked with PCMT. The resulting protein samples, enriched in PCMT substrates, were analyzed with 2D gel (Fig. 5 panel B). Protein spots, differentially expressed between sample and negative control, were considered for MALDI-TOF mass spectral analysis. Good matches were found for eight spots that were analyzed by mass spectrometry. All of them produced a good Z score, high sequence coverage and had molecular weights and isoelectric points consistent with the location of the protein on 2D gel. Several spots could not be identified, since relevant mass data did not yield a match with a high Z score or good sequence coverage (Table 1). 10.1371/journal.pone.0003258.g005Figure 5 Identification of PCMT substrates. Panel A: Experimental strategy for isolation and characterization of PCMT substrates. Step 1: human recombinant PCMT, purified to homogeneity, was immobilized onto sulfoSBED by N-hidroxysuccinimide chemistry; Step 2: cell extracts as a source of substrates were added and Step 3: proteins interacting with PCMT were immobilized upon UV photoactivation; Step 4: PCMT was released and biotin “transferred” onto the methyltrasferase substrate (Label Transfer Method); Step 5: purification was accomplished by exploiting streptavidin-biotin interactions. Panel B: 2D gel electrophoresis imaging of comparative proteomics. HUVEC were infected with antisense PCMT carrying retrovirus and then stressed with 0.3 mM of H2O2. Cells lysates were reacted with Sulfo-SBED previously cross-linked with recombinant PCMT (Panel B). Arrows indicate the protein spots which have been characterized as reported in Table 1. Background noise due to aspecific binding was subtracted by comparison with the 2D image obtained from a parallel sample reacted with non-cross-linked Sulfo-SBED. 10.1371/journal.pone.0003258.t001Table 1 PCMT substrates identified in endothelial cells. Spot no Protein Information and Sequence Analysis Tools (T) % pI kDa Est'dZ Function S1 T gi\|2507461\|pir\|\|P30101 protein disulfide-isomerase (EC 5.3.4.1) ER60 precursor - human 29 6.1 57.06 2.43 Thioreductase/isomerase activity S19 T gi\|16507237\|ref\|NP_005338.1\| heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa); Heat-shock 70 kD protein-5 (glucose-regulated protein, 78 kD); heat shock 70 kD protein 5 (glucose-regulated protein, 78 kD) [Homo sapiens] 36 5.1 72.43 2.39 Chaperone S20 T gi\|20149594\|ref\|NP_031381.2\| heat shock 90 kDa protein 1, beta; heat shock 90 kD protein 1, beta; Heat-shock 90 kD protein-1, beta [Homo sapiens] 29 5.0 83.59 1.97 Chaperone S5 T gi\|4557593\|ref\|NP_000131.1\| ferrochelatase [Homo sapiens] 53 9.3 48.39 2.01 ferrochelatase activity S10 T gi\|9910382\|ref\|NP_064628.1\| mitochondrial import receptor Tom22 [Homo sapiens] 25 4.3 15.50 2.32 receptor activity S12 CAA80661 Homo sapiens Bcl-xL 25 5.4 17.10 2.41 anti-apoptosis S 14 T gi\|54036678\|pir\|P63261\| gamma-actin [Homo sapiens] 39 5.3 41.99 2.43 Cytoskeletal structure and dynamics S18 T gi\|229674\|pdb\|1ALD\| Aldolase A (E.C.4.1.2.13) 18 8.8 39.73 2.43 Energy metabolism HUVEC cells were infected as described under “Methods”. The antisense negative dominants were treated with 0.3 mM H2O2. PCMT ligands were isolated by means of human recombinant methyltransferase immobilized onto SulfoSBED. Purification, 2D separation and MS analysis were accomplished as detailed in the online supplemental material. Previous work amply demonstrated that two structural features are essential for PCMT function: a) presence of an isoAsp residue; b) recognition by the methyltransferase. As for the first feature, the most deamidation-susceptible residues are asparagines followed by non bulky residues [1], [2], [22]. As for substrate-PCMT interaction, negative structural requirements are the presence of Cys or charged residues immediately following isoAsp [23] as well as N-terminal isoAsp residues [24]. Based on these criteria we therefore carefully reviewed published sequences of all proteins substrates we had identified. Results confirmed that all proteins indeed contained at least one deamidation site, thus allowing us to predict the position at which the actual methylatable isoaspartyls may occur. Protein disulfide isomerase (accession no P30101) displays one theoretically deamidation-susceptible site at Asn199Gly, another at Asn272Ala, plus two adjacent AsnThr sites at position 88. Molecular chaperone HSP70 [accession number (AN) NP_005338.1] contains several theoretical deamidation sites including: five Asn-Ala (at position 59, 280, 367, 515, 619), two Asn-Ser (407, 646), Asn62Thr. Asn41Ser occurs in HSP90 (AN NP_031381.2). Ferrochelatase (AN NP_000131.1) displays three of such features (Asn153Thr; Asn204Ala; Asn372Gly). As for mitochondrial import receptor (AN NP_064628.1) the only theoretical deamidation site is Asn124Thr. Actin (AN P63261) contains the following theoretically deamidation-susceptible asparagines: Asn12Gly, Asn-Thr at positions 128 and 296, Asn280Ser. In addition, we identified, as a PCMT substrate, the cleaved form of the antiapoptotic protein Bcl-xL [25]. Bcl-xL deamidation sites have been experimentally and unambiguously identified by previous work, as discussed below. It is now clear that this protein undergoes deamidation at two (Asn52 and Asn66) of the three Asn-Gly sequences, which are theoretically sensitive hot spots for this post-biosynthetic modification. Present results demonstrate that this protein actually is recognized by the repair methyltransferase PCMT. Discussion Our data demonstrate that PCMT overexpression confers resistance to apoptosis, induced by oxidative stress, in endothelial cells. Conversely, under conditions in which PCMT was suppressed, cells accumulated isoaspartyl-containing, deamidated proteins, upon oxidative stress. The identification of isoaspartyl-containing derivatives as the actual target-substrate for this protection was made possible by the utilization of PCMT as a specific enzymatic probe, which selectively recognizes the isoaspartyl moiety in deamidated proteins. In fact we were able to show that isomerized proteins increase significantly in the cells committed to apoptosis as the result of oxidative treatment. To clarify the mechanism involved in this resistance we employed a proteomics approach using, again, PCMT as a specific ligand to isolate deamidated-isomerized protein substrates. We can conclude that all the proteins we were able to isolate act as endogenous substrates for PCMT, under conditions in which cells undergo apoptosis. These substrates include various stress proteins (HSP70, HSP90, mitochondrial import receptor, protein disulfide isomerase), the cytoskeletal component actin (a long-established substrate for PCMT), ferrochelatase and Bcl-xL. The role in apoptosis of some of these proteins has been well established, in particular regarding chaperone HSP70 and HSP90 [26]. As for HSP70, this protein prevalently exerts antiapoptotic activity, both in the intrinsic and in the extrinsic pathways, according to a complex pattern throughout the apoptosis cascade [27], [28]. As for HSP90, this component, as well, is endowed with antiapoptotic activity through a multifaceted mechanism [27], [29]. Bcl-xL is perhaps the most interesting protein we were able to isolate, for at least two reasons: first it exerts powerful and direct antiapoptotic activity. Second, and more relevant to our aim, Bcl-xL undergoes deamidation in relation with cell damage, giving rise to its isoaspartyl derivative, and this process has profound implications on its functional antiapoptotic role. In fact it has been previously reported in the literature that Bcl-xL deamidation is critical in the signaling pathway leading from DNA damage to apoptosis. Data accumulating over the last few years led to the notion that Bcl-xL deamidation induces a profound structural modification, which, in turn, hampers the antiapoptotic function of this protein. In particular Deverman and coworkers used constructs where Asn52 and Asn66 in the wild type form of Bcl-xL, the two critical deamidation-susceptible asparagines, are replaced [15]. Results, as rediscussed subsequently, were consistent with the view that Asn deamidation to isoaspartate, as the real product, results in the loss of Bcl-xL function (Erratum for [15] in [30]. The mechanism for generation of the isoaspartyl derivative of Bcl-xL has been finally elucidated, as it involves an increase of the intracellular pH, consequent to cell injury, and transcriptional activation of a Na/H antiport, which constitutes a favorable microenvironment for asparaginyl deamidation through ASU formation [16]. It is worth noting that an oxidative stress, as the means we used to induce apoptosis, also represents a protein deamidation-favoring microenvironment. In this respect, it has been previously shown that a) human erythrocytes treated with either t-BHP or H2O2 accumulate deamidated isomerized proteins in the cell membrane, which are methyl esterified ex vivo by PCMT [31]; b) human erythrocytes from G6PD-deficient subjects are particularly prone to undergo deamidation at membrane protein level, upon oxidative stress, compared to normal red cells [9]; c) melanoma cells also accumulate PCMT substrates upon UV irradiation, according to a mechanism which is mediated by an oxidation [12]. Finally, we detected increased isoaspartyl formation in the erythrocytes from patients with Down Syndrome, a condition which is characterized by increased oxidative stress [32]. Now a more general question arises: is the occurrence of isoaspartyls upon deamidation a built-in destruction device or, rather, a fine tuning system acting in concert with methylation? The presence of deamidation-susceptible Asn in proteins appears to be selected against during evolution [33]. Some early experimental evidence [34], [35] supports a “Molecular Clock” hypothesis. According to this model, the presence of such deamidation-susceptible Asn may account for the occurrence of time-dependent structural modifications of functional meaning. For example, in the case of triosephosphate isomerase and serine hydroxymethyltransferase, deamidation of labile asparagines residues may represent specific signals for commitment of protein to degradation. On the other hand, it has been suggested that the non-random distribution of such labile residues in proteins may not only be related to their degradation. It has been argued that although deamidation could be actually considered as a “structural alteration”, at least in some instances, the persistence of such deamidation hot spots during evolution may otherwise serve to certain functions [5]. According to this view, deamidation may be interpreted as a molecular switch that regulates partitioning over time between two molecule subpopulations of a certain protein, which are also functionally modified. The existence of PCMT, as an enzyme involved in the “repair” of the isopeptide bond resulting from Asn deamidation, is also in agreement with the latter mechanism. In this respect protein deamidation to isoaspartyl-containing products, which are susceptible to recognition and repair by PCMT, has been found to occur in the extracellular matrix [36], [37]. More recently it has been reported that deamidation of susceptible proteins in the extracellular matrix may also serve as a molecular signal to unravel new integrin binding sites [38]. Deamidation of Bcl-xL, with consequent abolishment of its antiapoptotic function, thus represents a further example of such a molecular device to change functional properties of those proteins in which it occurs. Based on present results we then propose that PCMT is involved in the modulation of the apoptotic process, by regulating the balance, within the cell, between the isoaspartyl-containing and the repaired aspartyl form of Bcl-xL (Fig. 6). The repair mechanism, as it has been shown on various models, does not consist in the restoration of the asparaginyl residues, but, rather, in the conversion of the isopeptide bond at the level of the isoAsp into a normal α–peptide bond [1], [2]. 10.1371/journal.pone.0003258.g006Figure 6 Proposed role of Bcl-xL deamidation and methylation in apoptosis. Deamidation-isomerization of two critical Asn residues of Bcl-xL abolishes its antiapoptotic function. Methylation of the same residues can restore the functional integrity of Bcl-xL through repair of the isopeptide bonds. Regulation of apoptosis toward antiapoptosis is gained through fine balancing of Bcl-xL deamidation and methylation. Present results allow us to speculate on the possible implications of deamidation and methylation in the modulation of programmed cell death, under conditions in which this process is altered, such as in cancer. Some of the proteins we thus identified as PCMT substrates, which are known to be involved in apoptosis, have been implicated in pathogenesis of tumors, with particular regard to the acquisition of resistance by the transformed cell phenotypes. In this respect, it has been recently reported that HSP70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma [39]. Data also showed the role of HSP90 in the mechanism of metastasis [40] about the involvement of apoptosis in cancer, Bcl-xL perhaps provides the best example of how methylation of deamidated proteins may play a role in maintaining full functioning antiapoptotic proteins (Fig. 6). These latter, in turn, may contribute to the resistance of transformed, tumor cells, which otherwise would undergo programmed death. In line with this interpretation are previous results showing a significant reduction in the deamidation rate of Bcl-xL hepatomas, compared to normal liver tissue [17]. Our present results, on the ability of PCMT to recognize and methylate Bcl-xL, thus preserving the antiapoptotic features of this protein, in fact suggest a role of this methyltransferase as a potential target for anticancer intervention. Materials and Methods Materials The plasmid encoding the GFP was from Stratagene (USA). E. Coli strain DH5a was used as a host for the plasmids and cloning procedures, and BMH71-18 mutS was used in the mutagenesis step (Clontech, Palo Alto, CA, USA). Bl21, Sulfo-SBED and Immunopure immobilized monomeric avidin beads were purchased from Pierce (Rockford, IL, USA). DTT, ACTH, Renin, and Angiotensin I were from Sigma (St. Louis, MO, USA). DMEM, RPMI and FBS were purchased from Life technologies (Invitrogen S.R.L., Milan, Italy). Trypsin sequencing grade (Product number V5111) was from Promega (Madison, Wisconsin, USA). Protein standards and Iodoacetamide were from BioRad laboratories (Milan, Italy). Water and Acetonitrile were HPLC grade (Sigma). S-adenosyl-L-[methyl-14C]Met [specific activity (sp. act.) 50 mCi/mmol] was purchased from Amersham International (Buckinghamshire, UK). S-adenosyl-L-[methyl-3H]Met (sp. act. 500 mCi/mmol) and S-adenosyl-L-[methyl-14C]Met (sp. act. 50 mCi/mmol) were purchased from Amersham International (Little Chalfont, Buckinghamshire, UK). All standards and reagents were from Sigma Chemical Co. and were of the purest grade available. PCMT clone The pBluescript II SK-based plasmid construct pDm2X, containing human PCMT isoform II cDNA, and polyclonal antibodies against the C-terminal dodecapeptide of PCMT were generously provided by Dr. Steven Clarke (Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA). Cell Culture and Transient Expression PAEC were grown on 24–well plates, in DMEM supplemented with 10% FCS and 1× penicillin/streptomycin. Cells were maintained at 37°C in humidified air with 5%CO2 atmosphere. For transient transfection by electroporation, PAEC growing in 150 cm2 flasks were tripsinized before confluence, collected by centrifugation, washed with PBS and resuspended in 20 mM HEPES buffer at pH 7.4, containing 137 mM NaCl, 5 mM KCl, 0.7 mM NaH2PO4 6 mM glucose, at 4°C. HUVECs from American Type Culture Collection were maintained in RPMI 1640 with 10% FCS and 2 mM glutamine, and were used at passages 5 to 10. Co-transfections were performed by mixing 15 µg of each pcDNA3.1 construct and 2 µg EGFP control. Cells were exposed to a pulsed electric field that corresponded to a field strength of 0.75 kV/cm and a duration ranging from 9 to 12 ms using electroporation cuvette and Gene Pulser II (Bio-Rad Laboratories, Hercules,. CA, USA). Samples were incubated on ice for a further 5 min, then transferred to a 10 cm diameter tissue culture dish and incubated at 37°C under standard growth conditions. After transfection, cells were plated for 16 h in DMEM supplemented with 10% FCS. Medium was replaced after 24 h. Mutagenesis Mutagenesis was performed by Transformer Site-Directed Mutagenesis kit from Clontech (Palo Alto, CA). Of the three highly conserved methyltransferase sequence motifs we chose to perform mutagenesis of the invariable aspartyl site within sequence motif I (ALDVGSGSGI), involved in the AdoMet binding site [18], [19]. The mutagenesis primer was 5′-ggagctaaagctcttttcgtaggatctgg-3′, (Asp replaced by Phe); 5′ ggagctaaagctcttgtcgtaggatctgg3′ (Asp mutated into Val). The selection primer was 5′-caggaaagaagatctgagcaaaag-3′. A unique restriction site (AflIII) was replaced with a new unique restriction site (BglII). Sequencing of double-stranded plasmid DNA by the Sanger method was used to confirm the desired nucleotide substitution. Characterization of PCMT plasmid constructs PCMT activity in endothelial cells transfected with vectors carrying PCMT sense or antisense or either mutants, was detected by means of the radiochemical assay described by Macfarlane, using ovalbumin as a standard methyl-accepting substrate [41]. One enzyme unit is defined as 1 pmol methyl groups transferred×min−1. Plasmids pcDNA3.1 wild type Construction of mammalian expression plasmid encoding human PCMT was subcloned from construct pDM2X used for mutagenesis, into pcDNA3.1, using the KpnI and HindIII restriction sites. pcDNA3.1 antisense The cDNA was subcloned from pcDNA3.1 wild type. pcDNA3.1 mut phe pcDNA3.1 encoding for mutated PCMT Asp83→Phe was subcloned from construct pDM2X mut Phe into pcDNA3.1 using the KpnI and HindIII restriction sites. pcDNA3.1 mut Val pcDNA3.1 encoding for mutated PCMT Asp83→Val was subcloned from construct pDM2X mut Val into pcDNA3.1 using the KpnI and HindIII restriction sites. Apoptosis Assay Endothelial cells were grown to 70% to 80% confluence and incubated for 18 h in the absence or the presence of 0.1, 0.2, 0.3, 0.4 or 0.5 mM H2O2. Parallel samples were treated with 35 mM Cisplatin for 18 h as a positive apoptosis control. The optimal active H2O2 concentration and exposure time were selected by determining dose-response and time curve. Under these conditions, the assay was linearly dependent on H2O2 concentration and incubation time. Longer incubation periods or higher H2O2 concentrations resulted in massive cell death. Apoptosis was assayed in vitro by a combination of three distinct approaches on endothelial cells transfected with vectors carrying PCMT sense or antisense or either mutants. Caspase-3 activity and PARP cleavage After oxidative injury, cells were collected and lysed in the appropriate buffer. Total cell extracts (30 to 50 µg) were electrophoresed onto a 12.5% SDS-PAGE gel system and transferred onto polyvinylidene difluoride membrane (Millipore S.p.A, Milan, Italy). Blots were incubated with a polyclonal anti-caspase-3 antibody (BD-PharMingen Milan, Italy), or mouse polyclonal antipoly (ADP-ribose) polimerase (PARP). Total active caspase-3 and PARP bands were revealed by chemiluminescence (Amersham-Pharmacia Biotech). DNA ladder Cells treated as above were harvested and DNA was extracted using the Apoptotic DNA ladder Kit (Roche Diagnostics S.p.A., Monza, Italy). DNA Ladder profiles were detected upon electrophoresis on 2% agarose gel. FACS scan analysis Six hours after the removal of the stimulus, apoptosis was checked by detected fluorescence microscopy. FACS analysis (FACSCalibur; Becton-Dickinson, San Jose, CA, USA) was performed after treating cells pellets (approx 106 cells) with 50 µg/ml propidium iodide (PI) in 0.1% sodium citrate, in the presence of 0.1% Nonidet P40 and 100 µg/ml Dnase-free RNase A (Boeringer Mannheim, Milan Italy). Integration of area under the pre-G0 peak was measured to quantify percentages of apoptotic cells. Human recombinant PCMT purification E. Coli strain DH5a was used for cloning and propagation of plasmid constructs. For PCMT overexpression, E. Coli strain BL21 (DE3) was transformed with pDM2x expression plasmid and grown in Luria-Bertani medium, in the presence of 100 mg/mL ampicillin. PCMT was purified from transformed bacteria basically as described by MacLaren and Clarke 1995 [42], except that the original DEAE-cellulose chromatography final step, under non-equilibrium conditions, was replaced by Q-Sepharose HP chromatography, using a Hiload 26/10 column (Pharmacia, Uppsala, Sweden). Column was equilibrated with buffer A (20 mM Tris-HCl, 0.2 mM EDTA disodium salt, 10% wt/vol glycerol, 15 mM beta-mercaptoethanol, 25 mM phenylmethylsulfonyl fluoride, 0.1 M NaCl, pH 8.0). After sample loading (10 mL, 6 mg/mL protein concentration), the column was washed with 10 volumes of buffer A (at 1 mL/min flow rate), followed by a linear gradient from 0.1 to 0.7 M NaCl over 210 minutes. Quantitation of isoaspartyl residues in cells lysates after oxidative stress Cells transfected with PCMT wild type (sense), antisense PCMT, PCMT mutants (Asp83 →Phe and PCMT Asp83→Val) and untransfected endothelial cells were stressed with 0.3 mM of H2O2. Deamidated proteins were detected in each lysate preparation by an in vitro assay using recombinant PCMT. Damaged residues were specifically recognized and methyl esterified by PCMT, using radiolabeled AdoMet as the methyl donor, under conditions designed to insure complete labeling, on a 1∶1 molar ratio, of accessible damaged residues in proteins. This method has proven highly sensitive, specific, reproducible, and particularly suitable when analysis of deamidated protein mixtures is to be carried out [43]. Identification of PCMT substrates In order to identify specific PCMT substrates, HUVEC were infected with PCMT antisense and stressed with 0.1 mM H2O2. Cell lysates were prepared as above and analyzed by 2D-gel electrophoresis according to Zhu et al [44]. Generation of the retrovirus An 800-bp genomic sequence including either wild type PCMT or its antisense counterpart was cloned upstream from the IRES of the MSCV-IRES-GFP (MIGR) (murine stem cell virus-internal ribosome entry site-green fluorescent protein) retrovirus. Infectious defective virions were transiently produced by transfection of the 293 FT cell line with 3 plasmids: pCMV gag-pol, pCMV-VSV-G (vesicular stomatite virus envelope glycoprotein). Briefly, 293 FT cells were seeded at a concentration of 106 cells per well in 6-wells plates. The next day, 0.5 µg of each plasmid was cotransfected using Exgen reagent (Euromedex, Mundolshein, France) according to the manufacturer's recommendations. Supernatants were collected after 48, 72, and 96 h and concentrated 20-fold over an amicon membrane (Centricon Plus-80; Millipore, St-Quentin en Yvelines, France). Viral titers were determined by limiting dilution assay on NIH 3T3 cells. GFP fluorescence was analyzed by flow cytometry. Virus stocks containing 107 infectious particles per milliliter or more were used to infect HUVEC (ATCC- CRL-1730). Quantitative real-time -PCR RNA was extracted from the antisense PCMT-transduced cells and non-transfected HUVEC by a double Trizol- chloroform treatment and precipitated in isopropanol. Total RNA. 250 ng, was reverse transcribed using the Superscript II (Invitrogen), where the reaction mixture contained forward primer for PCMT (5′- TTAAAGCCCGGAGGAAGATT3′) and, as the reverse, the oligodT examer included in the kit. The specific PCMT cDNA was then amplified in the presence of 1× SYBR green by for quantification by real time PCR. using an iCycler iQ machine (BiorRad, inc.); primers pairs used were 5′-TTAAAGCCCGGAGGAAGATT3′ and 5′-ATCACTTCCACCTGGACCAC-3, designed to amplify 169 bp region of PCMT cDNA. Relative expression was calculated using the ΔCt method. To determine the quantity of PCMT transcript present in the antisense-PCMT-infected HUVEC, relative to uninfected ones, their respective Ct values were first normalized by subtracting the Ct value obtained from the evaluation of the GAPDH transcript, chosen as an housekeeping gene (ΔCt = CtPCMT−CtGAPDH). The relative abundance of PCMT transcript in the infected HUVEC compared with uninfected cells was calculated by subtracting the normalized Ct values obtained for uninfected cells from those obtained from antisense-PCMT infected cells (ΔΔCt = ΔCtinfected−ΔCtuninfected; the relative expression was then determined (2−ΔΔCt). The the value of 2−ΔΔCt>1 reflects increased expression of the target PCMT gene, and a value of 2−ΔΔCt<1 points to a decrease in the gene expression [45]. Crosslinking reaction PCMT was dissolved at 1 mg/mL in phosphate buffer (0.1 M phosphate, 0.18 M Na, pH 7.5). 1.12 mg of SulfoSBED was weighed and dissolved in DMSO under subdued light immediately before use. PCMT and sulfoSBED solutions were combined and maintained under subdued light with aluminum foil wrapping for 30–60 min at room temperature and nonreacted SulfoSBED was removed from solution by dialysis using Slide-A-Lyzer Dialysis Cassette (Pierce). 5 mg of proteins extracted from HUVEC cells line after infection with PCMT antisense carrying retrovirus and treated for 24 h with 0.1 mM H2O2, were dissolved in 0.5 ml PBS and incubated at room temperature for 3–5 minutes with the biotinylated complex. A 365 nm UV lamp (Rad-Free Model RF UV-365, Schleicher and Schuell, Keene, NH, USA), held at 5 cm distance from sample solutions, was used to activate the arylazide portion of the crosslinker. Samples were reduced by adding DTT to a final concentration of 50 mM and incubating for 1 h at room temperature. Binding biotinylated proteins Avidin affinity capture of biotinylated species was performed using immobilized monomeric avidin. Small batches (typically 100 µl) of beads in 50% aqueous suspension (MagPrep Streptavidin Beads purchased from Novagen, Merck chemicals Ltd, Nottingham, UK) were prepared for use into a clean 1.5 ml microcentrifuge tube and placed in magnetic tube rack, following wash with 400 µl of 0.1 M phosphate buffer (pH 7.5, 0.18 M Na) and the aqueous supernatant removed. The beads were washed twice and resuspended to their original volume then incubated for 30 min at RT with purified biotinylated target protein. After the binding, the excess of protein was taken away by magnetizing the beads and removing the aqueous phase. 2D analysis and MALDI-TOF Gels were stained with Coomassie blue and spots of interest were identified by comparing gel images as appropriate. Spots of interest were then cut and submitted to the Proteomics Core. The samples were transferred to the MassPrep station for automated in-gel protein digestion, following the protocol included with the WinPREP Multiprobe II software (WinPREP Multiprobe II; Perkin Elmer, Massachussets, USA). Briefly, gel pieces were de-stained with ammonium bicarbonate/acetonitrile and reduced with dithiothreitol. The reducing mixture was removed and iodoacetamide in ammonium bicarbonate added and incubated for 20 min at 37°C. The alkylation solution was removed, followed by washing with ammonium bicarbonate/water and dehydration with acetonitrile. In-gel digestion of the extracted proteins was carried out with 6 ng/µL trypsin in 50 mM ammonium bicarbonate for 5 h at 37°C. The digested peptides were extracted in a mixture of 1% formic acid/2% acetonitrile and applied onto a stainless steel MALDI plate (Micromass). Mass spectra of the resulting peptides were recorded on the MALDI-TOF spectrometer in reflectron mode (Perkin Elmer Mass Prep Station; Micromass Maldi-TOF RL). Prior to data collection, calibration was performed with Angiotensin I (Average molecular mass 1296.5 Da), Renin (Average molecular mass 1759.0, Da), and ACTH 18–39 clip (adenocorticotropic hormone clip 18–39, average molecular mass 2465.199 Da). Sofware MassLynx 4.0 (Micromass); ProteinLynx 2.0 (Micromass) was used for processing, background subtraction, and some supplemental analysis. Resulting peptides were matched with their corresponding proteins with XProteo (XProteo: fast, reliable protein identification), by searching the non-redundant database maintained at the NCBI (http://www3.ncbi.nlm.nih.gov/). In order to produce a putative protein identification and score, the following parameters were used for search: mass tolerance 0.07 Da, incomplete cleavages allowed, alkylation of Cys and oxidation of Met were considered as possible modifications. Percentage of sequence coverage (%) was indicated for each protein assignment. Z-score is defined as the distance to the population mean in unit of standard deviation. It also corresponds to the percentile of the search in the random match population (according to ProFound - Peptide Mapping Version 4.10.5 - The Rockefeller University Edition). The authors wish to thank Dr Vincenzo Nigro, Professor of General Pathology at S.U.N., for helpful hints and for kindly reviewing the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported in part by Grant 2005062199_003 from M.I.U.R.-P.R.I.N. to D. Ingrosso and by Grant 2004057129_003 from M.I.U.R.-P.R.I.N. to P.Galletti. ==== Refs References 1 Galletti P Ingrosso D Manna C Zappia V 1995 Protein damage and methylation-mediated repair in the erytrocyte. Biochem J 306 313 325 7887885 2 Clarke S 2003 Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair. Ageing Res Rev 2 263 285 12726775 3 Robinson NE Robinson AB 2001 Molecular clocks. Proc Natl Acad Sci 98 944 949 11158575 4 Aswad DW Paranadi MV Schurter BT 2000 Isoaspartate in peptides and proteins: formation, significance, and analysis. J Pharm Biomed Anal 21 1129 1136 10708396 5 Reissner KJ Aswad DW 2003 Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitions signals? 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==== Front J Autoimmune DisJournal of Autoimmune Diseases1740-2557BioMed Central 1740-2557-5-41866424910.1186/1740-2557-5-4ResearchAnalysis of the TCR alpha and beta chain CDR3 spectratypes in the peripheral blood of patients with Systemic Lupus Erythematosus Luo Wei [email protected] Li [email protected] Qian [email protected] Xin-Sheng [email protected] Na [email protected] Hong-Yun [email protected] Ming-Qian [email protected] Ying [email protected] Zhen-Qiang [email protected] Xiao-Wei [email protected] Ju-Fang [email protected] Xiao-Ning [email protected] Institute of Molecular Immunology, Southern Medical University, Guangzhou 510515, PR China2 College of Biosciences & Bioengineering, South China University of Technology, Guangzhou 510641, PR China3 Department of Dermatology & Rheumatology, Nan fang Hospital, Guangzhou 510515, PR China2008 29 7 2008 5 4 4 16 4 2007 29 7 2008 Copyright © 2008 Luo et al; licensee BioMed Central Ltd.2008Luo et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ==== Body BioMed Central have removed this article from the public domain as the article was published in error.	18664249	PMC2535584	CC BY	2021-01-04 17:12:53	yes	J Autoimmune Dis. 2008 Jul 29; 5:4
==== Front PPAR ResPPAR ResPPARPPAR Research1687-47571687-4765Hindawi Publishing Corporation 1881027510.1155/2008/649808Research ArticleRosiglitazone Suppresses the Growth and Invasiveness of SGC-7901 Gastric Cancer Cells and Angiogenesis In Vitro via PPARγ Dependent and Independent Mechanisms He Qing 1,2 Pang Ruiping 3 Song Xin 1 Chen Jie 1 Chen Huixin 1 Chen Baili 1 Hu Pinjin 1 Chen Minhu 1 1Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China2Department of Gastroenterology, Gastroenterology Institute, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China3Department of Physiology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, ChinaMinhu Chen: [email protected] by Dipak Panigrahy 2008 15 9 2008 2008 64980823 3 2008 1 7 2008 Copyright © 2008 Qing He et al.2008This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Although thiazolidinediones (TZDs) were found to be ligands for peroxisome proliferators-activated receptorγ (PPARγ), the mechanism by which TZDs exert their anticancer effect remains unclear. Furthermore, the effect of TZDs on metastatic and angiogenesis potential of cancer cells is unknown. Our results in this paper show that rosiglitazone inhibited SGC-7901 gastric cancer cells growth, caused G1 cell cycle arrest and induced apoptosis in a dose-dependent manner. The effects of rosiglitazone on SGC-7901 cancer cells were completely reversed by treatment with PPARγ antagonist GW9662. Rosiglitazone inhibited SGC-7901 cell migration, invasiveness, and the expression of MMP-2 in dose-dependent manner via PPARγ-independent manner. Rosiglitazone reduced the VEGF induced angiogenesis of HUVEC in dose-dependent manner through PPARγ-dependent pathway. Moreover, rosiglitazone did not affect the expression of VEGF by SGC-7901 cells. Our results demonstrated that by PPARγ ligand, rosiglitazone inhibited growth and invasiveness of SGC-7901 gastric cancer cells and angiogenesis in vitro via PPARγ-dependent or -independent pathway. ==== Body 1. INTRODUCTION Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the ligand-inducible nuclear receptor superfamily. After activation, PPARγ associates with the 9-cis retinoic acid receptor (RXR) to form functional heterodimers, which binds to the PPAR response element of the target genes and regulates the expression of these genes. Previous documents have shown that the PPARγ/RXR signal pathway plays critical role in a variety of biological processes, including adipogenesis, glucose metabolism, inflammation as well as inhibition of normal and tumor cells growth [1]. Thiazolidinediones (TZDs) are synthetic agonists for PPARγ. These PPARγ ligands were clinically used as antidiabetic drugs which could attenuate the insulin resistance associated with obesity, hypertension, and impaired glucose tolerance in humans [2]. Recent studies have suggested that PPARγ is a potential molecular target for anticancer drug development, due to the increased expression of PPAR in several cancer cells. It has been reported that TZDs could inhibit growth and induce apoptosis in a variety of cancer cell lines. More importantly, TZDs exhibited antitumor activities in vivo in the prevention of prostate, liver, and pituitary cancers. Although increasing evidence showed that TZDs are potential anticancer agents [3], the mechanisms underlying the antitumor effects are not well understood. TZDs were initially thought to inhibit the cancer cells proliferation through regulation of expression of PPARγ-mediated target genes. However, recent evidence revealed that the antitumor effects of TZDs exist via PPARγ-independent mechanisms in various types of cancers [4–6]. We previously found the expression of PPARγ decreased in primary and metastatic gastric carcinoma, compared with normal gastric tissues [7]. Recent studies in gastric cancer cells demonstrated that TZDs treatment resulted in significant growth arrest both in cultured cell and in nude mice models [8–12]; however, the effects of PPARγ ligands on invasiveness and angiogenesis of gastric cancer are still unclear. Therefore, this work was undertaken to investigate the effects of PPARγ agonists, such as rosiglitazone, on cell growth and the invasiveness in human cell line SGC-7901, as well as on angiogenesis in vitro. 2. METHODS 2.1. Cell culture Human gastric cancer cell line, SGC-7901, was obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from the Keygen Technology Company (Najing, China). SGC7901 cells and HUVECs were cultured in RPMI-1640 medium (GIBCO, Carlsbad, Calif, USA) containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, Sigma-Aldrich, Mo, USA). 2.2. RT-PCR Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, Calif, USA) according to the manufacturer's instructions. Reverse transcription reaction was performed with random hexamer primers and a SuperScript Reverse transcriptase kit (Invitrogen, Carlsbad, Calif, USA). The sequences of specific primers were as follows: PPARγ mRNA, forward, 5′-TCT CTC CGT AAT GGA AGA CC-3′, and reverse, 5′-GCA TTA TGA GAC ATC CCC AC-3′. MMP-2 mRNA, forward, 5′-GGC CCT GTC ACT CCT GAG AT-3′, and reverse, 5′-GGC ATC CAG GTT ATC GGG GA-3′. VEGF mRNA, forward, 5′-GAC AAg AAA ATC CCT GTG GGC-3′, and reverse 5′-AAC GCG AGT CTG TGT TTT TGC-3′. β-actin mRNA, forward, 5′-CTT CTA CAA TGA GCT GCG TA-3′, and reverse, 5′-TCA TGA GGT AGT CAG TCA GG-3′. PCR conditions were 94°C, 30 seconds, 55–57°C (depending on the primer set), 30 seconds, and 72°C, 1 minute with 35 cycles using Taq PCR MasterMix (Tianwei, Beijing, China). The resultant PCR products were 474 bp (PPARγ), 243 bp (β-actin), 474 bp (MMP-2), and 169 bp (VEGF). PCR products were electrophoresed on a 1.2% agarose gel and visualized by ethidium bromide staining. 2.3. Quantitative real-time RT-PCR analysis The PCR reactions were performed in a Brilliant SYBR Green QPCR master mix (Stratagene, Calif, USA) according to the manufacturer's instructions. The sequences of specific primers were the same as for RT-PCR. After 10 minutes at 95°C to denature the cDNA, the cycling conditions were 95°C, 1 minute, 55–57°C (depending on the primer set), 30 seconds, and 72°C, 1 minute with 40 cycles. The LightCycler software constructed the calibration curve by plotting the crossing point (Cp), and the numbers of copies in unknown samples were calculated by comparison of their Cps with the calibration curve. To correct differences in both RNA quality and quantity between samples, the data were formalized to those for β-actin. 2.4. Western blotting The cells proteins were extracted according to NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce, Rockford, Ill, USA). Protein concentration of each sample was assayed using BCA Protein Assay Reagent according to manufacturer's instructions (Pierce Biotechnology, Rockford, Ill, USA). Twenty micrograms of proteins of different groups were separated in 10% SDS-PAGE, and transferred onto PVDF membrane (Invitrogen, Carlsbad, Calif, USA). Five percent of milk (blocking solution) was loaded over the membrane and incubated for 1 hour at room temperature with agitation. The membranes were then incubated with the mouse antihuman PPARγ antibody at a dilution of 1:200 (Santa Cruz, Calif, USA), the mouse antihuman MMP-2 antibody (1:400, Neomarker, Calif, USA), the rabbit antihuman VEGF antibody (1:200, Zymed, Calif, USA), and the mouse antihuman β-actin (1:200, Xiaxin, China) overnight at 4°C with agitation. After being washed with 0.1% Tween 20 in Tris-saline, three times, the membranes were incubated with biotin-labeled antirabbit or mouse IgG for 1 hour at room temperature with agitation. Reactive protein was detected using ECL chemiluminescence system (Pierce, Rockford, Ill, USA). 2.5. ELISA of secreted VEGF The effect of RGZ on VEGF release in tumor cells was measured by ELISA. Cells grown in 90 mm plates were exposed to various concentrations of RGZ (1–20 μM) or vehicle with or without GW9662 (2.5 μM, pretreated 1 hour) for 24 hours. VEGF concentration in the supernatant was measured using a VEGF ELISA kit (R & D systems, Minneapolis, Minn, USA). 2.6. Cell viability The viability of the cells was assessed by MTT assay. Briefly, cells grown in 96-wells were exposed to various concentrations of RGZ with or without GW9662 (2.5 μM, pretreated 1 hour), for 24, 48, or 72 hours. Then, 20 μL of MTT (5 mg/mL) was added to each well, and cells were incubated continuously at 37°C for 4 hours. After removal of medium, the crystals were dissolved in DMSO, and absorbance was assessed at 570 nm with a microplate reader. 2.7. Cell cycle and apoptosis analysis Cells treated with RGZ (1–20 μM) or vehicle with or without GW9662 (2.5 μM, pretreated 1 hour) for 48 hours were collected and fixed in cold 70% ethanol. Then, the samples were treated with RNase, stained with 50 mg/mL propidium iodide (PI), and analysed by EPICS Elite flow cytometer (Coulter Electronics, Fla, USA). 2.8. Invasion assay The ability of cells to invade through a Matrigel-coated filter was measured in transwell chambers (Corning, NY, USA). Polyvinylpyrrolidone-free polycarbonate filters (pore size 8 μm) were coated with basement membrane Matrigel (50 μL/filter) (BD, Bedford, Ohio, USA). The membrane was washed in PBS to remove excess ligand, and the lower chamber was filled with 0.6 mL of RPMI-1640 medium containing 10% fetal bovine serum (FBS). Cells were serum-starved overnight (0.5% FBS), harvested with trypsin/EDTA, and washed twice with serum-free RPMI-1640 medium. Cells were resuspended in migration medium (RPMI-1640 medium with 0.5% FBS), and 0.1 mL migration medium containing 1 × 105 cells was added to the upper chamber. After incubation with RGC (1–20 μM) with or without GW9662 (2.5 μM, pretreated 1 hour) at 37°C for 24 hours, the cells on the upper surface of the membrane were removed using a cotton swab. The migrant cells attached to the lower surface were fixed in 10% formalin at room temperature for 30 minutes and stained with hematoxylin. The numbers of migrated cells were counted under a microscope. 2.9. Scratch wound-healing motility assays Gastric cancer cells were seeded on 60 mm plates and allowed to grow to confluence. Confluent monolayers were scratched with a pipette tip and maintained under RGZ (1–20 μM) with or without GW9662 (2.5 μM, pretreated 1 hour) for 24 hours. Plates were washed once with fresh medium to remove nonadherent cells and then photographed. The cell migration was evaluated by counting cells that migrated from the wound edge. 2.10. In vitro Angiogenesis assay The angiogenesis assays were performed as per the manufacturer's instructions, that is, transfer 50 μL of ECMatrixTM solution to each well of a precooled 96-well tissue culture plate on ice. Incubate at 37°C for 1 hour to allow the matrix solution to solidify. Harvest human umbilical vein endothelial cells (HUVECs) resuspend and Seed 5 × 103 cells per well onto the surface of the polymerized ECMatrixTM. Incubate with RGC (1–20 μM) with or without GW9662 (2.5 μM, pretreated 1 hour) at 37°C for 12 hours. Inspect tube formation under an inverted light microscope at 100 X magnification. 2.11. Zymography Cells were cultured for 24 hours in serum-free medium, washed twice, and finally treated with RGZ (1–20 μM) with or without GW9662 (2.5 μM, pretreated 1 hour) for a further 48 hours. The supernatants were collected and concentrated, using centrifugal filter devices (Millipore Corp., Bedford, Mass, USA) and the protein content was determined using BCA Protein Assay Reagent. Equal amounts of protein (20 μg) were mixed with SDS sample buffer without reducing agents and incubated for 40 minutes at 37°C. For gelatinolytic activity, the assay samples were separated on polyacrylamide gels containing 1mg/mL gelatin. After electrophoresis, the gels were stained for 1 hour in a 45% methanol/10% acetic acid mixture containing coomassie brilliant blue G250 and destained. Zymograms were photographed after 10 hours of incubation at 37°C. 2.12. Statistical analysis Data are expressed as mean ± standard deviation (SD) of three independent experiments, each done in triplicate. Differences between control and experiment groups were analyzed using the t-test. P < .05 was considered statistically significant. 3. RESULTS 3.1. RGZ inhibited proliferation and induced apoptosis in SGC-7901 cells through PPARγ-dependent mechanism In SGC-7901 cells, the expression of PPARγ was observed by RT-PCR and western blot (not shown). RGZ (0.1–100 μM) treatment for 24, 48, and 72 hours inhibited cells growth in a dose- and time-dependent manners in SGC-7901 gastric cancer cell line as determined by MTT assay. Pretreatment with the highly selective PPARγ antagonist GW9662 (2.5 μM) reversed the effect of RGZ on cell viability (see Figure 1(a)). To explore whether the growth inhibition of RGZ in SGC-7901 cells was caused by apoptosis, we analyzed the sub-G1 population of the cells after treatment with RGZ (1–20 μM) for 48 hours. RGZ induced apoptosis in a dose-dependent manner, which was also reversed completely by 2.5 μM GW9662 treatment (see Figure 1(b)). Furthermore, to determine whether the inhibitory effect of RGZ on cell viability is associated with the arrest of the cell cycle, we analyzed the cell cycle progression after treatment with RGZ (1–20 μM) for 48 hours. RGZ treatment increased the number of cells in the G1-G0 and decreased the number of cells in the S phases in dose-dependent manner. The effects of RGZ on cell cycle of SGC-7901 cells were also reversed by 2.5 μM GW9662 (see Figure 1(c)). 3.2. RGZ inhibited SGC-7901 cells migration and invasiveness through PPARγ-independent mechanism After treatment with RGZ (1–20 μM) for 48 hours, the number of cells migrated to the scratched area was 60 ± 3.1 cells/mm2, 58 ± 2.7 cells/mm2, 49 ± 2.8 cells/mm2, 27 ± 2.9 cells/mm2, and 20 ± 1.9 cells/mm2, respectively, which were significantly lower than those in control group (84 ± 3.4 cells/mm2). GW9662 treatment had no effects on the cells migration with inhibition induced by RGZ. The number of the cells migrated to the scratched area treated with GW9662 and RGZ (1–20 μM) for 48 hours was 61 ± 1.8 cells/mm2, 53 ± 3 cells/mm2, 47 ± 2.5 cells/mm2, 29 ± 2.8 cells/mm2, 18 ± 3.2 cells/mm2, respectively, which were not different from those in the groups treated with RGZ alone (see Figure 2(a)). The effect of RGZ on the cells invasion through reconstituted basement membranes was analyzed using Matrigel-coated invasion chambers. After treatment with RGZ (1–20 μM) for 48 hours, the cells attached to the lower surface of the filters were 256 ± 9 cells/mm2, 248 ± 7 cells/mm2, 219 ± 12 cells/mm2, 174 ± 11 cells/mm2, and 154 ± 10 cells/mm2, respectively, which were significantly lower than those in control group (279 ± 9 cells/mm2). After cotreatment of the cells with GW9662 and RGZ, the cells attached to the lower surface were 251 ± 29 cells/mm2, 238 ± 12 cells/mm2, 220 ± 7 cells/mm2, 166 ± 16 cells/mm2, and 148 ± 12 cells/mm2, respectively, which were not different from those in the groups treated with RGZ alone (see Figure 2(b)). Metalloproteases (MMPs) have been demonstrated to play a significant role in tumor cell invasion [13]. In this study, our results showed that RGZ inhibited the mRNA and protein expression levels of MMP-2 in a dose-dependent manner (see Figures 3(a), 3(c), and Tables 1, 2). Moreover, the gel zymography results demonstrated that the activity of MMP-2 decreased after RGZ (1–20 μM) treatment for 48 hours in dose-dependent manner (see Figure 4(a)). The inhibitory effects of RGZ on MMP-2 were not affected by GW9662 treatment (see Figures 3(b), 3(c), and 4(b)). 3.3. Effects of RGZ on angiogenesis in vitro Matrigel-plated HUVECs elongated and migrated in the presence of VEGF and formed tubular networks. RGZ markedly suppressed the formation of the tube-like structures of HUVEC cells in a dose-dependent manner (see Figure 5(a)), which was completely antagonized by GW9662 (see Figure 5(b)). These results suggested that rosiglitazone exhibits antiangiogenic activity via PPARγ-dependent mechanism. To further determine whether the effect of RGZ on angiogenesis is due to the down regulation of the tumor-secreted growth factors, we measured the expression levels of VEGF in SGC-7901 cell cultured medium, after treatment with various concentrations of RGZ. Our results demonstrated that RGZ (1–20 μM) did not change the expression of mRNA and protein of VEGF in SGC-7901 cells (see Figures 3(a), 3(c), and Table 1), but also the results were confirmed by ELISA (see Figure 6). 4. DISCUSSION As a potential molecular target for anticancer drug development, PPARγ and its ligands have been extensively studied in the past several years. Previous studies have shown that PPARγ is expressed in several human gastric-cancer cell lines, including MKN-7, MKN-28, MKN-45, and AGS. TZDs could inhibit these cancer cell lines growths in vitro and in vivo [9, 12]. Also, the growth inhibitory effects of TZDs on MKN45 cells depend on the PPARγ expression levels. The growth inhibition of TDZs was more significant in the higher PPARγ expressing cells. Moreover, Lu et al. [10] found that PPARγ (+/ − ) mice were more susceptible to MNU-induced gastric cancer than wild-type (+/+) mice, and troglitazone significantly reduced the incidence of gastric cancer in PPARγ (+/+) mice but not in PPARγ (+/ − ) mice. All these results indicated that TZDs inhibit the cancer cells growth via PPARγ-dependent mechanism. Our results demonstrated that RGZ, the most potent and selective synthetic ligand of PPARγ, inhibited SGC-7901 gastric cancer cells growth, caused G1 cell cycle arrest, and induced apoptosis in a dose-dependent manner. The effects of RGZ on SGC-7901 cancer cells were completely reversed by treatment with PPARγ antagonist GW9662. These results indicated that RGZ suppressed the SGC-7901 cancer cells growth in a PPARγ-dependent mechanism. In this study, we found that the RGZ inhibited invasion, migration, and the secretion of MMP-2 of SGC-7901 cells. The inhibitory effects of RGZ on metastases and MMP-2 activity were not directly mediated by PPARγ activation, since these effects were not reversed by GW9662 treatment. Our results were consistent with the previous works on human adrenocortical cancer cell line H295R [14], pancreatic cancer cells [15], and human myeloid leukemia cells [16], which showed that PPARγ ligands act independently of PPARγ activation in the invasion suppression and down-regulation of MMP-2 activity. Recent papers showed that PPARγ regulated E-cadherin expression and inhibited growth and invasion of prostate cancer [17], and PPARγ ligand troglitazone inhibited transforming growth factor-beta-mediated glioma cell migration and brain invasion [18]. But some studies have contrasting results that the PPARγ, ciglitazone, induced cell invasion, through activation of Pro-MMP-2, activation via the generation of ROS, and the activation of ERK [19], and that PPARγ antagonists induced vimentin cleavage and inhibited invasion in high-grade hepatocellular carcinoma [20]. Further studies are needed on the mechanism of PPARγ in cancer and invasion. Recent investigations suggested that PPARγ ligands had inhibitory effects on tumor cell lines, but the effects appear not to be entirely elicited by the direct action on tumor cells. Inhibition of the neovascularization may be another target of TZDs to suppress the growth of cancers. PPARγ is expressed in endothelial cells, and the PPARγ ligands can inhibit the proliferation of these cells induced by growth factors, or cause their apoptosis in vitro [21–23]. It has been reported that PPARγ ligands could inhibit choroidal, retinal, and corneal neovascularization when administered intraocularly [24–26]. In addition, systemic administration of rosiglitazone and troglitazone inhibits FGF2-induced angiogenesis; thereby inhibiting primary tumor growth and metastasis [27]. We observed that RGZ inhibited the angiogenesis of HUVECs in dose-dependent manner via PPARγ pathway. The effects RGZ on the endothelium suggest that RGZ may regulate tumor growth by targeting non-cell-autonomous mechanisms. Previous studies [5] showed that suppression of angiogenesis could result from a decrease in the local levels of stimulators (e.g., VEGF and FGF2) and/or an increase of endogenous inhibitors of angiogenesis (e.g., thrombospondin) produced by tumor cells. PPARγ ligands suppressed VEGF production in colon carcinoma [28], human breast cancer [29], and human renal cell carcinoma cells [30]. However, contradictory results have also been reported in bladder and prostate cancer cells in which PPARγ ligands increased VEGF production [31, 32]. Inconsistent with the above documents, our results showed that RGZ did not change the secretion of VEGF from SGC-7901. Taken together, our results demonstrated that RGZ inhibited growth and invasiveness of SGC-7901 gastric cancer cells and angiogenesis in vitro via PPARγ-dependent or -independent pathway. Further study is needed to elucidate the mechanisms by which RGZ exhibits different manner. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant no. 30671904, 30670949), China Postdoctoral Science Foundation (no. 2004035181), and The Doctor Station of Ministry of Education of China (no. 20060558010). Figure 1 (a) RGZ (0.1–100 μM) treatment for 24, 48, and 72 hours inhibited cell growth in a dose- and time-dependent manners in SGC-7901 gastric cancer cell line, as determined by MTT assay, which was reversed completely by 2.5 μM GW9662 pretreatment for 1 hour. Cell viability was expressed as the percentage of cells under control conditions (0 μM of RGZ or GW9662). (b) RGZ induced apoptosis in a dose-dependent manner, which was also reversed completely by 2.5 μM GW9662 pretreatment for 1 hour. (c) RGZ treatment increased the number of cells in the G1-G0 and decreased the number of cells in the S phases in dose-dependent manner, which was reversed completely by 2.5 μM GW9662 pretreatment for 1 hour. Values are the means ± SD of three representative experiments.Statistical significance (P < .05 or higher degree of significance) versus vehicle-treated controls. Figure 2 (a) Effect of RGZ on the migration and (b) invasion of SGC-7901 gastric cancer cells, which was reversed completely by 2.5 μM GW9662 pretreatment for 1 hour. Values are the means ± SD of three representative experiments.Statistical significance (P < .05 or higher degree of significance) versus vehicle-treated controls. Figure 3 (a) RGZ (1–20 μM) inhibited the mRNA and (c) protein expression levels of MMP-2 in a dose-dependent manner, which were not affected by 2.5 μM GW9662 pretreatment for 1 hour (b), (c). RGZ (1–20 μM) did not change the expression of VEGF in SGC-7901 cells (a), (c). Figure 4 (a)The activity of MMP-2 was decreased after RGZ (1–20 μM) treatment for 48 hours in dose-dependent manner. (b) The inhibitory effects of RGZ on MMP-2 were not affected by 2.5 μM GW9662 pretreatment for 1 hour. Figure 5 (a) RGZ markedly suppressed the formation of the tube-like structures of HUVEC cells in a dose-dependent manner, (b) which was completely antagonized by 2.5 μM GW9662 pretreatment for 1 hour. Figure 6 RGZ had no effect on the secretion of VEGF of SGC-7901 cell. Table 1 Expression of MMP-2 and VEGF after RZD treatment in SGC-7901 gastric cancers by real-time PCR. Rosiglitazone (μmol/L) PPARγ MMP-2 VEGF 0 0.132127 ± 0.045513 0.008912 ± 0.000133 0.61132 ± 0.078921 1 0.121878 ± 0.034219 0.006003 ± 0.000331* 0.620255 ± 0.054671 3 0.130134 ± 0.0521137 0.005486 ± 0.000541* 0.60728 ± 0.036799 5 0.137778 ± 0.046222 0.005048 ± 0.000346* 0.599438 ± 0.076541 10 0.141171 ± 0.038741 0.001924 ± 0.000189* 0.624165 ± 0.038966 20 0.143889 ± 0.061237 0.001298 ± 0.000267* 0.604246 ± 0.065679 Statistical significance (P < .05 or higher degree of significance) versus vehicle-treated controls. Table 2 Expression of MMP-2 and VEGF after RZD and GW9662 cotreatment in SGC-7901 by real-time PCR. Rosiglitazone (μmol/L) PPARγ MMP-2 0 0.14161 ± 0.055389 0.00975 ± 0.000533 1 0.137738 ± 0.030102 0.008974 ± 0.000113 3 0.134614 ± 0.029881 0.006003 ± 0.000401* 5 0.141156 ± 0.564569 0.00564 ± 0.000246* 10 0.135666 ± 0.034887 0.002182 ± 0.000364* 20 0.129278 ± 0.019262 0.001712 ± 0.000178* *Statistical significance (P < .05 or higher degree of significance) versus vehicle-treated controls. ==== Refs 1 Rosen ED Sarraf P Troy AE PPARγ is required for the differentiation of adipose tissue in vivo and in vitro Molecular Cell 1999 4 4 611 617 10549292 2 Saltiel AR Olefsky JM Thiazolidinediones in the treatment of insulin resistance and type II diabetes Diabetes 1996 45 12 1661 1669 8922349 3 Grommes C Landreth GE Heneka MT Antineoplastic effects of peroxisome proliferator-activated receptor γ agonists The Lancet Oncology 2004 5 7 419 429 15231248 4 Chaffer CL Thomas DM Thompson EW Williams ED PPARγ -independent induction of growth arrest and apoptosis in prostate and bladder carcinoma BMC 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induces vascular endothelial growth factor in the hormone-independent prostate cancer cell line PC 3 and the urinary bladder carcinoma cell line 5637 International Journal of Oncology 2002 21 4 915 920 12239635	18810275	PMC2542845	CC BY	2021-01-05 11:46:11	yes	PPAR Res. 2008 Sep 15; 2008:649808
==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central 1471-2202-9-921880865910.1186/1471-2202-9-92Research ArticleDifferential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors Wislet-Gendebien Sabine [email protected] Naomi P [email protected] Shawn N [email protected] Diana [email protected] Weimin [email protected] Daniel [email protected] Paul E [email protected] Steffany AL [email protected] Anurag [email protected] Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, M5S 3H2 Canada2 Centre for Cellular and Molecular Neurobiology, University of Liege, 4000 Liege, Belgium3 Neural Regeneration Laboratory, Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, K1H 8M5, Canada4 Institute for Biological Sciences, National Research Council of Canada, Ottawa, K1A 0R6, Canada2008 22 9 2008 9 92 92 6 5 2008 22 9 2008 Copyright © 2008 Wislet-Gendebien et al; licensee BioMed Central Ltd.2008Wislet-Gendebien et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Alpha-Synuclein (α-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the α-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of α-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood. Results In the present study, we analysed the ability of cytosolic factors to regulate α-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant α-syn. To characterize cytosolic factor(s) that modulate α-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate α-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T α-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance α-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P α-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol. Conclusion These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant α-syn membrane binding, and could represent potential targets to influence α-syn solubility in brain. ==== Body Background The synuclein family of intrinsically unfolded proteins is composed of three homologous and evolutionarily-conserved members with poorly defined physiological roles [1]. Of these, α-synuclein (α-syn) has gained particular prominence due to its abundance in nerve terminals and its association with multiple neurodegenerative disorders including Parkinson disease (PD) [2]. α-Syn behaves as a peripherally associated membrane protein and can stably interact with synthetic phospholipid vesicles containing negatively charged head groups [3] via its amino-terminal domain, an amphipathic region comprising almost two-thirds of the protein and containing seven copies of an 11-residue repeat sequence [4]. Whereas the freely diffusible form of α-syn is natively unfolded, the N-terminal repeat region adopts an α-helical conformation upon binding to artificial vesicles and detergent micelles [3]. Numerous studies have revealed that the interaction of α-syn with phospholipid membranes, fatty acids, or detergent micelles alters the kinetics of its aggregation [4-9]. We and others have previously reported that synaptic α-syn in vivo is partitioned between both cytosolic and membrane-bound fraction [10-14]. However, despite the understanding of the conformational properties of membrane-bound α-syn, the biochemical mechanisms that mediate α-syn interaction with biological membranes are poorly understood, thereby limiting our understanding of α-syn's physiological role, as well as potential therapeutic approaches to moderate its misfolding and aggregation in disease. In this study, we developed an in vitro assay to characterise the factor(s) involved in α-syn's binding to synaptic membranes (Figure 1A). Using this assay, we analysed the effects of cytosolic proteins, lipids, ATP and calcium on the modulation of α-syn membrane association. Our results revealed that ATP and lipids are two of the principal cytosolic components that modulate the α-syn binding to synaptic membranes. In addition, we report here that the binding of A30P α-syn to synaptic membranes improves significantly in the presence of endogenous cytosolic protein(s) and that the lower recovery of membrane bound A30P is likely due to a more transient interaction which can be stabilised by artificial cross-linking. Figure 1 (A) α-syn binding assay. Step 1. Synaptosomes are prepared from α-syn-/- mice and α-syn (human Wt and PD-linked A30P and A53T forms) is expressed and purified from E. coli. Step 2. Synaptic membranes (α-syn acceptor fraction) are prepared from intact synaptosomes using hypotonic buffer and incubated with purified α-syn (donor fraction) in presence or absence of α-syn-/- (KO) cytosol. Step 3. Membrane and cytosol fractions are separated by centrifugation and the membrane proteins are analysed by western blotting. (B) Using the binding assay, KO synaptic membranes were incubated, for 10 min at 37°C, with 3 μg of Wt, A30P or A53T purified α-syn in absence or presence of 1.5 mg/ml of KO cytosol. As shown on this graph, A30P purified α-syn has a lower binding compared to Wt and A53T α-syn in absence (One-Way ANOVA, p < 0.0001, n = 4; Bonferroni's multiple comparison test) or presence (One-Way ANOVA, p < 0.0001, n = 4; Bonferroni's multiple comparison test) of KO cytosol. (C) KO synaptic membranes were incubated, for 10 min at 37°C, with 0.1, 0.6 and 3 μg of Wt, A30P or A53T purified α-syn in absence or presence of 1.5 mg/ml of KO cytosol. Results are normalized to the maximal binding observed for each respective α-syn. These data show that the cytosol has a significant effect by increasing the binding of all types of α-syn (One-Way ANOVA: Wt: p < 0.0001, n = 4; A30P: p < 0.0001, n = 4; A53T p < 0.001, n = 4). Methods Synaptosome preparation Synaptosomes were prepared as described (Fischer von Mollard et al. 1991;Tandon et al. 1998a). Briefly, the cerebral cortices from mice α-syn KO mice were dissected and homogenized with 10 strokes at 500 rpm, in ice-cold buffer A (320 mM sucrose, 1 mM EGTA, and 5 mM HEPES [pH 7.4]). The homogenate was centrifuged at 1000 × g for 10 min. Next, the supernatant was spun for 10 min at 24000 × g and the resulting pellet (P2) resuspended in buffer A. The P2 fraction was loaded onto a discontinuous FICOLL gradient (13%, 9%, 5% in buffer A) and centrifuged for 35 min at 35,000 × g. The 13%-9% interface, containing intact synaptosomes, was resuspended in buffer B (140 mM NaCl, 5 mM KCl, 20 mM HEPES, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1 mM MgCl2, 1 mM EGTA, and 10 mM glucose). The sample was spun at 24000 × g for 10 min and the pellet was washed two times in buffer C (10 mM HEPES, 18 mM KOAc, [pH 7.2]), then spun at 24000 × g for 10 min and resuspended in buffer D (25 mM HEPES, 125 mM KoAc and 2.5 mM MgCl2). After centrifugation (24000 × g for 10 min), synaptosomes were resuspended in buffer D and were incubated with or without brain α-syn KO cytosol. Samples were incubated for 10 min at 37°C before separating membrane and supernatant by centrifugation at 24000 × g for 10 min. α-syn binding was quantified by western blotting. Cytosol preparation Mouse brains were thoroughly homogenized in 85 mM sucrose, 100 mM KOAc, 1 mM MgOAc, and 20 mM HEPES (pH 7.4). The homogenate was centrifuged for 10 min at 15,000 × g and the supernatant spun for 1 hr at 100,000 × g. The supernatant was subsequently dialyzed for 4 hr in 145 mM KOAc and 25 mM HEPES (pH 7.2) and frozen at -80°C. Protein concentration was determined by BCA protein assay (Pierce, Biolynx Inc., Canada). Lipid-free cytosol preparation Chloroform was added to the cytosol (v/v), vigorously vortexed and incubated for 30 min at room temperature. After centrifugation for 10 min at 14000 × g, two phases were obtained: upper phase (TOP) containing the gangliosides or small organic molecules, the interphase containing the proteins and the lower phase containing the lipids. In some experiments, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF, Biomol) was added alone or directly to delipidated cytosol to test effect on α-syn membrane binding. Cytosol digestion Cytosol digestion was done with trypsin or Proteinase K and proteolytic activity was terminated with trypsin inhibitor or PMSF, respectively prior to the incubation with membranes. The enzyme inhibition was controlled by a partial rescue of the digested cytosol after half-dilution with untreated cytosol. Expression and Purification of Recombinant α-synuclein Human Wt α-syn cDNAs were subcloned into the plasmid pET-28a (Novagen), using Nco I and Hind III restriction sites. α-Syn was overexpressed in Escherichia coli BL21 (DE3) via an isopropyl-1-thio-3/4-D-galactopyranoside-inducible T7 promoter. The bacterial pellet was resuspended in phosphate buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The bacterial suspension was then sonicated for 30 sec several times, boiled for 15 min, and ultracentrifuged at 150,000 × g for 30 min. The supernatant containing the heat-stable α-syn was dialyzed against 50 mM Tris, pH 8.3, loaded onto a Q-Sepharose column (Pharmacia Biotech), and eluted with a 0–500 mM NaCl step-gradient. The eluents were desalted and concentrated on a Centricon-10 (Millipore) in 5 mM phosphate buffer, pH 7.3. Aliquots of each purification step were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) to confirm purity. Protein concentration was determined by Lowry assay. Western blotting Proteins were boiled briefly in loading buffer (glycerol 10% v/v; Tris 0.05 M pH 6.8; SDS 2%, bromophenol blue and 2.5% v/v β-mercaptoethanol) and separated by electrophoresis using 12% Tris-glycine polyacrylamide gels. Proteins were transferred to nitrocellulose (Life Sciences) and probed by western blotting using: antibodies against α-syn (monoclonals 211 and Syn-1 at 1:1000, Neomarkers), our own rabbit polyclonal (LWS1, 1:1000) raised to a 24-mer α-syn-specific peptide, or synaptophysin (Mouse monoclonal antibody, dilution 1:10000, Biodesign International). Bound HRP-conjugated anti-mouse or anti-rabbit IgG (Sigma) were revealed by chemiluminescence using ECL Plus (GE Healthcare) and quantified with a Storm 860 fluorescent imager and ImageQuant software (Molecular Dynamics). Statistical comparisons were calculated with GraphPad InStat software using Student's T-test for comparisons between two groups or ANOVA (Bonferroni test) for multiple comparisons. Synaptic lipid raft preparation Lipid rafts were prepared from the synaptosomes or synaptic membrane isolated from cortices as described above. Synaptosomes or synaptic membrane were resuspended in 25 mM MES, pH 6.5, 50 mM NaCl, 1 mM NaF, 1 mM Na3VO4, and 1% TX-100 (lysis buffer) supplemented with phosphatase inhibitor cocktails (Sigma) and incubated on ice for 30 min with Dounce homogenization every 10 min. The cell lysate was then adjusted to 42.5% sucrose, overlayed with 35 and 5% sucrose in lysis buffer without TX-100 and sedimented at 275,000 × g for 18 hr at 4°C. Fractions were collected from the top of the gradient and stored at -80°C. Equal volumes of each fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with the different antibodies as described above. Lipid raft-containing fractions were identified by the presence of flotillin-1 (BD Biosciences, Canada). Glycerophospholipid extraction C13:0 lysophosphatidylcholine (C13:0 LPC) and 1-O-hexadecyl-2-[2H4]acetyl-glycerophosphocholine (d4-16:0 PAF) were purchased from Avanti Polar Lipids (Alabaster, AL). Stock chemicals were purchased from J.T. Baker (Phillipsburg, NJ) with the exception of bovine serum albumin (BSA) from Sigma (St. Louis, MO). Glycerophospholipids were extracted according to a modified Bligh/Dyer procedure [15] as we have previously published[16]. Briefly, lipids were extracted using a volumetric ratio of 0.95 of chloroform and 0.8 of 0.1 M Na acetate (aq) per volume of MeOH in acid-washed borosilicate glass tubes (Fisher, Ottawa, ON). Phospholipids were collected from the organic phase after layer separation by centrifugation. The aqueous phase was back-extracted three times in the organic phase of a wash solution prepared by combining RPMI+ 0.025% BSA, methanol, chloroform, and sodium acetate in the volumetric ratio of 1:2.5:3.75:1. The organic fractions were combined, evaporated under a stream of nitrogen gas, and dissolved in 300 μl EtOH. C13:0 lysophosphatidylcholine (C13:0 LPC), a lipid not naturally occurring in mammalian cells [17], was spiked into cytosol preparations at a concentration of 189 ng prior to extraction to control for variation in extraction efficiency. LC-ESI-MS Glycerophospholipids were analyzed as we have described previously [16]. Briefly, extracts were diluted 1:4 in EtOH with 13 μL of diluent brought to 40 μl with 0.1% formic acid in H2O. To validate the identity of target species, analytes were spiked with 1-O-hexadecyl-2-[2H4]acetyl-glycerophosphocholine (d4-16:0 PAF, 2.5 ng) in replicate LC-ESI-MS/MS analyses. Under these circumstances, 10 μl of diluted analyte was added to 5 μl of standard (2.5 ng) and brought to 40 μl with 0.1% formic acid in H2O. Samples were loaded onto a 96-well sampling plate, covered with a pre-slit well cap, and thermostated at 4°C. A micro flow 1100 HPLC system (Agilent, Palo Alto, CA) introduced the analytes onto a 200 um × 50 mm pre-column packed with 5 μm YMC ODS-A C18 beads (Waters, Milford, MA) at a flow rate of 10 μl/min in a 2000 Q TRAP mass spectrometer. The solvents used were water and acetonitrile each with 0.1% formic acid (J.T. Baker, Phillipsburg, NJ). The HPLC flow was split and the analyte was eluted through a 75 um × 50 mm picotip emitter (New Objective, Woburn, MA), interfaced with the mass spectrometer via electrospray ionization, at ~200 nL/min. The emitter was packed with the same beads as those of the pre-column. A linear gradient was used to separate glycerophospholipid species. The gradient of the HPLC increased from 5% to 30% acetonitrile in 2 minute, from 30 to 60% acetonitrile in 7 minute, from 60% to 80% acetonitrile over the next 33 minutes, and from 80% to 95% acetonitrile over the next 4 minutes. Data were collected on a 2000 Q-TRAP mass spectrometer operated with Analyst 1.4.1 (Applied Biosystems/MDS Sciex, Concord, ON). Total glycerophospholipids between m/z range of 450 to 600 were analyzed by enhanced MS scan. Specific glycerophosphocholine species were further analysed in positive ion mode using precursor ion scan for an MS/MS fragment with a mass to charge ratio (m/z) of 184.0, a diagnostic fragment of phosphocholine [18]. Extracted ion chromatogram (XIC) generated peak areas of LC-MS/MS data measured using Analyst 1.4.1 (Applied Biosystems/MDS Sciex). Peak areas were normalized to the spiked internal standard to standardize between MS runs and to control for variation in extraction efficiency. Individual species were identified based on theoretical mass validated by closer examination of retention time and following spiking with deuterated standards. Results Cytosol modulates α-syn membrane binding To identify novel co-factors of α-syn binding to presynaptic membranes, we assessed whether co-incubation with brain cytosol modifies α-syn's interaction with membranes. Our assay measured the binding of recombinant human α-syn purified from E. coli to synaptic membranes prepared from brains of α-syn-deficient (KO) mice, in the presence or absence of brain cytosol derived from α-syn-deficient mice (Figure 1A). We first analysed α-syn binding to synaptic membranes in the presence or absence of cytosol. As shown in Figure 1B, the binding of α-syn, with or without familial PD-linked mutations, was significantly improved by co-incubation with cytosol. Despite the deficient membrane binding of A30P as compared to that of Wt and A53T, all three forms of α-syn showed increased binding over a 30-fold range in concentration, with a pronounced augmentation of binding in the presence of cytosol (Figure 1C). The ratio of bound/unbound α-syn was higher at lower α-syn concentrations. These results suggest that endogenous cytosolic factors becoming limiting with increasing α-syn and can partially counterbalance the otherwise impaired binding induced by the A30P mutation. Characterization of cytosol action on α-syn binding We recently reported that the dissociation of the α-syn from synaptic membranes requires cytosolic proteins as defined by sensitivity to proteases. To further characterize cytosol action on α-syn binding, and because the data in Fig 1C suggests that cytosol activity becomes saturated at high α-syn concentration, we analysed α-syn binding with varying cytosol concentrations over a 6-fold range that we have previously shown to be effective in mobilizing reserve neurotransmitter from permeabilized synaptosomes [19] (Figure 2A). In accord with the data in figure 1B, both Wt and A30P α-syn binding was strongly up-regulated by increasing cytosol concentration, whereas only high cytosol concentration resulted in increased A53T α-syn binding. To determine whether the cytosolic factors act on α-syn or on the acceptor synaptic membranes, we first pre-incubated α-syn or synaptic membranes separately with KO cytosol. The membranes were subsequently washed briefly to remove unbound cytosolic factors. As shown on Figure 2B, exposure of the membranes alone to cytosol was sufficient to potentiate α-syn binding, which was equivalent to α-syn binding to membranes in the presence of cytosol. These results suggest that cytosolic activity can be mediated by affecting the acceptor membrane rather than soluble α-syn. Figure 2 Effect of cytosol on binding α-syn. (A) Recombinant α-syn (Wt, A30P and A53T) were incubated in presence of different concentrations of KO cytosol (0.5, 1.5, and 3 mg/ml), for 10 min at 37°C. Compared to the control condition (without cytosol), all cytosol concentrations had a significant effect on Wt and A30P α-syn binding, but only the highest concentration of cytosol had a significant effect on A53T α-syn binding (One way ANOVA test, p < 0.0001, Bonferroni's multiple comparison post-test). (B) KO synaptic membranes and α-syn were pre-incubated for 15 minutes at room temperature with KO cytosol. Membranes were then centrifuged at 14000 × g and washed with KOAc buffer to remove unbound factors. Binding of purified α-syn to KO membranes in the absence of cytosol (ctrl) was compared to its binding to cytosol-treated membranes without added cytosol (memb), and to cytosol-treated α-syn incubated with KO membranes (α-syn). No significant difference was observed between the two pre-incubated condition (Student T-test, p > 0.05). (C) KO cytosol was pre-incubated with trypsin or proteinase K for 15 min at 37°C. Enzymes were then respectively inactivated with trypsin inhibitor and PMSF for 10 min at room temperature. Compared to the cytosol condition (cyt) which, as a control, was incubated with the enzyme pre-inactivated by the inhibitor, only A30P α-syn binding was significantly affected by the cytosolic protein digestion (Student T-test, p < 0.0001), whereas no significant differences were observed for Wt and A53T proteins (Student T-test, p > 0.05). To determine the nature of the cytosolic factor(s), we assessed whether activity was affected by pre-digestion of cytosolic proteins by trypsin- or proteinase K-mediated proteolysis (Figure 2C). Digestion of cytosol was terminated by trypsin inhibitor and PMSF prior to incubation with α-syn and synaptic membranes, and the extent of proteolysis was verified by Coomassie blue stain (not shown). Although, no significant differences between undigested and digested cytosol were observed for either Wt or A53T α-syn binding, the A30P mutant showed significantly reduced binding in the presence of protease-treated cytosol, reaching a basal level similar to the control condition in absence of cytosol. This suggests that the A30P mutation confers a unique dependence on cytosolic protein(s) required to mediate α-syn interactions with synaptic membranes. Moreover, comparable levels of a non-specific protein, bovine serum albumin (BSA), did not affect A30P α-syn binding to synaptic membranes (data not shown), suggesting that A30P α-syn binding depends on specific cytosolic proteins. Involvement of cytosolic lipids in α-syn membrane binding Because Wt and A53T α-syn appear to require protease-insensitive cofactors for membrane binding, and α-syn conformation is known to be affected by lipids (Jo et al. 2002), we examined whether removal of cytosolic lipids by chloroform extraction can alter the proportion of α-syn able to bind synaptic membranes (Figure 3A). We observed that the binding of Wt α-syn and PD-linked mutants were decreased in the presence lipid-deficient cytosol, suggesting a role for cytosolic lipids in the binding of α-syn to synaptic membranes. These results are also consistent with our observation that heat-denatured cytosol retains its activity to potentiate Wt and A53T α-syn binding (data not shown). Moreover, consistent with the results in Fig 1B showing that A53T α-syn membrane binding is less dependent on cytosol, it was also the least affected by lipid extraction. It is also important to note that the chloroform extraction did not non-specifically denature cytosolic proteins because the protein-containing fraction partially rescued A30P α-syn binding, in accord with its dependence on a protease-sensitive cytosolic component. Figure 3 Effects of cytosolic lipid depletion on α-syn binding. (A) Using chloroform extraction to fractionate cytosol into three fractions: the top fraction contains the gangliosides or small organic polar molecules, the interface layer contains the proteins and the bottom phase contains lipids solubilised in chloroform. We incubated the synaptic membrane with the two lipid free-fractions, top and interphase (protein) layers, in presence of recombinant α-syn. The lipid-free fractions did not show any significant effects on the Wt and A53T α-syn binding compared to the control condition (α-syn incubated with synaptic membranes in absence of cytosol; Student T-test, p > 0.05) while the A30P α-syn binding was increased (compared to control condition, Student T-test, p < 0.01). (B) Recombinant α-syn (Wt, A30P and A53T) were incubated with synaptosomal membranes in the presence of 1.5 mg/ml cytosol from either KO mice (KO) or from non-transgenic mice (nonTg) for 10 min at 37°C. Binding of normal and mutant human α-syn, measured by the human α-syn specific monoclonal antibody 211, is normalized to that of Wt α-syn in the presence of KO cytosol. (C) Recombinant Wt α-syn was incubated with synaptosomal membranes and C16:0 PAF (0, 10, 100 nM) in the absence (open bars) or presence of delipidated cytosol (closed bars). Inclusion of 100 nM C16:0 PAF significantly increased α-syn binding only in the presence of the delipidated cytosol (compared to corresponding condition without C16:0 PAF, Two-Way ANOVA, p < 0.01, Bonferroni's multiple comparison test p < 0.01, n = 3). Several studies have noted significant changes in brain lipids, notably in the metabolism of neutral brain lipids, in α-syn-deficient animals [20-22]. Therefore, to test whether our results are specific to KO cytosol we compared human α-syn binding in the presence of KO cytosol or cytosol derived from nontransgenic animals with normal α-syn expression. In order to detect only the exogenously added human α-syn, and not endogenous murine α-syn present in normal cytosol, we used the human α-syn specific monoclonal antibody 211. We observed no significant differences in cytosol-dependent α-syn binding when KO versus normal cytosol was used (Figure 3B). We used LC-ESI-MS to identify lipid cofactors present in α-syn KO cytosol. Because previous studies have indicated that the strongest lipid interactions with α-syn are with either neutral or anionic phospholipids [3,8,23,24], we focused our initial analysis on the glycerophosphocholine species present in KO cytosolic extracts [18] as the detection methodologies are well-established in our laboratory [16]. As our extracts are dialyzed prior to testing, these lipid species are predicted to be in complexes with proteins greater than 12 kDa. Choline-containing lipids extracted from these complexes were separated and species with a mass to charge ratio (m/z) between 450 and 600 identified by MS scan for a protonated molecule at expected m/z followed by positive ion mode precursor ion scan for a phosphocholine product ion at m/z 184 [16]. Twenty-four candidate species were identified in the extracted ion chromatographs (Table 1). Because α-syn is known to play a role in regulating lipid metabolism in brain, notably in the regulation of the glycerophosphocholine fatty acid turn over, [21,22,25,26], we compared this profile to the lipids detected in Wt cytosol. We found that Wt cytosol contained the same glycerophosphocholines as KO preparations with the exception of two species detected de novo (Table 1). The overall abundance of the majority of small second messenger species was elevated in KO cytosol relative to Wt. Table 1 Elution time and parent ion masses of candidate glycerophosphocholine species bound to proteins in dialyzed α-synuclein KO cytosolic extracts identified by LC-ESI-MS Parent ion mass (± 0.15 m/z)a LC elution time (± 0.4 min)a Lipid species in complex with proteins in KO cytosolb Species abundance relative to Wt (Fold change) Species bound to α-syn following immunoprecipitation (Fold change above non-specific binding)c 494.7 12.29 C14:1-PAF ↑-fold 12.89 C16:1-LPC No change 496.8 13.79 C14:0-PAF ↑3-fold ↑9.2x 14.39 C16:0 LPC ↑2-fold ↑9.8y 520.7 13 C16:2-PAF ↑2-fold 13.41 C18:2-LPC No change 522.8 14.66 C16:1-PAF ↑5-fold 15.15 C18:1-LPC ↑2-fold 524.9 17.2 C16:0-PAFc ↑2-fold ↑1.7x 18.11 C18:0-LPC ↑4-fold ↑2.3y 545 C18:4-PAF ↑11-fold 13.12 C20:4-LPC 545.9 13.11 C18:3-PAF De novo detection 13.99 C20:3-LPC 568.8 13.12 C20:6-PAF ↑4-fold C22:6-LPC 581 15.32 C20:0-PAF ↑17-fold 16.04 C22:0-LPC ↑12-fold 594 16.52 C24:7-LPC ↑2-fold C22:7-PAF 23:7c 24:7d C20:0-acyl-PAF C24:0-lysoPAF a Variations between m/z and retention time between runs were established for all glycerophospholipid species and respresents mean ± standard deviation. b Identification is predicted based on the theoretical monoisotopic mass values. CX:Y refers to the number of carbon atoms and double bonds in the sn-1 chain with a predicted acetyl (PAF) or hydroxyl (LPC) group at the sn-2 position. Only the most likely species are indicated although multiple isoforms may be present with the double bond in the alkyl chain at different positions. Isobaric species with same m/z eluting at different times were not further distinguished with the exception of C16:0 PAF. c Replicate experiments were performed in which α-syn was immunoprecipitated from Wt cytosolx or recombinant α-syn was added to KO cytosoly. Immunoprecipitates were analysed by LC-ESI-MS. Data represent mean increase in relative abundance above background (non-specific) signal ± standard deviation as described in Materials and Methods. d Identity verified by based on its coelution with d4-C16-PAF spiked at time of analysis. To identify glycerophosphocholines interacting directly with α-syn in our binding assays, we performed two complementary analyses. First, we immunoprecipitated α-syn from Wt cytosol and identified the glycerophosphocholine present in protein complex after dialysis by LC-ESI-MS. Second, we incubated recombinant α-syn with KO cytosol and identified lipid binding partners following immunoprecipitation. Non-specific lipid binding was assessed by lipid analysis of immunoprecipitates for α-syn from KO cytosol. Data are expressed as fold change in lipid abundance above background (Table 1). Only two predicated species exhibited significant association with Wt and Wt recombinant α-syn: C14:0 PAF and C16:0 PAF. C16:0 PAF was definitively identified by based on its co-elution with d4-C16-PAF (m/z 528.7) (data not shown). C14:0 PAF was identified based on retention time and monoisotopic mass values. Definitive identification was not possible in the absence of a commercially available synthetic standard of suitable purity. To validate effects of C16:0 PAF on α-syn membrane interaction, we tested whether C16:0 PAF enhanced α-syn binding to synaptic membranes directly (Fig 3C). Incubation of α-syn with C16:0 PAF alone did not affect α-syn membrane binding. However, when C16:0 PAF was added in combination with delipidated cytosol, α-syn binding was significantly increased. This data are suggestive of a protein-lipid complex required to enhance α-syn's capacity to interact with neuronal membranes. Specificity was tested using C16:0 lyso-PAF that differs from PAF by the presence of an hydroxyl group in place of an acetyl group at the sn-2 position. C16:0 lyso-PAF was not detected by LC-ESI-MS analysis in complex with protein in KO cytosol or α-syn immunoprecipitates and did not enhance α-syn membrane binding alone or in combination with delipidated cytosol (data not shown). A30P Parkinson's disease-linked mutation interacts differently with synaptic membranes compared to Wt Our results above, though consistent with previous reports showing that the A30P mutation impairs membrane binding ability compared to Wt and A53T α-syn, notably indicate that A30P α-syn binding is also significantly enhanced by cytosol, albeit not to the extent of Wt α-syn. Because α-syn is prone to self-aggregation and changes to the secondary structure of α-syn could induce artifactual differences between Wt, A53T and A30P membrane binding, we assessed whether each of the α-syn proteins are structurally similar in their soluble form prior to exposure to membranes, and not dimerized or aggregated which could affect membrane binding ability. All three α-syn proteins eluted in the same fractions as monomers from a size-exclusion column, and their circular dichroism spectra showed the characteristic minima of a randomly structured protein near 200 nm (Figure 4). Figure 4 Purified E-coli α-syn is monomeric and unstructured. Each recombinant α-syn (Wt, A30P and A53T) was analyzed by size exclusion chromatography to determine the presence of monomeric, dimeric, or other higher order forms. Eluate peaks (fraction 27) were then assessed by circular dichroism spectra to define the secondary structure of the α-syn proteins (Inset). Far-UV circular dichroism spectra were recorded on an Aviv circular dichroism spectrometer model 62DS (Lakewood, NJ, USA) at 25°C using quartz cells with a path length of 0.1 cm. Spectra were obtained from 195 nm to 260 nm, with a 1.0-nm step, 1.0-nm bandwidth, and 4-s collection time per step. The experimental data were expressed as mean residue ellipticity (θ) (deg·cm2·dmol-1). Only monomeric forms of α-syn where identified by size exclusion chromatography, and all α-syn share similar random secondary structure. Previous in vitro studies using artificial or cellular membranes showed that α-syn interacts with lipids and preferentially associates with lipid raft fractions isolated from cultured HeLa cells or synaptic vesicles [27,28]. Moreover, in those studies the A30P mutation impaired interaction with rafts, and consequently, with the membrane. Because those studies evaluating α-syn membrane binding did not assess cytosolic co-factors that could ostensibly regulate α-syn behaviour in vivo, we analysed the proportion of purified α-syn recovered with the lipid raft fractions following binding in the presence or absence of KO cytosol (Figure 5A). In contrast to the previous report [28], we found that very little exogenously-added α-syn (< 5%) co-eluted with the flotillin-positive lipid raft fractions, and this was not affected by the presence of cytosol, although α-syn immunoreactivity in other fractions (6–9) was increased by cytosol. These results indicate that the cytosol-dependent change in α-syn membrane binding was not due to increased association with lipid rafts, and the A30P α-syn was not less likely to co-elute with flotillin-rich fraction than either Wt or A53T α-syn. Figure 5 α-Syn binding to synaptosomal lipid rafts. Using a 42-30-5% discontinuous sucrose gradient, we analysed the proportion of α-syn that co-localised with flotilin-1, a lipid-raft marker. (A) Less then 5% of α-syn co-eluted with flotillin-1 after binding (in vitro) to α-syn KO synaptic membranes, in absence or presence of KO cytosol, and proportionally, no significant differences were observed between Wt and PD-linked mutants (Student's T-test, p > 0.05). (B) The proportion of α-syn that co-localised with flotillin-1 in vivo, using intact synaptosomes from transgenic mice expressing the human α-syn (Wt, A30P or A53T). As observed in vitro, only a small proportion of α-syn co-eluted with lipid rafts and no significant differences were observed between Wt and PD-linked mutations (Student T-test, p > 0.05). (C) A30P α-syn was subjected to paraformaldehyde-induced cross-linking to potential interacting proteins in synaptic membranes after 1, 2, 3, 5 and 10 minutes of incubation with synaptic membranes. A significant increase of bound α-syn after 2, 3 and 5 minutes was observed compared to the control condition (without cross-linking) (One-Way ANOVA p < 0.001, Bonferroni's multiple comparison test). (D) The proportion of α-syn present in the lipid-raft fraction after cross-linking did not show any significant differences compared to incubations without cross-linking (Student t-test: 1 min: p > 0.05; 3 min: p > 0.05). (E) A significant increase of bound Wt and A53T α-syn after paraformaldehyde-induced cross-linking was observed compared to the control condition without cross-linking (One-Way ANOVA, p < 0.05, p < 0.01,**p < 0.001, Bonferroni's multiple comparison test). To assess whether endogenously expressed cytosolic factors might play a role in regulating α-syn association to lipid rafts in vivo, but are not fully reproduced in our in vitro assay, we also quantified the amount of α-syn that co-elutes with flotillin-1 in synaptosomes from brains of human α-syn Tg mice. Only a minor fraction of total α-syn co-eluted with the lipid raft fraction from mouse brain synaptosomes (Figure 5B) or from whole brains (not shown), and we observed no significant differences between both PD mutants and Wt α-syn. Thus, mouse brain-expressed A30P α-syn appears to show a similarly weak distribution (< 5%) to gradient fractions containing lipid raft marker flotillin-1 as Wt and A53T α-syn. We also considered the possibility that the lower binding of A30P α-syn to total membranes is due to a transient or low affinity interaction that is not stable during isolation. To test this hypothesis, we assessed whether covalent cross-linking using paraformaldehyde after different incubation periods with purified A30P α-syn might stabilize the bound α-syn. Under these conditions, cross-linking increased α-syn association at t = 2, 3 and 5 minutes (Figure 5C). This additional α-syn was mostly excluded from the gradient fractions containing lipid rafts (Figure 5D) suggesting that the α-syn binding to membranes may be stabilized by other membrane proteins but not those associated with lipid rafts. Similar to the A30P mutant, Wt and A53T α-syn binding to membrane was also increased by cross-linking (Fig 5E). However, maximal binding of the Wt α-syn occurred in the first minute and remained stable thereafter. The binding of A53T mutant also peaked in first minute, but then slowly declined. Thus, the binding kinetics of α-syn bearing either PD-linked mutation suggest a more transient membrane interaction. α-Syn interaction with synaptic membrane is regulated by ATP α-Syn membrane attachment may be regulated by nerve terminal activity initiated by membrane depolarization [27], a process which results in Ca2+ influx, and elevated metabolic energy consumption. Therefore, we tested whether the addition of Ca2+ and ATP influenced α-syn binding. Our results show that ATP, but not ATPγS, significantly increased the level of membrane bound Wt α-syn and PD-linked mutants in the absence or presence of KO cytosol, whereas Ca2+ had no affect the α-syn binding (Figure 6A–C). The effect of ATP was additive to cytosol action suggesting that they act independently, and this was supported by the fact that ATPγS did not reduce the cytosol-dependent binding. Figure 6 (A-C) Recombinant α-syn (Wt, A30P and A53T) were incubated with ATP (1 mM), ATPγS (3 mM), Ca2+ (1 mM), ATP/Ca2+ or ATPγS/Ca2+ in absence or presence of 1.5 mg/ml of KO cytosol, for 10 min at 37°C. Incubation with ATP (Student's T-test, p < 0.001), but not ATPγS or Ca++ (Student's T-test, p > 0.05), induced a significant increase in the binding of Wt and mutant forms of α-syn (compared to control condition without added cofactors). Discussion Aberrant aggregation of α-syn has been detected in an increasing number of neurodegenerative diseases, now collectively known as synucleinopathies. These include Parkinson's disease (PD), Dementia with Lewy bodies (DLB), Alzheimer's disease (AD), multiple system atrophy, and Down syndrome [29]. Accumulations of α-syn in all these disorders have a common fibrillar configuration, though they differ in the co-localisation with other proteins including tau, parkin, and synphilin [30]. While the physiological functions of normal α-syn remain to be fully elucidated, several studies suggested it may play a role in synaptic plasticity, regulate dopamine (DA) neurotransmission via effects on vesicular DA storage, and act as a co-chaperone with cysteine-string protein to maintain nerve terminals [31]. These roles may involve α-syn interactions with proteins in cytosol and on membranes, though little is known about the α-syn membrane interaction in vivo and how membrane-bound and freely-diffusible pools of α-syn are maintained. Therefore, to understand the regulation of α-syn interaction with synaptic membranes, we developed an in vitro assay which measures the binding of recombinant E. coli-derived α-syn to α-syn-deficient synaptosomal membranes and recapitulates many features of the interactions observed in vivo. Using this binding assay, we showed that approximately 60% of the Wt and A53T soluble α-syn interacts with membrane, whereas only a small amount of the PD-linked A30P mutant is able to stably bind to the membrane (Figure 1B). Reduced A30P binding has been well-reported in several studies using artificial membranes [32-34] and can be explained by the expected disruption of the α-helix induced by the mutation. Indeed, the secondary structure of α-syn is divided into an α-helical lipid-binding amino-terminal and an unstructured lipid-free carboxyl-terminal [35]. The replacement of an Ala by a Pro in the A30P variant restricts the conformational space available to the preceding residue, Ala29, implying the loss of two intra-helical hydrogen bonds modifying the backbone structure of the protein, while the backbone structure and dynamics of the A53T α-syn mutant is found to be virtually unchanged from the Wt [36]. Despite the predicted structural limitations induced by the A30P mutation, and its impaired membrane binding capacity noted in in vitro assays, we showed previously that the amounts of Wt, A53T, and A30P α-syn that segregate with synaptic membrane fractions derived from mouse brains are not significantly different [14]. These disparate findings suggest that additional factors in vivo control α-syn membrane binding and can be reconciled by our present results showing that the addition of mouse brain cytosol stimulated the membrane association of Wt and A53T α-syn and partially rescued the intrinsically poor binding of the A30P α-syn. These data provide evidence that the subcellular proportion of membrane-bound and soluble α-syn may be regulated by cytosolic factors in vivo, which are far more concentrated (~300 mg/ml) than the 0.5–3 mg/ml cytosol used in our assay, and might compensate for the low A30P α-syn membrane association observed in vitro. Surprisingly, we observed that pre-exposure of membranes to cytosol was sufficient to augment subsequent α-syn binding, suggesting a mechanism whereby membranes can be primed by cytosolic factors for α-syn recruitment. Moreover, unlike the cytosolic protein requirement for the dissociation of α-syn from synaptic membrane [14], the cytosol-dependent component of α-syn binding is resistant to digestion by proteinase K and trypsin for the Wt and A53T α-syn, though not for A30P α-syn. This suggests that although cytosolic proteins are not required for the Wt and A53T α-syn membrane interaction, α-syn with A30P mutation would require protein assistance. As it is unlikely that a specific protein interaction evolved to specifically maintain A30P α-syn binding, the protein interaction implied by our results may also interact with Wt α-syn as well, though it is not essential for its membrane binding. We previously reported that cytosolic proteins are required for the dissociation of membrane-bound α-syn [14], presumably by transient association with α-helical conformation of α-syn on lipid bilayers. The same factor(s) may also aid in the reverse reaction by coordinating the A30P α-syn amino terminal to configure into an amphipathic α-helix so as to overcome its conformational limitations or to stabilize the mutant in closer apposition to the lipid bilayer prior to membrane binding. Such a mechanism could also account for the transient interaction we observed for A30P mutant with the membrane fraction. By briefly inducing covalent cross-links at various times to stabilize bound α-syn, we observed that A30P α-syn binding was biphasic, peaking at 3 min and declining thereafter. It is important to note this was not due to non-specific cross-linking because the later incubation periods (i.e. 10 min) did not show increased recovery of A30P α-syn with membranes despite the substantial soluble A30P α-syn. These results suggest that α-syn membrane binding may be partially coordinated by local synaptic vesicle proteins. Indeed, proteins such as cysteine string protein or members of the Rab family may fulfil this role [31,37,38]. Cross-linking also increased the recovery of bound Wt and A53T α-syn, although the kinetics were different from the A30P mutant. Both Wt and A53T binding peaked in the first minute of incubation suggesting a quicker interaction with synaptic membranes. Interestingly, the Wt α-syn remained stably associated even when cross-linking was activated after 10 min of incubation, the A53T binding declined slowly. These results are consistent with our previous report [14] showing greater cytosol-dependent dissociation of both PD mutants from synaptic membranes. In the course of characterizing the protein-dependence, we noted that lipid-depleted cytosol lost its activity to induce α-syn binding. Because the cytosol used in these experiments is dialyzed using membranes with a 12 kDa molecular weight cut-off, only lipid-protein complexes larger than 12 kDa are retained. These results suggested that protein-bound polar lipids are likely the protease-insensitive cytosolic components responsible for assisting the membrane binding of α-syn. In accord with the conformational model of α-syn [39,40] whereby it acquires a folded helical structure in the N-terminal region in its membrane-bound state, our results suggested that endogenous cytosolic lipids transferred to membranes prior to α-syn recruitment or bound directly to cytosolic α-syn may aid α-syn folding at the lipid-cytoplasm interface so it is more amenable to binding directly to synaptic membranes. To provide further insight into this novel protein-lipid-protein interaction, we profiled glycerophosphocholines bound to proteins in α-syn-deficient cytosol by nanoflow LC-ESI-MS and precursor ion scan. Our analysis identified 24 species that can potentially affect α-syn membrane interactions. While this number clearly underestimates the cytosolic lipid content in vivo given our MS analyses were limited to polar glycerophospholipids with an m/z between 450 and 600, of which glycerophosphocholine-containing species were further analyzed, these data represent the first profile of candidate lipid interactors at the molecular level responsible for the enhanced α-syn binding. Further, we demonstrated that two glycerophosphocholines C14:0 PAF and C16:0 PAF interact with α-syn, with C16:0 PAF definitively identified at the molecular level. Importantly, C16:0 PAF was able to rescue the ability of delipidated cytosol to potentiate α-syn membrane binding but did not, in and of itself, enhance α-syn interaction with membranes. This result suggests the involvement of a cytoplasmic protein, and although appears inconsistent with data in Fig. 2C showing that α-syn binding does not require intact cytosolic proteins, a more likely possibility is that a cytoplasmic protein may be required to activate or modify the exogenously added lipid. For example, binding to GM2 activator protein elicits a conformational change in PAF [41]. Arguably, endogenous PAF in brain cytosol would be active prior to the cytosol depletion, and thus delipidation, but not protein depletion, would impact a-syn binding. Similarly, addition of exogenous PAF, presumably in an inactive conformation, would need prior activation by delipidated cytosol. These findings are also consistent with previous studies indicating that α-syn does not directly bind to palmitic acid (C16:0) [25], yet addition of 1,2-palmitoyloleoylphosphatidylcholine to α-syn containing protein lysates promotes self-association and formation of protein complexes [24]. Here, we further confirmed specificity of these interactions using C16:0 lyso-PAF. C16:0 lyso-PAF did not impact α-syn interaction with neuronal membranes. Clearly, the nature of these protein-protein-lipid complexes and their effects on α-syn binding to synaptic membranes will require further investigation and expansion of the analysis of lipid co-factors beyond the small second messenger neutral glycerophosphocholines tested in this study. Careful analysis of these lipids will also be relevant to aging and neurodegeneration because abundant data suggest that cumulative oxidative modification of biomolecules, including lipids, plays an important role in aging, and free radical damage to brain lipids is involved in neuronal death in neurodegenerative disorders [42]. There is also accumulating evidence that α-syn deficiency has complex effects on brain lipid metabolism and production of lipid second messengers although the underlying mechanisms are poorly understood [20,21,25]. Consistent with these data, we also detected differences in PAF and LPC glycerophosphocholine levels between KO and normal cytosols, but these did not impact α-syn binding in our assay. Altogether, our data suggest that brain-lipids regulate α-syn binding, and an imbalance in specific species could mediate α-syn accumulation in the cytosol leading to fibril formation. Despite previous studies suggesting that α-syn preferentially binds to lipid rafts in HeLa cells and to purified lipid raft fractions from rodent brain [27,28], we were unable to corroborate this interaction in our studies. We found that < 5% of total exogenously added α-syn co-eluted with the lipid raft marker flotillin-1, and this was unaffected by PD-linked mutations. Moreover, the same minor proportion of brain-expressed α-syn co-eluted with the flotillin-1 enriched fractions isolated from synaptosomes or whole brain, ostensibly reflecting negligible lipid raft associated α-syn in vivo. This low level of brain α-syn in lipid rafts was also noted by Fortin et al. [27], though they postulated that α-syn may dissociate from brain lipid rafts during the biochemical isolation. However, this explanation is inconsistent with the high recovery of overexpressed α-syn in lipid rafts from HeLa cells following the identical isolation procedure [27], and with our results showing that chemical cross-linking of A30P α-syn stabilized its membrane association, though not to lipid raft fractions. Two other explanations could account for the difference in the earlier studies and ours: First, lipid rafts in HeLa cells likely have a distinct lipid and protein composition compared to those in mammalian nerve terminals, possibly allowing them to bind overexpressed α-syn, which is not normally expressed in HeLa cells. Second, in the present work, lipid raft fractions were isolated only after α-syn was incubated with permeabilized synaptosomes, which retain sufficient internal architecture as to permit Ca2+-dependent exocytosis [19,43,44]. In contrast, the study by Kubo et al. [28] isolated lipid rafts before incubating with exogenous α-syn. The biochemical purification with 1% TX-100 likely modifies lipid rafts by altering lipid packing and/or loss of peripherally attached constituents, conceivably affecting subsequent α-syn binding capacity that is not normally present in vivo. Because calcium influx and metabolic energy are both critical for the normal function of nerve terminals, we examined whether α-syn binding can be affected by modulating the availability of either Ca2+ or ATP. We observed that α-syn binding has an ATP-dependant component that was not supported by ATPγS, and is insensitive to calcium. Because the increased α-syn binding in the presence of ATP and cytosol were additive and ATPγS did not affect cytosol-induced α-syn binding, it is likely that ATP and cytosolic factors act independently. One possibility is that ATP acts on a membrane protein whose interaction with membrane-bound α-syn is stabilized by chemical cross-linking, whereas cytosolic lipids modulate α-syn conformation either by direct interaction in cytosol or after intermediate transfer to a membrane component. Our results suggest that changes in synaptic ATP levels due to elevated metabolic consumption during exocytosis could modulate the α-syn solubility and may explain how neuronal depolarization can increase the level of freely-diffusible cytoplasmic α-syn in a Ca2+-independent manner [45]. The ATP sensitivity is also relevant to aging because neurodegenerative diseases are commonly associated with mitochondrial dysregulation and consequent impairment of energy production[46]. Under such pathological conditions, it is possible that lowered ATP levels may increase the cytosolic α-syn, which is significantly less constrained structurally than the membrane bound form. Concomitant oxidative stress could thereby promote β-sheet formation and accelerate α-syn aggregation. Conclusion In conclusion, while the identities of the cytosolic components that assist the membrane interaction of α-syn remain to be fully characterized, our study reveals that cytosolic lipids and ATP are two of the principal factors regulating α-syn interaction with synaptic membranes. In addition, the relatively poor membrane binding of A30P α-syn could be explained by a more transient interaction with synaptic membrane and was partially rescued by the presence of protease-sensitive factors in brain cytosol. Those results suggest that endogenous brain proteins moderate the otherwise inefficient membrane association of A30P α-syn mutant, and represent a potential targets to influence α-syn solubility in brain. Abbreviations α-syn: Alpha-synuclein; ESI-MS: electrospray ionization mass spectrometry; XIC: Extracted ion chromatogram; HPLC: High performance liquid chromatography; PD: Parkinson's disease; PAF: Platelet activating factor; Tg: Transgenic; Wt: Wild type. Authors' contributions SW-G conducted the majority of the binding assays and drafted the manuscript; NPV contributed to the cross-linking and cytosolic lipid activity assays; SNW characterized the cytosolic and alpha-synuclein bound lipids; DM contributed to the alpha-synuclein purification and binding assays; WH and DF provided the MS data; PEF contributed reagents and participated in the circular dichroism analyses; SALB and AT designed and coordinated the study. All authors read and approved the final manuscript. Acknowledgements AT is a CIHR New Investigator and was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Parkinson Society of Canada. Postdoctoral support was provided to SW-G by the CIHR, the Leon Frederick Foundation and the Journal of Cell Science travel grant, and to NPV by the Parkinson Society of Canada. PEF was supported by grants from the CIHR, Michael J Fox Foundation and Alzheimer Society of Ontario. DF would like to acknowledge a Canada Research Chair in Proteomics and Systems Biology. SALB is a CIHR New Investigator and an Ontario Mental Health Foundation (OMHF) Intermediate Investigator. SALB. and DF were supported by grants from the Ontario Mental Health Foundation (OMHF) and the Parkinson Research Consortium. 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10.1242/jcs.03443	18808659	PMC2562387	CC BY	2021-01-04 17:16:25	yes	BMC Neurosci. 2008 Sep 22; 9:92
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1895341308-PONE-RA-05048R110.1371/journal.pone.0003529Research ArticleOncologyCell Biology/Cell SignalingPhysiology/Cell SignalingVirology/Viruses and CancerWomen's Health/Gynecological CancersHPV16 E7-Dependent Transformation Activates NHE1 through a PKA-RhoA-Iinduced Inhibition of p38alpha PKA-RhoA-p38 in TransformationCardone Rosa A. 1 Busco Giovanni 1 Greco Maria R. 1 Bellizzi Antonia 2 Accardi Rosita 4 Cafarelli Antonella 1 Monterisi Stefania 1 Carratù Pierluigi 3 Casavola Valeria 1 Paradiso Angelo 2 Tommasino Massimo 4 Reshkin Stephan J. 1 * 1 Department of General and Environmental Physiology, University of Bari, Bari, Italy 2 Clinical Experimental Oncology Laboratory, National Cancer Institute Giovanni Paolo II, Bari, Italy 3 Department of Respiratory Medicine, University of Bari, Bari, Italy 4 Infections and Cancer Biology Group, IARC-WHO, Lyon, France Jin Dong-Yan EditorUniversity of Hong Kong, China* E-mail: [email protected] and designed the experiments: RAC MT SJR. Performed the experiments: RAC GB MRG AB RA AC SM. Analyzed the data: RAC PC VC AP MT SJR. Contributed reagents/materials/analysis tools: MT. Wrote the paper: RAC VC SJR. 2008 27 10 2008 3 10 e352911 6 2008 3 10 2008 Cardone et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/+H+ exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice. Methodology Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1. Principal Findings We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition. Conclusions All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation. ==== Body Introduction Neoplastic transformation is the first step of the carcinogenic process that involves the initial altered responses of the cells to normal regulatory influences and sets the stage for further alterations that result in carcinoma. A wide variety of altered phenotypes appear as a result of transformation. Hallmarks of epithelial transformation and carcinogenesis include loss of polarity, as well as uncontrolled, serum-independent and anchorage-independent proliferation and resistance to apoptosis [1]. Other fundamental hallmarks of epithelial carcinogenesis include an elevated intracellular pH (pHi) as well as their increased rate of glucose utilization over oxidative phosphorylation [2], [3]. However, our understanding of the sequence of early events mediating the initiation, development and regulation of malignant transformation is still incomplete. One major group of cellular signal transduction components implicated in carcinogenesis are the mitogen-activated protein kinases (MAPKs). Altered expression/activity of each of the MAPKs such as ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal kinase) and p38 has been linked to tumor progression in a wide variety of cellular contexts [4]–[6]. In particular, mounting evidence indicates a negative role for the p38alpha MAP Kinase in chemical-[7] and oncogene-[8] induced tumor formation and proliferation [9], in tumor cell directed cell polarity [10]–[12] and in malignant invasion [11], [12]. Conversely, a positive role of p38 has been shown in tumor suppression and delay of tumorigenesis [13], [14], in induction of apoptosis [15], [13], [16], in a specific tumor-suppressing defense mechanism of normal non transformed cells known as oncogene induction of senescence, [17], in dormancy [18], [19] and in the increased cell viability and enhanced growth of HPV-induced recurrent respiratory papillomatosis [20]. The importance of p38 as tumor suppressor is highlighted by recent attempts to identify cancer-associated mutations in protein kinase genes, which revealed that several components of the p38 pathway, including p38alpha, are mutated in human tumors [17]. Further, p38 is activated in cancer cells during paclitaxel-driven [21], cisplatin-driven [22] and ROS-driven [23] apoptosis. In liver cells chemically induced to form tumors, p38alpha negatively regulated tumor proliferation via a repression of the JNK-c-Jun pathway [24]. While the negative role for p38alpha in regulating carcinogenesis is well described, whether it plays a similar negative role in the initiation of neoplastic transformation and through which signaling pathways are still undetermined. In this context, important questions concern if p38alpha plays a role in the development of the initial transformed phenotype after oncogene expression, what is its pattern of involvement and what are its critical upstream and downstream components. Recent progress suggests possible candidate signal transduction pathways. As a regulator of gene expression, cell cycle progression and actin cytoskeleton organization, it is now clear that RhoA, a member of the Rho family of GTPases, plays a central role in carcinogenesis and tumor progression [25]–[27]. Recent studies indicate RhoA as a central upstream regulator of MAP kinase activity [26], [28]–[31] and specifically in breast cancer [11] and pancreatic carcinoma [32] cells. Importantly, forced expression of the E7 oncogene of HPV16 in keratinocytes has recently been shown to inhibit RhoA activity although the mechanism is still not completely clear [33]. The cAMP/PKA system has been demonstrated to be involved in both transformation/tumor progression [11], [34], [35] proliferation [35]–[37] and apoptosis [38]–[40]. This system is also involved in regulating p38 activity [11], [41]–[43] and there is evidence that direct PKA-dependent phosphorylation of RhoA at Ser 188 inhibits its activity in endothelial cells [44], in smooth muscle cells [45], in cytotoxic lymphocytes [46] and in tumor cells [11] suggesting that PKA and RhoA could regulate p38 activity through a common pathway. The goal of the present work is to determine which signal transduction systems are involved in transformation driven by a relevant oncogene. That little is known about the modifications occuring in the regulatory pathways during malignant transformation has been due, in part, to the lack of experimental models in which the transformation of normal cells by a relevant oncogene can be finely controlled and followed [17]. Since the human papilloma viruses, especially HPV16, are the prime cause of the majority of virus-associated carcinomas and the E7 and E6 proteins are the viral proteins responsible for malignant transformation [47], we chose the E7 oncogene of HPV16 to create an experimental cell model useful for the investigation of the alterations in signal transduction mechanisms underlying the initial phases of the shift from the normal to transformed state [48]. This cell model (2BN11 cells) consists of normal NIH-3T3 fibroblasts stably transfected with the gene for the HPV16 E7 oncoprotein in a vector in which its expression is under the control of a tetracycline (tet) inducible promoter such that the level of E7 expression and, therefore, transformation can be tightly regulated. This inducible expression permits the repeated following of the time-dependent development of the events in which the same cell constitutes its own control. Using the 2BN11 model system, we previously observed that intracellular alkalinization driven by an up-regulation of the Na+/H+-exchanger (NHE1) is a very early physiological event in transformation and is essential for the development of other transformed phenotypes such as increased glycolytic metabolism, increased growth rate and both serum-independent and anchorage-independent growth [48]. Here, we report that HPV16 E7-dependent transformation mediated by NHE1 is driven by the action of a RhoA-p38alpha module gated by the PKA dependent phosphorylation of RhoA. Materials and Methods Cell culture, transfections and reagents NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g of glucose per liter, supplemented with 10% calf serum. Normal human foreskin keratinocytes (HFKs) were isolated from neonatal foreskin as described previously [49] and were maintained in keratinocyte growth medium supplemented with bovine pituitary extract (Clonetics). Primary human keratinocytes infected with HPV16 [50] and cultured at low (HPK1a early) or high (HPK1A late) passage numbers were maintained as previously described [48]. These cells spontanously transform at high passages and high passage cells are tumorgenic in nude mice [48]. High-titer retroviral supernatants (106 virus particles/ml) were generated by transient transfection of Bosc23 cells (ecotropic viruses) or Phoenix (amphotropic viruses) and used to infect the cells as described previously [48]. Cells were grown to about 80% confluence in a 10-cm-diameter dish and infected with 5 ml of the recombinant retrovirus or parental virus in the presence of 8 µg of Polybrene per ml in order to enhance infection efficacy. After 6 h infected cells were fed with complete medium and kept at 37°C with 5% CO2 for 48 h. The cells were then transferred to four 10-cm-diameter dishes and selected with 2.0 µg of puromycin/ml for 3 to 4 days. Selected cells were expanded and used for experiments, generally when at 80% confluence. NIH3T3 or HFK cells were infected with recombinant retrovirus (pLXSN vector, Clontech, BD, Le Pont Claix, France) expressing non-tagged HPV16 E7 proteins. In some experiments, NIH3T3 cells constitutively expressing E7 genes were generated by infection with pBabepuro vector expressing HPV16 HA-tagged E7 proteins, wild-type or mutant. The 2BN11 cell line was created by infecting NIH3T3 cells with recombinant retrovirus expressing the HPV16 E7 gene under the control of a tetracycline repressed promoter. The tetracycline-controlled expression system consisted of regulator and response elements in a one vector system [48]. Cells were cultured in DMEM high glucose (4500 mg/l) supplemented with NaHCO3 (3700 mg/l), 10% (v/v) heat inactivated fetal bovine serum, L-glutamine (2 mM), Na-Pyruvate (1 mg/ml) and 1 µM tetracycline. The clones, after selection in puromycin, were selected on the basis of having low basal E7 expression in the presence of tetracycline and high inducibility as determined by Western blot and RT-PCR analysis. Transient transfections with the various mutated cDNAs were performed with the FuGENE HD Transfection Reagent (Roche) according to the manufacturer's instructions [12]. H89, SB203580 and IBMX were purchased from Sigma whereas Forskolin was from Calbiochem (Novabiochem Corp., La Jolla, CA). The effect of these clones or pharmacological agents on E7 expression were tested in an RT-PCR assay as described below and none of them had any effect on E7 message (Figure S1) RNA preparation and semiquantitative RT-PCR Total RNA was extracted from cells using the RNeasy system (Qiagen, Valencia, CA) and treated with RNAase-free Dnase (Qiagen) for 15 min at room temperature. Spectrophotometric ratios of A260 to A280 were greater than 1.8. Total RNA (500 ng) was reverse transcribed in 20 µl reaction system using Random Hexamers priming and MuLV Reverse Transcriptase (RT) with the RNA PCR Kit GeneAmp (Applied Biosystems) under conditions described by the supplier. Reverse transcription-PCRs (RT-PCRs) were carried out essentially as previously reported [12]. Quantification of HPV16 E7 expression levels was performed by comparison to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping gene. Primer sequences were as follows: HPV16 E7 gene, 5′-ATG CAT GGA GAT ACA CCT AC-3′ (forward) and 5′-TAT GGT TTC TGA GAA CAG ATG-3′ (reverse); GAPDH gene, 5′-ACC ACA GTC CAT GCC ATC AC-3′ (forward) and 5′-TCC ACC ACC CTG TTG CTG TA-3′ (reverse). PCRs for HPV16 E7 and the GAPDH gene were performed by a touchdown protocol with the following cycling conditions: 10 min at 95°C (initial denaturation), 6 cycles of step-down PCR consisting of 45 s at 95°C (denaturation), 60 s at 58°C (annealing) – decrease 1°C each cycle until 53°C; and 120 s at 72°C (extension). Amplification of the final product was completed for 26 cycles of 45 s at 95°C, 60 s at 53°C, and 120 s at 72°C, with a final extension of 10 min at 72°C. In the negative control, RNase free water (Gibco) was used instead of template RNA. The positive control included HPV16 E7 cDNA. Amplicons were separated on 1.5% agarose gel and visualized by ethidium bromide staining. NHE1 activity Intracellular pH was determined spectrofluorimetrically in cells loaded with the acetoxy-methyl ester derivative of the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (AM-BCECF, Invitrogen). NHE1 activity was determined by measuring the rate of pHi recovery from an acid load produced with the NH4Cl prepulse technique by evaluating the derivative of the slope of the time-dependent pHi recovery (dpHi/dt) as previously described [40], [48]. The use of CO2/HCO3 free solutions minimizes the likelihood that Na+-dependent HCO3 transport was responsible for the observed pHi changes. After each experiment trypan blue exclusion was also measured for each cover slip and when was more than 5% the experiment was not used. Construction of expression vectors containing RhoA mutants Site-directed mutagenesis of RhoA to create RhoAS188A was performed by PCR overlap extension as previously described [11]. The successful construction of the mutants was confirmed by DNA sequence analysis. The cDNA were cloned into the pBabe puro expression vector containing a hemagglutinin (HA) tag. RhoA RNA Interference Small interfering RNA (siRNA) duplex against RhoA (sc-29471) was obtained from Santa Cruz Biotechnology. The specificity of the silencing tecnique was verified by using a control, non targeting 20–25 nucleotide siRNA designed as a negative control (Control siRNA-A, sc-37007, Santa Cruz Biotechnology). siRNAs were used at 100 nM concentration and transfection with siRNA was performed using Lipofectamine 2000 (Invitrogen) 24 hr after plating cells. The efficiency of RhoA silencing was measured in immunofluroescence microscopy using a monoclonal anti-RhoA antibody (sc-418, Santa Cruz Biotechnology) and was found to be ∼75% after 72 hrs siRNA treatment (Fig. S2). RhoA expression levels were measured via pixel density analysis using the WCIF Image J 1.37c software (Wayne Rasband, NIH, USA). Therefore, NHE1 activity was analyzed 72 hrs post-transfection with either RhoA siRNA or control siRNA-A in the presence of tet or 24 hrs after tet removal. Adherent cell cAMP assay Cells (104 cells in 100 µl of media) were plated into each well of a Greiner Bio-one tissue culture grade 96-well white clear bottom microassay plate (PBI S.p.a., Italy) and incubated in 5% CO2 atmosphere at 37°C for 24 hr. Cells were washed once with 100 µl of DMEM serum free medium plus 100 µM IBMX and then treated with 20 µM of FSK and 1 mM of IBMX in 20 µl of reaction media at 37°C for 30 min. Levels of cytosolic cAMP were then measured with the cAMP-Glo™ assay (Promega, Italy) as per manufacturers instructions. Briefly, the medium was aspirated, 20 µl of lysis buffer and 40 µl of cAMP-Detection Solution was added and the plate was incubated with gentle rocking at room temperature for 20 min. Next, 80 µl of Kinase-Reagent were added, and luminescence was read after incubation for 10 minutes in a Fluorescence Spectophotometer Cary Eclipse VARIAN in its chemiluminescence mode where each well is read for 20 seconds. Increases in cAMP levels result in decreases in chemiluminescence. cAMP FRET measurements and cell imaging For time-lapse FRET experiments, cells were cotransfected with 1.5 µg each of pcDNA3Cat-YFP and pcDNA3RII-CFP plasmids using Fugene 6 (Roche, Switzerland) for 24 hr as described [51]. Twenty-four hours after transfection, monolayers of cells were cultured an additional 3 or 24 h in the presence or absence of tetracycline and imaged on a Nikon ECLIPSE TE 2000-S equipped with a charge-coupled device camera, a controlled DeltaRAM monochromator on the excitation side and a beam splitter (Optical Insight, St. Cloud, MN) on the emission side fitted with a 505DCRX dichroic and two emission filters, D480/30 and D535/40. Excitation was at 430 nm and the dichroic mirror was a 455DRLP. Acquisition and FRET analyses were performed using the Metafluor 4.6 software (Meta Imaging 4.6; Universal Imaging, Downingtown, PA). The live imaging was done with 4×4 binning to minimize exposure time, photobleaching and registration artifacts. To study agonist-induced changes in FRET: following the recording of a baseline, cells were first continuously superfused with the phosphodiesterase inhibitor IBMX (100 µM) and then treated with a mix of IBMX (100 µM) and the adenylate cyclase activator forskolin (25 µM). The change in FRET signal due to mobilization of cAMP was detected by FRET/CFP ratiometric processing in both non stimulated and agonist-stimulated cells, in which both FRET (CFP excitation–YFP emission) and CFP (CFP excitation–CFP emission) images were first background subtracted. Cells were thresholded to discard any portions of the image with insufficient intensity to provide reasonable signal/noise. The resulting background-subtracted FRET image was divided by that of CFP image to obtain a pixel-to-pixel FRET/CFP ratio image Increasing cAMP levels result in a reduction in FRET ratio (Em YFP/Em CFP). The final FRET images were displayed in pseudocolors scaled linearly from the lowest (red) to the highest (blue) signal to show relative increase in cAMP mobilization of levels within each cell at each treatment at both 3 and 24 hrs. Protein extraction and Western blotting Western Blotting was performed as described [11]. Samples were extracted in sodium dodecyl sulfate (SDS) sample buffer (6.25 mM Tris-HCl, pH 6.8, containing 10% (v/v) glycerol, 3 mM SDS, 1% (v/v) 2-mercaptoethanol and 0.75 mM of Bromophenol Blue), separated by 4–12% SDS-PAGE and blotted to Immobilon P. Analysis of phospho- and total RhoA was performed by using an antibody produced against a peptide of RhoA phosphorylated at serine 188 by PRIMM (Milan, Italy) diluted 1∶1000, against total RhoA (sc-418, Santa Cruz, CA) diluted at 1∶1000, against tubulin (T5293, Sigma) and against the HA tag (MMS-101R BabCo, diluted 1∶200). Molecular weights standards were ‘Biotinylated Protein Ladder Detection Pack’ (Cell Signaling Technology, MA). Phospho-Kinase Assays For the kinase phosphorylation measurements total cellular protein was extracted in SDS-sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol and 0.1% bromophenol blue) and approximately 50 µg was separated on 10% SDS-PAGE and transfered to Immobilon P, (Millipore). The relative amount of each phosphorylated kinase (ERK, JNK and p38) to its total expression was determined by Western Blotting with antibodies specific to each obtained from Cell Signaling Technology (MA, USA). p38 MAP kinase activity assay To assay p38 MAP kinase activity, cells were grown to approximately 70% confluence in 10 cm plates (GIBCO) and treated as decribed in figure legends. After treatment, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed by 5 minutes at 4°C in lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 20 mM Tris, pH 7.4) plus 1 mM PMSF. The cells were scraped into Eppendorf tubes and triturated by sonification. The cell lysate was centifuged at 4°C for 10 min at 14,000 rpm and the supernatant collected. Protein levels were equalized by normalizing them to the protein levels measured before the assay. p38 MAP kinase activity was quantified using an immune complex kinase assay kit according to the manufacturer's protocol (New England Biolabs). Briefly, cleared lysates were immunoprecipitated overnight at 4°C with p38 MAPK antibodies conjugated to agarose (New England BioLabs). Beads were washed three times with ice-cold lysis buffer and three times with kinase reaction buffer (25 mM Tris pH 7.5, 5 mM ß-Glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2) minus ATP and ATF-2. The pellet was resuspended in 50 µl kinase reaction buffer plus 200 µM ATP and 10 µg GST-ATF-2 as substrate and incubated at 30°C for 30 minutes. The reaction was stopped by addition of 2× Laemmli buffer. The sample was run on 10% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore) for immunoanalysis. The amount of ATF-2 phosphorylated by p38 was analyzed by Western blotting with a Phospho-ATF-2 (Thr71) antibody (Cell Signaling) that detects only catalytically activated ATF-2. Total p38 expression measured by immunoblotting was not found to vary under any experimental conditions. Analysis of RhoA serine phosphorylation state After treatment cell monolayers were washed twice with ice-cold PBS and lysed in ice-cold RIPA (the above lysis buffer plus 0.1% SDS and 0.2% Na-deoxycholate). The cellular lysate was centrifuged at 14,000 rpm for 5 min at 4°C. Protein levels were equalized by normalizing them to the protein levels measured before the assay and the supernatent pre-cleared with protein A-agarose for 2 hrs at 4°C. Cleared lysates were immunoprecipitated overnight at 4°C with a phosphoserine antibody conjugated to agarose (SIGMA). The agarose beads were washed four times with simple RIPA buffer. The pellet was resuspended in 50 µl Laemmli buffer. The sample was run on 12% SDS-PAGE and blotted onto Immobilon P (Millipore) for immunoanalysis of the amount of RhoA immunoprecipitated by antiphosphoserine with a RhoA antibody (sc-418, Santa Cruz). Analysis of the activity of RhoA RhoA activity was assessed using the RhoA-binding domain of Rhotekin in a kit supplied from Upstate Biotechnology (Lake Placid, NY). In brief, 3×106 cells were plated onto 10 cm cell culture dishes and after 24 hrs treated as indicated. After the indicated time, cells were extracted with RIPA buffer (50 mM Tris, pH 7.2, 500 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 10 mM MgCl2, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 4 µg/ml aprotinin, and 2 mM PMSF). After centrifugation at 14,000 g for 3 min, the extracts were incubated for 45 min at 4°C with glutathione beads coupled with GST–RBD (Rho-Binding Domain of Rhotekin) fusion protein (Upstate Biotechnology, Lake Placid, NY), and then washed three times with Tris buffer, pH 7.2, containing 1% Triton X-100, 150 mM NaCl, and 10 mM MgCl2. The RhoA content in these samples or in 50 µg protein of cell homogenate was determined by immunoblotting samples using anti-RhoA antibody from Santa Cruz (sc-418, Santa Cruz, CA). FRET assay for RhoA activity For these experiments, endogenous RhoA activity was measured in FRET microscopy using the Raichu 1297 probe as previously described [11]. In this sensor, the Rho Binding Domain (RBD) of the RhoA effector protein, Rhotekin, is sandwiched by Venus-YFP and CFP. The binding of endogenous GTP-RhoA to RBD generates a conformational change that displaces YFP and CFP, thereby decreasing fluorescence resonance energy transfer (FRET) efficiency between the two fluorophores, while a reduction of intracellular active RhoA results in the opposite effect. The CFP channel images were divided by the YFP-FRET channel images. The activity of RhoA is monitored by measuring CFP (480 nm)/YFP(545) fluorescence emission values upon excitation of the transfected cells at 430 nm. To eliminate the distracting data from regions outside of cells, the YFP channel is used as a saturation channel. The ratio images are presented in pseudocolor mode. Ratio intensity is displayed stretched between the low and high renormalization value, according to a temperature-based lookup table with blue (cold) and red (hot) indicating respectively high and low values of RhoA activity. Additionally, FRET was used to monitor RhoA activity due to the activity balance between endogenous guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs): cells were transfected with a plasmid with the cDNAfor Raichu-RhoA-1293, which consisted of truncated RhoA (aa 1–189), the RhoA-binding domain (RBD) of effectors, and a pair of GFP mutants, YFP and CFP. In these probes, the intramolecular binding of GTP-RhoA to the effector protein was expected to bring CFP in closer proximity to YFP, resulting in an increase in FRET from CFP to YFP. The set up of the microscope and the filters used for FRET excitation and emission were identical to those reported for the cAMP FRET measurements. Briefly, twenty-four hours after transfection, monolayers of cells were cultured an additional 24 h in the presence or absence of tetracycline and, images from filter sets dedicated for YFP, CFP, and FRET fluors were first captured, and then three to four random regions of interest within each cell, were chosen for analysis. Sensitized FRET measurements Off-line image analysis was performed using the Metafluor 4.6 software (Meta Imaging 4.6; Universal Imaging, Downingtown, PA). Correction of FRET measurements for spectral bleedthrough and cross excitation was calculated on a pixel-by-pixel basis for the entire image by estimating net FRET (nF) as follows: nF = IFRET−(IYFP×a)−(ICFP×b), where IFRET is sensitized YFP emission (excitation 430 nm, emission 545 nm) and IYFP and ICFP are YFP emission (545 nm) upon excitation at 480 nm and CFP emission (480 nm) upon excitation at 430 nm, respectively. a is a norm of the percentage of CFP bleed-through, and b is a norm of the percentage of direct excitation of YFP at 430 nm. a and b were determined by analyzing images of cells expressing only CFP or YFP as described previously [11], and for our system a and b values corresponded to 64 and 8%, respectively. Corrected FRET ratio was calculated as ICFP/nF. Statistical Procedures In the in vitro experiments, student's t-test was applied to analyze the statistical significance between treatments. All comparisons were performed with InStat (GraphPad Software). Results Our goal was to study the signal transduction mechanisms involved in neoplastic transformation driven by a biologically relevant oncogene. The E7 oncoprotein of HPV16 is known to induce transformation both in vitro and in vivo and to fully transform immortalized rodent fibroblasts (E7/NIH3T3 cells) or human keratinocytes which are tumorigenic in nude mice [48], [50]. HPV16 E7-induced transformation induces down-regulation of p38 but not JNK or ERK MAPKinase activity We first determined the effect of HPV16 E7 expression on MAP Kinase activity by transducing immortalized NIH3T3 cells or human foreskin kerotinocytes (HFK), the natural host of the virus, with a recombinant retrovirus expressing non-tagged, wild-type HPV16 E7 and measuring the phosphorylation state of ERK1/2, JNK and p38 MAP kinase as described in Material and Methods. As seen in Figures 1A (NIH3T3) and 1B (HFK), E7 expression reduced the phosphorylation state of p38 MAP kinase (p38) by approximately 90% while having no significant effect on the phosphorylation level of either the ERK or JNK MAP kinases. The same results were observed in NIH3T3 cells infected with HA-tagged wild-type E7 (data not shown). Further, two different protocols were used to determine whether it is simply E7 expression or E7-dependent transformation that leads to an inhibition of p38. First, we used human primary keratinocytes (HPKIA cells) immortalized by transfection of the entire HPV16 genome and which become spontaneously transformed at high passage number and are tumorigenic in nude mice [48], [50]. As shown in Figure 1C, p38 was much less phosphorylated in the transformed (late passage) keratinocytes than in the immortalized (early passage) cells while, also here, having no significant effect on the phosphorylation levels of either the ERK1/2 or JNK MAP kinases. To further confirm this dependance of p38 dephosphorylation upon HPV-induced transformation, we then extended our study to NIH3T3 cells infected with recombinant retroviruses expressing either wild-type HPV16 E7 or transformation-deficient mutants [53], [54]. As seen in Fig. 1D, the wild-type E7 reduced p38 phosphorylation as above while the transformation negative mutants had no effect on p38 phosphorylation. Altogether, these results demonstrate that HPV16-induced transformation reduces the p38 activity through E7 without affecting either the ERK or the JNK MAP kinases and are consistent with the reported specificity of p38 in oncogenic processes. 10.1371/journal.pone.0003529.g001Figure 1 Only HPV16 E7 expression and not other non-transforming HPV types decreases p38 MAPK phosphorylation in both NIH3T3 cells and human keratinocytes. A. Cell extracts from empty pLXSN vector NIH3T3 cells and HPV16 E7 infected cells were analyzed by Western Blot for both the phosphorylation state and total expression level of ERK, JNK and p38 MAPK. Western blot analysis was carried out using polyclonal anti-phospho-ERK1/2, anti-phospho-JNK and anti-phospho-p38 antibodies followed by polyclonal anti-total ERK1/2, total-JNK and total-p38 MAP Kinase antibodies. Tubulin expression was used as an additional loading control and 500 ng of each sample total RNA were subjected to a semi-quantitative RT-PCR for HPV16 E7 expression analysis. GAPDH: loading control, pc: positive control, nc: negative control. B. Cell extracts from empty pLXSN vector HFK cells and HPV16 E7 infected HKF cells were analyzed by Western Blot for both the phosphorylation state and total expression level of ERK, JNK and p38 MAPK as above. C. Cell extracts from early passage (p94) HPK1A cells and late passage (p396) HPK1A cells were analyzed by Western Blot for both the phosphorylation state and total expression level of ERK, JNK and p38 MAPK as above. D. NIH3T3 cells were infected with recombinant retroviruses expressing either empty vector (pBabe), wild type HPV16 E7-Ha tag (WT), or transformation-deficient HPV16 E7-Ha mutants (24, 10, 31/32), and total cell extracts were assayed in Western blot for phosphorylated and total p38 expression levels as above. All the cell lines expressed E7 as confirmed by using an Ab anti HA-tag (E7-HA). Preliminary experiments demonstrated that the HA tag does not interfer with the induction of transformation (data not shown). HPV16 E7-dependent down-regulation of p38 is an early event in transformation The above experimental systems do not permit the determination of the dynamic processes occurring during transformation. To determine whether inhibition of p38 is an early event in transformation, a cell model was constructed in which the transformation of normal cells can be rapidly induced and early events subsequently monitored. NIH3T3 cells were infected with a recombinant retrovirus in which HPV16 E7 gene expression is under the control of a promoter that is negatively regulated by tetracycline, clone 2BN11 [48]. The temporal sequence of the increase in E7 message (Fig. 2A) and the change in p38 phosphorylation state (Fig. 2B) and in activity, measured as the phosphorylation of its substrate, ATF2, (Fig. 2C) was monitored after induction of E7 expression. These experiments showed that the increase in E7 mRNA and reduction of both p38 phosphorylation and activity levels started approximately 2–3 hrs after tet removal, which is in agreement with the previously observed time course for the appearance of E7 mRNA and protein [48]. 10.1371/journal.pone.0003529.g002Figure 2 Time course of changes in HPV16 E7 expression and both phosphorylation and activity of p38 MAPK in 2BN11 cells after tetracycline (tet) removal. A. The activation of HPV16 E7 transcription upon tet removal from the culture medium was determined by RT-PCR as described in in Materials and Methods. After tet removal the cells were collected at the indicated times, total RNA prepared, cDNA generated and the levels of message were determined. The levels of GAPDH were also determined in each sample as internal control. nc: negative control in which the PCR was performed in the absence of a template; pc: positive control included HPV16 E7 cDNA as template. B. Time course of HPV16 E7 expression-dependent inhibition of p38 phosphorylation, total p38 and tubulin in 2BN11 cells after tetracycline removal. C. p38 activity during HPV16 E7 expression (tet removal) was determined by exposing p38 immunoprecipitated from 1 mg of cell lysate to 10 µg GST-ATF-2 as described in methods. The amount of ATF-2 phosphorylated by p38 was analyzed by Western Blotting with an Phospho-ATF-2 (Thr71) antibody and tubulin levels in the homogenate was used as loading control. HPV16 E7 expression activates the cAMP pathway As the cAMP/PKA system has been demonstrated to be involved in both transformation/tumor progression [12], [35], [55], [56] and regulation of p38 [57], we next measured the effect of E7-driven transformation on the levels of cellular cAMP at both early (3 hrs) and late (24 hrs) time points after tet removal. We started with a biochemical assay in which the bioluminescence intensity level inversely correlates with cAMP intracellular mobilization (see Materials and Methods). Cells were first treated with 100 µM of the phosphodiesterase inhibitor, IBMX, in order to observe the activity level of endogenous adenylate cyclase and then were treated with IBMX together with 20 µM of the pharmacologic adenylate cyclase activator, forskolin, to determine the dynamic range of increasing activity of the remaining non E7-stimulated enzyme. The left panel of Figure 3A shows a much greater decrease of bioluminescence after inhibition of intracellular phosphodiesterases by IBMX in the E7-transformed cells (−tet) while the subsequent decrease of bioluminescence after addition of the adenylate cyclase stimulator, forskolin, was similar in control and transformed cells. Calculation of cAMP concentration with a standard curve showed that transformation increased cellular cAMP levels (measured in the presence of IBMX), by approximately 2.5-fold at 3 hrs and 5-fold at 24 hrs without increasing total potential adenylate cyclase activity (Figure 3A right panel). Interestingly, a similar rise in cAMP was observed in late passage compared to early HPK1A cells and, further, blockage of NHE1 activity with 2 µM of its specific inhibitor, HOE642, had no effect on this E7-induced increase in cAMP in either 2BN11 or HPK1A cells (Figure S3). 10.1371/journal.pone.0003529.g003Figure 3 HPV16 E7 expression stimulates cAMP generation in 2BN11 cells. To determine the effect of E7 expression on cAMP production, cAMP levels were measured using A. the biochemical luciferase-based Kinase-Glo System where luminescence is inversely proportional to cAMP levels (for details see Methods). Cells were cultured with or without tet for either 3 or 24 hr and then either not treated (dark bars) or treated for 30 min with either 100 µM IBMX (light bars) or 100 µM IBMX plus 20 µM FSK (stippled bars). Left panel shows levels of luminescence in the various treatments while right panel shows the calculated increase in cAMP concentration with each treatment. Error bars represent the standard error of the mean (SEM) of three independent experiments. B. Time-lapse FRET imaging in live cells transfected with a PKA-based FRET probe, in which cyan and yellow mutants of GFP have been fused to its regulatory and catalytic subunits, respectively, so that cAMP-induced dissociation between the two is detected as FRET changes. Cells were transfected with the FRET cAMP biosensor for 24 hrs and then cultured with or without tet for either 3 or 24 hr. Cells were mounted in a perfusion chamber and the FRET ratio measured during superfusion with ringer alone followed by superfusion first with 100 µM IBMX and then 100 µM IBMX plus 20 µM FSK. Cells were imaged for CFP and YFP every 20 s and the YFP/CFP emission ratios (FRET ratios) obtained. Left panel shows levels of YFP/CFP emission ratios in the various treatments while right panel shows the calculated increase in YFP/CFP emission ratios with each treatment which is relative to increases in cAMP production. Data are the mean±SEM from 32 different cells. C. Pseudocolor images representing YFP/CFP emission ratios recorded under control conditions (plus and minus tet for either 3 or 24 hrs), after exposure to 100 µM IBMX and after stimulation with IBMX (100 µM) plus FSK (20 µM). Each image was scaled according to its high and low values at each time point to show relative level of cAMP at each time point. The dynamic range was 0.9–1.3. Warmer colors correspond to lower cAMP levels. To further verify this specific stimulation of cAMP mobilization in response to HPV16 E7 expression and visualize cAMP dynamics in vivo, we transfected 2BN11 cells with a PKA-based FRET construct composed of green fluorescent protein variants bound to either the PKA catalytic subunit (Cat-YFP) or the PKA regulatory II subunit (RII-CFP) and recorded FRET images with a dual-emission CCD camera. Increased levels of cAMP produce a decrease in FRET signal, measured as YFP-to-CFP emissions ratio, due to cAMP binding to the chimeric PKA reporter and the resultant separation of the catalytic and regulatory subunits. In time course experiments, when the cAMP signal was stable, cells were first treated with IBMX in order to observe the activity level of endogenous adenylate cyclase and then were perfused with IBMX together with forskolin to determine the dynamic range of increasing activity of the remaining non E7-stimulated enzyme. As can be seen in Figure 3B, IBMX superfusion stimulated cAMP production approximately 2-fold at 3 hrs and 4.5-fold at 24 hrs in the E7 transformed cells compared to the control cells, while the further cAMP elevation after forskolin (FSK) treatment was similar in both conditions. Altogether, these data suggest that, indeed, transformation of the cells by E7 stimulates adenylate cyclase-dependent production of cAMP above very low basal levels in the control cells. Figure 3C shows the relative increase in cAMP mobilization as pseudocolor changes in typical FRET experiments. Involvement of PKA and p38 in HPV16 E7-mediated transformation To assess the role and dynamics of PKA and p38 in mediating transformation, we utilized changes in NHE1 activity as the transformation readout because we have previously demonstrated that stimulation of NHE1 occurs early in transformation and is necessary and sufficient for the further development of transformed phenotypes [48]. As seen in Figure 4, the induction of cellular E7 expression and transformation by removal of tetracycline for 24 hrs stimulated NHE1 activity by 69.5±6.2%, n = 15, P<0.02 (blue cross-hatched bar) and this stimulation was blocked by 2 µM of the specific inhibitor of the NHE1, HOE642 (data not shown). Inhibition of PKA by its specific inhibitor, H89, completely abrogated the transformation-dependent increase in NHE1 activity (green stippled bar) while inhibition of p38 activity by either its pharmacological inhibitor, SB203580, or by the transient expression of the dominant negative (dn) mutant for p38alpha (p38AF) further potentiated the transformation-dependent increase in NHE1 activity by approximately 2-fold (black stripped bars). Furthermore, inhibition of p38 either with SB203580 or via transient expression of dnp38alpha blocked the abrogation of the transformation-dependent activation of NHE1 by H89 (red stripped bars). These data strongly suggest that PKA and p38alpha are part of the same pathway in regulating the transformation-dependent stimulation of NHE1 and that PKA is up-stream of p38. This conclusion is further supported by the ability of H89 to block and forskolin (Fsk) to potentiate the E7-dependent down-regulation of p38 phosphorylation (Figures 4B & C) while having no effect on the phosphorylation state of JNK and ERK (Figure S4). 10.1371/journal.pone.0003529.g004Figure 4 PKA is up-stream of p38 in regulating the HPV16 E7 expression-dependent stimulation of NHE1 activity. A. HPV16 E7 expression was induced by removing tetracycline from the culture medium for 24 hrs and the consequences on NHE1 activity of the specific inhibition for 24 hrs of PKA by 10−7 M of its specific inhibitor, H89, and/or p38 by either 10−9 M of its specific pharmacological inhibitor, SB203580, or by the transient expression of a dominant negative (dn) mutant for p38alpha (p38AF, 3 µg) was determined by spectrofluorometry using the pH sensitive probe BCECF-AM. Confluent monolayers were loaded with BCECF and placed in the perfusion cuvette and the monolayer perfused with 135 mM Na+ nominally bicarbonate free-HEPES ringer (pH 7.4) plus (Cont) or minus (E7) 2 µM tetracycline as previously described [48]. To analyze if PKA and p38 interact one each other in regulating NHE1 activity, cells were either first transfected with dnp38 for 48 hrs and then treated with H89 for 24 hrs or were simultaneosly treated with SB plus H89 for 24 hrs and NHE1 activity determined as above. Bars are mean±S.E. and the number of experiments ranged from 5 to 8. B. Non transformed (+tet) and transformed (−tet) cells were not treated (Cont) or treated with either 10−7 M H89 or 10−5 M FSK for 24 hrs and cells were homogenized as described in Materials and Methods. Aliquots containing 50 µg of protein were subjected to 10% SDS-PAGE and total and phosphorylated p38 was determined in Western Blot as in Figure 1. A representative immunoblot is shown. C. Summarized data of densitometrical analyses of p38 phosphorylation is represented as the relative ratio of the density of phospho-p38 against that of total p38. Relative ratio in control, non transformed cells was expressed as 1 arbitrary unit. Control cells are represented by dark bars, H89 treated cells by stippled bars and Fsk treated cells by cross-hatched bars. The data shown are mean values±S.E. (n = 4). p<0.05 () and p<0.01 () when compared with the control value by Student's t test. HPV16 E7-dependent transformation induces PKA-dependent phosphorylation and down-regulation of RhoA However, the steps that mediate the down-regulation of p38 by PKA still need to be identified. RhoA is a potential candidate since it has been shown to be an upstream regulator of p38 in enhancing migration and invasion of breast cancer [11] and pancreatic carcinoma cells [32] and to be inhibited by PKA-dependent phosphorylation on serine 188 [11], [58]. If this is occurring during transformation then we would expect RhoA to be phosphorylated by PKA and to be inhibited by E7-dependent transformation. To test this hypothesis, we first measured the effect of transformation on the phosphorylation state of RhoA via Western Blotting analysis with antibodies specific for phosphorylated Ser-188 of RhoA (Fig. 5A) or via co-immunoprecipitation with anti-phosphoserine followed by anti-RhoA Western Blotting (Fig. 5B) at different times of HPV16 E7 expression (t0, 1 hr, 3 hr, 6 hr, 12 hr). Indeed, transformation induced a significant increase in RhoA phosphorylation in 2BN11 cells. A similar increase in RhoA phosphorylation was observed in E7 expressing HFK cells and late passage HPK1A cells (Figure S5) and blockage of NHE1 actvity with HOE642 had no effect on this process in either 2BN11 or HPK1A cells (Figure S3). To verify that this phosphorylation of RhoA observed upon transformation was due to PKA, we removed tet for 24 hrs in the absence or presence of a pharmacological PKA inhibitor (H89, 100 nM) or activator (forskolin, 10 µM) and we measured RhoA phosphorylation as above with the anti-phosphoSer188. The induction of phosphorylation was blocked by incubation with H89 and potentiated by incubation with forskolin (Fsk) during the time of induction of E7 expression (Fig. 5C). 10.1371/journal.pone.0003529.g005Figure 5 HPV16 E7 induces PKA-dependent phosphorylation of RhoA. A. Western Blotting analysis of the phosphorylation state of RhoA at different times after tet removal (+tet, 1 hr, 3 hr, 6 hr and 24 hr) with antibodies specific for phosphorylated Ser-188 of RhoA (phospho RhoA) followed by polyclonal anti-RhoA antibody (total RhoA). B. Co-immunoprecipitation with anti-phosphoserine antibody followed by anti-RhoA Western Blotting (phospho RhoA) at different times of HPV16 E7 expression (t0, 1 hr, 3 hr, 6 hr, 24 hr). Expression of total RhoA in cell lysates was analyzed by immunoblot analysis using the polyclonal anti-RhoA antibody. C. To test if RhoA-phosphorylation induced by HPV16 E7 expression was dependent on PKA, tetracycline was removed and the cells either not treated or treated with either the pharmacological PKA inhibitor (H89, 100 nM) or activator (forskolin, 10 µM) and RhoA phosphorylation was measured as above (A). As PKA can inhibit RhoA activity via its phosphorylation of serine 188, we next examined the effect of transformation on RhoA activity, first by a pulldown analysis of its binding to GST-fusion protein to the Rho binding domain of Rhotekin which associates preferentially with GTP-bound RhoA [59]. Figure 6A shows that the activity of RhoA was reduced upon E7-dependent transformation with a time course similar to that reported for the up-regulation of NHE1 activity by the same treatment [48] and for RhoA phosphorylation shown above and that H89 treatment blocked while forskolin (Fsk) treatment stimulated this reduction in RhoA activity. However, the biochemical detection of active RhoA via pull-down requires cell disruption and is performed in the presence of detergents which can lead to dissociation of preexisting complexes and, therefore, cause incorrect estimation of the extent of RhoA-GTP association to its down-stream effectors. In contrast, FRET microscopy permits the direct detection of the amount of active RhoA in intact living cells during E7-transformation. For this reason, we next measured RhoA activity state by using a a single-chain CFP/YFP FRET biosensor for RhoA (pRaichu 1297×) [60], which directly monitors the level of the endogenous RhoA-GTP by measuring FRET between the two pairs of GFP mutants fused to the Rho-Binding-Domain (RBD) of Rhotekin. Specifically, in this probe the binding of endogenous GTP-RhoA to RBD displaces YFP and CFP, thereby decreasing FRET efficiency. Using this FRET-based probe we verified that 24 hrs after tet removal, RhoA activity, which was assessed as the ratio of the CFP signal to the YFP signal, was significantly reduced (Figure 6B). Further, treatment with H89 during the transformation period completely reversed this E7-dependent inhibition of RhoA, again indicating that E7-induced RhoA inhibition was dependent on PKA. 10.1371/journal.pone.0003529.g006Figure 6 HPV16 E7 expression activates NHE1 via a PKA-mediated-reduction in RhoA activity. A. Representative Western Blot of three GST-RBD pull-downs showing RhoA activity in 2BN11 cells at different times after tet removal and treated with 100 nM H89 and 10 µM FSK for the indicated times. The lower gel shows the amount of total RhoA in cell lysates. B. Sensitized FRET measurements of 2BN11 cells to determine the effect of E7 expression on RhoA activity in live cells. Cells were transfected with the RhoA biosensor pRaichu-1297× and then cultured with or without tet for 24 hr and treated or not with 100 nM H89 during the 24 hr period. Cells were imaged for CFP and FRET and the relative decreases in CFP/FRET ratios obtained (presented in the central column) indicate a decrease in active RhoA. Data are the mean±SEM from 32 to 37 different cells. **P<0.005. CFP/FRET ratio images are in pseudocolor, with the color indicating the relative value at each pixel (lateral images), such that blue indicates the highest EmCFP/EmYFP ratio (highest RhoA activity), and red reflects the lowest EmCFP/EmYFP ratio (lowest RhoA activity). Scale bar is 10 µm. C. Role of PKA-mediated RhoA signalling on HPV16 E7-induced up-regulation of NHE1 activity. 2BN11 cells were transfected transiently with cDNA for either a phosphorylation dead (pd) RhoA mutant (blue cross-hatched bars), with dominant negative RhoA (dn) mutant (green stippled bars), with constitutively active RhoA (ca) (red striped bars) mutant or with siRNA against RhoA (brown reverse stippled bars). Control cells were transfected with the empty plasmid or non-specific siRNA transfected cells (scrambled) served as the control for RhoA silencing. After 24 hrs for cDNA construct or 48 hrs for siRNA transfection, tetracycline was then removed (−tet) or not (+tet) for a further 24 hrs and NHE1 activity was measured as described in Materials and Methods. Expression of these constructs had no effect on basal NHE1 activity in control, +tet cells. Inactivation of RhoA with the dn mutant and siRNA significantly potentiated the E7-induced stimulation of NHE1 activity while both activation of RhoA with the ca mutant and the block of RhoA phosphorylation by PKA with the pd mutant abrogated the E7-induced stimulation of NHE1 activity. Efficiency of siRNA-mediated RhoA knock-down was analyzed with immunofluorescence assay by using a monoclonal anti-RhoA antibody (green) and the blue fluorescent dye DAPI for staining nuclei (Figure S2). In addition to phosphorylation by PKA, RhoA activity is controlled by the activity balance between other class of RhoA regulating proteins, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) [29]. To assess if E7 alters the balance of GEFs and GAPs thus changing RhoA-GTP loading, we monitored the relative level of GEFs and GAPs in the presence and absence of tet for 24 hr, by using another FRET probe, pRaichu 1293×, consisting of a chimera of RhoA and the RhoA binding domain (RBD) of PKN, sandwiched between YFP and CFP. The relative increase in GEF activity increases the amount of GTP-RhoA and the intramolecular binding of GTP-RhoA to RBD and brings CFP in close proximity to YFP, resulting in an increase in FRET from CFP to YFP [60]. With this probe, we did not find any difference in the FRET ratio during E7-dependent transformation (0.8±0.031 vs 0.79±0.030, n = 20, n.s., for +tet and −tet, respectively) demonstrating that transformation did not influence GEF or GAP activity. These data suggest that a PKA-dependent phosphorylation of RhoA is the critical mechanism of E7 induced-RhoA inhibition. HPV16 E7 expression activates NHE1 through a reduction in RhoA activity via its phosphorylation on serine 188 While the preceding experiments indicate that RhoA is a substrate for PKA in these cells and that its phosphorylation at serine-188 is increased with E7-driven transformation, a critical question in the context of the current study is if the observed PKA-dependent phosphorylation and inhibition of RhoA is necessary for the transformation-dependent up-regulation of NHE1 activity. The phosphorylation of RhoA at serine 188 by PKA has been shown to block its action [46], [61], suggesting that this serine could be the PKA target also in our case. Therefore, as an approach to assess the role of PKA-dependent phosphorylation of RhoA in the activation of NHE1, we mutated the PKA phosphorylation site, serine188, to alanine to create a PKA phosphorylation dead (pd) RhoA mutant [11], [12]. Transfection of control 2BN11 cells with this pd RhoAS188A mutant did not affect basal NHE1 activity levels of +tet cells, while it completely abrogated the increased NHE1 activity induced by expression of HPV16 E7 (Figure 6C, blue cross-hatched bars), confirming the requirement for PKA-dependent phosphorylation of RhoA at serine 188 for the up-regulation of NHE1 activity by E7-dependent transformation. We next determined if it is the alteration in RhoA activity utilizing either dominant negative (dn) mutants, constitutively active (ca) mutants [11] and/or expression utilizing an siRNA against RhoA that underlies stimulated NHE1 activity in transformed cells. Transient transfection of these constructs or the siRNA had no effect on NHE1 activity in +tet cells. Both inactivation of RhoA with the dn N19RhoA mutant (Figure 5C, green stippled bars) or the knock-down of its expression (Figure 6C, brown reverse stippled bars) significantly potentiated the E7-induced stimulation of NHE1 activity while activating RhoA with the ca V14RhoA mutant reduced this stimulation by approximately 80% (Figure 6C, red striped bars). Altogether, these data demonstrate that the PKA-dependent phosphorylation of RhoA is a critical mechanism of HPV16 E7 induced-RhoA inhibition and it is an integral part of the PKA to p38alpha signal transduction module involved in the activation of the NHE1 during transformation. Discussion There still is a dearth of data concerning the very early signal transduction events regulating neoplastic transformation- the first step of the carcinogenic process that is limited to the altered cell. The elucidation of the underlying alterations in signal transduction events mediating the initiation, development and regulation of transformation is a necessary prerequiste for understanding the origin and early development of cancer. As discussed in [17], to accomplish this it is necessary to use an experimental model in which the transformation of a normal, immortalized cell by a single oncogene can be highly controlled, thus permitting the dissection of the sequence of events occuring after the initial alteration and the role of a particular gene or signal transduction pathway. These cells, unlike cancer-derived cell lines, have not aquired genomic instability nor unspecific genetic and epigenetic alterations that can mask the specific transformation-dependent alterations. We utilized this type of inducible cell model with the E7 oncogene of HPV16 to dissect the sequence of the very early steps in signal transduction underlying “the moment” in which a still normal cell becomes transformed. To accomplish this, NIH-3T3 cells were transfected with a construct in which E7 gene expression is under the control of a promoter that is negatively regulated by tetracycline so that E7 is expressed only after tetracycline removal [48] (clone 2BN11). Using this model system, we previously reported that the development of transformed phenotypes (e. g. increased proliferative rate, anchorage-independent growth, serum independence and increased glycolytic metabolism) are under the strict control of E7 expression in these cells and, further, that intracellular alkalinization driven by an up-regulation of the Na+/H+-exchanger (NHE1) is a very early physiological event in transformation and which, in turn, is necessary for the development and maintenance of many of the cellular events occurring later in the transformation process such as serum independence, increased growth rate, anchorage-independent growth and in vivo tumour development in nude mice. For this reason, we here utilized changes in NHE1 activity as the readout for the analysis of the role of a particular signal transduction pathway in transformation in the dissection of the early signaling events occurring up-stream of NHE1 activation during E7-dependent transformation. This experimental model enabled us to recognise and follow a strictly defined sequential progression arising from the initial expression of E7 that rapidly transforms the cells. Our data demonstrate that an intracellular mobilization of cAMP is one of the first signaling events occurring during transformation (Figure 3). Further, we provide evidence that this initial rise in cAMP is followed by a PKA-dependent inhibition of RhoA activity (Figures 6A and 6B) which is necessary for the inactivation of p38alpha (Figure 4B) and which, in turn, regulates the subsequent E7-mediated transformation–dependent early stimulation of NHE1 activity (Figures 4A and 6C). Although the individual importance of each of these signal systems in tumor induction both in vitro and in vivo is well documented, their interrelations in mediating transformation were heretofore still unknown. Indeed, while there is now much data demonstrating the tumor suppressor role of p38 MAP Kinase (see introduction), to date it has not been demonstrated whether the down-regulation of p38alpha plays a role in the early induction of cellular events leading to transformation. Further, if it is involved in transformation, we asked at which point is it located in the transformation process, which up-stream mechanisms regulate its inhibition and what is its down-stream target. We first demonstrated in NIH3T3 cells and in human primary (HPK1A) and secondary (HFK) keratinocytes that E7-dependent transformation specifically reduces p38 phosphorylation. Then, using the inducible experimental model we show that p38alpha plays a key role in the acquisition of the increased activity of the NHE1 which sets the stage for the development of the other transformed phenotypes [48]. Interestingly, p38alpha has also been found to have a negative role in regulating both migration and invasion of pancreatic and breast carcinoma cells [11]–[12], [32] and its activity is reduced in hepatocellular carcinomas in comparison to adjacent normal tissue [62], suggesting that its down-regulation is not limited to just transformation and early tumorigenesis but also in later metastatic/aggressive stages. The involvement of the cAMP/PKA pathway in mediating tumor progression [35] together with the demonstrated integration of p38 with cAMP/PKA signaling in different cell systems [43], [57], [63], [64] focused our attention on the possibility that E7 transformation-dependent down-regulation of p38 involves the cAMP/PKA system. Transformation induced substantial increase in adenylate cyclase-dependent cellular cAMP mobilization (Fig. 2) and incubation with the PKA selective inhibitor, H89, during E7-dependent transformation blocked the down-regulation of p38 phosphorylation while stimulation of the cAMP/PKA system by forskolin (Fsk) enhanced this E7-dependent down-regulation of p38 phosphorylation (Fig. 4B and 4C). Further, inhibition of p38 with either SB203580 or via transient expression of a dominant negative p38alpha mutant (dn p38alpha) blocked the H89-dependent abrogation of the transformation-dependent activation of NHE1 (Figure 3A), further supporting the suggestion that PKA and p38alpha belong both physically and functionally to a common signalling unit, in which PKA acts up-stream to p38 in regulating the transformation-dependent stimulation of NHE1 activity. As RhoA is known be a PKA-signalling effector in a plethora of cell responses [11], [58], [44] and to play an important role in regulating p38 kinase activity in several cellular regulatory contexts [11], [32], [63], [64], we analysed its involvement in underlying the down-regulation of p38 by PKA and in their regulation of E7-dependent activation of NHE1. We observed that RhoA was rapidly phosphorylated at Ser-188 upon E7 expression (Figs. 4A & B) and PKA is involved in this phosphorylation since inhibition of the kinase with H89 or its stimulation by forskolin (Fsk) during the time of induction of E7 expression respectively blocked or potentiated the E7-induced phosphorylation (Fig. 4C). Further, RhoA was inhibited upon E7 expression with a time course parallel to its E7-induced phosphorylation (Figures 6A & B) and inhibition of PKA by H89 reversed the decrease in RhoA activity only in transformed cells (Figure 6B). Interestingly, FRET measurements of Rho-GEFs/GAP activity revealed that the inhibition of RhoA via changes in their activity could be excluded during E7 transformation. Altogether these data suggest that the PKA that is activated in HPV16 E7 transformed cells inhibits RhoA activity through its phosphorylation of RhoA at serine 188. RhoA has been recently reported to be inhibited as a result of transformation driven by TGFbeta [67] or, importantly, HPV16 E7 expression [33] suggesting that this might be a common mechanism. An important question was if the observed PKA-dependent phosphorylation and inhibition of RhoA is necessary for the transformation-dependent up-regulation of NHE1 activity. Analysis of NHE1 activity after transfection of a dn RhoA mutant or siRNA to block RhoA function/expression or a ca RhoA mutant to enhance RhoA function (Figure 6C) demonstrated the importance of the inhibition of RhoA activity in E7 transformation-induced NHE1 activity while transfection of the phosphodead (pd) RhoA mutant revealed the important role for its PKA-dependent phosphorylation in E7 transformation-induced NHE1 activity. The activity of many enzymes is strictly controlled by intracellular pH (pHi) and, therefore, an important question is what extent this E7-induced signal cascade described herein might be altered via feedback by the changes in pHi driven by the stimulated NHE1 [48]. To address this question, we measured cAMP production, phosphorylation of RhoA and the inhibition of p38 in the presence and absence of 2 µM HOE642, a potent and specific inhibitor of the NHE1, in both 2BN11 cells and in early and late passages of HPK1A cells. This treatment has been previously shown to block the development of phenotypes down-stream of the NHE1 in both cell lines [48]. As can be seen in Figure S3, this treatment had no effect on any of these process in either of the cell lines suggesting a strict unidirectionality of this signal cascade both in the rat fibroblast and human keratinocyte models. In conclusion, in this study we have recognised a strictly defined sequential progression arising from the initial expression of E7 that rapidly transforms the cells. Our data demonstrate that an intracellular mobilization of cAMP is one of the first signaling events occurring during transformation and that this initial rise in cAMP is followed by a PKA-dependent inhibition of RhoA activity which is necessary for the inactivation of p38alpha which, in turn, regulates the subsequent E7-mediated transformation–dependent early stimulation of NHE1 activity. An important question is whether this signal cascade is engaged by simple E7 expression or is transformation dependent. The data in Figure 1D shows that only transformation competent E7 is able to inhibit p38 and the use of the HPK1A model system in this study, in which the original viral infection (early passage) simply immortalizes the cells and with time they become transformed (late passage), further validated the concept of transformation specific effects. Importantly, finding the same signal transduction cascade turned on in the late passage HPK1A cells also demonstrated that this signal cascade is engaged during the natural progression of primary human keratinocyte cells that were infected with the actual HPV16 virus and is not just a consequence of the expression of a single viral gene as was also observed previously for NHE1 activity [48]. The elucidation of these signal transduction systems and, more importantly, their interrelations in the early stages of the HPV-dependent transformation processes could provide indications for novel therapeutic strategies and/or a potential marker test for the clinical determination of pre-cancer, HPV-transformed cells in the cervix. Supporting Information Figure S1 Effect of pharmacological treatment and transfection of cDNA plasmids on E7 message expression by RT-PCR analysis. 2BN11 cells were treated with each of the pharmacological agents or transfected with the indicated cDNA encoding vectors as described in Results. RNA was extracted and 500 ng of each sample total RNA were subjected to a semi-quantitative RT-PCR for HPV16 E7 expression analysis. GAPDH: loading control. (0.07 MB TIF) Click here for additional data file. Figure S2 Analysis by immunofluorescence microscopy of the decrease in RhoA expression by RNAi in 2BN11 cells. A. Cells were transfected with either control, non-targeting siRNA (left panel) or RhoA specific siRNA (right panel) as described in Materials and Methods. RhoA was visualized with Alexa Fluor 488 (green) and the nuclei with DAPI (blue). B. Quantification of the intensity of RhoA signals through the cell area normalized to cell number. Data represent mean±SE of three independent experiments. (1.35 MB TIF) Click here for additional data file. Figure S3 Monolayers of either 2BN11 or HPK1A were treated or not with 2 µM of the specific NHE1 inhibitor, HOE642, and analyzed for cAMP, phospho-RhoA or phospho-p38 levels as described in Materials and Methods. In 2BN11 cells the measurements were made in the presence of tetracycline (+tet) or 3 or 24 hours after its removal while in HPK1A cells the comparison was between early and late passage cells. (0.21 MB TIF) Click here for additional data file. Figure S4 Non transformed (+tet) and transformed (−tet) cells were not treated (Cont) or treated with either 10−7 M H89 or 10−5 M FSK for 24 hrs and cells were homogenized as described in Materials and Methods. Aliquots containing 50 µg of protein were subjected to 10% SDS-PAGE and total and phosphorylated JNK (upper blot) or ERK1/2 (lower blot) was determined in Western Blot as in Figure 1. A representative immunoblot is shown for each MAP kinase. (0.39 MB TIF) Click here for additional data file. Figure S5 Western Blotting analysis of the phosphorylation state of RhoA in HFK or HPK1A cells with antibodies specific for phosphorylated Ser-188 of RhoA (phospho RhoA) followed by polyclonal anti-RhoA antibody (total RhoA). A representative immunoblot is shown for each. HFK cells were infected with empty pLXSN vector or pLXSN vector containing wild-type, non-tagged E7 while HPK1A early passage cells were compared with late passage cells. (0.13 MB DOC) Click here for additional data file. We dedicate this paper to the memory of Dr. Antonella Cafarelli whose courage and faith in the face of insurmountable odds taught us the importance of the true values of life. We thank Prof. M. Matsuda (Osaka University, Osaka, Japan) for the Raichu 1297x and 1293x plasmids, Drs. M. Dürst and H. Zur Hausen (German Cancer Research Center, Heidelburg) for the gift of the HPK1A cell lines, Prof. M. Zaccolo (University of Glasgow, UK) for the FRET plasmids and Prof. S. Lugwig (University of Munster, Germany) for the dominant negative p38 plasmid (p38AF). We thank Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario for providing the FRET microscope. Competing Interests: The authors have declared that no competing interests exist. Funding: Italian Association For Cancer Research, l'AIRC. This non-profit organization had no role in any of these activities. ==== Refs References 1 Hanahan D Weinberg RA 2000 The hallmarks of cancer. 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==== Front Br J Cancer Br J Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 6604727 10.1038/sj.bjc.6604727 18854833 Molecular Diagnostics Effect of the chemokine receptor CXCR7 on proliferation of carcinoma cells in vitro and in vivo CXCR7 promotes proliferation of carcinoma cells Meijer J 1 Ogink J 1 Roos E 1* 1 Division of Cell Biology, The Netherlands Cancer Institute, 121 Plesmanlaan, 1066CX Amsterdam, The Netherlands * E-mail: [email protected] 04 11 2008 14 10 2008 99 9 14931501 29 05 2008 18 09 2008 19 09 2008 Copyright © 2008 Cancer Research UK 2008 Cancer Research UK https://creativecommons.org/licenses/by/4.0/ This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. The chemokine CXCL12/SDF-1 and its receptor CXCR4 have been implicated in invasion, survival and proliferation of carcinoma cells. Recently, CXCR7 was identified as a second receptor for CXCL12. We observed that CXCL12 promoted proliferation of CT26 colon and KEP1 mammary carcinoma cells, and this was blocked when CXCR7 was downregulated by ‘intrakines’ or RNAi, but not by CXCR4 inhibitors. The K1R mutant of CXCL12, which acts as a CXCR4 antagonist, also promoted proliferation through CXCR7 and is therefore a selective CXCR7 agonist. The effect of CXCR7 was not due to reduced apoptosis, and CXCR7 mediated chemotaxis of the carcinoma cells towards CXCL12. These results differ from those in a previous report on other carcinoma cells. We conclude that CXCL12 can be a potent growth factor for carcinoma cells by acting on CXCR7. Nevertheless, we observed no effect of complete and stable CXCR7 suppression on the growth of s.c. tumours or lung metastases of KEP1 and CT26 cells. A CXCR7 inhibitor has been reported to reduce growth of other tumours. Our results indicate that this inhibitor may not be applicable to therapy of all carcinomas. tumour growth chemokines carcinoma migration ==== Body pmcCXCR4, the receptor of the chemokine CXCL12/SDF-1, is expressed in many tumours of multiple types including mammary (Muller et al, 2001), colon (Zeelenberg et al, 2003) and pancreatic (Saur et al, 2005) carcinoma, melanoma (Scala et al, 2005) and brain tumours (Rubin et al, 2003). Expression often correlates with the degree of malignancy and metastasis formation (Kaifi et al, 2005; Katayama et al, 2005; Kim et al, 2005; Scala et al, 2005; Schimanski et al, 2005). Indeed, CXCR4 was demonstrated to be required for experimental metastasis of mammary (Muller et al, 2001) and colon (Zeelenberg et al, 2003) carcinomas in the lungs and liver. In addition, CXCR4 was among a set of proteins that, in various combinations, promoted bone metastasis of mammary carcinoma cells (Kang et al, 2003). Furthermore, CXCR4 is induced by hypoxia and was proposed to be responsible for enhanced malignancy of tumour cells in hypoxic areas (Schioppa et al, 2003; Staller et al, 2003). This enhanced malignancy was generally attributed to a role of CXCR4 in invasion, as the ligand CXCL12 is present in many tissues and thought to promote migration into those tissues (Muller et al, 2001). We and others have shown, however, that chemokine receptors can also promote survival and proliferation of tumour cells (Barbero et al, 2003; Rubin et al, 2003; Zeelenberg et al, 2003; Katayama et al, 2005; Sutton et al, 2007; Yang et al, 2007). For CT26 colon carcinoma cells, we provided evidence that CXCR4 is required for the formation of liver and lung metastases, not because of enhanced invasion but rather due to an essential role in the outgrowth of the metastases (Zeelenberg et al, 2003). To demonstrate this involvement of CXCR4 in the CT26 cells, we had expressed an ‘intrakine’ (Chen et al, 1997) that is the ligand CXCL12 extended with a C-terminal KDEL sequence. The intrakine binds to the KDEL receptor that retains resident proteins in the endoplasmic reticulum (ER). The intrakine is therefore retained in the ER, where it binds to newly synthesised CXCR4, which is also trapped. Thus, we generated cells without cell surface CXCR4 (Zeelenberg et al, 2001, 2003) that did not grow out in the lungs and liver. This intrakine approach was based on the generally held assumption that CXCR4 was the only receptor for CXCL12. Recently, however, the RDC1 protein was shown to be a second CXCL12 receptor and was renamed CXCR7 (Balabanian et al, 2005; Burns et al, 2006). It was described to be present on the surface of many tumour cell types and on activated endothelial cells. CXCR7 promoted the survival of tumour cells in minimal medium by preventing apoptosis and did not mediate chemotaxis towards CXCL12. Importantly, a CXCR7 inhibitor greatly reduced the growth of tumours generated from three different tumour cell lines (Burns et al, 2006). The CXCL12-KDEL intrakine should bind CXCR7 and, similar to CXCR4, also trap CXCR7 in the ER. The results we obtained with the CT26 colon carcinoma cells might thus involve CXCR7 rather than, or in addition to, CXCR4. We therefore investigated this possible role of CXCR7 in the CT26 cells as well as in Panc02 pancreatic carcinoma and in KEP1 (knockout/E-cadherin/P53) mouse mammary carcinoma cells. The KEP tumours arose in p53 knockout mice in which the E-cadherin gene was disrupted in the skin and mammary glands. They are quite similar to human invasive lobular carcinomas with high metastatic capacity, and this behaviour is replicated by isolated KEP cells (Derksen et al, 2006). We found that CXCR4 is in fact the receptor required for metastasis. The cells do express CXCR7, however, and we observed CXCR7-mediated effects of CXCL12 that we describe here. We found that CXCL12 strongly enhanced proliferation, mediated by CXCR7, and on some cells, also by CXCR4. This chemokine is therefore a growth factor for carcinomas. Despite the strong in vitro effects, however, we observed no difference in growth rate in vivo of cells in which CXCR7 was completely and stably suppressed, at least not in s.c. tumours and lung metastases. This contrasts with the previously described effects of a CXCR7 inhibitor on other tumour cells, including a carcinoma (Burns et al, 2006). These observations are relevant for the consideration to use this receptor as a target for cancer therapy. Materials and methods Cell culture The source of CT26 and Panc02 cells was described before (Meijer et al, 2006). KEP1 cells were kindly provided by Dr J Jonkers of the Netherlands Cancer Institute. All cells were cultured in DMEM with 10% FCS or in Keratinocyte medium with 1% FCS, both with 100 IU ml−1 of penicillin and 100 mg ml−1 of streptomycin. The Phoenix virus-packaging cell line (http://www.stanford.edu/group/nolan) was cultured in DMEM with 10% FCS and 0.584 g l−1 L-glutamine, the T-cell hybridoma TAM2D2 as described (Zeelenberg et al, 2001). Media, FCS and supplements were from Life Technologies Ltd, Paisley, UK. Generation and transduction of DNA constructs The generation of the CXCL12-KDEL construct was described previously (Zeelenberg et al, 2001). CXCL12 cDNA was generated by PCR from this construct and the mutant K1R-CXCL12 from the K1R-CXCL12-KDEL construct (Zeelenberg et al, 2001). Both were cloned into the pLZRS-IRES-hygroEGFP vector (Zeelenberg et al, 2001). CXCL11-KDEL and CXCL10-KDEL were generated using a one-step RT–PCR kit (Qiagen, Valencia, CA, USA) on RNA isolated from mouse spleen, and inserted into the same vector. All constructs were checked by sequencing. Vectors were transfected into the virus-packaging Phoenix cells and the supernatant used to infect tumour cells. Transduced cells were selected with 0.05 (CT26) or 0.1 mg ml−1 (Panc02 and KEP1) hygromycin (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) and cells with high GFP levels were isolated by FACS sorting. Control cells were transduced with the empty vector and sorted similarly. Luciferase was introduced as described (Meijer et al, 2006). The RNAi sequences GTGTTTCAATTCCAGCATA (CXCR4), CCCACTGTCTACTCAGGAA (CXCR7 RNAi.1: effective) and GAAATCATGTCCTTATCTT (CXCR7 RNAi.3: ineffective) were incorporated into the microRNA-30 sequence (Silva et al, 2005) and cloned into the LMP (MSCV/LTRmiR30-PIG) vector (Open Biosystems, Huntsville, AL, USA) that also contains the GFP cDNA under the control of the PGK promoter. For details see the literature provided by the supplier: http://www.openbiosystems.com/RNAi/RetroviralCloningVectors. The chosen sequences were derived from the RNAi Codex (http://codex.cshl.edu/scripts/newmain.pl). For transduction, we used Phoenix cells as above. Puromycin (1.75 μg ml−1; Sigma-Aldrich, St Louis, MO, USA) was used for selection and cells with high GFP levels were FACS sorted. Generation of CXCR7 antiserum The peptide DYAEPGNYSDINWP (mouse CXCR7, 7–21) was synthesised by the in-house facility and coupled to keyhole limpet haemocyanin. A polyclonal antiserum was generated in rabbits by standard procedures. Flow cytometry Cultures were trypsinised and 106 cells were incubated for 60 min with CXCR4 mAb 12G5 (R&D systems, Minneapolis, MN, USA) or with the CXCR7 antiserum and next with PE-conjugated antibodies against mouse or rabbit IgG, respectively, and analysed using a Becton Dickinson FACSCan using CellQuest software. Cells from tumours were isolated and treated as described (Meijer et al, 2006) and gated to exclude dead and GFP-negative cells so that only tumour cells that are GFP positive were analysed. Proliferation assay CT26 cells (5 × 103 or 104 per well) or Panc02 or KEP1 cells (5 × 104 per well) were seeded in several 96-well plates in DMEM with 10% FCS or Keratinocyte medium with 1% FCS. Luciferase activity was measured every 24 h after the addition of 10 μl of 15 mg ml−1 D-luciferin, using a Xenogen IVIS imaging system (see below: in vivo bioluminescence). Each time, only one plate was measured and a different plate was assessed for each of the time points, so that each plate was measured only once. The data were normalised to 1 at day 0. As this assay did not involve any washing steps, it was quite reproducible, with standard deviations of triplicates of ∼0.02%. To some wells, 100 ng ml−1 CXCL12 (PeproTech Inc., Rocky Hill, NJ, USA) was added or supernatants of CT26 cells transfected with either CXCL12 or K1R-CXCL12 that had been grown in either 10 or 1% FCS. Supernatants of similarly cultured CT26 cells were used as controls. In some experiments, 125 ng ml−1 AMD3100 (Sigma, St Louis, MO, USA) or 1 ng ml−1 TC14012 was added. The TC14012 was synthesised by the in-house peptide facility. Apoptosis assay Adherent cells were trypsinised and both the detached cells and those floating in the medium were collected, fixed with 70% ethanol, stained with propidium iodide and analysed by FACS, without gating. Cells in the G1 (n) and G2/M (2n) phases of the cell cycle could be distinguished. Apoptotic cells have a DNA content lower than n. As a positive control, apoptosis was induced by UV irradiation (50 J m−2, 16 h before the assay). Chemotaxis For the carcinoma cells, chemotaxis was assayed as described (Meijer et al, 2006) with 100 ng ml−1 of CXCL12 in the lower well. Chemotaxis of T-cell hybridoma cells was measured as described (Zeelenberg et al, 2001). Tumour formation in vivo All procedures involving animals were approved by the Animal Welfare Committee. For CT26 cells, we used syngeneic Balb/c mice and for KEP1 cells, nude mice, both 6–8 weeks old. Cells (103) were dispersed in 0.5 ml Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) at 0°C and injected s.c. into mice anaesthetised with 3% 1-chloro-2,2,2-triflouroethyl-diflouromethyl-ether (isoflurane). Alternatively, we injected 0.2 ml PBS containing 105 cells into a tail vein or 0.1 ml containing 106 cells subcutaneously. In vivo bioluminescence imaging D-Luciferin (Xenogen, Alameda, CA, USA) was dissolved at 15 mg ml−1 in sterile PBS and stored at −20°C. Animals were anaesthetised with 3% isoflurane. Luciferin solution was injected i.p. (0.01 ml per g body weight). Light emission was measured 5 min later, using a cooled CCD camera (IVIS; Xenogen), coupled to Living Image acquisition and analysis software over an integration time of 2 min. Signal intensity was quantified as the total counts measured over the region of interest. Results CXCL12 promotes proliferation of CT26 carcinoma cells, but not through CXCR4 We have previously shown that CT26 colon carcinoma cells require CXCR4 for outgrowth of metastases (Zeelenberg et al, 2003). The cells have no surface CXCR4 in vitro (see Figure 1C) but acquire it in vivo. The cells do not express the CXCR4 ligand CXCL12, as determined by RT–PCR (data not shown). We reasoned that if these cells would produce this ligand, in vivo growth of metastases might be further promoted, and therefore we transfected the CXCL12 cDNA. Much to our surprise, the resulting CXCL12-producing cells proliferated faster than control CT26 cells in vitro, despite the absence of CXCR4. In addition, we transfected the K1R mutant of CXCL12, which is a potent inhibitor of CXCR4 (Crump et al, 1997). Remarkably, the mutant also increased proliferation. This indicated that the increase in proliferation was mediated by an, at the time, unknown second CXCL12 receptor. To demonstrate that the effect was due to the expressed proteins, the supernatants were added to wild-type CT26 cells. Proliferation was increased by both supernatants to a similar extent as recombinant CXCL12 (Figure 1A). This was not only seen in minimal medium (1% FCS) but also in optimal medium (10% FCS) in which proliferation was already quite fast. In either of these circumstances, the percentage of apoptotic cells was very low, as assessed by FACS analysis of propidium iodide-stained cells (Figure 1C). The percentage varied between 1 and 4%, as compared with ∼50% in UV-irradiated cells that were used as positive controls, and was not lower in CXCL12-treated cells, showing that the effect could not be due to the suppression of apoptosis. To confirm that the K1R mutant is a CXCR4 inhibitor, we studied chemotaxis of TAM2D2 T-cell hybridoma cells (Zeelenberg et al, 2001) towards CXCL12. As shown in Figure 1B, this is blocked by the CXCR4 inhibitors AMD3100 (Donzella et al, 1998) and TC14012 (Tamamura et al, 2001), and therefore mediated by CXCR4. K1R-CXCL12 did not induce chemotaxis and it inhibited chemotaxis towards intact CXCL12, confirming that it is a CXCR4 antagonist. The two CXCR4 inhibitors did not influence the CXCL12-induced enhancement of proliferation of CT26 cells at concentrations at which they completely inhibited chemotaxis mediated by CXCR4 (Figure 1B). The CXCL12 concentration in the supernatant was ∼100 ng ml−1, based on a comparison of dose–response curves with recombinant CXCL12 (data not shown). The mutant was expressed at similar levels, as deduced from co-expressed GFP, and its concentration in the supernatant is likely roughly comparable, as deduced from ∼35% inhibition of chemotaxis towards 100 ng ml−1 CXCL12 (Figure 1B). For proliferation, concentrations were saturating, as the effect was the same at 50% dilution. For both, the effect was reduced by ∼60% at 10% dilution (data not shown), indicating that the affinity for CXCR7 was comparable. Effect of CXCL12 on proliferation of CT26 cells is mediated by CXCR7 While this work was in progress, the identification of a second CXCL12 receptor was reported: the orphan receptor RDC1/CMKOR1, which was renamed CXCR7 (Balabanian et al, 2005; Burns et al, 2006). We established by RT–PCR that CT26 and KEP1 cells expressed CXCR7 (data not shown). Next, we generated a polyclonal antiserum against a peptide that is the mouse homologue of the epitope of an anti-human CXCR7 mAb (Balabanian et al, 2005). Using this antiserum, we detected CXCR7 on the surface by FACS analysis. As shown in Figure 1C, CT26 cells that were devoid of CXCR4 did express CXCR7 in vitro. Next, we tested CXCL12 on KEP1 mammary carcinoma cells (Derksen et al, 2006). The KEP1 cells did express not only CXCR4 in vitro, but also CXCR7 (Figure 2A). They grow slower than CT26 cells, but again we observed enhanced proliferation although the effect was somewhat less pronounced than in CT26 cells (Figure 2B), and the extent of stimulation was again similar in 1 and 10% FCS. The data shown in Figure 2B were from cells in 1% FCS. The K1R mutant had a similar effect (Figure 2B), indicating that CXCR4 was not involved. The concentration of the ligands was saturating, as a 50% reduction led to identical results. CXCL12 similarly enhanced the proliferation of human carcinoma cell lines, including CAPAN1 pancreatic carcinoma and MDA-MB-231 mammary carcinoma (data not shown). The MDA-MB-231 variant we used did not express CXCR4 in vitro, again showing that CXCR4 was not involved. RNAi would have been the most straightforward approach to discriminate between functions of CXCR4 and CXCR7. As this was initially not successful, and also to obtain independent evidence, we again used the intrakine approach (see Introduction). As CXCR7 also binds CXCL11, whereas CXCR4 does not (Burns et al, 2006), a CXCL11-KDEL intrakine should trap CXCR7 in the ER selectively. However, CXCL11 also binds the CXCR3 chemokine receptor that may be expressed by carcinoma cells (Goldberg-Bittman et al, 2004). We therefore used a CXCL10-KDEL intrakine as control, because CXCL10 also binds CXCR3 but not CXCR7 (Burns et al, 2006). We could not express either of these intrakines in CT26 cells even though we did successfully transfect several other intrakines: CXCL12-KDEL (Zeelenberg et al, 2003), CXCL13-KDEL (Meijer et al, 2006) and recently, also CXCL16-KDEL (Meijer et al, 2008), in these cells. This suggested that CXCR3 is required for their survival and/or growth. However, we did express the two intrakines in KEP1 cells and both at similar levels, as determined by FACS analysis of GFP that was co-expressed from the bicistronic vector. CXCL11-KDEL completely blocked transport to the cell surface of CXCR7, but not CXCR4, resulting in cells without cell surface CXCR7 (Figure 2A). CXCL10-KDEL had no such effect (Figure 2A). The CXCR7-deficient cells did not respond at all to intact or mutant CXCL12 (Figure 2B), providing strong evidence that CXCR7 was the involved receptor. CXCL12-KDEL downregulated both CXCR4 and CXCR7 (Figure 2A), and because of the latter effect also blocked the (K1R)-CXCL12-induced proliferation (Figure 2B). Later, we did achieve knockdown of CXCR7 using an shRNAmir vector that encodes a natural microRNA containing the RNAi sequence (Silva et al, 2005). Sorting for high levels of co-expressed GFP yielded KEP1-RNAi.1 cells in which CXCR7 was completely suppressed (Figure 2A). As a control, we used another CXCR7 sequence (RNAi.3) that was completely ineffective (Figure 2A), as well as a CXCR4 RNAi that completely suppressed CXCR4 but not CXCR7 (Figure 2A), all expressed at similar levels as based on GFP fluorescence (not shown). Again, the CXCR7-deficient cells did not respond at all to CXCL12, whereas the control cells did (Figure 2B), demonstrating that CXCR7 is the involved receptor. CXCR7 RNAi.1 had the same effect in CT26 cells (not shown). Both CXCR7 and CXCR4 enhance proliferation in Panc02 pancreatic carcinoma cells We next tested Panc02 pancreatic carcinoma cells that express both CXCR4 and CXCR7 (Figure 3A). However, the CXCR7 levels were lower than on KEP1 cells, whereas the CXCR4 levels were comparable or higher (compare Figures 2A and 3A). Again, CXCL12 enhanced proliferation (Figure 3B and C). In these cells, this was mediated by both CXCR7 and CXCR4, as a complete knockdown of either (Figure 3A) caused a partial inhibition (Figure 3B and C), whereas the CXCL12-KDEL intrakine, which completely eliminated both from the surface (Figure 3A), reduced proliferation to control levels (Figure 3B). In line with this notion, K1R-CXCL12 had a smaller effect than intact CXCL12, and this was fully blocked by CXCR7 but not CXCR4 knockdown (Figure 3D), again showing that the K1R-CXCL12 mutant activates only CXCR7. We conclude that both receptors can trigger growth-promoting signals. In all these experiments, proliferation was measured by bioluminescence. As this involves no washing steps, the data are quite reproducible with s.e.m. of ∼0.2%. The CXCL12-induced increases in cell number observed after 7 days were therefore always highly significant (P<0.0001). To demonstrate that bioluminescence reflects cell number, we trypsinised the cells from the wells after measuring bioluminescence and counted the cells. An example is given in Figure 3C. The results were similar and the P-values of the differences were <0.0001. CXCR7 mediates chemotaxis towards CXCL12 CXCR7 was reported to differ from CXCR4 in that it did not induce chemotaxis towards CXCL12 (Burns et al, 2006). We tested this using the CXCR4-negative CT26 cells and found that CXCL12 potently induced chemotaxis, which was not inhibited by the CXCR4 inhibitors AMD3100 and TC14012 (Figure 4A). Chemotaxis was maximal at 100 ng ml−1 and half-maximal at 40 ng ml−1 (not shown). The K1R CXCR4 mutant was equally effective, showing again that it is a CXCR7-specific agonist. Migration was inhibited by 60% in cells in which Gi protein signalling was fully blocked by pertussis toxin, showing that it is mediated by both Gi-dependent and Gi-independent mechanisms (not shown). Cells expressing CXCL12-KDEL or CXCR7-RNA.1, in which CXCR7 was suppressed, did not migrate towards CXCL12 (Figure 4B). CXCR4 RNAi had no effect, as expected for cells that do not express CXCR4 in vitro. This chemotaxis was comparable to the extent to that induced by CXCL11 (not shown). This was reduced by ∼50% by CXCR7 RNAi, the remainder likely due to CXCR3, the presence of which was not further studied. We conclude that CXCR7 signals are clearly able to trigger migration but that this apparently depends on the cell type. No effect of CXCR7 on tumour growth in vivo To test the effects on growth in vivo, we subcutaneously injected cells in Matrigel. On account of the instant solidification at 37°C, cells remained dispersed and initially grew as separate single tumours. All cells expressed luciferase and growth was monitored by measuring bioluminescence after the injection of luciferin. As shown in Figure 5A, KEP1 control cells (expressing GFP from an empty vector) and other CXCR7-positive KEP1 controls (the ineffective CXCR7 RNAi.3 and CXCL10-KDEL) grew at the same rate as the CXCR7-deficient cells (CXCR7 RNAi.1 and CXCL11-KDEL). The Td for controls was 54.3 h (s.e.m., 0.7) and for the CXCR7-deficient cells 53.1 h (s.e.m., 0.8). The difference was clearly not significant (P=0.27). In cells isolated from tumours 4 weeks after injection, CXCR7 was still completely suppressed (Figure 5B). (Please note that the FACS profiles differ from those in vitro (see Figure 2), quite likely due to the collagenase treatment. The relevant comparison is with control cells of which the CXCL10-KDEL cells are shown). This lack of effect might be due to the Matrigel that might contain factors that compensate for the lack of CXCR7 signalling. We therefore injected cells subcutaneously, without Matrigel, similarly as the Lewis lung carcinoma cells of which the growth was reported to be reduced by a CXCR7 inhibitor (Burns et al, 2006). Again, we saw no effect of CXCR7 deficiency (Figure 5C). The growth rate was remarkably similar to that in Matrigel: Td=54.0 h (s.e.m., 1.1) for controls and 54.0 h for the CXCR7-deficient cells (s.e.m., 1.4; P=0.48). Finally, we injected CT26 cells into a tail vein and found that lung metastases of CXCR7-deficient cells grew at the same rate as control cells (Figure 6A). The Td for controls was 73.2 h (s.e.m., 3.6) and for the CXCR7-deficient cells, it was 77.2 h (s.e.m., 1.2). The difference was not significant (P=0.34). We conclude that the presence of CXCR7 does not lead to enhanced proliferation in vivo, at least not in s.c. tumours and in lung metastases. The tumour cells were isolated from lung metastases after 24 days and analysed by FACS (Figure 6B). Thus, we confirmed our previous observation (Zeelenberg et al, 2003) that CT26 cells, which are devoid of CXCR4 in vitro, do express CXCR4 in vivo. Furthermore, we found that CXCR7 was still completely suppressed in the CXCR7-RNAi cells, so that the lack of effect on growth is not due to loss of the RNAi effect during the experiment. Discussion CXCR7, the newly discovered second receptor of CXCL12, was reported to be expressed by several different tumour cell types and to mediate a CXCL12-induced increase of survival of mammary carcinoma cells in minimal medium. CXCR7 did not promote proliferation (Burns et al, 2006). We show here that these conclusions are not generally valid for carcinomas. In mouse CT26 colon, KEP1 mammary and Panc02 pancreatic carcinoma cells, CXCL12 increased proliferation and this was mediated by CXCR7, as we conclude based on the following observations. First, CT26 cells express CXCR7 but not CXCR4 in vitro. Second, the K1R mutant of CXCL12, which is a CXCR4 antagonist (Crump et al, 1997), also induced proliferation. Third, downregulation of CXCR7, by either the CXCL11-KDEL intrakine or RNAi, abolished the effects. CXCL10-KDEL (that should affect CXCR3, if present, similarly as CXCL11-KDEL) and an ineffective RNAi sequence served as controls that were expressed at similar levels but did not inhibit. It is particularly noteworthy that the K1R mutant of CXCL12 triggers CXCR7. Lysine at position 1 in CXCL12 is not required for binding to CXCR4, but is essential for the triggering of CXCR4 signals (Crump et al, 1997). The K1R mutant binds CXCR4 but does not signal and therefore acts as a powerful antagonist. Indeed, it strongly inhibited the chemotaxis of T-cell hybridoma cells towards CXCL12. In contrast, it enhanced proliferation, by activation of CXCR7, to a similar extent as the intact chemokine. K1R-CXCL12 is therefore a selective agonist for CXCR7. CXCR7 is not the only CXCL12 receptor that can trigger enhanced growth. CXCR4 can also mediate this effect, as demonstrated using specific inhibitors and antibodies (Barbero et al, 2003; Rubin et al, 2003; Katayama et al, 2005; Sutton et al, 2007; Yang et al, 2007). In fact, we show here that in Panc02 pancreatic carcinoma cells, CXCR4 contributes to the CXCL12-induced enhancement of proliferation. In KEP1 cells, the effect is mediated exclusively by CXCR7. This may be due to the relatively high CXCR7 levels (compare Figures 2A and 3A). It was previously reported that the number of MDA-MB-435 human mammary carcinoma cells expanded in the presence of CXCL12, an effect mediated by CXCR7 (Burns et al, 2006). This occurred in minimal medium containing only 1% FCS, in which extensive apoptosis was observed in the absence of CXCL12. The increase in cell number was due to the prevention of apoptosis by the CXCR7 signals, and apparently not due to an enhanced proliferation. Our results with the CT26 and KEP1 cell lines were quite different. We observed only minimal apoptosis in 1% FCS. Moreover, an equally strong increase in proliferation occurred even in ‘optimal’ medium containing 10% FCS. Furthermore, it was reported that CXCR7 did not mediate chemotaxis of MCF-7 mammary carcinoma cells towards CXCL12 (Burns et al, 2006). This is, however, not generally true as the CXCR4-negative CT26 colon carcinoma cells did migrate towards CXCL12, mediated by CXCR7, as was in fact previously demonstrated for T lymphocytes (Balabanian et al, 2005). We conclude that CXCR7 is able to initiate powerful proliferation- and migration-inducing signals, but that the response apparently differs between cell types. Furthermore, we conclude that the chemokine CXCL12 can act as a powerful growth factor by signals transmitted by either or both CXCR4 and CXCR7. This adds to our previous observation that the chemokine CXCL13 can act as a growth factor for carcinoma cells that express its receptor CXCR5 (Meijer et al, 2006), suggesting that chemokines may be more generally involved in tumour growth. Surprisingly, despite the strong in vitro effects, CXCR7 did not influence tumour growth in vivo, at least not in s.c. tumours and in lung metastases. Both the CXCL11-KDEL intrakine and RNAi caused complete and stable downregulation of CXCR7, but neither had any effect on growth rates in s.c. Matrigel plugs in vivo. This might be attributed to the absence of CXCL12 in these artificially induced tumours, which may lack the proper tumour environment. Supplying the ligand did not seem to have an effect either, however, as the CT26 cells in which CXCL12 or the selective CXCR7 agonist K1R-CXCL12 was expressed did not grow faster in vivo (data not shown), although we can not be sure that production of these chemokines was actually maintained in vivo. Importantly, we also found no evidence for CXCR7 involvement in s.c. tumours, established in a manner comparable with the Lewis lung carcinomas that were shown to be affected by a CXCR7 inhibitor (Burns et al, 2006). Finally, also growth of lung metastases was not influenced by the suppression of CXCR7. Recently, in vivo effects of CXCR7 RNAi on other tumours were reported (Miao et al, 2007). Tumour growth was reduced to different extents. For instance, the final tumour weight of s.c. Lewis lung carcinomas was reduced by ∼50%, whereas the effect on 4T1 mammary carcinoma was larger. Our results clearly indicate that this is not generally applicable to carcinomas, at least not in s.c. tumours or lung metastases. This does not exclude an effect of CXCR7 in other circumstances. In fact, the expression of CXCR7 on many tumour cells and the ubiquitous presence of CXCL12 in tissues suggest that this should occur, perhaps in tissues that are particularly rich in CXCL12 or in particular stages of tumour development. It may also depend on whether, and to what extent, the tumour cells produce autocrine CXCL12. It should, however, not be expected that CXCR7 inhibitors would be applicable to therapy of all carcinomas. We thank Tania Maidment for expert technical assistance with animal experiments, Dr O van Tellingen for help with the IVIS bioluminescence assay system, and Anita Pfauth and Frank van Diepen for FACS sorting. We are grateful to Dr IJ Fidler, Dr T Sauerbruch and Dr J Jonkers for providing cells. This research was supported by Dutch Cancer Society Grant NKI 2003–2856. Figure 1 (A) Proliferation of CT26 colon carcinoma cells in supernatants of CT26 cells, either or not supplemented with 100 ng ml−1 recombinant CXCL12, or in supernatants of CT26 cells expressing CXCL12 or the K1R-CXL12 mutant. The supernatants contained either 1 or 10% FCS. Changes in cell numbers were measured by bioluminescence of these luciferase-expressing cells, after the addition of luciferin. Shown are the results of one of the two experiments with virtually identical results. The s.e.m. are extremely small (∼0.02%) and error bars are therefore not shown. For clarity, we used markers that are far larger than the error bars, and they were slightly displaced when overlapping to make them all visible. (B) Verification of effects on CXCR4 by CXCR4-mediated chemotaxis of TAM2D2 T-cell hybridoma cells in 2 h towards different concentrations (ng ml−1) of recombinant CXCL12, towards supernatants (sup) of control CT26 cells or of CXCL12- or K1R-CXCL12-expressing CT26 cells, or to 100 ng ml−1 CXCL12 added to the K1R-CXCL12-containing supernatant. AMD3100 (125 ng ml−1) and TC14012 (1 ng ml−1) inhibit completely at these concentrations, whereas they have no effect on CXCL12-induced proliferation. Shown are the results of one of the four experiments with similar results. (C) FACS analysis of CXCR4 and CXCR7 on CT26 cells grown in vitro and of PI-stained cells, either UV-irradiated or not 16 h before. Figure 2 (A) Effects of intrakines or RNAi on CXCR4 and CXCR7 surface levels of KEP1 mammary carcinoma cells. Open histograms: negative controls (second antibody only for CXCR4 or preimmune serum and second antibody for CXCR7). Grey filled histograms: either CXCR4 mAb (upper row) or CXCR7 antiserum (lower row). (B) Effect of intrakines and RNAi on proliferation of KEP1 cells in 1% FCS-containing supernatants, as in Figure 1. Concentrations of both CXCL12 and mutant were saturating, as the effects were similar at 50% dilution (not shown). Results are from one of the two experiments with virtually identical results. Figure 3 (A) Effects of intrakines or RNAi on CXCR4 and CXCR7 surface levels of Panc02 pancreatic carcinoma cells. Open histograms: negative control (preimmune serum and second antibody for CXCR7). Grey filled histograms: CXCR4 mAb; black filled histograms: CXCR7 antiserum. For clarity, only the negative control for the polyclonal antiserum is shown. Therefore, the fluorescence of CXCR4-suppressed cells seems lower than this control. In reality, they coincide with the proper control. (B) Proliferation of these cells in the presence or absence of 100 ng ml−1 CXCL12, as measured by bioluminescence. (C) The number of cells per well after 7 days in the same experiment as in panel B, manually counted after trypsinisation. (D) The effect of CXCL12-KDEL intrakine and CXCR4 or CXCR7 RNAi on the proliferation of Panc02 cells in supernatants of control or CXCL12- or K1R-CXCL12-expressing CT26 cells, as in Figure 1A. Results in panels B–D are of one of the three experiments with almost identical results. Figure 4 Chemotaxis of CT26 colon carcinoma cells in 6 h towards 100 ng ml−1 CXCL12. (A) In the presence or absence of the CXCR4 inhibitor AMD3100 (125 ng ml−1) or TC14012 (1 ng ml−1). Data shown are of one experiment of two with similar results. (B) The effect of CXCL12-KDEL intrakine and CXCR4 or CXCR7 RNAi. Results are averages of two experiments. Error bars represent s.e.m. Figure 5 Effect of CXCR7 suppression on growth of KEP1 mammary carcinoma cells in vivo. (A) In Matrigel plugs. CXCR7-positive controls: untransfected, CXCL10-KDEL and CXCR7-RNAi.3 (see Figure 2A), n=2 for each, total n=6. CXCR7-negative cells: CXCR7-RNAi.1, CXCL11-KDEL (see Figure 2A), n=2 for each, total n=4. Error bars represent s.e.m. (B) CXCR7 levels on cells isolated from plugs. Note that background is higher for these collagenase-treated cells than for untreated cultured cells (compare negative controls with Figure 2A). The profile of CXCR7-deficient cells coincides with the negative controls, showing that the effects of intrakine and RNAi are stable for at least 4 weeks in vivo. (C) The growth of s.c. injected cells (without Matrigel), as in panel A. CXCR7-deficient cells now also include cells expressing CXCL12-KDEL. Total of both control and deficient cells, n=6. Figure 6 (A) Proliferation of lung metastases of CT26 colon carcinoma cells after tail vein injection of mice with control cells (n=5) or CXCR7-deficient cells (CXCR7 RNAi.1, n=5). Error bars represent s.e.m. 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==== Front PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1902342108-PLPA-RA-0993R210.1371/journal.ppat.1000210Research ArticleGenetics and Genomics/Gene FunctionInfectious Diseases/Bacterial InfectionsMicrobiologyMicrobiology/Cellular Microbiology and PathogenesisThe Bicarbonate Transporter Is Essential for Bacillus anthracis Lethality Bacillus anthracis Bicarbonate MetabolismWilson Adam C. Soyer Magali Hoch James A. Perego Marta * The Scripps Research Institute, Department of Molecular and Experimental Medicine, Division of Cellular Biology, La Jolla, California, United States of America Gilmore Michael S. EditorSchepens Eye Research Institute, United States of America* E-mail: [email protected] and designed the experiments: ACW JAH MP. Performed the experiments: ACW MS. Analyzed the data: ACW JAH MP. Wrote the paper: ACW JAH MP. 11 2008 21 11 2008 4 11 e100021027 8 2008 20 10 2008 Wilson et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO2/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO2 and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO2, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO2/bicarbonate, and suggests it may be a target for antibacterial intervention. Author Summary Hospital-acquired bacterial infections are a growing public health concern. The bacteria responsible for these infections are often resistant to multiple antibiotics, making the problem of nosocomial infections even more dramatic and the need for new antibacterial treatment more urgent. Bacteria rely on a variety of mechanisms in order to trigger an infection, but the first step must be the recognition of the host environment. In this work, we have identified the first component of a pathway that allows a bacterial pathogen, Bacillus anthracis, to recognize the environment in which to thrive during an infection, i.e. the blood of the host. The molecule sensed is bicarbonate, a critical component in the blood for maintaining its correct pH. Bicarbonate is essential to induce the virulence factors of B. anthracis and is most likely relevant in infections by other organisms such as Streptococci, E. coli, Borrelia, Clostridium botulinum, and Vibrio cholera. Our identification of the B. anthracis transporter responsible for the internalization of bicarbonate and the activation of virulence factor production provides a new target for new antibacterial intervention that could be effective on a variety of bacterial pathogens. ==== Body Introduction Bacillus anthracis is a Gram-positive, endospore-forming bacterium that is the etiological agent of anthrax. Anthrax is primarily a disease of grazing herbivores with human infections as the result of either direct contact with infected animal products or intentional dispersion of anthrax spores as a biological weapon. Anthrax can manifest as localized, cutaneous infections or as systemic infections resulting from spore inhalation, ingestion, or spread of cutaneous infections. While localized, cutaneous infections are curable, systemic infections are almost uniformly fatal with death occurring within days of initial infection [1]. Virulence in the mammalian host requires expression of both the anthrax toxin and the antiphagocytic capsule. The tripartite anthrax toxin is encoded by three non-contiguous genes, lef, cya and pagA, carried on the virulence plasmid pXO1 [2]. lef encodes Lethal Factor (LF), a zinc metalloprotease targeting host MAP-kinase signaling [3], cya encodes Edema Factor (EF), an adenylate cyclase that increases cellular cAMP levels [4], and pagA encodes Protective Antigen (PA), which forms a pore allowing entry of toxin components [5]. The antiphagocytic, poly-D-glutamic acid capsule, which is essential for bacterial dissemination in the host [6], is encoded by genes in the cap operon carried on virulence plasmid pXO2 [7],[8]. The regulatory protein AtxA, encoded by the atxA gene on pXO1, is required for the transcription of both the toxin genes and the capsule operon [9],[10]. Control of AtxA, in turn, is integrated into several metabolic regulatory circuits, including the sporulation phosphorelay through AbrB [11] and the phosphoenolpyruvate-dependent phosphotransferase system via regulated phosphorylation/dephosphorylation of histidine residues [12]. Many environmental cues influence the expression of B. anthracis virulence factors, one of the earliest identified being the effect of CO2/bicarbonate levels on capsule production and virulence [13]. Elevated CO2/bicarbonate levels are thought to serve as a signal of the mammalian host environment and a cue to induce expression of virulence factors. Incubation of B. anthracis in media supplemented with sodium bicarbonate and grown under elevated CO2 levels (above 5%) results in an approximately 10-fold increase in transcription of all three toxin genes [14] and a more than 20-fold increase in capsule operon transcription [15]. AtxA is required for CO2/bicarbonate induction of toxin and capsule genes, however, AtxA expression is unaffected by increased CO2/bicarbonate levels [16],[17]. The presence of additional CO2/bicarbonate regulatory components on the main chromosome is suggested by the observation that pagA transcription is induced by CO2/bicarbonate in a pXO1− pXO2− strain when atxA and pagA only are supplied on multicopy plasmids [18]. Additionally, an uncharacterized gene carried on pXO1 may also play a role in CO2/bicarbonate regulation of toxin expression [19]. Notwithstanding these indirect suggestions of more extensive regulation, additional CO2/bicarbonate regulatory components have yet to be directly identified. Without a mechanistic basis for the CO2/bicarbonate regulation of virulence factor expression, our focus turned to identifying conserved responses to CO2/bicarbonate homeostasis and relating these pathways to B. anthracis. Study of CO2/bicarbonate metabolism is complicated by its labile nature, with CO2, H2CO3, HCO3 −, and CO3 2− existing in equilibrium depending on pH, temperature, and partial pressure of CO2. Under typical biological conditions, CO2 generally diffuses across membranes; once inside the cell, carbonic anhydrases can actively interconvert CO2 and bicarbonate. On the other hand, bicarbonate is impermeable across lipid bilayers, and many cellular systems rely on dedicated transporters to import bicarbonate [20]. One of the best-studied bacterial bicarbonate transporters is the CmpABCD ABC transport system in the cyanobacterial species Synechococcus PCC 7942 (Figure 1) [21]. In this bacterium, elevated CO2 concentration around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is essential for efficient carbon fixation. Synechococcus uses this high affinity bicarbonate transporter to import and accumulate inorganic carbon (such as HCO3 −), which can then be converted by carbonic anhydrase to CO2 in the presence of Rubisco in a specialized compartment called the carboxysome [22]. 10.1371/journal.ppat.1000210.g001Figure 1 Schematic representation of the ABC-type transporters of bicarbonate in Synechococcus. The Substrate Binding Protein CmpA is presumably anchored to the periplasmic face of the cytoplasmic membrane via a lipid anchor attached to a conserved cystine at the end of a lipoprotein signal peptide [45]. The SBP domain is composed of two domains organized in a C-clamp shape [23]. In Synechococcus, a SBP domain is also present intracellularly at the carboxy terminal end of the CmpC ATPase subunit. The CmpC protein is absent in B. anthracis and in S. pyogenes. Also, in S. pyogenes, the SBP domain is fused at its C-terminal end to the CmpB-like permease domain. Here we report the identification of an ABC transporter with similarity to the Synechococcus CmpABCD system that is essential to virulence in B. anthracis. Deletion of the genes for the transporter reduced bicarbonate uptake and eliminated toxin gene induction ex vivo in response to bicarbonate. More importantly, the strain lacking the transporter was avirulent in a mouse model of anthrax infection, demonstrating the importance of this pathway for recognition of the host environment and pathogenesis. Results Identification of a putative bicarbonate ABC transporter Despite the recognized role of CO2/bicarbonate in toxin synthesis, the mechanism linking CO2/bicarbonate levels to toxin regulation and virulence of B. anthracis remains to be characterized. As a reverse genetic approach to identify components of the CO2/bicarbonate regulatory pathway, we searched the B. anthracis Sterne strain genome (GenBank: AE017225) for protein sequences similar to the products of the cmpABCD operon encoding the bicarbonate transporter of Synechococcus elongatus PCC 6301 (GenBank: AP008231). Unlike many ABC transporters, which are characterized largely based upon multisubunit organization including proteins with ABC-type ATP-binding domains in association with hydrophobic permease domains, identification of CmpABCD-like bicarbonate ABC transporters is aided by structural features of the substrate binding domain for bicarbonate and the highly similar nitrate transporters [23],[24]. A BLASTP search revealed similarity between components of CmpABCD system and the products of the BAS2714-12 and BAS4675-77 genes (Table 1). Both operons had yet to be characterized but appeared to encode components of ABC transporters. BAS2714 and BAS4676 encode ATP-binding proteins, BAS2713 and BAS4675 are predicted to encode substrate binding proteins, and BAS2712 and BAS4677 are likely transmembrane permease proteins. Unlike cmpABCD, which encodes two ATP-binding proteins (CmpC, also containing a CmpA-like substrate-binding protein, and CmpD) (Figure 1), the two B. anthracis loci encode only one single-domain ATP-binding protein (BAS2714 or BAS4676). A role in bicarbonate transport was suggested by the presence in the BAS2713 and BAS4675 proteins of a TauA domain (NCBI Accesion Number COG0715), a conserved element associated with periplasmic substrate binding components of ABC transporters specific for nitrate, sulfonate, or bicarbonate. Furthermore, a fold-recognition bioinformatic analysis by the FFAS03 server revealed a highly significant score (−60.5 to −64.8) between BAS2713 or BAS4675 and the bicarbonate (CmpA) and nitrate (NrtA) substrate binding protein, suggesting a structural and functional homology despite the limited sequence conservation. Based on similarity to the CmpABCD proteins and conserved features shared by bicarbonate transporters, the BAS2714-12 and BAS4675-77 systems appeared to be good candidates to function as a B. anthracis bicarbonate ABC transporter. 10.1371/journal.ppat.1000210.t001Table 1 Similarity analysis between the B. anthracis BAS2714-12 and BAS4675-77 transporter and the Synechococcus bicarbonate transporter CmpABCD. CmpC CmpD CmpA CmpB Gene BAS2714 BAS2714 BAS2713 BAS2712 P-value BAS4676 BAS4676 BAS4675 BAS4677 Identity 5e-49 1e-43 6e-46 5e-37 .11 5e-06 7e-26 1e-09 % 40 38 40 34 23 25 31 25 P-value and percentage of identity were obtained using BlastP. Deletion of BAS2714-12 eliminates bicarbonate-induced toxin gene expression To investigate the role of BAS2714-12 and BAS4675-77 in bicarbonate metabolism and virulence, B. anthracis 34F2 (pXO1+ pXO2−) derivative strains were generated containing a markerless deletion of the three genes annotated as BAS2714-12 or BAS4675-77. As described in the Experimental Procedures, using plasmid pAW091, a region from 97 nucleotides upstream of the translation start site of BAS2714 to 33 nucleotides upstream of the termination codon of BAS2712 was deleted. This completely eliminated the coding regions of BAS2714 and BAS2713 while leaving a small portion of the 3′ end of the BAS2712 coding sequence and the entire intergenic region between BAS2712 and BAS2711 intact so as to leave potential regulatory sequences controlling expression of the downstream gene, BAS2711. Similarly, for the deletion of BAS4675-77, the integration of plasmid pAW093 resulted in the deletion of a region from 70 nucleotides downstream of the translation start site of BAS4675 to 52 nucleotides upstream of the termination codon of BAS4677. This completely eliminated the coding regions of BAS4676 while leaving a small portion of the 5′ end of the BAS4675 coding sequence and a small portion of the 3′ end of the BAS4677 coding sequence intact so as to leave potential regulatory sequences controlling expression of genes upstream and downstream of the operon. Under all conditions tested, deletion of BAS2714-12 or BAS4675-77 had no significant effect on growth relative to the parental strain 34F2 (Figure 2 and data not shown). 10.1371/journal.ppat.1000210.g002Figure 2 Cell growth and virulence gene expression in B. anthracis 34F2 and 34F2ΔBAS2714-12 under various growth conditions. For β-galactoside assays, cells carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in medium supplemented with Kanamycin. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. (A) pagA-lacZ reporter strains grown in LB broth in air at 37°C. (B) pagA-lacZ reporter strains grown in R Media with 0.8% NaHCO3 under 5% CO2 at 37°C. (C) atxA-lacZ reporter strains grown in R Media with 0.8% NaHCO3 under 5% CO2 at 37°C. Symbols in all panels: -□- 34F2 cell growth; -○- 34F2ΔBAS2714-12 cell growth; -▪- 34F2 LacZ expression; -•- 34F2ΔBAS2714-12 LacZ expression. Expression of pagA, encoding the PA subunit of anthrax toxin, was monitored in different growth conditions, simulating host and non-host environments, known to affect virulence gene expression. A pagA-lacZ reporter on the replicative vector pTCV-lac [25] was transformed into the parental, the ΔBAS2714-12 and ΔBAS4675-77 strains and used to monitor pagA expression levels through β–galactosidase activity. To replicate non-host conditions that result in low-level expression of toxin genes, the strains were grown in LB broth in air under standard laboratory conditions (Figure 2A) while growth in defined R-medium in the presence of 0.8% NaHCO3 in a 5% atmosphere was used to mimic the host environment (Figure 2B). Deletion of the BAS4675-77 genes did not affect pagA expression in either growth condition tested indicating that this transport system did not have a role in bicarbonate transport and/or regulation of toxin gene expression and therefore was not further analyzed (data not shown). The deletion of the BAS2714-12 genes did not affect pagA expression when cells were grown in LB in air suggesting that this system does not contribute to toxin expression under non-host growth conditions (Figure 2A). In contrast, when the strains were grown in defined R-medium under conditions known to induce toxin expression (0.8% NaHCO3 and 5% CO2), induction of pagA in the BAS2714-12 deletion strain was abolished compared to the parental strain (Figure 2B). These observations suggested that BAS2714-12 is required for induction of toxin expression under CO2/bicarbonate growth conditions believed to mimic the mammalian host. The primary regulatory protein of toxin gene expression in B. anthracis, AtxA, is required for the observed induction of toxin expression in response to CO2/bicarbonate [18],[19]. Previous studies demonstrated that transcription of atxA is not directly induced in response to elevated CO2/bicarbonate [16]. To investigate the contribution of BAS2714-12 to atxA transcriptional regulation, an atxA-lacZ reporter carried on the pTCV-lac vector was electroporated in the 34F2 and 34F2ΔBAS2714-12 strains. Under the growth conditions that induced toxin expression and under which we observed a substantial difference in pagA expression, atxA expression was unchanged in 34F2ΔBAS2714-12 relative to the parental strain (Figure 2C). Thus, consistent with the lack of effect on atxA by the growth in the presence of CO2/bicarbonate [16], disruption of bicarbonate metabolism through deletion of the putative bicarbonate transporter BAS2714-12 did not affect atxA transcription. To ensure that deletion of BAS2714-12 was responsible for the observed phenotypes, the BAS2714-12 deletion strain was complemented with these genes carried on a replicative plasmid. The BAS2714-12 locus, as well as a region 640 base pairs upstream of BAS2714 that may carry potential promoter and regulatory sequences, was cloned in the multicopy vector pHT315 to generate plasmid pAW144, and both plasmids were electroporated into strain 34F2ΔBAS2714-12. Expression of protective antigen was monitored by Western blotting on culture supernatants (Figure 3). When grown under toxin-inducing conditions, 34F2 supernatant samples contained detectable amounts of PA while 34F2ΔBAS2714-12 supernatant samples did not contain detectable levels of PA. When carrying the empty plasmid pHT315, PA remained undetectable in supernatant samples of the BAS2714-12 mutant strain while the presence of pAW144 restored PA expression, demonstrating that deletion of BAS2714-12 was, in fact, responsible for the elimination of toxin induction. 10.1371/journal.ppat.1000210.g003Figure 3 Western blotting using α-PA antibody. Strains grown in R Media with 0.8% NaHCO3 under 5% CO2 at 37°C supplemented with Erythromycin and Lincomycin as appropriate. Amount of sample loaded on 10% SDS-PAGE gel was normalized relative to cell density. Lane 1, MagicMark XP; Lane 2, 34F2 strain; Lane 3, 34F2ΔBAS2714-12 strain; Lane 4, 34F2ΔBAS2714-12 strain carrying plasmid pHT315; Lane 5, 34F2ΔBAS2714-12 strain carrying plasmid pAW144. Deletion of BAS2714-12 reduced bicarbonate uptake The sequence similarity to known bicarbonate transporters and the elimination of bicarbonate-induced toxin expression following deletion suggested that BAS2714-12 may function as a bicarbonate transporter. To directly test the function of BAS2714-12 in bicarbonate transport, we compared the uptake of radiolabeled NaH14CO3 in the parental and mutant strain. Strains 34F2 and 34F2ΔBAS2714-12 were grown in R-media without added NaHCO3 in the presence of 5% CO2 to an OD600 of 0.4. NaH14CO3 was added to each culture, and uptake of NaH14CO3 was measured at several time points by liquid scintillation counting (Figure 4). The uptake of 14C in the 34F2ΔBAS2714-12 strain occurred at a significantly lower rate (6 fold) than in the parental 34F2 strain, indicative of disruption of bicarbonate uptake and providing further evidence that BAS2714-12 functions as a bicarbonate transporter. 10.1371/journal.ppat.1000210.g004Figure 4 Uptake of H14CO3 − by B. anthracis 34F2 and 34F2ΔBAS2714-12. Cells were grown in R Media without added NaHCO3 under 5% CO2 at 37°C to OD600 = 0.4. NaH14CO3 was added at time 0 and cell samples collected at times indicated. H14CO3 − uptake was determined by 14C accumulation in cells as measured by liquid scintillation counting; -▪- parental 34F2 strain, -•- 34F2ΔBAS2714-12 strain. Data was obtained from 3 independent cultures and error bars represent standard deviation from the mean. Carbonic anhydrase inhibitors do not significantly affect bicarbonate induction of toxin expression Bicarbonate transporters import membrane-impermeable bicarbonate while carbonic anhydrase enzymes interconvert bicarbonate and CO2 and, thus, are able to convert membrane-permeable CO2 to bicarbonate [20]. Induction of toxin expression in B. anthracis is influenced by both bicarbonate and CO2, and, given the interconversion between the two compounds, separation of the relative influence of each compound on virulence has been difficult. The identification and deletion of the bicarbonate transporter essential to toxin induction now provided a tool to further probe the mechanism of induction. A panel of available carbonic anhydrase inhibitors was tested including acetazolamide, ethoxyzolamide, hydrochlorothiazide and topiramate. Hydrochlorothiazide was found most efficacious as measured by reduced toxin expression levels (data not shown). In the presence of NaHCO3 and CO2, expression of pagA-lacZ in the parental strain 34F2 was identical with or without hydrochlorothiazide (Figure 5A). However, the residual pagA-lacZ expression in strain 34F2▵BAS2714-12 was completely inhibited by hydrochlorothiazide. Without added NaHCO3 but in the presence of atmospheric 5% CO2, hydrochlorothiazide reduced the expression of pagA-lacZ in the parental strain 34F2 and hydrochlorothiazide further reduced the level of pagA-lacZ expression in the 34F2ΔBAS2714-12 strain (Figure 5B). These data suggest that the residual pagA-lacZ expression in the absence of added NaHCO3 is due to the conversion of CO2 to −HCO3 by carbonic anhydrase. These data also reinforce the concept that it is bicarbonate, and not CO2, that directly signals induction of virulence factor expression under host growth conditions. 10.1371/journal.ppat.1000210.g005Figure 5 Toxin gene expression in B. anthracis 34F2 and 34F2ΔBAS2714-12 in the presence of the carbonic anhydrase inhibitor hydrochlorothiazide. Cells carrying a pagA-lacZ fusion on the replicative vector pTCV-lac or promoter-less pTCV-lac were grown in medium supplemented with Kanamycin. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. (A) Strains grown in R Media with 0.8% NaHCO3 under 5% CO2 at 37°C. (B) Strains grown in R Media without added NaHCO3 under 5% CO2 at 37°C. Symbols in all panels: -▪- 34F2 pagA-lacZ expression; -□- 34F2 pagA-lacZ expression with 900 µM hydrochlorothiazide; -•- 34F2ΔBAS2714-12 pagA-lacZ expression; -○- 34F2ΔBAS2714-12 pagA-lacZ expression with 900 µM hydrochlorothiazide; -▴- 34F2 promoter-less pTCV-lac expression; -▵- 34F2 promoter-less pTCV-lac expression with 900 µM hydrochlorothiazide. Deletion of BAS2714-12 rendered B. anthracis avirulent in a mouse model of infection While the role of CO2/bicarbonate in the induction of virulence gene expression is well demonstrated in laboratory batch cultures (ex vivo), no evidence has been provided yet that this role also is relevant in B. anthracis cells growing in the infected host (in vivo). In order to investigate whether the inability to import bicarbonate had any effect on the virulence of B. anthracis, an animal model of infection was used that employs a mouse strain highly susceptible to the unencapsulated Sterne strains [26]. Six week old female mice of the complement deficient strain A/J (The Jackson Laboratory) were injected subcutaneously with 106 spores of the parental strain 34F2 or the 34F2▵BAS2714-12 mutant strain and monitored over the course of 12 days. A group of five A/J mice were infected for each strain. Within 54 hours, a mouse in the 34F2 control group infected with the parental strain showed significant swelling and reduced physical activity. Death occurred within the following 10 hours. In this group, two other mice became symptomatic and died within 72 hours, a fourth after 84 hours and the fifth mouse after 96 hours from the infection (Figure 6). In contrast, all 5 mice in the group infected with the mutant survived up to 12 days with no obvious signs of swelling or disease. While the parental strain is fully virulent in this mouse model, the ΔBAS2714-12 is avirulent, demonstrating for the first time that bicarbonate transport is essential to B. anthracis pathogenesis in vivo. 10.1371/journal.ppat.1000210.g006Figure 6 34F2 and 34F2ΔBAS2714-12 in a mouse model of infection. A total of 10 6-week-old female A/J mice, 5 for 34F2 (-•-) and 5 for 34F2ΔBAS2714-12 (-▪-), were subcutaneously injected with 106 spores and monitored over the course of 12 days. Mice were visually monitored daily for activity, and data expressed as percentage of survival. Discussion B. anthracis must integrate numerous environmental signals to effectively replicate and induce disease. B. anthracis relies on a multiphasic lifestyle: non-metabolically active spores are necessary for infection and spread between hosts but are themselves incapable of replication while the metabolically active vegetative cells replicate and cause disease in the host but are incapable of dissemination between hosts. During the course of an infectious cycle, the pathways leading either to development through sporulation or to pathogenesis through toxin and capsule production are mutually exclusive suggesting the existence of a regulatory balance between the two pathways [27]. What the bacterium recognizes in the host as signals to induce pathogenesis mechanisms and the nature of the mechanisms necessary for commitment to development or pathogenesis remain poorly understood. Herein, we have identified an essential component for the induction of virulence gene expression in response to host bicarbonate levels and have exploited this finding to understand the extracellular and intracellular signals controlling virulence. Our data demonstrated that the BAS2714-12 genes encode a previously uncharacterized bicarbonate ABC transporter. Similar ABC transporters have been identified and characterized in photosynthetic bacteria [21], but this is the first report of an ABC transporter involved in virulence in a pathogenic bacterial species. The BAS2714-12 system was originally annotated (Gen Bank: AE017225) as a putative sulfonate transporter, largely due to similarity to the characterized Ssu ABC transporter in B. subtilis. However, given the conservation between bicarbonate, nitrate, and sulfonate ABC transporters, the lack of characterized bicarbonate transporters in Gram-positive bacteria, and the difficulty in predicting the function of ABC transporters based upon nucleotide sequence, assignment of substrate specificity is ambiguous. Here we have shown that the ABC transporter encoded by BAS2714-12 is required for internalization of 14C-labeled bicarbonate. Together with the observation that the addition of taurine, a substrate of the B. subtilis Ssu system [28] and a commonly available sulfonate compound in the host, does not affect toxin gene expression and does not compete with bicarbonate induction (unpublished data) argues for the BAS2714-12 system as being specific for bicarbonate transport. The additional observation that the deletion of the BAS2714-12 genes eliminated the bicarbonate-dependent induction of toxin gene expression confirms a role for this transporter system in bicarbonate metabolism in B. anthracis. Deletion of BAS2714-12 eliminates bicarbonate induction of pagA expression in growth conditions that mimic the animal host environment but does not significantly alter expression under non-inducing conditions. In LB media without added bicarbonate or CO2, conditions which mimic non-host and non-toxin inducing conditions, pagA expression is unaltered in the deletion strain. In contrast, when grown in R-media with added bicarbonate and CO2, conditions which mimic host and toxin inducing conditions, pagA expression remains very low in the mutant strain while pagA expression is strongly induced in the parental strain. These observations suggest that basal levels of pagA expression are unaffected by BAS2714-12 deletion, but, instead, the specific induction by bicarbonate requires the presence of BAS2714-12. Despite the strong effect of the BAS2714-12 deletion on toxin induction, the deletion strain shows no difference in growth under any condition tested relative to the parental strain, suggesting bicarbonate uptake through BAS2714-12 does not significantly contribute to non-virulence metabolic pathways under laboratory growth conditions. The deletion of BAS2714-12 can be complemented by supplying the locus in trans on a replicative plasmid. Small differences in pagA expression between the parental and deletion-complementation strains are likely due to differences in expression or gene copy number of BAS2714-12 carried on a relatively high copy number plasmid (15 copies/cell [29]). The presence of the AtxA regulator is required for high-level expression of pagA in response to CO2/bicarbonate, but transcription of atxA is not directly regulated by CO2/bicarbonate [16]. Consistent with these observations, deletion of BAS2714-12 did not affect atxA transcription (Figure 2C). The effect of BAS2714-12 on virulence in an in vivo animal model is drastic: while, as expected, infection of A/J mice with spores of the parental 34F2 strain quickly resulted in extensive edema followed by death [30], the infection with spores of the deletion strain showed no visual signs of infection and all mice survived to the end of the experiment (Figure 6). These results confirmed a correlation between the bicarbonate-dependent induction of toxin gene expression ex vivo and the virulence of B. anthracis in vivo. The A/J mouse model of infection was selected due to sensitivity to infection with the toxin-producing Sterne strain 34F2 (pXO1+ pXO2−). However, virulence in human hosts requires expression of both the toxin and the capsule. Capsule expression, like toxin production, is induced by bicarbonate [15]. Given the similar dependence of bicarbonate-induced capsule induction on both AtxA and genomic sequences [10],[15], it is likely that a BAS2714-12 deletion would abolish also the induction of capsule production and thus the virulence of a fully virulent, toxin- and capsule-producing strain. Bicarbonate, and not CO2, appears to be the primary molecule regulating induction of virulence gene expression. If CO2 were the primary signaling molecule, one would expect a reduction of toxin expression in the presence of the carbonic anhydrase inhibitor as bicarbonate in solution can no longer be quickly converted into CO2 inside the cell. Instead, we observed the opposite phenomenon: inhibition of carbonic anhydrase activity only affects toxin expression in the absence of added bicarbonate or when bicarbonate can no longer be imported into the cell (Figure 5), conditions under which carbonic anhydrase would convert CO2 into bicarbonate. Induction of toxin expression ex vivo by high atmospheric CO2 levels in the absence of added bicarbonate is likely the result of spontaneous or carbonic anhydrase-driven conversion of CO2 into bicarbonate which then signals an increase in toxin expression. The sensor or metabolic pathway that directly responds to bicarbonate and induces toxin gene expression remains unknown, but its identification is an ongoing focus of our work. Bicarbonate is a major element in the mammalian body. It is present in all body fluids and organs and plays a major role in acid-base homeostasis. The normal concentration of bicarbonate in the blood ranges between 22–26 mM and its presence, together with carbonic acid (H2CO3), hydrogen ions and carbon dioxide forms the buffering system required to provide resistance to drastic changes in pH values. Bicarbonate released from the pancreas also acts to regulate the pH in the small intestine to neutralize the acid entering the duodenum from the stomach [31],[32]. Thus bicarbonate may be a virulence signaling molecule for enterobacteria pathogenesis as well as blood borne pathogens. B. anthracis is not alone among bacteria in regulating virulence gene expression in response to CO2/bicarbonate. CO2 and/or bicarbonate increases toxin production in Vibrio cholerae [33] and Staphylococcus aureus [34], induces expression of attachment genes in Escherichia coli O157:H7 [35], alters the antigenic profile of Borrelia burgdorferi [36], and activates a virulence regulatory protein in Citrobacter rodentium [37]. In any of these systems, a bicarbonate transport system similar to BAS2714-12 may be directly involved in bicarbonate transport and stimulation of virulence factor expression. Most directly relevant to bicarbonate regulation in B. anthracis is the stimulation of the antiphagocytotic M protein in Streptococcus pyogenes [38]. M protein expression is controlled by the regulatory protein Mga, a transcription factor that is similar to the B. anthracis AtxA regulatory protein, because it contains two PRD domains and may be subject to regulation by phosphorylation/dephosphorylation through the PTS carbohydrate utilization system [12],[39]. The apparent overlap of CO2/bicarbonate metabolic systems and regulation of PRD domains in regulatory proteins in response to carbohydrate utilization invites speculation of similar bicarbonate transport and regulatory systems between these pathogenic species. Interestingly, a BLAST search of the S. pyogenes M6 strain (Accession number NC_006086) using the BAS2713 substrate binding protein as query, identified the product of the Spy1045 gene as the protein with the strongest similarity (21%) and a TauA domain in its amino terminal half of the protein. In the C-terminal portion, Spy1045 contains an ABC-type permease domain (Binding-Protein-dependent transport system inner membrane component superfamily cl00427) that, together with the ATPase domain encoded by the Spy1046 gene (35% identity to BAS2714) may constitute the bicarbonate transporter of S. pyogenes (Figure 1). The regulation of B. anthracis virulence factor requires a complex interaction between overlapping metabolic systems, but, for the first time, we have unraveled the dedicated transport components of the CO2/bicarbonate regulatory pathway. This has allowed us to directly separate the influences of multiple signaling molecules to discover that bicarbonate is directly responsible for the in vivo as well as ex vivo induction of virulence factor expression that is essential to B. anthracis pathogenesis. Notably, the availability of the 3-dimensional structure of the bicarbonate binding domain of the Synechococcus CmpA protein in the presence and absence of ligand may be exploited to uncover specific inhibitors of this domain and provide new avenues for antibacterial intervention [23]. In light of these findings, investigation of bicarbonate regulation and transport should be of much greater significance to a large number of pathogenic organisms. Materials and Methods Bacterial strains and growth conditions B. anthracis Sterne 34F2 (pXO1+ pXO2−) and its derivatives were routinely grown in LB broth supplemented with the appropriate antibiotics at the following concentrations: chloramphenicol 7.5 µg/ml, tetracycline 5 µg/ml, erythromycin 5 µg/ml, lincomycin 25 µg/ml, or kanamycin 7.5 µg/ml. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was added at a final concentration of 40 µg/ml to LB agar to monitor β-galactosidase activity in solid media. All carbonic anhydrase inhibitors (Sigma) were freshly prepared in DMSO immediately before addition to cultures. To induce high-level toxin expression, B. anthracis was grown in LB-agar plates or LB liquid media containing 0.8% sodium bicarbonate and 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) [pH 8.0] or in R-Media [40] under 5% CO2. Electrocompetent B. anthracis cells were prepared following the method of Koehler et al [18]. The E. coli TG1 strain was used for plasmid construction and propagation. E. coli strain SCS110 was used for the production of unmethylated DNA for transformation in B. anthracis. E. coli transformation was performed by electroporation using the Bio-Rad Gene Pulser according to the supplier. Transformants were selected on LB broth supplemented with ampicillin (100 µg/ml), chloramphenicol (7.5 µg/ml), or kanamycin (30 µg/ml). Markerless gene deletion Gene deletions in B. anthracis were generated essentially by the method of Janes and Stibitz [41]. A 738 bp region upstream of BAS2714 was amplified using primers BAS2714U5'Bam and BAS2714U3'Sal (Table S1) while an 828 bp region downstream of the BAS2712 was amplified using primers BAS2712D5'Sal and BAS2712D3'Pst. The sequenced products were then cloned into the temperature sensitive plasmid pORI-I-SceI [42] to generate plasmid pAW091. For deletion of BAS4675-77, a 624 bp region upstream of BAS4675 was amplified using primers BAS4675U5'Bam and BAS4675U3'Sal while a 750 bp region downstream of the BAS4677 was amplified using primers BAS4677D5'Sal and BAS4677D5'Pst. The sequenced products were also cloned in plasmid pORI-I-SceI to generate plasmid pAW093. Plasmids pAW091 and pAW093 were electroporated into B. anthracis 34F2 and grown at the permissive temperature of 28°C in the presence of chloramphenicol. Bacteria were then shifted to the non-permissive temperature of 37°C in the presence of chloramphenicol to achieve targeted plasmid integration by homologous recombination. Following plasmid integration, the protocol of Janes and Stibitz [41] was followed to generate the markerless deletion. Diagnostic PCR was carried out to ensure that the entire coding sequence had been correctly deleted. Diagnostic PCR was also carried out on genomic DNA using atxA-specific primers to ensure that the pXO1 plasmid was not lost during the process (Table S1). Complementation analysis The BAS2714-12 region, including a region 640 base pairs upstream of BAS2714 containing potential regulatory sequences, was amplified using primers BAS27145'Xba and BAS27123'Hind and introduced into the pCR4Blunt-TOPO vector (Invitrogen). Following sequencing, the insert was removed by XbaI - HindIII digestion and ligated into XbaI – HindIII digested pHT315 multicopy plasmid vector [29], generating plasmid pAW144. pAW144, as well as pHT315 vector plasmid, was electroporated into both parental 34F2 and 34F2ΔBAS2714-12 strains. Diagnostic PCR was carried out on genomic DNA using atxA specific primers to ensure that the pXO1 plasmid was not lost during the process. β-Galactosidase assays B. anthracis strains harboring the pagA-lacZ [12] or atxA-lacZ (pAtxA12 [17]) fusions on the replicative transcriptional fusion vector pTCV-lac [25] were grown at 37°C in LB or R medium supplemented with the appropriate antibiotics. β-galactosidase activity was assayed as described previously and specific activity was expressed in Miller units [43],[44]. SDS-PAGE and Western blotting B. anthracis strains were grown in R Media to approximately OD600 1.0 for 8 hours in 5% CO2 atmosphere at 37°C, and supernatant samples were isolated by microcentrifugation of cell suspensions. SDS sample buffer was added to each supernatant, and samples were boiled for 5 minutes and loaded on 10% SDS-PAGE gels. The amount loaded was normalized relative to cell density. The gels were run at 30 mA for approximately 2 hr. The gels were transferred to a PVDF membrane (BioRad) in transfer buffer (25 mM Tris base, 192 mM glycine, 20% methanol) at 20 V overnight. The membranes were incubated for 30 minutes at room temperature in blocking buffer (5% dried milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20)) followed by addition of a polyclonal protective antigen antibody diluted 1∶10,000. The blots were washed 5 times and then incubated for 1 hour at room temperature with horseradish peroxidase-conjugated goat anti-rabbit antibody (BioRad) diluted 1∶10,000 in blocking buffer. Following washing of the membrane, binding of the antibodies was probed using the ECL Plus kit (GE) and the protein bands were visualized by PhosphorImager (Molecular Dynamics). Bicarbonate uptake analysis Overnight cultures of B. anthracis 34F2 and 34F2ΔBAS2714-12 strains were diluted 1∶100 in R Media without added NaHCO3 and grown in 60 ml sterile culture bottles at 37°C in 5% CO2 atmosphere. When cultures reached an OD600 of 0.4, 50 µCi of NaH14CO3 (MP Biomedical) was added to each culture. At time intervals indicated, cells were separated from 1 ml of culture by vacuum filtration onto glass filters (Millipore) and immediately washed in 10 ml of cold medium. The filters were then placed in glass vials containing 5 ml of Bio-Safe II counting cocktail (RPI corp.), and radioactivity retained on the filter was measured in a Packard 1600 TR Liquid Scintillation Analyzer. Spore preparation B. anthracis 34F2 and 34F2ΔBAS2714-12 strains were grown in Schaeffer's sporulation medium for approximately 72 hours until over 80% of spores were single and free by phase-contrast microscopy. Cells were collected by centrifugation at 10,000 g for 30 minutes, the medium was aspirated, and cell pellets resuspended in 20 ml of sterile distilled water. The cells were washed twice daily for 5 days by centrifugation at 12,000 g and resuspension in 20 ml fresh, sterile water in order to eliminate most vegetative cells. The cell pellets were then resuspended in 20% renografin (Squibb) and carefully layered over 50% renografin in a 30 ml Corex centrifuge tube. Tubes were then centrifuged at 13,000 g for 30 minutes. The supernatant containing vegetative forms was removed and the purified spore pellets were resuspended in 1 ml of sterile water. The spore pellets were washed twice daily for 3 days by microcentrifugation at 14,000 RPM followed by resuspension of spore pellet in 1 ml sterile water. Total spore counts were measured using a hemacytometer while live spore counts were measure by serial dilution followed by plating on LB-agar. Mouse infection 6-week-old female A/J mice (The Jackson Laboratory) were injected subcutaneously with 106 renografin-purified spores. Progression of disease was monitored visually over 12 days. All mice were housed and maintained at The Scripps Research Institute animal facility under the approval of the Institutional Animal Care and Use Committee. Supporting Information Table S1 Oligonucleotide primers used in this study (0.04 MB PDF) Click here for additional data file. This is manuscript number 19701 from The Scripps Research Institute. The authors have declared that no competing interests exist. This study was supported in part by grant AI055860 from the National Institute of Allergy and Infectious Diseases and grants GM019416 and GM055594 from the National Institute of General Medical Sciences, National Institutes of Health. Oligonucleotide synthesis and DNA sequencing were supported in part by the Stein Beneficial Trust. ==== Refs References 1 Dixon TC Meselson M Guillemin J Hanna PC 1999 Anthrax. 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==== Front Nutr Metab (Lond)Nutrition & Metabolism1743-7075BioMed Central 1743-7075-5-271892856010.1186/1743-7075-5-27ReviewRole of cytochrome P450 in drug interactions Bibi Zakia [email protected] Department of Chemistry, University of Karachi, Karachi-75270, Pakistan2008 18 10 2008 5 27 27 12 6 2008 18 10 2008 Copyright © 2008 Arayne et al; licensee BioMed Central Ltd.2008Arayne et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. ==== Body Introduction The cytochrome P450 (P450 or CYP) isoenzymes are a group of heme-containing enzymes embedded primarily in the lipid bilayer of the endoplasmic reticulum of hepatocytes, it takes part in the metabolism of many drugs, steroids and carcinogens [1]. The most intensively studied route of drug metabolism is the P450-catalysed mixed-function oxidation reaction which conforms to the following stoichiometry NADPH + H+ + O2 + RH → NADP+ + H2O + ROH where, RH represents an oxidisable drug substrate and ROH is the hydroxylated metabolite, the overall reaction being catalysed by the enzyme P450. At the present time a number of CYP isoenzymes are expressed in each mammalian species including humans [2], many of these have specific role involving anabolic steroids and are localized in the liver. The present system of nomenclature for the various CYP isozymes employs a three-tiered classification based on the conventions of molecular biology: the family (members of the same family display > 40% homology in their amino acid sequences), subfamily (55% homology), and individual gene [3]. This pedigree is indicated by, respectively, an Arabic numeral (family), a capital letter (subfamily) and another Arabic numeral (gene), e.g. CYP1A2. The enzymes transforming drugs in humans belong to the CYP families 1–4 and more than 30 human CYP isozymes have been identified to date. It has been estimated that 90% of human drug oxidation can be attributed to six main enzymes (CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A4/5). The activities of the CYP2C19 [4-7] and CYP2D6 [8-14] enzymes are biomedically distributed in the population, allowing classification of individuals as either extensive (EM) or poor metabolizers (PM). The concept that most drug oxidations are catalysed primarily by a small number of P450 enzymes is important in that the approaches to identifying drug-drug interactions are feasible, both in vivo and in vitro. More side-effects of drugs and drug-drug interactions are being reported, as highly effective drugs are developed and multiple-drug therapies are increasingly used. Drug interactions involving the P450 isoforms generally are of two types: enzyme induction or enzyme inhibition. Common substrates, inhibitors and inducers of P450 isozymes. Enzyme inhibition reduces metabolism, whereas induction can increase it. In general, high-extraction drugs are less affected by these interactions than low-extraction drugs. As have been shown in recent deaths [15,16] caused by dysrhythmia or bone marrow (haematopoietic) inhibition due to combined administration of terfenadine and ketoconazole [17,18], erythromycin [19] and itraconazole [20], and sorivudine and fluoropyrimidines, are clinically important and severe interactions do occur. Furthermore, side-effects due to drug-drug interactions in elderly patients because of their reduced physiological functions are reportedly becoming more frequent and associated with more severe symptoms; thus, much importance is being attached to information about drug-drug interactions when giving any drug therapy. A number of reviews of these interactions have been published [21-63]. In recent years, access to human tissue samples was not possible in Japan. However, characterization of P450 reactions catalysed by human P450s have been carried out in the United States and Europe. The availability of the recombinant human P450s expressed in various systems has also facilitated studies of their catalytic selectivity [64]. Thus, it is now relatively straightforward to determine in vitro interactions in which P450s oxidizes a particular drug and which drugs can inhibit oxidations catalysed by this P450. Thus, it is possible to perform logical in vivo studies to test the relevance of in vitro findings [65,66]. This review discusses interactions and their clinical management. P450 enzyme classification In man there are around 30 CYP enzymes which are responsible for drug metabolism and these belong to families 1–4. It has been estimated, however, that 90% of drug oxidation can be attributed to six main enzymes: CYP 1A2, 2C9, 2C19, 2D6, 2E1 and 3A4 [6]. The most significant CYP isoenzymes in terms of quantity are CYP3A4 and CYP2D6. CYP3A4 is found not only in the liver but also in the gut wall, where it may serve as a primary defence mechanism. The bulk of drugs acting on the CNS (Central Nervous System), with the exception of volatile anaesthetic agents, are metabolized by this enzyme. CYP1A subfamily CYP1A1 and CYP1A2 The CYP1A family consists of two enzymes, 1A1 and 1A2. CYP1A1 is not significantly expressed in the liver. It is found mainly in the lungs, mammary glands, placenta and lymphocytes. It is an enzyme involved in the inactivation of procarcinogens and is highly induced by polycyclic aromatic hydrocarbons (PAHs), which are found in cigarette smoke [7]. There is a strong association between the activity of CYP1A1 and the risk of lung cancer [8]. CYP1A2 is expressed mainly in the liver and is induced by cigarette smoking [9]. It is also induced by the ingestion of some foodstuffs such as cruciferous vegetables as well as barbecued or charbroiled food [10]. Some drugs such as omeprazole may induce CYP1A2 activity [11]. Drugs which are known to be metabolized by CYP1A2 include theophylline, caffeine, imipramine, paracetamol and phenacitin [12]. Alteration in CYP1A2 activity, for example by smoking, may alter the requirements for theophylline among asthmatics [13] and haloperidol among psychiatric patients [14]. Caffeine metabolism is also induced by smoking and explains the increased tolerance to caffeine among smokers [15]. CYP2 family CYP2A6, previously known as coumarin hydroxylase, is a relatively unimportant enzyme in terms of the number of substrates which it metabolizes, one of the substrates broken down by this enzyme is nicotine. Differences, both racial and inter individual, in expression levels are thought to be related to the propensity to develop nicotine dependence [16]. CYP2C subfamily: is one of the most important families and consists primarily of two enzymes, CYP2C9 and CYP2C19. CYP2C9: Among the substrates of CYP2C9 is the anticoagulant warfarin, which exists in two distinct isoforms of which the S-form is the most important and this is metabolized by CYP2C9 [17]. There are a number of polymorphisms of the gene that encode this enzyme resulting in poor metabolic status. These patients may be difficult to stabilize on standard warfarin regimens [18]. Other drugs metabolized by CYP2C9 include non-steroidal anti-inflammatory drugs (NSAIDs) (including COX-2 selective inhibitors), the hypoglycemic agent tolbutamide, phenytoin and the angiotensin-II receptor antagonist losartan. CYP2C19 has a number of commonly used substrates including the benzodiazepine diazepam, the proton pump inhibitor omeprazole, propanolol and the antidepressive amitriptyline [19]. A number of important abnormal variants of this enzyme exist, one of these has important clinical consequences. It has been demonstrated that poor metabolizers who are prescribed proton-pump inhibitor omeprazole as part of therapy against Helicobacter pylori infection may have significantly better clinical outcomes as compared to a group of patients homozygous for the normal, i.e. wild-type, alleles [20]. CYP2D subfamily CYP2D6. A large number of drugs are metabolized by this enzyme including a number of anti-arrhythmics such as flecanide and encainide, tricyclic antidepressants, some beta-blockers and a number of selective serotonin re-uptake inhibitors. It is of particular relevance to anesthetics because a number of commonly used analgesics, including codeine and tramadol, are broken down by this enzyme [21]. Previously named debrisoquine hydroxylase [22], was one of the first to be categorised following recognition that the metabolism of the hypotensive agent debrisoquine was abnormal in a proportion of individuals. It was renamed CYP2D6 after the parent gene was cloned and the enzyme categorized [23]. To date, more than 70 polymorphisms of CYP2D6 have been catalogued. The majority of these enzymes result in a poor metaboliser phenotype as opposed to the normal, i.e. extensive metabolize phenotype. In addition, a number of genotypes exist where gene duplication results in an ultra rapid metabolize status. These patients eliminate CYP2D6 substrates faster than normal and in case of pro-drugs such as codeine are at greater risk of opiate related side-effects [24]. CYP2E1 The CYP2E family contains only one enzyme, CYP2E1 (previously dimethylnitrosamine N-demethylase), which is responsible for the metabolism of small organic compounds such as alcohol and carbon tetrachloride as well as the halogenated anaesthetic agents halothane, enflurane, diethyl ether, trichloroethylene, chloroform, isoflurane and methoxyflurane [25]. It is also responsible for the breakdown of many low molecular weight toxins and carcinogens, many of which are used in manufacturing and dry cleaning industry, including benzene, styrene, acetone, vinyl chloride and N-nitrosamines. Some of these substances are pro carcinogens which are activated by CYP2E1. There are gender differences in the expression of the enzyme, obesity and fasting may also affect its activity [26]. This may provide a putative explanation for obesity related cancers [27]. Because of the key role of CYP2E1 in the biodegradation of a number of environmental carcinogens, the enzyme has been studied closely in relation to the causation of neoplasia. For example, in China an association was detected between polymorphisms of CYP2E1 and oesophageal and gastric cancer [28]. There is also mounting evidence that CYP2E1 may be a key factor in the pathogenesis of alcoholic liver disease [29]. The exact role of CYP2E1 is unclear, although the enzyme is induced by both alcohol and nicotine [30], and may explain the higher ethanol elimination rates among smokers [31]. CYP3A subfamily CYP3A4 is the most abundantly expressed drug metabolizing enzyme in man responsible for the breakdown of over 120 different medications and is thus an important area for study with respect to enzyme based drug interactions. Among the drugs metabolized are sedatives such as midazolam, triazolam and diazepam, the antidepressives amitriptyline and imipramine, the anti-arryhthmics amiodarone, quinidine, propafenone and disopyramide, the antihistamines terfenadine, astemizole and loratidine, calcium channel antagonists such as diltiazem and nifedipine and various antimicrobials and protease inhibitors [6]. Mechanism of pharmacokinetic drug-drug interaction Inhibition Inhibition is reduced enzyme activity due to direct interaction with a drug. This process usually begins with the first dose of the inhibitor, and the start and finish of inhibition correlate with the half-lives of the drugs involved [67]. There are three basic types of enzyme inhibition (competitive, non-competitive and uncompetitive), and clinical effects are influenced by these basic mechanisms [68,69]. The first type is competitive inhibition between inhibitor and substrate for the same binding site on an enzyme. The size and flexibility of the binding site of the microsomal P450 with which we are concerned here are unknown. For example, when single oral doses of metoprolol (50 mg), a beta-adrenoceptor blocking agent and/or propafenone (150 mg) were administered, or when the two drugs were given in combination to healthy subjects, an approximately two-fold reduction in the oral clearance of etoprolol was observed when propafenone was included. The dose of metoprolol should be reduced when propafenone is also given [70]. Similar drug-drug interactions are seen in the combined administration of thioridazine and propranolol (CYP2D6) [71], fluoxetine and desipramine (CYP2D6) [72], omeprazole and diazepam (CYP2C19) [73-75], tolbutamide and phenytoin (CYP2C9) [32], and diltiazem and cyclosporin (CYP3A) [76-78]. The most typical example of the second type of drug-drug interaction includes that of terfenadine and erythromycin [19]. The combined use of these drug, terfenadine, and macrolides (antibiotics) or ketoconazole prolongs electrocardiographic QT intervals, thereby triggering a specific cardiac dysrhythmia known as torsades de pointes' [18]. The mechanism of this interaction is considered to occur when a nitro compound, namely a metabolite demethylated by P450, forms a complex with P450. Since macrolides are catalysed by CYP3A, metabolites selectively form CYP3A and a stable enzyme-substrate complex [34,79-81]. In consequence, it has been reported that the metabolism of drugs like carbamazepine [81-83], midazolam [84-86] and cyclosporin [87] are catalysed by CYP3A, and their plasma concentrations are increased when its metabolism is inhibited by combined use with erythromycin. A P450 species that catalyzes the metabolism of terfenadine was identified recently as CYP3A [88,89] during investigations of the mechanism of interactions with macrolides. Another type is non-competitive inhibition, where the inhibitor binds at a site on the enzyme distinct from the substrate, as happens in classical studies of enzymology. Such examples include interactions between cimetidine and a number of drugs. The duration of this type of inhibition may be longer if new enzymes are synthesized after the inhibitor drug is discontinued. Cimetidine is bound to P450 and produces a stable cytochrome-substrate complex. It is the formation of this complex which prevents access of other drugs to the P450 system. Cimetidine does not inhibit conjugation mechanisms including glucuronidation, sulphation and acetylation, or deacetylation or ethanol dehydrogenation. It binds to the haem portion of P450 and is, thus, an inhibitor of phase I drug metabolism reactions (i.e. hydroxylation, dealkylation) [90-92]. Although generally recognized as a nonspecific inhibitor of this type of metabolism, cimetidine does demonstrate some degree of specificity. Since every molecular species of P450 has a haem portion, it is possible for cimetidine to nonspecifically inhibit any drug that is metabolized by any molecular species. However, in a recent study that compared the inhibitory effect of several P450 isozymes in a study using drugs such as ketoconazole, clotrimazole, miconazole, fluconazole, secnidazole and metronidazole, all imidazole derivatives [92,93], it was reported that isoniazid, an antituberculous drug, inhibits the metabolism of phenytoin [94]. As for its inhibitory mechanism, it is conceivable that there is an interaction between the hydrazino group of isoniazid and the haem portion of P450. As far as ethinylestradiol, an oral contraceptive, is concerned, the CYP3A isozyme is one of the major forms involved in its 2-hydroxylation [93,95,96]. Guengerich reported that in vitro it is a relatively effective and selective mechanism-based inactivator of CYP3A4 [96]. This inactivation is due in part to the presence of an ethynyl moiety, which is also found in many inactivators [93-97]. Induction The effect of induction is simply to increase the amount of P450 present and speed up the oxidation and clearance of a drug [67]. It is rather difficult to predict the time-course of enzyme induction because of several factors, including the drug half-life and enzyme turnover, which determine the time-course of induction. A complicating factor is that the time-course of induction depends on the time required for enzyme degradation and new enzyme production. The short half-life of rifampicin results in enzyme induction (CYP3A4, CYP2C), apparent within 24 h, whereas phenobarbital, which has a half-life of 3–5 days, requires ≅1 week for induction (CYP3A4, CYP1A2, CYP2C) to become apparent. These enzyme-induction reactions also occur with smoking and long-term alcohol or drug consumption and can reduce the duration of action of a drug by increasing its metabolic elimination. Of all these drugs, the clinically most problematic drug involves the rifampicin series [98-106] which includes antiepileptic drugs such as phenobarbital [107,108], carbamazepine [109,110] and phenytoin [108,110] and antituberculous drugs [110]. The CYP1A2 enzyme can be induced by exposure to polycyclic aromatic hydrocarbons, such as are found in char-grilled foods and cigarette smoke [111,112]. Most human CYP2C and 3A subfamily proteins are induced by barbiturates [113], while human CYP2E1 is inducible by ethanol and isoniazid, although the mechanism involved is complex [114,115]. One example has been described by Lee et al. [99] who reported that changes in the pharmacokinetics of prednisolone were caused by administration or discontinuation of rifampicin. Pharmacokinetic studies of prednisolone (1 mg/kg) in patients over a 1-month period of rifampicin co-treatment or after its withdrawal revealed significant changes in the area under the curve (AUC), total body clearance, non-renal clearance and half-life. As mentioned earlier, rifampicin is possibly associated with plural molecular species of P450 (several isozymes), but mainly, a large increase in the CYP3A content often becomes a problem, while phenobarbital, carbamazepine and phenytoin, antiepileptic drugs, also induce CYP3A [32]. Thus, appropriate therapeutic effects can hardly be obtained unless the doses are increased significantly, since plasma concentrations are not elevated in patients receiving these drugs which are metabolized by CYP3A. The P450 isoenzymes induced by exposure to polycyclic aromatic hydrocarbons, such as those found in char-grilled foods and cigarette smoke, are CYPlAl and CYP1A2 [116,117]. CYPIA2 is a molecular species of P450 which participates in the metabolism of several important drugs such as theophylline and propranolol and, since its activity is enhanced by smoking and eating grilled meat or cruciferous vegetables, it is difficult to obtain therapeutic effects. Although CYP2C9, CYP2C19 and CYP2E1 are also induced, no specific inducers of CYP2D6 have yet been identified clearly. However, it appears to be inducible. Mechanism of non-microsomal pharmacokinetic drug-drug interactions Sixteen Japanese patients died when given both sorivudine and fluoropyrimidines orally. Sorivudine is a potent inhibitor of hepatic dihydropyrimidine dehydrogenase, the enzyme responsible for the catabolism of fluoropyrimidines. Therefore, the fluoropyrimidine levels in these patients reached toxic levels due to the inhibition of dihydropyrimidine dehydrogenase by sorivudine [15,16]. Clinical example of P450-based interactions Terfenadine Terfenadine is the first non-sedating H1-antihistamine drug. It is rapidly oxidized by CYP3A4 to two metabolites, acyclinol and an alcohol derived from the oxidation of a t-butyl methyl group [118]. The alcohol is further oxidized to a carboxylic acid by either CYP3A4 or dehydrogenase [119]. This carboxylic acid then binds to the H1 histamine receptor and should relieve allergy symptoms. The oxidation of terfenadine by CYP3A4 can be inhibited strongly by azole antifungal or antimicrobial agents such as ketoconazole [17,18] and erythromycin [19]. For example, Honing et al. [18] performed experiments on six healthy volunteers (four men and two women, aged 24–35 years). After achieving a steady-state while taking terfenadine (60 mg every 12 h for 7 days), daily concomitant oral ketoconazole (200 mg every 12 h) was added to the regimen. Pharmacokinetic profiles were obtained while subjects were taking terfenadine alone and after the addition of ketoconazole. Electrocardiograms were obtained at baseline, after 1 week of taking terfenadine alone, and at the time of the second pharmacokinetic profile after the addition of ketoconazole to the regimen. Serum concentrations of terfenadine and its acid metabolite and corrected QT intervals were obtained. All subjects had detectable levels of unmetabolized terfenadine after the addition of ketoconazole, associated with QT prolongation. Only two of the six subjects were able to complete the entire course of ketoconazole coadministration. Four subjects received a shortened period of ketoconazole therapy because of significant electrocardiographic repolarization abnormalities. There was a significant reduction in the AUC of the acid metabolite of terfenadine during ketoconazole administration. Therefore, the blood concentration of terfenadine increased. High blood levels of terfenadine have been associated with cardiac problems including dysrhythmias, torsade de pointes, and abnormal ventricular rhythms. For this reason, very carefully controlled co-administration of terfenadine is advised. Cimetidine Cimetidine inhibits antihistamine H2-receptor binding and is used in the treatment of gastric ulcers. The mechanism of inhibition appears to involve the imidazole ring of cimetidine with competitive binding, which is not present in ranitidine [90,91]; it also exhibits selective inhibition of reactions catalysed by CYP2D6 and 3A4 [90,92-120]. For example, unlike ranitidine, cimetidine significantly increased the maximum plasma concentration (Cmax), AUC and the total amount of disopyramide excreted unchanged in the urine, but the serum profile of mono-N-dealkyldisopyramide, a metabolite of disopyramide, was not affected significantly. Ranitidine had no significant effect on the pharmacokinetics of disopyramide and mono-N-dealkyldisopyramide. These results indicate that cimetidine, but not ranitidine, significantly increases the absorption of oral disopyramide [121]. Tanaka and Nakamura also investigated the effects of H2-receptor antagonists (cimetidine, ranitidine, and famotidine) on ethanol metabolism. In both aldehyde dehydrogenase (ALDH)-1 deficient subjects and in those with normal ALDH-1, the three H2-receptor antagonists and placebo had similar effects on the pharmacokinetic parameters of ethanol, i.e. peak time (tmax), metabolic rate, Cmax, volume of distribution (Vd) and AUC. The AUC of acetaldehyde was slightly (P < 0.05) but significantly greater only after treatment with cimetidine; the Cmax and tmax of acetaldehyde were unchanged [122]. As mentioned above, there are a number of drugs whose metabolism is inhibited when cimetidine is administered in combination [90-92]. Grapefruit juice The opportunity for a food-drug interaction is an everyday occurrence, which can be particularly important when total drug absorption is altered. Recently, a chance observation led to the finding that grapefruit juice could markedly increase the oral bioavailability of a number of medications [123]. This article retraces discovery of this novel interaction and reviews the mechanism of action, summaries studied and predicted medications for an interaction, discusses possible active ingredient in the juice and considers clinical implications. In 1989 it was reported that coadministration of grapefruit juice with the calcium channel antagonist felodipine resulted in a large increase in serum felodipine concentrations, as well as an enhancement of the pharmacodynamic effects of the drug [124]. Some drugs exhibit a significantly increased (up to three-fold) mean oral bioavailability when co-administered with grapefruit juice. Bailey et al [125] reported that the inhibitory effect of grapefruit juice was discovered rather serendipitously in an interaction study with ethanol and felodipine, a 1,4-dihydropyridine calcium entry blocker. Flavonoids (e.g. quercetin, naringenin, kaempferol) found in large amounts in oranges, grapefruit and their juices are known to alter the activity of P450 enzymes (P450 isoenzyme). The mechanism of inhibition of drug oxidation probably involves intestinal CYP3A4. The major grapefruit-specific flavonoid is naringin, which can account for up to 10% of the dry weight. It is believed that this naringin mainly inhibits the enzyme (CYP3A) that metabolizes calcium antagonists. For example, interactions between benzodiazepines (e.g. midazolam, triazolam), antihistamines (e.g. terfenadine), immuno-suppressive drugs (e.g. cyclosporin) and grapefruit juice have been reported [125]. For example, Hukkinen et al. [126] studied 10 healthy young subjects who received a single 0.25 mg dose of triazolam with either 250 ml grapefruit juice or water. The plasma concentrations and effects of triazolam were measured up to 17 h. Grapefruit juice increased the AUC of triazolam in each subject and the Cmax in nine out of 10 subjects. The mean AUC of triazolam increased 1.5-fold (P < 0.001) and the peak concentration increased 1.3-fold (P < 0.05) following grapefruit juice. Grapefruit juice also postponed the peak time of triazolam from 1.6 to 2.5 h (P < 0.05). Grapefruit juice also increased the effects of triazolam, drowsiness being significantly (P < 0.05) enhanced. However, as it has been described in a paper [127] that other flavonoids (quercetin for example) may be major inhibitors of metabolism, the results of future studies are awaited with interest. On the other hand, it is reported that naringin also inhibits the demethylation (N-demethylation) of caffeine, metabolized by CYPIA2 [125]. It has already been established that, grape fruit juice is well known as potent inhibitors of cytochrome P450 3A4 activity. It increases bioavailability of several drugs known to be metabolized by CYP3A4, while on the other hand interact and block the activity of ciprofloxacin, ofloxacin, cefazolin and ceftizixime. Owing to clinical relevance of grapefruit juice-drug interactions, an investigation of drug interactions of two quinolones, ciprofloxacin and ofloxacin were investigated in vitro with all the fruit juices available locally at human body temperature [128]. A single glass of grapefruit juice has the potential to augment the oral bioavailability and to enhance the beneficial or adverse effects of a broad range of medications, even by juice consumed hours beforehand. Grapefruit juice acts by inhibiting presystemic drug metabolism mediated by CYP3A isoforms in the small bowel. The interaction appears particularly relevant for medications with at least a doubling of plasma drug concentration or with a steep concentration-response relationship or a narrow therapeutic index. Patients that appear particularly susceptible have high small bowel CYP3A4 content, hepatic insufficiency or a pre-existing medical condition, which predisposes to enhanced, excessive or abnormal drug effects [129]. Omeprazole Omeprazole is a proton-pump inhibitor used widely for the treatment of gastric ulcers [53,62]. Omeprazole is converted to hydroxyomeprazole and omeprazole sulphone primarily by CYP2C19 and CYP3A4, respectively. Gugler and Jensen first reported that omeprazole reduced the plasma clearance and prolonged the half-life of phenytoin and diazepam but did not affect the apparent volume of distribution and plasma protein binding of either diazepam or phenytoin [130]. Recently, in a pharmacogenetic study, Anderson et al. [131] studied the effect of omeprazole treatment on diazepam plasma levels in 6 EM and 4 PM of omeprazole. Single i.v. doses of diazepam (0.1 mg/kg) were administered after 1 week of oral omeprazole (20 mg) and placebo. The slow metabolizers of omeprazole also metabolized diazepam slowly, exhibiting only half the diazepam plasma clearance of the others. The mean clearance of diazepam fell 26% after omeprazole in the rapid metabolizers, whereas the slow group showed no apparent interaction. Desmethyldiazepam was formed more rapidly in the rapid compared with the slow metabolizers, which is a logical consequence of the rate of diazepam metabolism. In the light of these results, omeprazole appears to be a competitive inhibitor of CYP2C19, and involved in its metabolism. These data show that omeprazole interferes with the elimination of other drugs by inhibiting the mixed function oxidases of human liver. Other acid pump inhibitors (lansoprazole or pantoprazole) are also mainly metabolized by CYP2C19. For drugs metabolized by CYP2C19, such as 5-mephenytoin, imipramine or diazepam, their metabolism is inhibited [132]. Erythromycin Erythromycin, an antimicrobial agent, is known to inhibit a number of drug oxidation reactions catalysed by CYP3A4 [80]. It inhibits the oxidation of terfenadine [19,133], cyclosporin [134] and numerous other drugs both in vivo and in vitro and erythromycin N-demethylation itself is catalysed by CYP3A4/5 [135,136]. However, not all CYP3A4 reactions are inhibited by erythromycin. As far as the above results are concerned, Guengerich [56] has made the following two proposals: i lack of inhibition of a reaction by erythromycin may not always be a reliable indication that the reaction is not catalysed by CYP3A4 and (ii) not all CYP3A4-catalysed reactions may be prone to erythromycin interactions. The reasons for this are not clear at the moment. Cyclosporin Cyclosporin is the most popular immunosupressant used in organ transplantation. The major pathway of cyclosporin metabolism is via CYP3A4 [137,138], with three major metabolites being formed [139]. Since cyclosporin is mainly used as an immunosuppressant for organ transplantation, the CYP3A4 level in the donor's liver as well as the recipient's liver, small intestine and other tissues must always be taken into consideration. For example, Lucey et al. [140] reported that a 40-year-old male liver allograft recipient had neurological dysfunction and renal failure while his cyclosporin blood levels were in the therapeutic range. CYP3A activity, using the [14C] erythromycin breath test, was reduced compared with that in controls, including other liver transplant recipients. Pretreatment with rifampicin, an inducer of CYP3A, increased enzyme activity. After treatment with rifampicin the patient was able to be rechallenged with cyclosporin at a dose almost twice that which had previously been toxic. The patient died during a second transplantation and the microsomal CYP3A content was found to be low in the first transplant liver. Lower blood levels of cyclosporin may have been achieved when the drug used for enzyme induction (rifampicin) has been given to the transplant patient for a long period [140]. Rifampicin Rifampicin [98-106,141,142] and isoniazid [101] are key drugs used in the treatment of tuberculosis, while rifampicin is highly effective in inducing hepatic, drug metabolic P450 enzyme. When enzyme induction is achieved, the pharmacological effects of a specific drug may be reduced, since not only the metabolism of rifampicin itself, but also the metabolism of the other drug is accelerated [136]. The problem arises when doses are increased to reduce the effects of the combined drugs: increased serum concentrations of the combined drugs may possibly produce side-effects because of the lost enzyme induction if rifampicin is discontinued. Rifampicin is also known to induce CYP3A4 and CYP2C9 (e.g. cyclosporin, diazepam and steroids). As for dihydropyridine calcium channel blockers, it is quite possible that interactions with rifampicin may develop, since most of these drugs are metabolized by CYP3A4 [143-150,129]. Conclusion There are two main types of drug interaction: pharmacokinetic and pharmacodynamic. Pharmacokinetic interactions involve the effect of one drug on the absorption, metabolism, excretion or protein binding of another drug. On the other hand, pharmacodynamic interactions are caused by several effects (additive, synergistic or antagonistic effects) of the combined treatment at the site of biological activity, changing the pharmacological action of the drugs, even at standard blood concentrations. Pharmacokinetic interactions focused on P450 are described in this paper. The incidence of side-effects is markedly higher in the elderly and those with more severe symptoms. Thus, understanding the mechanism underlying drug interactions is useful, not only in preventing drug toxicity or adverse effects, but also in devising safer therapies for disease. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1905264208-PONE-RA-05876R210.1371/journal.pone.0003856Research ArticleNeuroscience/Neuronal Signaling MechanismsPhysiology/Integrative PhysiologyDiabetes and Endocrinology/ObesityCentral Exercise Action Increases the AMPK and mTOR Response to Leptin Exercise and Leptin ActionRopelle Eduardo R. Fernandes Maria Fernanda A. Flores Marcelo B. S. Ueno Mirian Rocco Silvana Marin Rodrigo Cintra Dennys E. Velloso Lício A. Franchini Kleber G. Saad Mario J. A. Carvalheira José B. C. * Department of Internal Medicine, FCM, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil Calbet Jose A. L. EditorUniversity of Las Palmas de Gran Canaria, Spain* E-mail: [email protected] and designed the experiments: ERR MFAF MBF MJS JBC. Performed the experiments: ERR MFAF MBF MU SR RM DEC. Analyzed the data: ERR MFAF LAV MJS JBC. Contributed reagents/materials/analysis tools: SR RM DEC LAV KGF MJS. Wrote the paper: ERR MFAF JBC. 2008 4 12 2008 3 12 e38567 8 2008 6 11 2008 Ropelle et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exercise is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exercise ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest period. Intracerebroventricular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitor prevented IL-6-induced anorexia. Activators of AMPK and fasting increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitor of AMPK had the opposite effect. Furthermore, the reduction of AMPK and ACC phosphorylation and increase in phosphorylation of proteins involved in mTOR signal transduction, observed in the hypothalamus after leptin infusion, were more pronounced in both lean and diet-induced obesity rats after acute exercise. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in responsiveness to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Thus, the effects of leptin on the AMPK/mTOR pathway, potentiated by acute exercise, may contribute to appetite suppressive actions in the hypothalamus. ==== Body Introduction Prolonged exercise of medium to high intensity is known to profoundly affect energy balance [1]–[3]. Studies of individuals who have maintained significant weight loss for >1 year have demonstrated that dieters who achieve long-term success are often those who engage in regular and extensive exercise programs [4]. Although the energy expenditure aspects of such exercise may contribute to the effects of weight maintenance, it has been suggested that exercise may also contribute to the energy balance by altering food intake [5], [6]. Rodents submitted to exercise have increased sensitivity to leptin, conversely animals with diet-induced obesity and most obese humans are resistant to leptin [5], [7], [8]. Thus, the mechanism for leptin increased responsiveness in exercise is of great interest and understanding this mechanism could lead to new approaches to prevent or treat obesity. The hypothalamus plays a central role in integrating hormonal (leptin and insulin) and nutritional signals from the periphery and modulating food intake, energy expenditure, and peripheral metabolism [9]. Multiple factors control food intake, including hormones, fuels and behaviour. AMPK is the downstream component of a kinase cascade that acts as a sensor of cellular energy charge, being activated by rising AMP coupled with falling ATP. Once activated, AMPK phosphorylates acetyl-CoA carboxylase (ACC) and switches on energy-producing pathways at the expense of energy-depleting processes [10]–[12]. Another target molecule for the control of food intake and energy homeostasis is represented by the phosphoprotein mammalian target of rapamycin, mTOR, in which the PI(3)K/Akt pathway has been suggested to affect the mTOR phosphorylation state and catalytic activity [13]. Activated signaling through mTOR phosphorylates the serine/threonine kinase p70S6K and the translational repressor eukaryotic initiation factor (eIF) 4E binding protein (4EBP1) [14], [15]. mTOR signaling is inhibited under conditions of low nutrients, such as glucose and amino acids and low intracellular ATP levels [16]. While mTOR was presumed to serve as the direct cellular sensor for ATP levels [17], mounting evidence has implicated AMPK in the regulation of mTOR activity [15], [18]–[20]. The level of circulating interleukin-6 (IL-6) increases dramatically in response to exercise [21], with IL-6 being produced by working muscle [22], [23] and adipose tissue [24], [25] and its concentration correlates temporally with increases in AMPK in multiple tissues [26]. In addition, AMPK activity is diminished in IL-6 deficient mice at rest and the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice [27]. It also appears that centrally-acting IL-6 plays a role in the regulation of appetite, energy expenditure, and body composition [5], [28]. The signaling mechanism of IL-6 in the hypothalamus is, however, not fully understood. In cells, binding IL-6 to the α subunit of its receptor triggers the recruitment of gp130, subsequently leading to the activation of the gp130-associated JAK [29]–[31]. JAK links cytokine receptor to the STAT3 and MAP kinase pathway [29], [30], [32]. In addition to JAK/STAT and MAP kinase pathways, IL-6 also activates the PI(3)K/Akt pathway [33]. In this study, we sought to determine whether the improved response of the AMPK and mTOR pathways to leptin could contribute to the increased molecular response of leptin in rats submitted to exercise in an IL-6-dependent manner. We therefore, examined hypothalamic modulation of AMPK/ACC and mTOR signaling pathways, induced by IL-6, as well as the role of IL-6 in those signaling pathways induced by leptin in rats after acute exercise. Results IL-6 decreases hypothalamic AMPK and increases mTOR signaling To determine whether IL-6 modulates hypothalamic AMPK/ACC signaling, we injected IL-6 into the third ventricle of rats and evaluated food intake and AMPK signaling. IL-6 caused a significant reduction in food intake (Figure 1a). We next investigated whether the microinfusion of IL-6 modulates the hypothalamic ATP concentration. Figure 1b shows that IL-6 (200 ng) changed ATP, ADP and AMP concentrations in the hypothalamus of rats, whereas, sixty minutes after IL-6 injection, the ATP content increased by ∼88% (Figure 1c) and decreased AMP∶ATP ratio by ∼54% in Wistar rats (Figure 1d). Consistent with the modulation of the AMP∶ATP ratio, we observed reduced hypothalamic AMPK and ACC phosphorylation induced by IL-6 (Figures 1e and f); whilst IL-6 increases p70S6K and 4EBP1 phosphorylation (Figures 1g and h). The αAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 1e–h, lower panels). 10.1371/journal.pone.0003856.g001Figure 1 Effects of IL-6 on food intake, hypothalamic AMPK/mTOR activity and ATP content. (a) Effect of i.c.v. administration of IL-6 on food intake; pretreatment with AICAR blocks IL-6-induced anorexia (n = 12–15 animals per group). (b, c, d) Typical chromatographic run (b) depicting the ATP, ADP, and AMP fractions in control (black line) and in i.c.v. IL-6 treated animals (red line), as mean ATP content and as AMP∶ATP ratio (c, d). (e, f, g, h) Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (e); phospho-ACCser79, serine phosphorylated ACC and total ACC (f); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (g); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (h). Confocal microscopy was performed using IL-6 receptor (IL-6R)-specific antibody (green) and DAPI (blue), with 50× magnification (i). Co-localization of IL-6R (green) and AMPK (red) and IL-6R (green) and phospho-p70S6K (red) 60 minutes after i.c.v. saline or IL-6 infusion in the arcuate nuclei of rats, with 200× magnification (j). Data are the means±SEM. p<0.05, vs. control group; p<0.01, vs. control group; # p<0.05, vs. other groups. If the effect of IL-6 on food intake is mediated by AMPK inhibition, AICAR infusion at doses that do not change food intake, but still increase AMPK and ACC phosphorylation, could be sufficient to block the effects of IL-6. Thus, to determine whether the effects of IL-6 on food intake are AMPK-dependent, we first identified a dose of AICAR that did not alter food intake (0.5 mM) when administered at the onset of the dark cycle. We then evaluated the effect of i.c.v. pretreatment with this dose of AICAR, or its vehicle, on the anorectic response to i.c.v. IL-6 (200 ng) and we observed that the anorectic response to i.c.v. IL-6 was reversed by AICAR at the time points studied (figure 1a). These findings indicate that inactivation of neuronal AMPK is necessary for some of the effects of IL-6 on food intake. Immunohistochemistry with anti-Interleukin-6 Receptor (IL-6R)-specific antibody showed that IL-6R is expressed in a majority of neurons in the arcuate nucleus (Figure 1i). Double-staining confocal microscopy showed the positive immunoreactivity of IL-6R and AMPK in the hypothalamic arcuate nucleus of rats 60 minutes after i.c.v. saline or IL-6 (200 ng) infusion. The immunoreactivity of IL-6R and pp70S6K was not detected in the hypothalamic arcuate nucleus of rats after i.c.v. saline infusion. However, we observed the positive double-staining of IL-6R and pp70S6K 60 minutes after i.c.v. IL-6 (200 ng) infusion (Figure 1j). IL-6 induction of hypothalamic mTOR does not require changes in PI(3)K signaling If the effect of IL-6 on food intake is mediated by mTOR activation, Rapamycin infusion at doses that do not change food intake, but still decrease p70S6K and 4EBP1 phosphorylations could be sufficient to block the effects of IL-6. Thus to determine whether IL-6 modulates hypothalamic mTOR signaling, we injected IL-6 (200 ng) into the third ventricle of rats and evaluated food intake and mTOR signaling. IL-6 caused a significant reduction in food intake (Figure 2a) and induced hypothalamic p70S6K and 4EBP1 phosphorylation (Figures 2b and c). The p70S6K and 4EBP1 protein levels were not different between the groups (Figures 2b and c, lower panels). To determine whether the effects of IL-6 on food intake are mTOR-dependent, we first identified a dose of Rapamycin that did not alter food intake (25 µg) when administered at the onset of the dark cycle. We then evaluated the effect of i.c.v. pretreatment with this dose of Rapamycin or its vehicle, on the anorectic response to i.c.v. IL-6 and we observed that IL-6 reduction of food intake was reversed by Rapamycin. 10.1371/journal.pone.0003856.g002Figure 2 Effects of IL-6 on food intake and hypothalamic PI(3)K/mTOR and JAK/STAT activity. (a) Pretreatment with Rapamycin blocks IL-6-induced anorexia (n = 10–12 animals per group). (b, c) Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (b); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (c). (d) Pretreatment with LY294002 had no effect on anorectic response to IL-6 (n = 10–12). (e) Representative western blot of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-Aktser473, serine phosphorylated Akt and total Akt. (f) Pretreatment with AG490 blocks IL-6-induced anorexia (n = 10–12 animals per group). (g, h) Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-JAK2, tyrosine phosphorylated JAK2 and total JAK2 (g); phospho-STAT3, Tyrosine phosphorylated STAT3 and total STAT3 (h). Data are the means±SEM. p<0.01, vs. control group; # p<0.05, vs. other groups. We next examined whether PI(3)K signaling is required for the IL-6-dependent reduction of food intake, by IL-6 administration in LY294002 pretreated (i.c.v.) animals. Pretreatment with LY294002 at a dose that did not alter food intake (1 nmol) had no effect on anorectic response to i.c.v. IL-6 (Figure 2d). Consistent with these data, we observed that a single IL-6 i.c.v. injection did not change Akt phosphorylation status in the hypothalamus (Figure 2e). The Akt protein levels were not different between the groups (Figure 2e, lower panel). These findings indicates that activation of neuronal mTOR is necessary for some of the effects of IL-6 on food intake and that these effects of IL-6 do not require any change in PI(3)K signaling. We also injected IL-6 (200 ng) into the third ventricle of rats and evaluated food intake and JAK2/STAT3 signaling. IL-6 caused a significant reduction in food intake (Figure 2f) and induced hypothalamic JAK2 and STAT3 phosphorylation (Figures 2g and h). The JAK2 and STAT3 protein levels were not different between the groups (Figures 2g and h, lower panels). To determine whether the effects of IL-6 on food intake are also JAK2/STAT3-dependent, we first identified a dose of AG490 that did not alter food intake when administered at the onset of the dark cycle. We then evaluated the effect of i.c.v. pretreatment with this dose of AG490 or its vehicle, on the anorectic response to i.c.v. IL-6 and we observed that IL-6 reduction of food intake was reversed by AG490 (Figure 2f). Physiological parameters measured in basal conditions after exercise protocol The plasma glucose level was lower in the exercised group compared to the control group (3.6±0.8 vs 4.6±0.5 mmol/L; n = 5; p<0.05) and the insulin levels were also lower (88±12 vs 193±17 pmol/L, n = 5; p<0.05). Exercise did not, however, reduce plasma leptin (2.6±0.5 vs 2.3±0.7 ng/ml). Insulinemia and leptinemia were not altered by third ventricle microinjection of leptin (data not shown). Exercise partially reverses the effects of AMPK agonists and fasting on food intake through modulation of the AMPK-mTOR signaling pathway in the hypothalamus To test the role of a single session of exercise on AICAR-increased food intake, AICAR (2 mM) or its vehicle were administered (i.c.v.) to control and exercised animals. 12-hour total food intake was measured after exercise. In exercised rats, AICAR (2 mM) did not cause any acute change in food intake but, in the control group, AICAR (2 mM) increased food intake by 32% (Figure 3a), suggesting that AICAR is not effective in exercised rats. Comparing AICAR-treated groups (control vs. exercise), exercised animals showed a 33% reduction in 12-hour total food intake (Figure 3a). 10.1371/journal.pone.0003856.g003Figure 3 AICAR, 2-DG and fasting effects on 12-h cumulative food intake and AMPK/mTOR signaling, in the hypothalami of control and exercised rats. (a) AICAR (2 mM) was administered in control and exercised rats. Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (b, c, d, e) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. (f) 2-DG (500 mg/Kg) was administered in control and exercised rats. Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (g, h, i, j) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. (k) Exercise decreases food intake in the fasting state (48 h fasting). Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (l, m, n, o) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK; phospho-ACCser79, serine phosphorylated ACC and total ACC; phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K; phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1. Data are the means±SEM. p<0.05, ** p<0.01, vs. control group; # p<0.05, ## p<0.001 vs. AICAR- or 2-DG-stimulated control group. Consistent with food intake, AICAR increased AMPK threonine and ACC serine phosphorylation levels in the hypothalami of control rats, whilst in exercised animals, AICAR did not change AMPK/ACC phosphorylation status (Figures 3b and c). Comparing AICAR treated groups (control vs. exercise), exercised animals showed reductions in AMPK threonine and ACC serine phosphorylation of 52% and 31%, respectively. AICAR reduced p70S6K and 4EBP1 threonine phosphorylation levels in the hypothalamus of control and exercised rats. However, comparing AICAR treated groups (control vs. exercise), exercised animals showed increases in p70S6K and 4EBP1 threonine phosphorylation of 230% and 310%, respectively (Figures 3d and e). The αAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 3b–e, lower panels). Similar results were observed after intraperitoneal treatment with 2-DG, another pharmacological activator of AMPK (Figure 3f–j). To be sure that the experiments represent a physiological condition we measured food intake in control and exercised animals after fasting. In the control animals, prolonged fasting (48 hours) increased ∼35% of food intake during 12-hours of refeeding period compared to control group. However, in the fasted rats, acute exercise session prevented fasting-induced hyperphagic response (Figure 3k). Comparing fasting treated groups (control vs. exercise), exercised animals showed reductions in AMPK threonine and ACC serine phosphorylation of 65% and 41%, respectively (Figures 3l and m). Comparing fasting groups (control vs. exercise), exercised animals showed increases in p70S6K and 4EBP1 threonine phosphorylation of 261% and 240%, respectively (Figures 3n and o). The αAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 3l–o, lower panels). Intracerebroventricular α-LA reduces food intake by modulating AMPK-mTOR hypothalamic phosphorylation levels to a greater extent in exercised animals α-LA is an essential cofactor of mitochondrial respiratory enzymes and exerts potent anti-obesity effects by suppressing hypothalamic AMPK activity [34]. The effects of α-LA (3 µg) intracerebroventricular administration, or its vehicle, on food intake control were studied by measuring the 12-hour total food intake after an acute exercise bout. In exercised rats, α-LA (3 µg) reduced food intake by 86% while control group showed a reduction of 58%. Comparing α-LA treated groups (control vs. exercise), exercised animals showed a 64% reduction in 12-hour total food intake (Figure 4a). 10.1371/journal.pone.0003856.g004Figure 4 α-LA effects on 12-h cumulative food intake and AMPK/mTOR signaling, in the hypothalami of control and exercised rats. (a) α-LA (3 µg) was administered in control and exercised rats. Animals were immediately exposed to food for a 12-hour period (n = 8–10 animals per group). (b, c, d, e) Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (b); phospho-ACCser79, serine phosphorylated ACC and total ACC (c); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (d); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (e). Data are the means±SEM. * p<0.05, vs. control group; # p<0.05, vs. exercised non-stimulated group. To determine the effects of exercise on the AMPK-mTOR signaling pathway, α-LA was i.c.v. administered and AMPK, ACC, p70S6K and 4EBP1 phosphorylation levels were assessed in the hypothalamus of all rats. α-LA reduced AMPK and ACC phosphorylation levels, in the hypothalami of control and exercised rats. Comparing α-LA treated groups (control vs. exercise), in exercised animals α-LA reduced both AMPK threonine phosphorylation and ACC serine phosphorylation of 39% and 57%, respectively (Figures 4b and c). α-LA induced p70S6K and 4EBP1 threonine phosphorylation in the hypothalami of control and exercised rats. Comparing α-LA treated groups (control vs. exercise), in exercised animals, α-LA increased p70S6K and 4EBP1 threonine phosphorylation of 19% and 11%, respectively (Figures 4d and e). α-LA did not change αAMPK, ACC, p70S6K and 4EBP1 protein expression (Figures 4b–e, lower panels). Intracerebroventricular leptin reduces food intake by modulating AMPK-mTOR hypothalamic phosphorylation levels to a greater extent in exercised animals The effects of leptin (10−6 M) i.c.v. administration or its vehicle on food intake control were studied by measuring the 12-hour total food intake after an acute exercise bout. In exercised rats, leptin (10−6 M) reduced food intake by 43%, when compared with exercised plus vehicle treated group, while the control group showed a reduction of 25%, when compared with vehicle treated group. Comparing leptin-treated groups (control vs. exercise), exercised animals showed a 31% reduction in 12-hour total food intake (Figure 5a). 10.1371/journal.pone.0003856.g005Figure 5 Leptin effects on 12-h cumulative food intake and AMPK/mTOR signaling, in the hypothalami of control and exercised rats. (a) Leptin (10−6 M) was administered in control and exercised rats. Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (b, c, d, e) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (b); phospho-ACCser79, serine phosphorylated ACC and total ACC (c); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (d); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (e). Data are the means±SEM. * p<0.05, ** p<0.01, vs. control group; # p<0.05, ## p<0.01 vs. leptin-stimulated control group. To determine the effects of exercise on AMPK-mTOR signaling pathway, leptin was i.c.v. administered and AMPK, ACC, p70S6K and 4EBP1 phosphorylation levels were assessed in the hypothalamus of all rats. Leptin reduced AMPK and ACC phosphorylation levels in the hypothalami of control and exercised rats. Comparing leptin-treated groups (control vs. exercise), in exercised animals, leptin reduced both AMPK threonine phosphorylation and ACC serine phosphorylation of 57% and 45%, respectively (Figures 5b and c). Leptin induced p70S6K and 4EBP1 threonine phosphorylation in the hypothalamus of control and exercised rats. Comparing leptin treated groups (control vs. exercise), in exercised animals, leptin increased both p70S6K and 4EBP1 threonine phosphorylation of 30% and 40% respectively (Figures 5d and e). The αAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 5b–e, lower panels). Role of IL-6 in anorectic response to leptin Hypothalamic IL-6 expression was detected in control animals; however, a 420% increase was observed in the exercised group (Figure 6a). We tested whether the inhibitory effects of leptin on food intake depends on IL-6, by i.c.v. infusion of anti–IL-6 antibody into exercised rats. Treatment with leptin (10−6 M) markedly reduced 12-h food intake in exercised rats pretreated with vehicle, although pretreatment with anti–IL-6 antibody blocked exercise-induced leptin responsiveness in a concentration-dependent manner (Figure 6b). 10.1371/journal.pone.0003856.g006Figure 6 Blockade of leptin induced inhibition of food intake by anti–IL-6 antibody. Hypothalami from rats were prepared as described in Research Design and Methods. (a) Tissue extracts from control and exercised rats were immunoblotted with anti–IL-6 antibody. (b) Leptin was injected intracerebroventricularly in control rats, exercised rats and exercised rats pretreated with anti–IL-6 at the doses indicated, and the animals were immediately exposed to food for a 12-hour period (n = 10–12 animals per group). (c, d, e, f) Representative Western blots of four independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (c); phospho-ACCser79, serine phosphorylated ACC and total ACC (d); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (e); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (f). Data are the means±SEM. p<0.05, vs. control, p<0.05, vs. control plus leptin, #p<0.05, vs. exercise plus leptin. Both AMPK and ACC phosphorylation levels, reduced by exercise, were reversed by anti–IL-6 antibody (Figure 6c and d). We also observed that the increased phosphorylations of p70S6K and 4EBP1, induced by exercise, were also reversed by anti–IL-6 infusion (Figures 6e and f). The αAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 6c–f, lower panels). The role of IL-6 on leptin responsiveness in the hypothalamus of diet-induced obesity (DIO) rats We next investigated the effect of IL-6 on leptin responsiveness in the hypothalamus of diet-induced obesity (DIO) rats after acute exercise. Hypothalamic IL-6 expression was detected in the hypothalamus of diet-induced obesity (DIO) rats; however, a 156% increase was observed in the DIO rats after acute exercise (Figure 7a). The effects of leptin (10−6 M) i.c.v. administration on energy intake control were studied by measuring the 12-hour total food intake after an acute exercise bout in DIO rats. Comparing leptin-treated rats (control vs. DIO), the energy intake was 28% higher in DIO rats after leptin infusion. However, the i.c.v. infusion of leptin was able to reduce the energy intake by about 31% in DIO rats after the exercise protocol, compared to DIO rats at rest (Figure 7b). Interestingly, the i.c.v. pretreatment with anti-IL-6 antibody (25 ng) blunted the anorexigenic effects of leptin in exercised DIO rats. 10.1371/journal.pone.0003856.g007Figure 7 Leptin effects on 12-h cumulative food intake and AMPK/mTOR signaling in the hypothalami of control, DIO and exercised DIO rats. (a) Representative Western blots of five independent experiments showing hypothalamic IL-6 expression in DIO Wistar rats at rest and immediately after acute exercise. (b) Leptin (10−6 M) was administered in control, DIO and exercised DIO rats. Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (c, d, e and f) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (c); phospho-ACCser79, serine phosphorylated ACC and total ACC (d); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (e); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (f). Data are the means±SEM. p<0.05, vs. control group, # p<0.05, vs. DIO group. To determine the effects of exercise on the AMPK-mTOR signaling pathway in the hypothalamus of DIO rats, leptin was i.c.v. administered and AMPK, ACC, p70S6K and 4EBP1 phosphorylation levels were assessed in the hypothalamus. The suppressive effects of i.c.v. infusion of leptin on AMPK and ACC phosphorylation were impaired in the hypothalamus of DIO rats by about 58 and 54%, respectively, when compared to the control group (Figure 7c and d). In exercised DIO rats, leptin reduced the phosphorylation of AMPK by 65% and ACC by 37%, compared to DIO rats at rest and the i.c.v. pretreatment with anti-IL6 antibody before the exercise protocol, reversed the suppressive effects of leptin on AMPK/ACC pathway in the hypothalamus of exercised DIO rats (Figures 7c and d). The AMPK and ACC protein levels did not differ between the groups (Figures 7c and d, lower panels). In addition, p70S6K and 4EBP1 phosphorylation in the hypothalamus of DIO rats after i.c.v. leptin infusion was reduced by about 46% and 45%, respectively, when compared to the control group. In exercised DIO animals, leptin increased the phosphorylation of p70S6K by 62% and 4EBP1 by 59%, compared to DIO rats at rest. I.c.v. pretreatment with anti-IL6 antibody before the exercise protocol blocked these effects in the hypothalamus of exercised DIO rats (Figures 7e and f). The p70S6K and 4EBP1 protein levels were not different between the groups (Figures 7e and f, lower panels). Blocking effects of AICAR and Rapamycin on leptin-induced anorexia We tested whether the i.c.v. administration of AICAR or Rapamycin, 60 minutes before the administration of leptin (10−6 M), prevents the anorexigenic effects of leptin. Leptin (10−6 M) treatment markedly reduced 12-h food intake in both control and exercised groups, although leptin was much more effective in exercised rats. AICAR (0.5 mM) or Rapamycin (25 µg), at doses that do not alter ingestion, completely blocked the suppression of food intake induced by an i.c.v. injection of leptin (10−6 M) (Figure 8a). The i.c.v. administration of leptin (10−6 M) to exercised rats pretreated with vehicle reduced AMPK and ACC phosphorylation in the hypothalamus by 63% and 60% respectively, compared with the control group. The administration of AICAR increased AMPK threonine and ACC serine phosphorylation, although at this dose, AICAR was not sufficient to induce significant an increase in food intake in exercised animals (data not shown). Comparing exercised animals, i.c.v. administration of leptin (10−6 M) to rats pretreated with AICAR increased both AMPK and ACC phosphorylation levels in the hypothalamus (Figures 8b and c). 10.1371/journal.pone.0003856.g008Figure 8 Blocking effects of AICAR and Rapamycin on leptin-induced anorexia. (a) Effect of i.c.v. administration of leptin on exercised rats; pretreatment with AICAR or Rapamycin (n = 8–10 animals per group). (b, c, d, e) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (b); phospho-ACCser79, serine phosphorylated ACC and total ACC (c); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (d); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (e). Data are the means±SEM. p<0.05, vs. other groups. The i.c.v. administration of leptin (10−6 M) to exercised rats pretreated with vehicle induced p70S6K and 4EBP1 phosphorylation in the hypothalamus of 60% and 70%, respectively, compared with the control group. Comparing exercised animals, i.c.v. administration of leptin (10−6 M) to rats pretreated with AICAR reduced both p70S6K and 4EBP1 phosphorylation levels in the hypothalamus. Exercised animals pretreated with Rapamycin also reduced hypothalamic p70S6K and 4EBP1 phosphorylation (Figures 8d and e). The αAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 8b–e, lower panels). Discussion During the last decade, a substantial number of studies have investigated the role of physical activity in the control of energy intake in rodents [5], [6], [35] and in humans [36]–[38]. However, the molecular mechanisms by which exercise controls food intake are still unsolved. Several experimental studies have demonstrated that neither acute [5], [36] nor chronic [6], [39] exercise per se change food intake, on the other hand, accumulating evidence shows that both acute and chronic exercise potentiate the anorexigenic effects of leptin in the hypothalamus. Our data indicate that IL-6 signaling through AMPK and mTOR reduces food intake in a dose-dependent manner. Leptin, as well as α-LA infusion, reduced food intake in exercised rats to a greater extent than that observed in control animals. Conversely, AICAR, 2-DG and fasting increased food intake in exercised rats to a lower extent than that observed in control animals. Exercise was associated with the effects of leptin on the AMPK/mTOR pathway activity in the hypothalamus. In addition, we investigated the regulatory role of IL-6 in mediating the increase in leptin responsiveness in the hypothalamus. Treatment with leptin markedly reduced food intake, AMPK activity and increased mTOR activity in exercised rats that were pretreated with vehicle, although no increase in response to leptin-induced anorexia and modulation of AMPK/mTOR pathway were detected after i.c.v. pretreatment with anti-IL-6 antibody. Taken together, these results suggest that IL-6 is a major component of the effects of exercise on the control of food intake. Increasing evidence shows that leptin and IL-6 activates AMPK in the peripheral tissues, such as skeletal muscle and adipose tissue, increasing fatty acid oxidation and glucose uptake in these tissues [40]–[42], however, leptin has an opposing effect in hypothalamic tissue, reducing neuronal AMPK activity [12], [43]. As well as in response to leptin, in the present study, we demonstrated that IL-6 alone reduced AMPK phosphorylation in the hypothalamus of rats. We also show that, IL-6 increased ATP levels and decreased the AMP/ATP ratio in the hypothalamus (figures 1b–d), but the mechanisms by which IL-6 modulates the ATP levels require further investigations. A number of recent studies have shown that AMPK plays a key role in regulating both energy intake and expenditure [12], [34], [44], [45]. In peripheral tissues, such as skeletal muscle, activation of AMPK switches on energy producing pathways and switches off energy consuming pathways. In the hypothalamus, activation of AMPK leads to increased feeding, thereby increasing energy intake. Conversely, inhibition of AMPK in the hypothalamus reduces food intake. These dual functions of AMPK suggest that it may act to coordinate energy expenditure with energy intake. There is already some evidence that this may be the case in one situation. More recently, Gao and colleagues [10] demonstrated that the hypothalamic ACC activation makes an important contribution to the anorexigenic effects of leptin that are mediated by AMPK. The aim of this study was to investigate whether IL-6 could affect AMPK activity in the hypothalamus, thereby providing a potential mechanism for the coordination of energy expenditure and energy intake during, or following exercise. Beyond STAT3 activation, we detected changes in the hypothalamic AMPK activity in rats after i.c.v. infusion of IL-6; we show that, IL-6 markedly decreased phospho-AMPK abundance (an index of activity) in the hypothalamus. In accordance with the reduction in AMPKthr172 phosphorylation, we observed that, after IL-6 administration, hypothalamic AMP∶ATP ratio was decreased. Wallenius et al [28] elegantly showed that long-term peripheral IL-6 treatment to Il6−/− mice caused a decrease in body weight. In addition to increasing energy expenditure, IL-6 may prevent obesity by inhibiting feeding as obese IL-6−/− mice had increased absolute food intake. However, central IL-6 treatment at the same dose that we used does not influence food intake in mice. In concordance with our results another study of the same group showed a reduced daily food intake over a two-week ICV treatment period with IL-6 in rats indicating a different pattern of response between rats and mice [46]. The mTOR, an evolutionary conserved serine-threonine kinase, central to integrating similar signals to control food intake, has now emerged as a detector of hormonal and nutritional signals in the hypothalamus [13], [15]. In this study, we investigated whether IL-6 activates mTOR. IL-6 increased mTOR activity; moreover inhibition of central mTOR reversed the anorectic effect of IL-6. In addition, the anorexigenic effect of IL-6 was absent in AICAR- and Rapamycin-pretreated rats, however, pretreatment with LY294002 - a PI(3)K inhibitor - had no effect on IL-6 induced anorexia, indicating that, in the hypothalamus, the effect of IL-6 is independent of the PI(3)K pathway. Signaling through gp130 commonly results in activation of PI(3)K, and IL-6 can activate PI(3)K [47] and its downstream target Akt [48]–[51], but it should be noted that this effect has not been observed in all studies [52], suggesting a tissue-dependent effect. Next, we investigated whether the increased sensitivity of the leptin action on food intake induced by exercise, could be due to the modulation of AMPK activity. As previously shown [43], exercise, per se, does not alter AMPK activity in the hypothalamus; however, we observed that the normal inhibition of AMPK phosphorylation and activity in the hypothalamus, induced by leptin administration, was improved in both lean and diet-induced obesity rats after acute protocol of exercise. In addition, we did not observe any normal stimulation of AMPK activity by AICAR in the hypothalamus of exercised rats, indicating that AMPK pathway is disrupted. This observation agrees with data from aging studies in which acute stimulation with AICAR was blunted in skeletal muscle of old rats [53]. Furthermore, fasting and the use of another activator of AMPK (2-DG) in exercising rats resulted in a lower activation of AMPK when compared to the control animals. In contrast, the pharmacological inhibition of AMPK by α-LA results in a greater inhibition of AMPK activity, compared to control animals. The mechanism by which exercise inhibits AMPK-induced food intake in the hypothalamus is not clear. Several lines of evidence point to a possible link between inhibited AMPK-induced food intake in the hypothalamus and IL-6 signaling through the AMPK/mTOR pathway. Firstly, we found that the leptin-inhibited food intake enhanced by exercise was blunted by anti-IL-6 antibody. Secondly, exercise induced increased response of leptin-inhibited AMPK signaling was reverted by AICAR. Finally, exercise induced increased responsiveness of leptin stimulated mTOR signaling was reverted by rapamycin. Our data are in accordance with earlier studies demonstrating that IL-6 treatment enhances energy expenditure in both rodents and humans [28], [54]–[56]. In exercising rats, hypothalamic leptin and insulin responsiveness are increased in an IL-6-dependent manner [5], [6]. It has been previously shown that IL-6 treatment stimulates energy expenditure at the level of the brain in rodents [28], [55], [57], and it might be assumed that endogenous IL-6 also acts on the brain during exercise. The IL-6 exerting this effect during exercise could be produced by the brain itself, which has been shown to have increased IL-6 production during exercise [58]. Alternatively, the large quantities of endocrine IL-6 produced from working skeletal muscle [58] might reach appropriate sites in the brain [21], [59], [60]. Interestingly, we did not observe any difference in food intake over a 12-h period, although the levels of hypothalamic IL-6 dramatically increased after exercise. At first glance, these data appear to be contradictory. However, a large decrease in insulin level was observed after exercise, and we have previously shown a synergic effect of IL-6 on the insulin and leptin signaling pathway, in the rat hypothalamus [5]. Since we did not observe modifications in the phosphorylation of JAK2 and the downstream targets of mTOR after exercise, these data suggest that the cross-talk between insulin, IL-6 and leptin have an essential role in controlling food intake after exercise. In this case, it is possible that increases in IL-6 levels were counterbalanced by the reduction in insulin levels. However, the present study has certain limitations. Exercise per se did not evoke any meaningful effect in terms of food intake; rather, it seemed to enhance the anorectic effect of exogenous leptin. Thus, the data presented herein may suggest but do not establish the mechanism by which long term exercise decreases leptin levels; whilst increases the response to leptin, contributing to its food-suppressive actions. Furthermore, settings of activation of AMPK or inactivation of mTOR were selected to induce changes in target protein phosphorylation, but not food intake. Such dissociation does not preclude a pharmacological rather than physiological effect of our data. Increased responsiveness of leptin action in the hypothalamus, through modulation of the AMPK-mediated pathway by exercise, could be pathophysiologically important in the prevention of obesity. Recent studies have shown that modulation of leptin signaling through the AMPK pathway could be involved in the development of obesity [61]. Taken together, these data indicate that the anti-obesity actions, induced by leptin, could be increased due to the more pronounced inhibition of the AMPK pathway observed after leptin infusion in the hypothalamus of both lean and diet-induced obesity rats after acute exercise. If the mechanism used by IL-6 to reduce food intake is AMPK-dependent, as our results suggest, the defective activation of AMPK in the hypothalamic neurons induced by exercise may increase the ability of leptin to reduce food intake. In conclusion, exercise improved the AMPK and mTOR responses to leptin administration and contributed to appetite-suppressive actions. This increased dynamic responsiveness of the AMPK/mTOR pathway to leptin could provide information regarding the molecular mechanism underlying the biological sensitivity to leptin in exercise. Furthermore, these findings provide support to the hypothesis that AMPK and mTOR interact in the hypothalamus to control feeding in exercised rats, in an IL-6-dependent manner. Methods Antibodies and Chemicals Reagents for SDS-polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad (Richmond, CA, USA). Tris[hydroxymethyl]amino-methane (Tris), aprotinin, ATP, dithiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, Tween 20, glycerol, and bovine serum albumin (fraction V) were from Sigma Aldrich (St. Louis, MO, USA). Protein A-Sepharose 6 MB and Nitrocellulose paper (Hybond ECL, 0.45 µm) from Amersham Pharmacia Biotech United Kingdom Ltd. (Buckinghamshire, United Kingdom). Ketamin was from Parke-Davis (São Paulo, SP, Brazil) and diazepam and thiopethal were from Cristália (Itapira, SP, Brazil). Anti-phospho-[Ser79] ACC (rabbit polyclonal, #07-184) and anti-phospho- [Tyr1007/1008] JAK2 (rabbit polyclonal, AB3805) antibodies were from Upstate Biotechnology (Charlottesville, VA, USA). Anti-ACC (goat polyclonal, sc-26816), anti-JAK2 (rabbit polyclonal, sc 278), anti-STAT3 (rabbit polyclonal, sc 483) and anti-IL-6 (rabbit polyclonal, sc-7920) antibodies were from Santa Cruz Biotechnology, Inc. Anti-phospho-[Thr172] AMPKα (rabbit polyclonal, #2531), anti-AMPKα (rabbit polyclonal, #2532), anti-phospho- [tyr705] STAT3 (rabbit polyclonal, #9135), anti-phospho- [Thr389] p70S6K (rabbit polyclonal, #9205), anti-p70S6K (rabbit polyclonal, #9202), anti-phospho- [Thr70]4EBP1 (rabbit polyclonal, #9455), anti-4EBP1 (rabbit polyclonal, #9452), anti-phospho- [Ser 473]Akt (rabbit polyclonal, #9271), and anti-Akt (rabbit polyclonal, #9272) were from Cell Signalling Technology (Beverly, MA, USA). Leptin, LY294002, and Interleukin-6 were from Calbiochem (San Diego, CA, USA); 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), 2-Deoxy-D-glucose and α-lipoic acid were from Sigma Chemical Co. (St. Louis, MO). Rapamycin was from LC Laboratories (Woburn, MA, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Serum insulin and leptin quantification Plasma was separated by centrifugation (1100 g) for 15 minutes at 4°C and stored at −80°C until assayed. RIA was employed to measure serum insulin, according to a previous description [62]. Leptin concentrations were determined using a commercially available Enzyme Linked Immuno Sorbent Assay (ELISA) kit (Crystal Chem Inc, Chicago, IL). Experimental Animals Male Wistar rats (8 weeks old/250–300 g) obtained from the University of Campinas Animal Breeding Center were used in the experiments. The investigation was approved by the ethics committee and followed the University guidelines for the use of animals in experimental studies and conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no. 85-23 revised 1996). The animals were maintained on 12h∶12h artificial light–dark cycles and housed in individual cages. Diet induced obesity (DIO) Male 4-wk-old Wistar rats from the University of Campinas Breeding Center were randomly divided into two groups, control, fed standard rodent chow (3948 kcal.Kg−1) and DIO, fed a fat-rich chow (5358 kcal.Kg−1) ad libitum for 3 months and then submitted to the different experimental protocols. This diet composition has been previously used [63]. Intracerebroventricular (i.c.v.) cannulation The animals were stereotaxically instrumented under intraperitoneal injection of a mix of ketamin (10 mg) and diazepam (0.07 mg) (0.2 ml/100 g body weight) with a chronic 26-gauge stainless steel indwelling guide cannula, aseptically placed into the third ventricle (0.5 mm posterior and 8.5 mm ventral to bregma), as previously described [64]. After a 1-wk recovery period, cannula placement was confirmed by a positive drinking response after administration of Angiotensin II (40 ng/2 µL); animals that did not drink 5 mL of water within 15 minutes after treatment were not included in the experiment. Exercise Protocol Rats were acclimated to swimming for 2 days (10 min per day). On the day of the experiment, animals swam in groups of four, in plastic barrels of 45 cm in diameter, filled to a depth of 50 cm. Water temperature was maintained at 34–35°C. They performed two 3-h exercise bouts, separated by one 45-min rest period, as previously described [65]. After the last exercise bout, some rats were injected into the cannula and food intake was determined over the next 4 and/or 12 h; the other rats were injected into the cannula and then anesthetized with intraperitoneal injection of sodium thiopethal (5 mg/100 g body weight) and hypothalamus was removed. Treatments For acute treatments, rats were deprived of food for 6 h with free access to water and i.p. injected (200 µl bolus injection) with either vehicle or 2-DG (500 mg/kg) or i.c.v. injected (3 µl bolus injection) with either vehicle, IL-6 (100 ng or 200 ng), AICAR (0.5 or 2.0 mM), Rapamycin (25 µg), α-LA (3 µg), leptin (10−6 M), LY294002 (50 µM) or anti-IL-6 antibody. Similar studies were carried out in rats that were initially pre-treated with i.c.v. microinjection of vehicle, AICAR, Rapamycin, anti-IL-6 antibody or LY294002, and after 60 min with i.c.v. microinjection of IL-6 or leptin. Thereafter, standard chow was given and food intake was determined by measuring the difference between the weight of chow given and the weight of chow at the end of 4 and/or 12-h periods. All acute treatments were performed at 5:00 and 6:00 p.m. Western Blot Analysis After exercise and i.c.v. treatments, rats were anaesthetized with intraperitoneal injection of a mix of ketamin (10 mg) and diazepam (0.07 mg) (0.2 ml/100 g body weight), and used as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The rats were killed, and hypothalamus was quickly removed, minced coarsely and homogenized immediately in a freshly prepared ice-cold buffer (1% Triton X-100, 100 mmol/l Tris pH 7.4, 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium vanadate, 2 mmol/l phenyl methylsulphonyl fluoride and 0.1 mg aprotinin) suitable for preserving phosphorylation states of enzymes and Western blot was performed, as previously described [66]. Insoluble material was removed by centrifugation (50 000 g) for 25 minutes at 4°C. Total extracts of hypothalamus were prepared and 0.25 mg total proteins were separed by SDS-PAGE. After SDS-PAGE (15% resolving gels for phospho-4EBP1 and 4EBP1; 12% resolving gels for phospho-AMPK, AMPKα2; phospho-p70S6K, p70S6K, phospho-Akt, Akt, phospho-STAT3 and STAT3; 8% resolving gels for phosphor-JAK2 and JAK2; 6.5% resolving gels for ACC and phospho-ACC), proteins were transferred from gel to nitrocellulose membrane. Membranes were blocked in 5% nonfat dried milk in PBST (139 mM NaCl, 2.7 mM KH2PO4, 9.9 mM Na2HPO4, and 0.1% Tween 20) for 2 hours and then incubated overnight with specific antibodies. After incubation with the relative second antibody, immune complexes were detected using the ECL method. Results were visualized by autoradiography using preflashed Kodak XAR film (Eastman Kodak, Rochester, N.Y., USA) with Cronex Lightning Plus intensifying screens at −80°C for 12–48 h. (Mass., USA). Band intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software - Scion Corporation, Frederick, Md., USA). Confocal microscopy Paraformaldehyde-fixed hypothalami were sectioned (5 µm). The sections were obtained from hypothalamus of six rats per group in the same localization (antero-posterior = −1.78 from bregma) and used in regular single- or double-immunofluorescence staining using DAPI, anti-IL6 receptor (IL6R), anti-AMPK, anti-phospho-p70S6K (1∶200; Santa Cruz Biotechnology), antibodies, according to a previously described protocol [67]. Analysis and photodocumentation of results were performed using a LSM 510 laser confocal microscope (Zeiss, Jena, Germany). The anatomical correlations were made according to the landmarks given in a stereotaxic atlas [68]. Chromatography After exercise, i.c.v. infusion of IL-6 or vehicle, rats were anaesthetized, and used as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The animals were killed and, within 40 seconds, the cranium was opened, the brain was removed and the hypothalamus was quickly dissected and frozen in liquid N2 for chromatographic analyses. Chromatographic analyses were carried out on a Waters Alliance equipment series 2695 (Milford, MA, USA) equipped with a quaternary pump, an autosampler, a degasser, and a Waters 2475 fluorescence detector model. The fluorescence of derivatized compounds (ATP, ADP, AMP, and ADO) was monitored with excitation and emission wavelengths set at 280 and 420 nm, respectively. Chromatographic separations of the compounds were achieved at room temperature, using a reversed-phase Cosmosil 5C18-MS column (150×4.6 mm i.d.; 5 µm particle size) with a Cosmosil guard column (5C18-MS 10×4.6 mm) purchased from Phenomenex (Torrance, CA, USA). The mobile phase composition was 50 mmol/L KH2PO4, 25 mmol/L citric acid (pH 4.5), and methanol (90∶10, v/v), which was prepared immediately before use and filtered through a 0.45 µm filter (Millipore, Milford, MA, USA). The column was equilibrated and eluted under isocratic conditions using a flow rate of 1.0 ml/min. The chromatographic run time for each analysis was 20 min. Aliquots of 25 µl were injected into the HPLC system. System control, data acquisition, and processing were performed with a PC-Pentium IV Processor personal computer from Dell, operated with Microsoft Windows XP version 2003 and Waters Empower 2002 chromatography software. A validation chromatographic run included a set of calibration samples assayed in duplicate and quality control samples at four levels in triplicate. The standard calibration curves for known amounts of ATP, ranging from 0.025 to 10.0 µmol/L, were linear (R>0.999) and could be described by the linear regression equation: y = 0.4992x−0.0463 (n = 4, P<0.0001, r = 0.9997), in which y is the ATP concentration in micromoles and x is the chromatogram peak area. Statistical Analysis All numeric results are expressed as the means±SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands or spots in autoradiographs and quantified by optical densitometry (Scion Image). Statistical analysis was performed by employing the ANOVA test with Bonferroni post test. Significance was established at the p<0.05 level. We thank Dr Nicola Conran for English language editing. Competing Interests: The authors have declared that no competing interests exist. 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==== Front Mol VisMVMolecular Vision1090-0535Molecular Vision 2562008MOLVIS203Research ArticleNeuroprotective effects of cannabidiol in endotoxin-induced uveitis: critical role of p38 MAPK activation El-Remessy A.B. 145Tang Y. 3Zhu G. 1Matragoon S. 45Khalifa Y. 1Liu E.K. 2Liu J-Y. 1Hanson E. 1Mian S. 2Fatteh N. 2Liou G.I. 11 Department of Ophthalmology, Medical College of Georgia, Augusta, GA2 School of Medicine, Medical College of Georgia, Augusta, GA3 Department of Neurology, The Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P. R. China4 Program in Clinical and Experimental Therapeutics, University of Georgia, Athens, GA5 V.A. Medical Center, Augusta, GACorrespondence to: Gregory I. Liou, Department of Ophthalmology, Medical College of Georgia, 1120 15th Street, Augusta, GA, 30912; Phone: (706) 721-4599; FAX: (706) 721-7913; email: [email protected] 03 12 2008 14 2190 2203 17 6 2008 24 11 2008 2008Molecular VisionThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose Degenerative retinal diseases are characterized by inflammation and microglial activation. The nonpsychoactive cannabinoid, cannabidiol (CBD), is an anti-inflammatory in models of diabetes and glaucoma. However, the cellular and molecular mechanisms are largely unknown. We tested the hypothesis that retinal inflammation and microglia activation are initiated and sustained by oxidative stress and p38 mitogen-activated protein kinase (MAPK) activation, and that CBD reduces inflammation by blocking these processes. Methods Microglial cells were isolated from retinas of newborn rats. Tumor necrosis factor (TNF)-α levels were estimated with ELISA. Nitric oxide (NO) was determined with a NO analyzer. Superoxide anion levels were determined by the chemiluminescence of luminol derivative. Reactive oxygen species (ROS) was estimated by measuring the cellular oxidation products of 2’, 7’-dichlorofluorescin diacetate. Results In retinal microglial cells, treatment with lipopolysaccharide (LPS) induced immediate NADPH oxidase-generated ROS. This was followed by p38 MAPK activation and resulted in a time-dependent increase in TNF-α production. At a later phase, LPS induced NO, ROS, and p38 MAPK activation that peaked at 2-6 h and was accompanied by morphological change of microglia. Treatment with 1 μM CBD inhibited ROS formation and p38 MAPK activation, NO and TNF-α formation, and maintained cell morphology. In addition, LPS-treated rat retinas showed an accumulation of macrophages and activated microglia, significant levels of ROS and nitrotyrosine, activation of p38 MAPK, and neuronal apoptosis. These effects were blocked by treatment with 5 mg/kg CBD. Conclusions Retinal inflammation and degeneration in uveitis are caused by oxidative stress. CBD exerts anti-inflammatory and neuroprotective effects by a mechanism that involves blocking oxidative stress and activation of p38 MAPK and microglia. GalleyStatusExport to XML ==== Body Introduction Degenerative retinal diseases such as uveitis, glaucoma, macular degeneration, and diabetic retinopathy all involve inflammation with activated microglia [1]. Inflammation is an active defense reaction against diverse insults, designed to remove or inactivate noxious agents and to inhibit their detrimental effects. Although inflammation serves as a protective function in controlling infections and promoting tissue repair, it can also cause tissue damage and disease. Following brain injury, inflammation occurs in response to glutamate, reactive oxygen species (ROS), nitric oxide (NO), and cytokines including tissue necrosis factor (TNF)-α, released from activated microglia or macrophage, leading to neurodegeneration [2]. To understand how inflammation affects retinal function in degenerative retinal diseases, it is necessary to examine the processes and signaling pathways during inflammation with in vivo and in vitro models. Endotoxin-induced uveitis (EIU) in rodents is an in vivo model for acute ocular inflammation induced by systemic or local injection of lipopolysaccharide (LPS) [3,4]. EIU is characterized by a breakdown of the blood–ocular barrier [2] with inflammatory cell infiltration involving the anterior and posterior segments of the eye [4] and accelerated death of retinal ganglion cells [5]. To further elucidate the molecular events of retinal inflammation, LPS-activated cultured retinal microglial cells have been used as a model to simulate neuroinflammation [6]. The p38 mitogen-activated protein kinase (p38 MAPK), a stress-activated serine/threonine protein kinase, is a downstream target of proinflammatory cytokines and oxidative stress. In addition, activation of p38 MAPK has been also implicated in both induction of inflammatory mediators and transcription-independent effects such as induction of actin reorganization and cellular motility [7-9]. The neuroprotective effects of a nonpsychoactive cannabinoid, cannabidiol (CBD), are largely mediated by its ability to scavenge ROS [10]. We have shown that CBD reduces diabetes- and glutamate-induced ROS formation, p38 MAPK activation, blood–retina barrier breakdown, and retinal degeneration [11,12]. Cannabinoids are known to serve as an anti-inflammatory by modulating the activity of cerebral microglia during inflammation [13]. To date, however, the cellular and molecular mechanism by which CBD reduces inflammation in degenerative retinal diseases is still unclear. In the present study, we test the hypothesis that retinal inflammation and degeneration are initiated by oxidative stress, which activates p38 MAPK, and causes cytokine release that eventually leads to the activation of microglial cells and neurodegeneration. We also show that the neuroprotective and anti-inflammatory effects of CBD involve reducing oxidative stress and modulating p38 MAPK activation in EIU model and LPS-treated retinal microglial cells. Methods Animal preparation and experimental design This study used inbred male, 8-10-week-old Sprague-Dawley (SD) rats, each weighing approximately 250 g (Charles River, Durham, NC). The animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three sets of animals were prepared for a total of 72 rats to study the effect of CBD on EIU. The CBD-treated control or uveitis group received one intraperitoneal injection of CBD (National Institute of Drug Abuse, Research Triangle Park, NC) at 5 mg/kg bodyweight in a 0.25 ml solution that contained 1 part alcohol to 1 part Cremophor EL to 18 parts Ringer solution. This dose was selected based on previous studies showing maximum protection of CBD [11,12]. Control groups received vehicle injections. One hour after CBD or vehicle injection, rats were anesthetized with an intraperitoneal injection of 40 mg/kg Nembutal (Abbott Laboratories, Abbott Park, IL). LPS (Sigma, St. Louis, MO), from Salmonella typhimurium prepared in sterile saline at 1 mg/ml, was given at 0.35 mg/kg bodyweight [13,14]. Experiments were performed 24 h after LPS injection, using 3–6 rats for each group in each experiment. Immunohistochemistry Immunohistochemistry was performed to identify macrophages or activated microglia on the flat-mounted retinas [14] using monoclonal antibody CD11b. This antibody reacts with the CR3 complement receptor expressed on monocytes, granulocytes, macrophages, dendritic cells, natural killer (NK) cells, and a subset of lymphocytes. Incubation with the primary and Texas-red fluorescence-labeled secondary antibodies at 4 °C was performed overnight. Immunostained, flat-mounted retinas were placed on slides with the inner side of the retina facing up, covered, and examined with confocal microscopy. Primary retinal microglia culture Microglial cells were isolated from retinas of newborn SD rats according to a previous procedure [15], with minor modifications. Briefly, retinas were dissected within 48 h from newborn SD rat pups. Tissues were collected into 0.01 M PBS (0.01 M NaH2PO4/Na2HPO4, 0.15 M NaCl, pH 7.4) and washed with ice-cold 0.01 M PBS, then digested with 0.125% trypsin for 3–5 min before mixing with a 1:1 blend of DMEM and F12 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) and 1% penicillin/streptomycin (Mediatech, Herndon, VA). Retina pieces were triturated by passing through a disposable pipette several times until cells were dispersed. Cells were then filtered through a 100 μm mesh, collected by centrifugation, resuspended in culture medium, and plated onto T150 cell culture flasks (Corning, Corning, NY) at a density of 2×105 cells/cm2. All cultures were maintained in a humidified CO2 incubator at 37 °C and 5% CO2 and fed on the third day, then once every 4 days. After 2 weeks, microglial cells were harvested by shaking the flasks at 100 rpm for 1 h. The cell suspension was centrifuged and the detached cells were replated in a 1:1 mixture of DMEM and F12 medium plus 10% FBS overnight and then in serum-free, low-protein medium (Cellgro Complete containing 0.1% BSA; Mediatech) at designated densities for various experiments. Immunocytochemical studies showed that more than 95% of the cultured cells stained positively for CD11b (Figure 1), with staining localized to the cell membrane. Almost none of these cells showed positive staining for glial fibrillary acidic protein (GFAP), indicating that the majority of the isolated cells were microglia and were not contaminated with astrocytes or Müller cells (data not shown). Figure 1 Cannabidiol prevents retinal microglial activation or macrophage infiltration and inhibits serum and retinal tumor necrosis factor release in the uveitic rat. A-E: Confocal micrographs of retina whole-mounts or sections that show activated microglia or infiltrated macrophages as stained by the microglia/macrophage-specific marker CD11b and by Texas red- or Oregon green-conjugated secondary antibody. A: Normal rat. B: Twenty-four h after lipopolysaccharide (LPS) injection. C: Cannabidiol (CBD)-pretreated and LPS-injected. D: Microglia in normal eye sections, Oregon green, counter-stained with propidium iodide (red). E: Microglia in normal retinal whole-mounts, Oregon green. F: Serum TNF-α levels in 3 rats, 24 h after LPS injection with or without CBD treatment (mean±SEM; asterisk represents that it is significantly different when compared with the control at p<0.05). G: Retinal TNF-α levels in 3 rats, 24 h after LPS injection with or without CBD treatment (mean±SEM; asterisk represents that it is significantly different when compared with the control at p<0.05). Drug treatment effects on cultured microglial cells Microglia collected from culture flasks were seeded at a density of 5×105 cells/well in 24 well tissue culture plates, or 1×105 cells/well in 96 well plates. One day after seeding, the culture wells were washed with Cellgro Complete (Mediatech) and incubated in the same medium with various treatments. For microglia treatment, LPS at the final concentration of 30 ng/ml (Escherichia coli 0111:B4; Sigma) was added to each well for 24 h. CBD (Cayman Chemical, Ann Arbor, MI) was dissolved in dimethyl sulfoxide (DMSO) as 30 mM stock solution and used at a final concentration of 1 μM. Apocynin, an NADPH oxidase assembly inhibitor, was dissolved in DMSO as 3M stock solution and a range of concentrations of 15, 100, and 200 µM was used. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial oxidase, was used at 15 μM. Finally, SB 203580, a specific inhibitor of p38 MAPK, was supplied as a 1 mg/ml solution in DMSO and used at a final concentration of 10 μM. At indicated time points, 50 μl aliquots of incubation medium were taken and analyzed for ROS, TNF-α, and NO. Cells were used for superoxide anion assay by chemiluminescence. Also at each time point, cells were washed, homogenized, and subjected to western analysis as will be described. Morphological analysis of cultured microglial cells Microglia collected from culture flasks were seeded at a density of 2×105 cells/chamber in 4 chambered slides. The purity of the microglial cultures and their morphological responses to LPS and CBD treatments were ascertained using immunocytochemical staining analysis for CD11b, a microglial marker, or for F-actin distribution using fluorescent phallotoxins (Molecular Probes, Eugene, OR). The purity and morphology of microglia in culture was examined with confocal microscopy. Protein extraction and western blot analysis Dissected individual rat retinas or washed cultured cells were homogenized in a Mini-Bead beater with treated Ottawa sand in modified RIPA buffer (Upstate, Lake Placid, NY), containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% deoxycholate, supplemented with 40 mM NaF, 2 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride and 1:100 (v/v) of proteinase inhibitor cocktail (Sigma). Insoluble material was removed by centrifugation at 12,000x g at 4 °C for 30 min. Protein was determined by DC Protein Assay (Bio-Rad, Hercules, CA). Antibodies for β-actin (Sigma), phospho-p38 MAPK, p38 MAPK, and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). Protein of 50−100 μg was boiled in Laemmli sample buffer, separated by SDS–PAGE on a 10% or gradient gel (4 to 20%; Bio-Rad), transferred to nitrocellulose membrane and incubated with specific antibodies. The primary antibody was detected using a horseradish peroxidase-conjugated goat antirabbit antibody and ECL advanced chemiluminescence (Amersham BioSciences, Buckinghamshire, UK). Intensity of immunoreactivity was measured by densitometry (n=3–5 in each group). Enzyme-linked immunosorbent assay for TNF-α in culture medium TNF-α levels in rat serum or supernatants of culture medium were estimated with ELISA kits (R&D, Minneapolis, MN) per the manufacturer’s instructions. Standards and samples were added and bound by the immobilized antibody. Samples were washed by the washing solution provided in the ELISA kit, and an enzyme-linked polyclonal antibody specific for the cytokine was added to the wells followed by a substrate solution, yielding a colored product. The intensity of the color was measured at 450 nm. The sample levels were calculated from a standard curve and were corrected for protein concentration. Nitric oxide measurement Nitrite (NO2-), the stable breakdown product of NO, was quantitatively reduced to NO and could be quantified by a chemiluminescence detector after reaction with ozone in an NO analyzer (Sievers, GE Analytical Instruments, Boulder, CO). Briefly, medium of cultured microglial cells were deproteinized, and samples containing NO2- were refluxed in glacial acetic acid containing sodium iodide. The amount of NO generated was calculated as the difference in basal and LPS-stimulated NO levels. Chemiluminescence The superoxide anion levels were determined in the LPS-treated cells in the presence or absence of 1 μM CBD, 15 μM apocynin (an NADPH oxidase assembly inhibitor), 15 μM TTFA (a mitochondrial oxidase inhibitor), or 100 U/ml superoxide anion dismutase. Cell medium in 96 well plates was replaced with 50 μl of Earle’s balanced salt solution. An equal volume of the same solution containing 800 μM of highly sensitive luminol derivative L-012 (Pure Chemical Industries, Osaka, Japan) [16] was added to each well. Chemiluminescence was measured with a Synergy-2 plate reader (Bio-Tek Instruments, Winooski, VT). Dichlorofluorescein (DCF) assay Dichlorofluorescein (DCF) is the oxidation product of the reagent 2’,7’-dichlorofluorescin diacetate (H2DCFDA; Molecular Probes), a marker of cellular oxidation by hydrogen peroxides and peroxynitrite [17]. Briefly, a 5 mM solution of H2DCFDA was prepared in absolute ethanol and stored under N2 at −20 °C, in the dark. Earle’s balanced salt solution containing 5 μM H2DCFDA was directly applied to and incubated with frozen eye sections for 30 minutes in the dark at room temperature. The sections were then washed with Earle’s balanced salt solution [12]. The fluorescence of DCF was measured and analyzed in eye sections or in cell lysates. The average retinal fluorescence intensity (6 fields/retina) was analyzed using fluorescence microscopy and Ultra-View morphometric software (Molecular Devices, Sunnyvale, CA). The fluorescence of the cell lysates was determined using a spectrofluorometer (Synergy-2 plate reader; BioTek Instruments Inc, Winooski, VT) with 488 nm excitation and 530 nm emission. Measurement of retinal nitrotyrosine Nitrotyrosine immunoreactivity was measured as an indicator for peroxynitrite formation. The distribution of nitrotyrosine in frozen eye sections was analyzed using immunolocalization techniques with confocal microscopy using antibodies specific for nitrotyrosine [12]. Frozen eye sections were fixed with 4% paraformaldehyde then reacted with a polyclonal rabbit antinitrotyrosine antibody (Upstate Biotechnology) followed by Oregon green-conjugated goat antirabbit antibody (Molecular Probes). Data (6 fields/retina; n=6 in each group) were analyzed using fluorescence microscopy and Ultra View Morphometric software to quantify intensity of immunostaining. Terminal dUTP nick end-labeling (TUNEL) Eyes were mounted in optimal cutting temperature (OCT) and 10 μm sections were collected and stored at −80 °C. TUNEL was performed in these frozen sections using the Apop TAG in situ cell death detection kit (TUNEL-FITC; Chemicon International) [11]. Data analysis The results are expressed as mean ±SEM. Differences among experimental groups were evaluated by ANOVA, and the significance of differences between groups was assessed by the posthoc test (Fisher’s PLSD) when indicated. Significance was defined as p<0.05. Results CBD inhibits retinal microglial activation in uveitic rats We examined the retinas from LPS-treated rats to test the hypothesis that activated retinal microglia or infiltrated macrophages are present in the retina during inflammation. Serum and retinas were collected for TNF-α measurement 24 h after footpad injection of LPS. The retinas were also examined by immunofluorescence using an antibody for CD11b, a marker for microglia or macrophages. As shown in Figure 2A, in the normal, untreated retina, the microglia, mainly localized in the ganglion and inner plexiform layers, appear to have long, thin processes. There were few CD11b-positive round cells attached to the retinal blood vessels. Upon LPS treatment, there was a dramatic increase in the number and intensity of the CD11b-positive cells in the flat-mounted retina. CD11b+ cells that attached to the retinal blood vessels began to migrate into the retina. At 24 h, we frequently observed attachment of an increasing number of round cells to the retinal blood vessels, accompanied by infiltration of some cells into the retina and occasionally by focal accumulation of these cells around the blood vessels. We found few CD11b+ round cells in 5 mg/kg CBD-pretreated uveitic rats. Figure 2 Effects of cannabidiol (CBD) on lipopolysaccharide (LPS)-induced morphological changes in rat retinal microglial cells. Confocal micrographs were made of cells identified by microglial- and macrophage-specific marker CD11b followed by Oregon green-conjugated secondary antibody. Cell nuclei were counter-stained by propidium iodide. A: Cells were cultured in serum-free medium for 12 h. B: Cells were treated with 30 ng/ml lipopolysaccharide in serum-free medium for 12 h. C: Cells were cannabidiol (CBD, 1 μM)-pretreated and lipopolysaccharide-treated for 12 h. D-F: Cells similarly treated were also stained with fluorescent phallotoxins for Filamentous actin distribution and cellular morphology. Scale bar represents 20 μm. In response to LPS challenge, the immune cells, including macrophages, released proinflammatory cytokines to amplify the inflammation reactions, both locally and systematically. As shown in Figure 2B,C, the serum or retinal levels of TNF-α were 30–40% higher than control 24 h after LPS injection. In LPS-treated rats that were pretreated with CBD, the rise of TNF-α was prevented. CBD reduces morphological changes in retinal microglial cells Activated microglia has been reported to play a key role in causing inflammation and neurodegeneration in uveitis and other models [2]. Therefore, we examined the behavior of cultured rat retinal microglial cells in response to LPS treatment to determine how inflammation occurs, and how CBD blocks this event. Rat retinal microglial cells were prepared according to an established protocol [15]. Immunocytochemical studies showed that the prepared cells express CD11b (Figure 2A). Treatment with 30 ng/ml LPS induced microglia activation as indicated by enhanced expression of CD11b and morphological changes (Figure 2B). These effects were prevented by pretreatment of cells with 1 μM CBD (Figure 2C). The activation effect of LPS and the prevention effect of CBD in microglial cells were further confirmed by the increased expression and re-arrangement of F-actin, as compared with the control (Figure 2D-F). CBD prevents TNF-α increase in retinal microglial cells To test the hypothesis that LPS treatment induces TNF-α production, we treated retinal microglial cells with LPS. We then collected culture medium at 5, 15, 20, and 30 min and 1, 2, 4, 6, 12, and 24 h after treatment and assayed for TNF-α. In the medium of LPS-treated cultures, levels of TNF-α began to increase significantly at or after 4 h of LPS treatment, peaking at 6 h, then leveled off within the 24 h period (Figure 3A). Early treatment (0–30 min) with LPS did not induce TNF-a production in microglial cells (data not shown). The levels of TNF-α in control culture medium without LPS treatment did not change throughout the experimental period (Figure 3A and data not shown). We next evaluated the effects of CBD in reducing TNF-α production. As shown in Figure 3B, pretreatment with 1 µM CBD blocked TNF-α production after 6 h of LPS treatment in retinal microglial cells. Figure 3 Cannabidiol reduces tumor necrosis factor-α levels in lipopolysaccharide-treated rat retinal microglial cells. Culture medium of retinal microglial cells were collected at different time points after lipopolysaccharide (LPS) treatment and assayed for tumor necrosis factor-α (TNF-α) with ELISA. A: TNF-α levels were measured at different times in LPS-treated microglial cells and were compared with the control. B: TNF-α levels were compared in the presence or absence of 1 μM cannabidiol (CBD). Data shown is the mean of 6 samples measured at 6 h±SEM. Asterisk represents that it is significantly different when compared with the 0 time or control at p<0.05. CBD blocks superoxide formation via NADPH oxidase inhibition It is known that many stress factors, including oxidative stress, lead to inflammation. Therefore, we determined ROS formation after LPS treatment in the presence or absence of CBD. Chemiluminescence assay showed that stimulation of the cells with LPS caused immediate and significant increases in superoxide anion formation (0–30 min), gradually decreasing at 60 min (Figure 4A). Pretreatment of microglial cells with CBD significantly reduced ROS formation during this period. To determine the source of superoxide anion formation, we treated microglia cells were pretreated with 15 μM apocynin (a specific NADPH oxidase assembly inhibitor), 15 μM TTFA (a mitochondrial oxidase inhibitor), or 100 U/ml superoxide anion dismutase (SOD). Chemiluminescence assay showed that stimulation of the cells with LPS caused about 50% increase in the ROS formation at 30 min (Figure 4B). Apocynin at 15 μM significantly reduced LPS-induced superoxide anion formation similar to CBD effects. In contrast, inhibiting mitochondrial oxidase with 15 μM TTFA did not alter LPS-induced superoxide anion formation. While CBD or apocynin did not alter superoxide levels in the controls, treatment with TTFA increased superoxide levels in the control. This may be due to a nonspecific effect of TTFA. The signal was blocked completely by SOD, indicating specificity of the detection of superoxide anion. Figure 4 Cannabidiol blocks lipopolysaccharide-induced early superoxide formation A: In microglial cells treated with lipopolysaccharides (LPS), maximal reactive oxygen species (ROS) formation measured by chemiluminescence assay was observed at 30 min. Cannabidiol (CBD) reduced ROS formation during this period. Data shown is the mean of 5 samples±SEM; Asterisk represents that it is significantly different when compared with CBD-treated at p<0.01. B: Comparison of ROS formation measured by chemiluminescence assay on microglial cells pretreated with CBD, apocynin, TTFA, or PEG-SOD and treated with vehicle (control) or LPS. Results suggested that superoxide component of ROS in LPS-treated retinal microglia is generated from NADPH oxidase. Data shown is the mean of 6 samples measured at 30 min±SEM; Asterisk represents that it is significantly different when compared with CBD-treated at p<0.01. P38 MAPK mediates LPS-induced release of TNF-α in retinal microglial cells Cytokine release has been reported to be mediated by p38 MAPK or oxidative stress in macrophages [7]. Immunoblot assays showed that stimulation of the cells with LPS caused minimal activation of p38 MAPK at 15 min, slowly increased at 30 min, and further increased at 60 min (Figure 5A). Pretreatment of CBD at 1 μM or apocynin at 200 μM blocked p38 MAPK activation in response to LPS. These results suggest that in response to LPS treatment, oxidative stress causes p38 MAPK activation in retinal microglial cells. To understand the regulation of cytokine release in LPS-treated retinal microglial cells, we sought to determine whether the release of TNF-α was mediated by oxidative stress as well as p38 MAPK. The roles of NADPH oxidase and p38 MAPK were examined by using apocynin and the p38 MAPK inhibitor SB203580, respectively [18]. Pretreatment of rat retinal microglial cells with SB203580 at 10 μM significantly decreased the LPS-induced release of TNF-α (Figure 5B). Pretreatment with apocynin at 200 μM, although less effective as compared with SB203580, also significantly decreased the LPS-induced release of TNF-α. Pretreatment with both apocynin and SB203580 did not further decrease TNF-α beyond the effect of SB203580 alone, suggesting that the effects of oxidative stress and p38 MAPK activation are related, and not independent, events. Figure 5 Oxidative stress and p38 MAPK activation are causally related and are involved in release of tumor necrosis factor-α. A: Lipopolysaccharide (LPS) caused a time-dependent activation of p38 MAPK with highest activation at 60 min. Cannabidiol (CBD; 1 μM) or apocynin (200 μM) reduced the activation of p38 MAPK (phospho-p38) throughout the 60 min. Data shown is the mean of 2-3 experiments±SEM; asterisk represents that it is significantly different when compared with the controls at p<0.05. B: Pretreatment of microglial cells with apocynin (200 μM) or the p38 MAPK inhibitor, SB203580 (10 µM), inhibited the LPS-induced release of tumor necrosis factor-α (TNF-α). Treatment with both apocynin and SB203580 did not further decrease the release of TNF-α. Data shown is the mean of 4-8 experiments±SEM. Asterisk represents that it is significantly different at p<0.001 when compared with vehicle control or with apocynin-treated and SB203580-treated control; hash mark represents that it is significantly different at p<0.05 when compared with LPS alone. Treatment with both apocynin and SB203580 did not alter TNF-α levels in the controls. In the same study, pretreatment with CBD at 1 μM almost completely blocked the release of TNF-α (Figure 3 B), suggesting that CBD has a potent effect in blocking oxidative stress and p38 MAPK activation. CBD inhibits NO, peroxynitrite, and p38 MAPK in microglial cells To study the effects of LPS and CBD on the release of NO, we collected culture medium at 5, 15, 20, and 30 min and 1, 2, 4, 6, 12, and 24 h after LPS treatment. NO levels in the culture medium were determined using an NO analyzer. The levels of NO in control culture medium without LPS treatment did not change throughout the experimental period. In the LPS-treated cultures, LPS-induced activation of microglia involved a significant late increase in the NO level in culture medium (Figure 6A). The increment of NO levels became significant only after 6 h of LPS treatment and continued for 12 h. LPS-treated rat retinal microglial cells pretreated with CBD for 60 min at 1 µM reduced NO at 6 or 12 h. Peroxynitrite, the combination product from superoxide and NO, increased oxidative and nitrative stresses in the cells. To study the effects of LPS and CBD on the oxidative and nitrative stresses, we collected culture medium at various time points after LPS treatment for fluorescence measurement of DCF (Figure 6B). LPS induced DCF fluorescence as early as 15 min, followed by a further increase at a later phase of 6-12 h. CBD at 1 µM reduced DCF fluorescence during this period. We next evaluated the effects of LPS on activation of p38 MAPK in the late phase (at 2, 6, or 12 h). Immunoblotting of phospho-p38 MAPK showed a time-dependent activation that peaked at 2-6 h in rat retinal microglial cells. Pretreatment with 1 μM CBD significantly reduced it (Figure 6C). LPS treatment did not affect the levels of p38 MAPK or actin proteins (data not shown). Together, these results suggest that LPS induces a second phase of increased oxidative stress and p38 MAPK activation, which coincides with cytokine release and morphological changes with F-actin rearrangement in the microglial cells (Figure 2E). Figure 6 Cannabidiol reduces lipopolysaccharide-induced late increases of nitric oxide and peroxynitrite and p38 MAPK activation. A: Lipopolysaccharide (LPS) caused maximal increase in nitric oxide (NO) formation at 6 h and after. Cannabidiol (CBD) reduced NO formation during this period. Data shown is the mean of 4-6 experiments±SEM. Asterisk represents that it is significantly different at p<0.05 when compared with 0 time. B: LPS caused peroxynitrite formation as early as 15 min, followed by a further increase at 6-12 h. CBD reduced peroxynitrite formation during this period. Data shown is the mean of 6 experiments±SEM; asterisk represents that it is significantly different at p<0.005 as compared to CBD-treated. C: LPS treatment of microglial cells for 0-12 h induced a second peak of phospho-p38 MAPK at 2-6 h. CBD significantly reduced p38 MAPK activation level during this period. Data shown is the mean of 3 experiments±SEM; asterisk represents that it is significantly different at p<0.05 when compared with 0 time. CBD reduces oxidative and nitrative stresses in the uveitic retina To elucidate the mechanism and the consequences of inflammation in the uveitic retina and the neuroprotective effects of CBD, we determined the oxidative and nitrative stresses in the rat retina in the presence or absence of CBD 24 h after the injection of LPS. As shown in Figure 7A, images from uveitic retinas of 6 rats showed increased DCF fluorescence in the inner and outer plexiform and the outer segment layers. Quantitative analysis showed a 1.5 fold increase (p<0.05) in the fluorescence intensity in the uveitic retinas compared to normal controls (Figure 7B). Treatment with CBD blocked uveitis-induced oxidative stress as indicated by significant inhibition of fluorescence accumulation in these retinas. We further confirmed the antioxidant effects of CBD by measuring tyrosine nitration. As shown in Figure 7C, images from uveitic retinas of 6 rats showed significant tyrosine nitration within retinal layers with strongest immunoreactivity within the plexiform layers. Quantitative analysis showed that levels of tyrosine nitration were increased 2.8 fold in the uveitic retinas in comparison with the controls. This tyrosine nitration was almost completely eliminated by CBD (Figure 7D). Figure 7 Cannabidiol reduces oxidative and nitrative stresses in the uveitic retina. A: Cannabidiol (CBD) reduces reactive oxygen species (ROS) in the retinas of uveitic rats as represented by DCF fluorescence in rat retina. Representative image shows the fluorescence distribution in different retinal layers (magnification ×100). Abbreviations: Ganglion cell layer (GCL); inner nuclear layer (INL); outer nuclear layer (ONL). B: Morphometric analysis of fluorescence intensity in serial sections of rat eyes shows that uveitic rats had a significant increase in fluorescence (1.5-fold) compared with controls. Treatment with CBD (5 mg/kg) inhibited ROS formation in uveitic rats. Data shown is the mean±SEM of 4-5 animals in each group (asterisk represents that it is significantly different when compared with the controls at p<0.05). C: CBD reduces nitrotyrosine in the retina of uveitic rats. A representative image shows the nitrotyrosine distribution mainly in the retinal plexiform layers and outer segments (magnification ×200). D: Morphometric analysis of fluorescence intensity in serial sections of rat eyes showing that uveitic rats had a significant increase in fluorescence (2.8-fold) compared with controls. Treatment with CBD (5 mg/kg) inhibited nitrotyrosine formation in the uveitic rats. Data shown is the mean±SEM of 6 animals in each group (asterisk represents that it is significantly different when compared with the controls at p<0.05). CBD reduces p38 MAPK activation and cell death in the uveitic retina Our results in cultured retinal microglial cells suggest that endotoxin-induced oxidative stress induces retinal inflammation via activation of p38 MAPK. Activated p38 MAPK is not only an important factor for microglial activation, it also plays an important role in cell survival and death. We therefore examined the role of p38 MAPK in uveitic retinas and determined whether CBD treatment blocks this pathway. Western blot analysis of the phosphorylation of p38 MAPK in uveitic retinas showed an estimated 1.8 fold increase at 24 h after LPS injection (Figure 8A). Treatment with CBD significantly blocked the increases in phosphorylation of p38 MAPK. Accelerated death of retinal ganglion cells and neurons has been reported to be significant in uveitis in vitro [5]. Therefore, we tested the hypothesis that CBD prevents death of neurons in LPS-induced uveitis in rat retinas. Retinal neuronal cell death was determined using TUNEL-FITC analysis on frozen eye sections and western analysis of retinal caspase-3. As shown in Figure 8B, LPS administration in rats resulted in the induction of neuronal death as indicated by the significant number of TUNEL positive cells (green, see white arrows) in the retinal ganglion cell layer. In these LPS-treated rats, the activated p38 MAPK was colocalized with TUNEL positive neurons (Figure 8C). CBD blocked the activation of p38 MAPK and neuronal cell death in LPS-injected animals. Caspase-3 is an intracellular cysteine protease that exists as a pro-enzyme and becomes activated during the cascade of apoptosis. We confirmed LPS-induced apoptosis by significant increases of cleaved caspase-3 expression in LPS-treated animals (Figure 8D). Treatment with 5 mg/kg CBD significantly reduced the number of TUNEL-positive cells and expression of caspase-3. Figure 8 Cannabidiol reduces p38 MAPK activation and prevents cell death in the uveitic retina. A: Lipopolysaccharide (LPS) treatment of rats resulted in a significant increase in p38 MAPK activation at 24 h. CBD (5 mg/kg) treatment reduced this effect. Data from 2 animals in each group are shown (asterisk represents that it is significantly different at p<0.05 as compared to control), Similar results were obtained in 2 independent experiments. B: CBD blocked neuronal cell death, as detected by TUNEL analysis. Data shown is the mean±SEM of 3 animals in each group (asterisk represents that it is significantly different at p<0.05 as compared to control). C: Colocalization of phospho-p38 MAPK (red) and TUNEL+cells (green) in the retinal ganglion cell layer. CBD blocked LPS-induced activation of p38 MAPK and blocked neuronal cell death. D: CBD blocked LPS-induced caspase-3 expression that was detected by Western analysis in the uveitic retina. Data from 2 animals in each group are shown (asterisk represents that it is significantly different at p<0.05 compared to control). Similar results were obtained in 2 independent experiments. Discussion In the present study, we have demonstrated that stress-activated retinal microglial cells play a key role in mediating retinal inflammation. Using primary culture of rat retinal microglia, we have shown that treatment with LPS first induces oxidative stress, then p38 MAPK activation, leading to accumulation of TNF-α. Retinal inflammation was maintained with nitrative stress, another phase of p38 MAPK activation, and resulted in neurodegeneration. These effects were blocked by treatment with the nonpsychoactive CBD in vitro and in vivo. To our knowledge, this is the first work to study the molecular and cellular processes involved in the stress-activated retinal inflammation and the antiinflammatory and neuroprotective effects of CBD in the retina. Inflammatory retinal diseases including uveitis, glaucoma, macular degeneration, and diabetic retinopathy are characterized by microglial activation. Microglial cells are commonly described as the central nervous system equivalent of tissue macrophages, which, upon tissue injury, become activated to release glutamate, ROS, and cytokines, leading to inflammation, vascular dysfunction, and neurodegeneration [1]. To test the hypothesis that activated microglia, similar to those in the injured brain, are present in the retina during inflammation, we examined the effects of LPS on rat retinas. Indeed, there was a dramatic increase in infiltrated macrophages in the retina and serum levels of TNF-α in response to LPS injection. In agreement, immune cells including macrophages are known to release proinflammatory cytokines to amplify the inflammatory reactions both locally and systematically. Moreover, our results showed that LPS-treated microglial cells exhibited significant increases in TNF-α and NO levels, indicating that retinal microglial cells provide a feasible model for studying the molecular mechanism and the role of microglia in mediating retinal inflammation. In addition, the use of primary culture of retina microglia offers the advantage of studying the response of a pure population of microglia to controlled and designated treatments. Using primary culture of rat retinal microglial cells, we showed that treatment with low levels of LPS induced an immediate and significant increase in superoxide anion formation which was blocked by treatment with 1 μM CBD. To identify the source of superoxide anion formation, we compared the antioxidant effects of CBD with TTFA, an inhibitor of mitochondrial oxidase or apocynin, an inhibitor of NADPH oxidase. The results showed comparable effects of CBD to apocynin in inhibiting superoxide anion formation. However, TTFA did not prevent superoxide anion formation in LPS-treated cells. Together, these results suggest that in response to LPS, activation of NADPH oxidase is a major source of superoxide anion. In agreement, other studies have shown that microglial NADPH oxidase is a major source of ROS formation in the LPS model of inflammation [6,19,20]. The antioxidant effects of CBD have been attributed to its ability to directly scavenge ROS [10]. However, this scavenging activity requires higher levels of CBD (2.5–5 μM) [21]. Thus, the antioxidant effects of CBD at 1 μM could be at least partially attributed to its effects in inhibiting NADPH oxidase activity rather than entirely ascribed to ROS scavenging activity. The receptor mainly responsible for LPS recognition is the toll-like receptor 4 (TLR4), which triggers a variety of intracellular signaling cascades, including MAPK cascade, leading to the induction of transcription of target genes involved in the innate immune response [19]. Therefore, we examined the effects of LPS on p38 MAPK activation in cultured microglial cells. Our results showed an early (30 min) and time-dependent effect of LPS in activating p38 MAPK, which was blocked by treatment with CBD or apocynin (Figure 5A). These experiments have clearly demonstrated the causal relationship of oxidative stress and p38 MAPK activation. Activation of p38 MAPK has been recognized as a major event involving MAPK in the signaling cascade of TNF-α induction in stimulated macrophages and cerebral microglia [7,9]. In agreement, our results showed that LPS treatment induced a time-dependent induction of TNF-α release that peaked after 6 h and was reduced by specific inhibitors of p38 MAPK (10 μM SB203580) and NADPH oxidase (200 µM apocynin). Furthermore, we demonstrate that LPS-induced release of TNF-α release is blocked almost totally by treatment with 1 µM CBD, clearly showing that CBD exerts more potent effects than apocynin or SB203580. Induction of inducible nitric oxide synthase and production of NO have been implicated as a mechanism by which activated microglia kill neurons [22]. Therefore, we investigated the effects of LPS on NO production in cultured microglial cells. Our results showed significant and dramatic increases in NO after 6–12 h but not at the early phase of LPS treatment (0–30 min). This effect was in parallel with a second wave of increased formation of ROS and peroxynitrite as indicated by increased DCF fluorescence and nitrotyrosine levels in vitro and in vivo. The late phase effects of LPS were prevented by CBD treatment in cultured microglial cells and in vivo. p38 MAPK, a stress-activated protein kinase, is a downstream target of proinflammatory cytokines including TNF-α [23] and oxidative stress [18]. The activation of p38 MAPK has been described to induce transcription-independent effects such as induction of actin reorganization and cellular motility [8]. In agreement, our results showed increased expression and redistribution of F-actin in LPS-activated microglia (Figure 2E) within the same time frame as late phase activation of p38 MAPK after 6 h (Figure 6C). Our findings indicate a biphasic pattern of p38 MAPK activation, including an early phase (30–60 min) and a late phase (2–6 h). This suggests that the first phase occurs as a result of oxidative stress to induce cytokine release and the second phase is induced by TNF-α stimulation and maintained by ROS formation, forming the autoregulatory loop of TNF-α, sustaining its own biosynthesis. Similar observations for p38 MAPK have been reported after stimulation with cytokines or vasoactive agents [24-26]. Figure 9 illustrates the pathways of LPS-induced oxidative stress leading to TNF-α release, biphasic p38 MAPK, and microglial activation as well as the mechanism by which CBD blocks these processes. Figure 9 Schematic figure summarizes the proposed mechanism of lipopolysaccharide-induced retinal degeneration. Lipopolysaccharide (LPS)-induced oxidative stress leads to p38 MAPK activation, tumor necrosis factor-α (TNF-α) release, and another phase of p38 MAPK activation. The autoregulatory loop of TNF-α release and oxidative stress leads to microglial activation and retinal neurodegeneration. Suggested sites where cannabidiol (CBD) blocks this pathway are indicated. The anti-inflammatory effect of CBD is not mediated by the known cannabinoid receptors because CBD does not bind well to these receptors. Here, we show that CBD blocks retinal inflammation and microglia activation via inhibition of NADPH oxidase activity and early p38 MAPK phosphorylation, resulting in blocking TNF-α release. The superior effect of CBD over inhibitors of NADPH oxidase and p38 MAPK could be due to modulation of microglial activity by inhibiting an unidentified cannabinoid receptor [27], or by enhancing endogenous adenosine signaling [28]. Ongoing studies by our group are in progress to further elucidate the role of adenosine signaling in the anti-inflammatory effect of CBD in models of retinal inflammation. Our results have demonstrated a critical role of oxidative stress in activating microglial cells and causing inflammation in vitro. To determine the consequences of such cellular events in vivo, we determined steady-state levels of inflammatory and oxidative stress markers in relation to retinal neuronal death. The neural retina has a high content of polyunsaturated fatty acids and hence is extremely susceptible to oxidative and nitrative insults [29]. NO and peroxynitrite formation have been reported to play a critical role in the pathogenesis of LPS-induced uveitis [30]. In agreement, our results showed a 1.5 fold increase in oxidative stress and a 2.8 fold increase in peroxynitrite formation as indicated by DCF fluorescence and nitrotyrosine, respectively (Figure 7). These effects were associated with increased inflammation (Figure 1), p38 MAPK activation, and retinal neurodegeneration (Figure 8). Similarly, accelerated death of retinal cells has been reported in rats after LPS treatment [31]. These results confirm our findings that LPS-induced oxidative and nitrative stress activate microglial cells, which causes neuronal degeneration. Our present finding that CBD blocked oxidative and nitrative stress, macrophage infiltration, TNF-α production, and prevented retinal neurodegeneration suggest that CBD represents novel therapeutics in the treatment of inflammation-mediated retinal damage. Furthermore, CBD is an attractive medical alternative to smoked marijuana or plant extract because of its lack of psychoactive effect [32] and is well tolerated in humans [33]. In conclusion, the data presented here provide evidence that stress-activated retinal microglial cells and their inactivation by CBD represent a central player in retinal inflammation and neuroprotection, respectively. Acknowledgment This work was supported by Knights Templar Educational Foundation of Georgia (G.I.L.), American Diabetic Association (G.I.L.), a Scientist Development Grant from the American Heart Association (A.B.E.), Juvenile Diabetic Research Foundation (A.B.E.), and University of Georgia Alliance Research Foundation (A.B.E.). ==== Refs Reference 1 Langmann T Microglia activation in retinal degeneration. J Leukoc Biol 2007 81 1345 51 17405851 2 Medana IM Chan-Ling T Hunt NH Redistribution and degeneration of retinal astrocytes in experimental murine cerebral malaria: relationship to disruption of the blood-retinal barrier. Glia 1996 16 51 64 8787773 3 Hoekzema R Verhagen C van Haren M Kijlstra A Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6. Invest Ophthalmol Vis Sci 1992 33 532 9 1544781 4 McMenamin PG Crewe J Endotoxin-induced uveitis. 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==== Front Environ Health PerspectEnviron. Health PerspectEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Sciences 1907971510.1289/ehp.11297ehp-116-1648ResearchG-Protein–Coupled Receptor 30 and Estrogen Receptor-α Are Involved in the Proliferative Effects Induced by Atrazine in Ovarian Cancer Cells Albanito Lidia 1Lappano Rosamaria 1Madeo Antonio 1Chimento Adele 1Prossnitz Eric R. 2Cappello Anna Rita 1Dolce Vincenza 1Abonante Sergio 1Pezzi Vincenzo 1Maggiolini Marcello 11 Department of Pharmaco-Biology, University of Calabria, Rende, Italy2 Department of Cell Biology and Physiology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico, USAAddress correspondence to M. Maggiolini, Department of Pharmaco-Biology, University of Calabria, 87030 Rende (CS), Italy. Telephone: 390984493076. Fax: 390984493458. E-mail: [email protected]* These authors contributed equally to this work. The authors declare they have no competing financial interests. 12 2008 22 7 2008 116 12 1648 1655 28 1 2008 18 7 2008 2008Publication of EHP lies in the public domain and is therefore without copyright. All text from EHP may be reprinted freely. Use of materials published in EHP should be acknowledged (for example, ?Reproduced with permission from Environmental Health Perspectives?); pertinent reference information should be provided for the article from which the material was reproduced. Articles from EHP, especially the News section, may contain photographs or illustrations copyrighted by other commercial organizations or individuals that may not be used without obtaining prior approval from the holder of the copyright. Background Atrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17β-estradiol and environmental estrogens. Objectives Given the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells. Methods and results Atrazine did not transactivate the endogenous ERα in different cancer cell contexts or chimeric proteins encoding the ERα and ERβ hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERα nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30–epidermal growth factor receptor transduction pathway and the involvement of ERα. Conclusions Our results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action. 17β-estradiolatrazineestrogen receptorGPR30ovarian cancer cells ==== Body Atrazine belongs to the 2-chloro-s-triazine family of herbicides (Figure 1) and is the most common pesticide contaminant of ground-water and surface water (Fenelon and Moore 1998; Kolpin et al. 1998; Lode et al. 1995; Miller et al. 2000; Müller et al. 1997; Solomon et al. 1996; Thurman and Cromwell 2000). Among the endocrine-disrupting effects, atrazine interferes with androgen- and estrogen-mediated processes (Babic-Gojmerac et al. 1989; Cooper et al. 1999, 2000; Cummings et al. 2000; Friedmann 2002; Kniewald et al. 1979, 1995; Narotsky et al. 2001; Shafer et al. 1999; Simic et al. 1991; Stoker et al. 1999, 2000). The interference of atrazine with androgen and estrogen action does not occur by direct agonism or antagonism of cognate receptors for these steroids as shown by binding affinity studies (Roberge et al. 2004; Tennant et al. 1994a, 1994b). In this respect, previous investigations have suggested that atrazine reduces androgen synthesis and action (Babic-Gojmerac et al. 1989; Kniewald et al. 1979, 1980, 1995; Simic et al. 1991) and stimulates estrogen production (Crain et al. 1997; Heneweer et al. 2004; Keller and McClellan-Green 2004; Sanderson et al. 2000, 2001, 2002; Spano et al. 2004). The latter ability is exerted through at least two mechanisms that converge on increasing aromatase expression and activity. First, inhibiting phosphodiesterase, atrazine up-regulates cAMP, which induces the expression of SF-1, an important regulator of the PII promoter of aromatase gene CYP19. The enhanced transcription of the aromatase gene increases both enzymatic activity of aromatase and estrogen production (Heneweer et al. 2004; Lehmann et al. 2005; Morinaga et al. 2004; Roberge et al. 2004; Sanderson et al. 2000, 2001). Next, atrazine binds to SF-1 and facilitates the recruitment of this factor to the PII promoter of the aromatase gene, further stimulating the biological effects described above (Fan et al. 2007a, 2007b). Epidemiologic studies have associated long-term exposure to triazine herbicides with increased risk of ovarian cancer in female farm workers in Italy (Donna et al. 1989) and breast cancer in the general population of Kentucky in the United States (Kettles et al. 1997). In addition, atrazine leads to tumor development in the mammary gland and reproductive organs of female F344 rats (Pintér et al. 1990), whereas in Sprague-Dawley rats it causes an earlier onset of mammary and pituitary tumors (Wetzel et al. 1994), a typical response to exogenously administered estrogens (Brawer and Sonnenschein 1975). Given the potential ability of atrazine to interfere with reproduction and to cause cancer, the European Union banned its use. However, the U.S. Environmental Protection Agency has approved the use of atrazine because of the lack of a clear association between the levels of exposure and cancer incidence in pesticide applicators (Gammon et al. 2005; McElroy et al. 2007; Rusiecki et al. 2004; Sass and Colangelo 2006; Young et al. 2005). Regarding the apparent estrogenic effects of atrazine, previous studies have demonstrated that triazine herbicides do not bind or activate the classical estrogen receptor (ER) (Connor et al. 1996; Tennant et al. 1994a, 1994b). In recent years, increasing evidence has demonstrated in different experimental models that steroid hormones, including estrogens, can exert rapid actions interacting with receptors located within or near the cell membrane (Falkenstein et al. 2000; Norman et al. 2004; Revelli et al. 1998). The importance of this signaling mechanism is becoming more widely recognized as steroid membrane receptors have been implicated in a large number of physiologic functions. Moreover, it has been suggested that nongenomic estrogen actions, like genomic ones, are susceptible to interference from environmental estrogens (Thomas 2000). Of note, these compounds compete with [3H]17β-estradiol ([3H]E2) for binding to estrogen membrane receptors (Loomis and Thomas 2000) and exert agonist effects on nongenomic transduction pathways in different cell contexts (Loomis and Thomas 2000; Nadal et al. 2000; Ruehlmann et al. 1988; Watson et al. 1999). However, the precise identity and function of many steroid membrane receptors are still controversial in terms of their specific molecular interactions with endogenous and environmental estrogens. A seven-transmembrane receptor, G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the nuclear ER, has been recently shown to mediate rapid actions of estrogens (Filardo et al. 2002; Revankar et al. 2005). Recombinant GPR30 protein, produced in ER-negative HEK-293 cells, exhibited all the steroid binding and signaling characteristics of a functional estrogen membrane receptor (Thomas et al. 2005; Thomas and Dong 2006). Our studies and others have also demonstrated that GPR30 mediates the rapid response to E2 in a variety of estrogen-responsive cancer cells by activating the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) transduction pathway (Albanito et al. 2007; Bologa et al. 2006; Filardo et al. 2000; Maggiolini et al. 2004; Revankar et al. 2005; Thomas et al. 2005; Vivacqua et al. 2006a, 2006b). In the present study, for the first time we have demonstrated that atrazine stimulates gene expression and growth effects in estrogen-sensitive ovarian cancer cells through GPR30 and the involvement of ERα. Moreover, we show that GPR30 mediates the stimulatory effects of atrazine in ER-negative SkBr3 breast cancer cells. Materials and Methods Reagents We purchased atrazine [2-chloro-4-(ethylamine)-6-(isopropylamine)-s-triazine], 17β-estradiol (E2), N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), wortmannin (WM), and PD98059 (PD) from Sigma-Aldrich (Milan, Italy); AG1478 (AG) from Biomol Research Laboratories (DBA, Milan, Italy); ICI 182,780 (ICI) from Tocris Chemicals (Bristol, UK); and GF109203X (GFX) from Calbiochem (VWR International, Milan, Italy). All compounds were solubilized in dimethyl sulfoxide (DMSO), except E2 and PD, which were dissolved in ethanol. Cell culture Human BG-1 and 2008 ovarian cancer cells as well as human Ishikawa endometrial cancer cells were maintained in phenol red–free Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). H295R adrenal carcinoma cells were cultured in DMEM/F12 1:1 supplemented with 1% ITS Liquid Media Supplement (Sigma-Aldrich), 10% calf serum, and antibiotics. Human MCF-7 breast cancer cells were maintained in DMEM with phenol red supplemented with 10% FBS, and human SkBr3 breast cancer cells were maintained in phenol red–free RPMI 1640 supplemented with 10% FBS. Cells were switched to medium without serum the day before experiments for immunoblots and reverse transcription-polymerase chain reaction (RT-PCR). Plasmids Firefly luciferase reporter plasmids used were XETL for ERα (Bunone et al. 1996) and GK1 for yeast transcription factor Gal4 fusion proteins (Webb et al. 1998). XETL contains the estrogen response element (ERE) from the Xenopus vitellogenin A2 gene (nucleotides −334 to −289), the herpes simplex virus thymidine kinase promoter region (nucleotides −109 to +52), the firefly luciferase coding sequence, and the SV40 splice and polyadenylation sites from plasmid pSV232A/ L-AA5. Gal4 chimeras Gal-ERα and Gal-ERβ were expressed from plasmids GAL93.ER(G) and GAL.ERβ, respectively. They were constructed by transferring the coding sequences for the hormone-binding domain (HBD) of ERα (amino acids 282–595) from HEG0 (Bunone et al. 1996), and for the ERβ HBD (C-terminal 287 amino acids) from plasmid pCMV5-hERβ into the mammalian expression vector pSCTEVGal93 (Seipel et al. 1992). We used the Renilla luciferase expression vector pRL-TK (Promega, Milan, Italy) as a transfection standard. Transfection and luciferase assays BG-1, MCF-7, Ishikawa, and SkBr3 cells (1 × 105) were plated into 24-well dishes with 500 μL/well DMEM (BG-1, MCF-7, and Ishikawa cells) or RPMI 1640 (SkBr3 cells) containing 10% FBS the day before transfection. We replaced the medium with phenol red–free DMEM or RPMI 1640, both supplemented with 1% charcoal-stripped (CS) FBS, on the day of transfection. Transfections were performed using FuGENE 6 Reagent as recommended by the manufacturer (Roche Diagnostics, Mannheim, Germany) with a mixture containing 0.3 μg of reporter plasmid, 1 ng pRL-TK, and 0.1 μg effector plasmid where applicable. After 5–6 hr, the medium was replaced again with serum-free DMEM lacking phenol red and supplemented with 1% CS-FBS; ligands were added at this point, and cells were incubated for 16–18 hr. We measured luciferase activity with the Dual Luciferase Kit (Promega) according to the manufacturer’s recommendations. Firefly luciferase values were normalized to the internal transfection control provided by Renilla luciferase activity. The normalized relative light unit values obtained from cells treated with vehicle were set as 1-fold induction, from which the activity induced by treatments was calculated. RT-PCR Using semiquantitative RT-PCR as described previously (Maggiolini et al. 1999), we evaluated gene expression for ERα [GenBank accession no. NM 000125 (National Center for Biotechnology Information 2008)], c-fos (NM 005252), progesterone receptor (PR; NM 000926), pS2 (NM 003225), cathepsin D (NM 001909), cyclin A (NM 001237), cyclin D1 (NM 053056), cyclin E (NM 001238), and the acid phosphoprotein P0 (36B4) (NM 001002) used as a control gene. We used the primers 5′-AATTCA-GATAATCGACGCCAG-3′ (ERα forward) and 5′-GTGTTTCAACATTCTCCCTC-CTC-3′ (ERα reverse); 5′-AGAAAAGGA-GAATCCGAAGGGAAA-3′ (c-fos forward) and 5′-ATGATGCTGGGACAGGAAG-TC-3′ (c-fos reverse); 5′-ACACCTTGC-CTGAAGTTTCG-3′ (PR forward) and 5′-CTGTCCTTTTCTGGGGGACT-3′ (PR reverse); 5′-TTCTATCCTAATAC-CATCGACG-3′ (pS2 forward) and 5′-TTTGAGTAGTCAAAGTCAGAGC-3′ (pS2 reverse); 5′-AACAACAGGGTG GGCTTC-3′ (cathepsin D forward), and 5′-ATGCACGAAACAGATCTGTGCT-3′ (cathepsin D reverse); 5′-GCCATTAGTT-TACCTGGACCCAGA-3′ (cyclin A forward) and 5′-CACTGACATGGAAGACAG GAACCT-3′ (cyclin A reverse); 5′-TCTAA-GATGAAGGAGACCATC-3′, (cyclin D1 forward) and 5′-GCGGTAGTAGGACAG GAAGTTGTT-3′ (cyclin D1 reverse); 5′-CCTGACTATTGTGTCCTGGC-3′ (cyclin E forward) and 5′-CCCGCT-GCTCTGCTTCTTAC-3′ (cyclin E reverse); and 5′-CTCAACATCTCCCCCTTCTC-3′ (36B4 forward) and 5′-CAAATCCCA-TATCCTCGTCC-3′ (36B4 reverse) to yield products of 345, 420, 196, 210, 303, 354, 354, 488, and 408 bp, respectively, with 20 PCR cycles for ERα, c-fos, PR, pS2, cathepsin D, cyclin A, and cyclin E and 15 PCR cycles for both cyclin D1 and 36B4. Western blotting Cells were grown in 10-cm dishes, exposed to ligands, and then lysed in 500 μL of 50 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), and a mixture of protease inhibitors containing 1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride, and 200 mmol/L sodium orthovanadate. We then diluted samples 10 times and determined protein concentration using Bradford reagent according to the manufacturer’s recommendations (Sigma-Aldrich). Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel and transferred to a nitro-cellulose membrane (Amersham Biosciences, Milan, Italy). Membranes were probed overnight at 4°C with the antibody against ERα (F-10), c-fos (H-125), β-actin (C-2), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2; E-4), and ERK2 (C-14), all purchased from Santa Cruz Biotechnology, DBA (Milan, Italy), and human P450 aromatase (MCA 2077S; Serotec, Milan, Italy), and then revealed using the ECL Western Blotting Analysis System (GE Healthcare, Milan, Italy). ER binding assay BG-1 cells were stripped of any estrogen by keeping them in medium without serum for 2 days. Cells were incubated with 1 nM [2,4,6,7-3H]E2 (89 Ci/mmol; Amersham Biosciences) and increasing concentrations of nonlabeled E2 or atrazine for 1 hr at 37°C in a humidified atmosphere of 95% air/5% CO2. After removal of the medium, cells were washed with ice-cold phosphate-buffered saline/0.1% methylcellulose twice, harvested by scraping and centrifugation, and lysed with 100% ethanol, 500 μL/60-mm dish, for 10 min at room temperature (Lee and Gorski 1996). We measured the radioactivity of extracts by liquid scintillation counting. Aromatase assay In subconfluent BG-1 or H295R cells, we measured aromatase activity in the cell culture medium by tritiated water release using 0.5 μM [1β-3H(N)]androst-4-ene-3,17-dione (25.3 Ci/mmol; DuPont NEN, Boston, MA, USA) as a substrate (Lephart and Simpson 1991). The cells were treated in a six-well dish in culture medium in the presence of atrazine or DMSO for 40 hr and then incubated with [1β-3H(N)]androst-4-ene-3,17-dione. Incubations were performed at 37°C for 6 hr under a 95%/5% air/CO2 atmosphere. The results were calculated as picomoles per hour, normalized to milligrams of protein (pmol/hr per 1 mg protein), and expressed as percentages of untreated cells (100%). GPR30 and ERα silencing experiments Cells were plated onto 10-cm dishes, maintained in antibiotic-free medium for 24 hr, and then transfected for additional 24 hr before treatments with a mixture containing Opti-MEM, 8 μL/well LipofectAMINE 2000 (Invitrogen, Milan, Italy), and 0.5 μg/well vector or short hairpin GPR30 (shGPR30) (Albanito et al. 2008), control small interfering RNA (siRNA), or ERα siRNA (Sigma-Aldrich). Proliferation assay For the quantitative proliferation assay, we seeded 10,000 cells in 24-well plates in regular growth medium. Cells were washed once they had attached and then incubated in medium containing 2.5% CS-FBS with the indicated treatments. Medium was renewed every 2 days (with treatments), and cells were trypsinized and counted in a hemocytometer on day 6. The day before treatments, 200 ng/L of the indicated short hairpin RNA was transfected using FuGENE 6 Reagent as recommended by the manufacturer, and then renewed every 2 days before counting. Statistical analysis Statistical analysis was performed using analysis of variance followed by Newman-Keuls testing to determine differences in means. p-Values < 0.05 are considered statistically significant. Results Atrazine does not activate ERα in cancer cells Based on the evidence that atrazine produces early onset and increased incidence of estrogen-sensitive tumors in different experimental models (Cooper et al. 2007), we first evaluated whether atrazine could activate a transiently transfected ER reporter gene in estrogen-sensitive ovarian (BG-1), breast (MCF-7), and endometrial (Ishikawa) cancer cells. Exposure to 100 nM E2 induced a strong ERα transactivation that was absent in the presence of 10 μM of the ER antagonist ICI in all cell contexts evaluated (Figure 2A–C). In contrast, treatments with 1 μM atrazine and even concentrations ranging from 1 nM to 10 μM (data not shown) failed to stimulate luciferase expression or to block that observed upon addition of E2 (Figure 2A–C). Moreover, atrazine did not activate an expression vector encoding ERα transiently transfected in ER-negative SkBr3 breast cancer cells (Figure 2D). To confirm that atrazine is not an ERα agonist and to examine whether ERβ could respond to atrazine, we turned to a completely heterologous system. Chimeric proteins consisting of the DNA binding domain of the yeast transcription factor Gal4 and the ERα or ERβ HBD transiently transfected in SkBr3 cells were strongly activated by E2 but not upon atrazine treatment (Figure 2E,F), further corroborating the aforementioned results. Atrazine neither regulates ERα expression nor competes with estrogen binding to ERα Considering that the down-regulation of ERα induced by an agonist has been considered an additional hallmark of receptor activation (Santagati et al. 1997), we further investigated whether atrazine could modulate ERα expression in BG-1 cells, which lack ERβ (data not shown), and express a receptor expression pattern similar to that found in primary ovarian tumors (Bardin et al. 2004; Geisinger et al. 1989). As shown in Figure 3A,B, 100 nM E2 down-regulated ERα at both mRNA and protein levels, whereas 1 μM atrazine did not produce any modulatory effect. In agreement with these results and those obtained in transfection experiments, atrazine showed no binding capacity for ERα (Figure 3C), as previously reported (Cooper et al. 2007). Altogether, our findings rule out that the estrogen action of atrazine occurs through binding and direct activation of ERα. Aromatase activity is not induced by atrazine Given that atrazine is able to up-regulate aromatase expression and function in different cell contexts (Cooper et al. 2007; Fan et al. 2007a, 2007b; Roberge et al. 2004; Sanderson et al. 2000, 2001), we then determined aromatase activity by tritiated water release assays in BG-1 cells. As shown in Figure 4, 1 μM atrazine did not stimulate aromatase activity, which in contrast was strongly induced in human H295R adreno-corticocarcinoma cells previously used as a model system to assess aromatase catalytic activity (Heneweer et al. 2004; Sanderson et al. 2001). In addition, the low aromatase protein expression detected in BG-1 cells did not increase upon exposure to 1 μM atrazine (data not shown). Hence, atrazine is neither an ERα activator nor an aromatase regulator in estrogen-sensitive ovarian cancer cells. ERK phosphorylation is stimulated by atrazine In recent years, numerous reports have demonstrated that estrogens and xeno-estrogens can generate rapid signaling via second messenger systems such as Ca2+, cAMP, nitric oxide, and G-proteins, which in turn leads to activation of different downstream kinases (Bulayeva and Watson 2004; Watson et al. 2007). To evaluate whether the potential estrogenic activity of atrazine is exerted through a rapid cellular response, we investigated its ability to produce ERK phosphorylation in BG-1 cells. Interestingly, atrazine stimulated ERK phosphorylation, although a higher concentration and prolonged time period were required to trigger this biochemical response compared with E2 (Figures 5A,B, 6A). ERK activation was also delayed in the presence of 1 μM atrazine compared with 100 nM E2 in 2008 ovarian cancer cells (Figure 6D), which present a receptor expression similar to that of BG-1 cells (Safei et al. 2005). To determine the transduction pathways involved in ERK activation by atrazine, cells were exposed to 100 nM E2 and 1 μM atrazine along with specific inhibitors widely used to pinpoint the mechanisms contributing to ERK phosphorylation (Bulayeva and Watson 2004). Of note, the ER antagonist ICI, the EGFR inhibitor AG and the ERK inhibitor PD prevented ERK activation induced by both E2 and atrazine, whereas GFX, H89, and WM, inhibitors of protein kinase C (PKC), protein kinase A (PKA), and phosphoinositide 3-kinase (PI3K), respectively, did not (Figure 6B,C,E,F). Considering that in a previous study ICI was able to trigger ERK phosphorylation (Filardo et al. 2000), we exposed SkBr3 cells to increasing concentrations of ICI. We observed no ERK activation after either 5 min (data not shown) or 20 min of treatment (Figure 7). Hence, in our experimental conditions, ICI showed only ERK inhibitor activity. Atrazine up-regulates the mRNA expression of estrogen target genes Having determined that atrazine signals through a rapid ERK activation, we evaluated in BG-1 cells its ability to regulate the expression of c-fos, an early gene that responds to a variety of extracellular stimuli, including estrogens (Maggiolini et al. 2004; Nephew et al. 1993; Singleton et al. 2003; Vivacqua et al. 2006a, b), along with other estrogen target genes. To this end, we performed semiquantitative RT-PCR experiments comparing mRNA levels after standardization with a housekeeping gene encoding the ribosomal protein 36B4. A short treatment (1 hr) with 1 μM atrazine enhanced c-fos and cyclin A levels, although to a lesser extent than 100 nM E2, which also stimulated PR, pS2, and cyclin D1 expression (Table 1). After a 24-hr treatment, atrazine increased PR, pS2, and cyclin A levels, whereas E2 additionally induced the expression of c-fos, cathepsin D, cyclin D1, and cyclin E (Table 1). We obtained results similar to those described above in 2008 cells (data not shown). Hence, atrazine is able to stimulate the expression of diverse estrogen target genes without an apparent activation of ERα. Transduction pathways involved by atrazine in the up-regulation of c-fos protein levels. Using c-fos expression as a molecular sensor of atrazine action at the genomic level, we sought to determine whether c-fos protein levels are also regulated by atrazine in a rapid manner and the transduction pathways involved in this response (Figure 8). Interestingly, the up-regulation of c-fos observed in BG-1 and 2008 cells after a short treatment (2 hr) was abolished by the ER antagonist ICI, the EGFR inhibitor AG, or the ERK inhibitor PD (Figure 8). On the contrary, GFX, H89, and WM, inhibitors of PKC, PKA, and PI3K, respectively, did not interfere with c-fos stimulation (Figure 8). Thus, in ovarian cancer cells, atrazine involves ERα and the EGFR/MAPK pathway to trigger c-fos protein increase. On the basis of these and our previous results showing that c-fos stimulation by E2 occurs through GPR30 and requires ERα and EGFR-mediated signaling in cancer cells expressing both receptors (Albanito et al. 2007; Maggiolini et al. 2004; Vivacqua et al. 2006a, 2006b), we examined whether atrazine could act in a similar manner. Interestingly, both E2 and atrazine were no longer able to induce c-fos expression after silencing either ERα or GPR30 in BG-1 and 2008 cells (Figure 9). To evaluate whether atrazine could induce a rapid response in a cell context expressing GPR30 alone, we turned to ER-negative SkBr3 breast cancer cells. Notably, both ERK phosphorylation and c-fos induction stimulated by atrazine were abolished after silencing GPR30 (Figure 10), indicating that the response to atrazine is differentially regulated according to cancer cell type. The proliferation of ovarian cancer cells induced by atrazine occurs through GPR30 and requires both ERα and EGFR/MAPK-mediated signaling. The aforementioned results were recapitulated in a more complex physiologic assay such as cell growth. We observed that both E2 and atrazine induced the proliferation of BG-1 and 2008 cells in a concentration-dependent manner (Figure 11A,E). Moreover, the growth effects elicited by E2 and atrazine were no longer evident in the presence of AG and PD (Figure 11B,F) or after silencing the expression of either GPR30 or ERα (Figure 11C,D,G,H), indicating that both receptors, along with the EGFR/MAPK transduction pathway, are involved in the growth effects as well as in the c-fos expression profile described above. Discussion In the present study, we demonstrated for the first time that atrazine exerts an estrogen-like activity in ovarian and breast cancer cells through GPR30, which is recently of interest because of its ability to mediate rapid estrogen signals (Albanito et al. 2007, 2008; Filardo et al. 2006, 2007; Revankar et al. 2005, 2007). Previous studies have demonstrated that atrazine elicits estrogen action by up-regulating aromatase activity in certain cancer cells with elevated aromatase levels (Fan et al. 2007a, 2007b; Heneweer et al. 2004; Sanderson et al. 2000, 2001) but not by binding to or activating ERα (Connor et al. 1996; Roberge et al. 2004; Tennant 1994a). Using different tumor cells and reporter genes, we confirmed that atrazine did not interact directly with ERα, yet it did not stimulate aromatase activity in our model system, likely as a consequence of a very low aromatase expression. Nevertheless, atrazine induced the expression of diverse estrogen target genes, recalling previous studies that demonstrated the recruitment of ERα by distinct compounds and growth factors to gene promoter sequences different from the classical estrogen response element (reviewed by Dudek and Picard 2008). Interestingly, we showed that GPR30 and ERα, together with the EGFR/MAPK pathway, are involved in the biological response to atrazine in ovarian cancer cells, which is in accordance with our recent investigation showing that the selective GPR30 ligand G-1 exerts biological activity similar to that of atrazine without binding or activating ERα (Albanito et al. 2007). Hence, our data indicate that a complex interplay between different ERs and transduction pathways contributes to atrazine activity, which nevertheless is still noticeable in the presence of GPR30 alone, as demonstrated in SkBr3 breast cancer cells. Although E2 exhibited an exclusive up-regulation of target genes through direct activation of ERα, the GPR30–EGFR transduction pathway was involved in estrogen-induced proliferation of ovarian tumor cells, as evidenced by silencing GPR30 and using specific pharmacologic inhibitors. A variety of environmental contaminants exhibit binding affinities for GPR30 and agonist activities similar to those for ERs (Thomas and Dong 2006). In the present study atrazine triggered rapid biological responses through GPR30 in both ovarian and breast cancer cells irrespective of ERα expression and despite a low binding affinity for GPR30 ectopically expressed in HEK-293 cells (Thomas and Dong 2006). In line with these findings, an efficient competitor of E2 for endogenous GPR30 in SkBr3 cells, such as an ortho, para-dichlorodiphenyldichloro-ethylene (DDE) derivative, was ineffective in binding to recombinant GPR30 (Thomas et al. 2005; Thomas and Dong 2006). Likely, the interaction of atrazine with GPR30 is facilitated by the relative abundance of this membrane receptor in cancer cells with respect to cells engineered to express recombinant GPR30, and/or yet unknown factors may contribute to the binding to GPR30 by these contaminants. Regarding the role of ERα, we proved that a complex interplay with GPR30 exists, as previously reported with some growth factor receptors (Migliaccio et al. 2006), but the molecular mechanisms involved remain to be elucidated. Our study and previous investigations indicate that environmental estrogens exert pleiotropic actions by directly binding to ERα as well as through GPR30–EGFR signaling, which can engage ERα depending on the receptor expression pattern present in different cell types. This mode of action of xenoestrogens fits well with the results obtained after silencing GPR30 or ERα expression in ovarian cancer cells, because silencing each gene prevented the growth response to atrazine. Our data recall the results of previous studies showing that xenoestrogens mimic rapid estrogen action in several animal and cell models (Bulayeva and Watson 2004; Loomis and Thomas 2000; Nadal et al. 2000; Ruehlmann et al. 1988; Watson et al. 1999, 2007). Particularly, in GH3/B6/F10 pituitary tumor cells, diverse xenoestrogens induced ERK phosphorylation with a temporally distinct activation pattern compared with E2 (Bulayeva and Watson 2004). In the latter study, on the basis of the inhibitory activity exerted by ICI, the authors hypothesized that an ER localized to the plasma membrane could mediate the ERK phosphorylation response by xenoestrogens, depending on their different ER binding affinities. Moreover, the authors suggested that the signaling cascades leading to ERK activation may involve the nature of membrane ERs and their ability to interact with various signaling partners (Bulayeva and Watson 2004). Interestingly, our findings have provided evidence that ERα may be involved by xenoestrogens without a direct binding activity and produce relevant responses such as ERK phosphorylation, gene expression, and cell growth. A subset of estrogen-sensitive cell tumors can proliferate independently from ER expression (i.e., ER-negative cells). In this condition, well represented by SkBr3 breast cancer cells, GPR30–EGFR signaling may still allow for environmental estrogen activity as we have shown in the present study as well as in a previous study (Maggiolini et al. 2004). Hence, multiple transduction pathways triggered simultaneously at the membrane level, as well as within each cell type, may contribute to the nature and magnitude of biological responses to distinct estrogenic compounds. These consequently should be examined individually for their complex mechanistic and functional outcomes that result from interaction with a different repertoire of receptor proteins. Atrazine, a potent endocrine disruptor, is the most common pesticide contaminant of groundwater and surface water. Here, we have provided novel insight regarding the potential role of GPR30 in mediating the action of atrazine in endocrine-related diseases, such as estrogen-sensitive tumors. This research was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, Ministero dell’Università e Ricerca Scientifica e Tecnologica, and Regione Calabria. Figure 1 Structures of E2 and atrazine. Figure 2 ERα transactivation in BG-1 (A), MCF-7 (B), and Ishikawa (C) cells transfected with the ER luciferase reporter plasmid XETL (ERE-luc) and treated with 100 nmol/L E2 or 1 μmol/L atrazine (Atr), with and without 10 μmol/L ER antagonist ICI. Luciferase activities were normalized to the internal transfection control, and values of cells receiving vehicle (−) were set as 1-fold induction, from which the activity induced by treatments was calculated. (D–F) SkBr3 cells were transfected with ER luciferase reporter gene XETL and ERα expression plasmid (D) and with Gal4 reporter gene (GK1) and the Gal4 fusion proteins encoding the HBD of ERα (GalERα; E) and or ERβ (GalERβ; F) and treated with 100 nmol/L E2 or 1 μmol/L atrazine, with and without 10 μmol/L ICI. Values shown are mean ± SD of three independent experiments performed in triplicate. p< 0.05 compared with vehicle. Figure 3 mRNA expression and binding of ERα in BG-1 cells treated for 24 hr with vehicle (−), 100 nmol/L E2, or 1 μmol/L atrazine (Atr). (A) mRNA expression of ERα was evaluated by semiquantitative RT-PCR; the values of housekeeping gene 36B4 were determined as a control. (B) Immunoblot of ERα from BG-1 cells, with 100 nmol β-actin serving as a loading control. Results in (A) and (B) are representative of three independent experiments. (C) ERα binding assay using increasing concentrations of atrazine. Figure 4 Aromatase activity assessed by tritiated water release in BG-1 and H295R cells treated with vehicle (−) or 1 μmol/L atrazine (Atr). Results are expressed as percentages of untreated cells (100%). Values are mean ± SD of three independent experiments, each performed in triplicate. p < 0.05 compared with vehicle. Figure 5 ERK1/2 phosphorylation (pERK1/2) in BG-1 cells exposed to increasing concentrations of E2 or atrazine (Atr) for 20 min. Figure 6 BG-1 (A–C) and 2008 (D–F) cells treated with vehicle (−) or 100 nmol/L E2 with or without 1 μmol/L atrazine (Atr) for 5, 10, 20, or 30 min (A,D), or for 20 min with vehicle E2 (B and E), or 1 μmol Atr in combination with 10 μmol/L ICI, AG, PD, GFX, H89, or WM, inhibitors of ER, EGFR, MEK (MAP/ERK kinase), PKC (protein kinase C), PKA (protein kinase A), and PI3K (phosphoinositide 3-kinase), respectively. pERK1/2, phosphorylated ERK 1/2. Figure 7 ERK1/2 phosphorylation (pERK1/2) in SkBr3 cells treated for 20 min with vehicle (−) or increasing concentrations of ICI. Figure 8 Immunoblots of c-fos from BG-1 (A,B) and 2008 (C,D) cells treated for 2 hr with vehicle (−), 100 nmol/L E2, or 1 μmol/L atrazine (Atr) in combination with 10 μmol/L ICI, AG, PD, GFX, H89, or WM, inhibitors of ER, EGFR, MEK, PKC, PKA, and PI3K, respectively. β-Actin served as a loading control. Figure 9 Immunoblots of c-fos from BG-1 (A,B) and 2008 (C,D) cells after silencing ERα and GPR30 expression. Cells were transfected with control siRNA or siRNA-ERα (A,C) or with vector or shGPR30 (B,D) and treated for 2 hr with vehicle (−) or 100 nmol/L E2 or 1 μmol/L atrazine (Atr). Efficacy of ERα and GPR30 silencing was ascertained by immunoblots, as shown in side panels. β-Actin served as a loading control. Figure 10 ERK1/2 phosphorylation (A) and c-fos expression (B) after silencing GPR30 in SkBr3 cells treated with vehicle (−) or 1 μmol/L atrazine (Atr). (C) The efficacy of GPR30 silencing was ascertained by immunoblots. β-Actin served as a loading control. Figure 11 Proliferation of BG-1 (A–D) and 2008 (E–H) cells exposed to E2 or atrazine (Atr). (A,D) Proliferation of cells in response to increasing concentrations of E2 or Atr. (B–H) Proliferation of cells treated with vehicle (−), 100 nmol/L E2, or 1 μmol/L Atr with or without 10 μmol/L AG or PD (B,F) (C,D, G, H) or transfected with vector or shGPR30 (C,G) or with control siRNA or siRNA-ERα(D,H). See “Materials and Methods” for details of experiments. Proliferation of cells receiving vehicle was set as 100%, and the cell growth induced by treatments was calculated. Values shown are mean ± SD of three independent experiments performed in triplicate; Efficacy of ERα and GPR30 silencing was ascertained by immunoblots (Figure 9). p< 0.05 compared with treated cells. Table 1 mRNA expression (mean percent variation ± SD) induced by 100 nM E2 and 1 μM atrazine in BG-1 cells. E2 Atrazine Gene 1 hr 24 hr 1 hr 24 hr c-fos 423 ± 28 239 ± 17* 269 ± 21* 120 ± 9 PR 228 ± 18* 298 ± 18* 122 ± 18 180 ± 11* pS2 175 ± 17* 270 ± 21* 99 ± 19 187 ± 20* Cathepsin D 106 ± 9 217 ± 16* 102 ± 5 109 ± 6 Cyclin A 262 ± 22* 293 ± 23* 220 ± 20* 190 ± 22* Cyclin D1 258 ± 19* 242 ± 19* 107 ± 4 118 ± 8 Cyclin E 120 ± 11 343 ± 21* 118 ± 8 119 ± 10 The values calculated by optical density in cells treated with vehicle were set at 100%, and the expression induced by treatments is presented as percent variation. * p < 0.05 compared with vehicle. ==== Refs References Albanito L Madeo A Lappano R Vivacqua A Rago V Carpino A 2007 G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells Cancer Res 67 1859 1866 17308128 Albanito L Sisci D Aquila S Brunelli E Vivacqua A Madeo A 2008 Epidermal growth factor induces G protein-coupled receptor expression in estrogen receptor-negative breast cancer cells Endocrinology 149 3799 3808 18467441 Babic-Gojmerac T Kniewald Z Kniewald J 1989 Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence J Steroid Biochem 33 141 146 2761262 Bardin A Hoffman P Boulle N Katsaros D Vignon F Pujol P 2004 Involvement of estrogen receptor beta in ovarian carcinogenesis Cancer Res 64 5861 5869 15313930 Bologa CG Revankar CM Young SM Edwards BS Arterburn JB Kiselyov AS 2006 Virtual and biomolecular screening converge on a selective agonist for GPR30 Nat Chem Biol 4 207 212 16520733 Brawer JR Sonnenschein C 1975 Cytopathological effects of estradiol on the arcuate nucleus of the female rat. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1911499808-PONE-RA-05040R210.1371/journal.pone.0004070Research ArticleCell BiologyCell Biology/Cell SignalingCell Biology/Cellular Death and Stress ResponsesAkt Regulates Drug-Induced Cell Death through Bcl-w Downregulation AKT and Cell DeathGarofalo Michela 1 2 Quintavalle Cristina 1 3 Zanca Ciro 1 De Rienzo Assunta 5 Romano Giulia 4 Acunzo Mario 1 3 Puca Loredana 1 Incoronato Mariarosaria 4 Croce Carlo M. 2 Condorelli Gerolama 1 3 6 * 1 Department of Cellular and Molecular Biology and Pathology, “Federico II” University of Naples, Naples, Italy 2 Department of Molecular Virology, Immunology and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America 3 IEOS, CNR, Naples, Italy 4 Fondazione SDN, Naples, Italy 5 IRGS, Biogem s.c.ar.l., Ariano Irpino (AV), Italy 6 Facoltà di Scienze Biotecnologiche, “Federico II” University of Naples, Naples, Italy Hotchin Neil EditorUniversity of Birmingham, United Kingdom* E-mail: [email protected] and designed the experiments: MG GC. Performed the experiments: MG CQ CZ ADR GR MA LP MI. Contributed reagents/materials/analysis tools: CC. Wrote the paper: GC. 2008 30 12 2008 3 12 e407010 6 2008 22 11 2008 Garofalo et al.2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Akt is a serine threonine kinase with a major role in transducing survival signals and regulating proteins involved in apoptosis. To find new interactors of Akt involved in cell survival, we performed a two-hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the 80 clones obtained, two were identified as Bcl-w. Bcl-w is a member of the Bcl-2 family that is essential for the regulation of cellular survival, and that is up-regulated in different human tumors, such as gastric and colorectal carcinomas. Direct interaction of Bcl-w with Akt was confirmed by immunoprecipitation assays. Subsequently, we addressed the function of this interaction: by interfering with the activity or amount of Akt, we have demonstrated that Akt modulates the amount of Bcl-w protein. We have found that inhibition of Akt activity may promote apoptosis through the downregulation of Bcl-w protein and the consequential reduction in interaction of Bcl-w with pro-apoptotic members of the Bcl-2 family. Our data provide evidence that Bcl-w is a new member of the Akt pathway and that Akt may induce anti-apoptotic signals at least in part through the regulation of the amount and activity of Bcl-w. ==== Body Introduction Akt is a serine–threonine kinase downstream of PTEN/PI3K, involved in cellular survival pathways [1], [2]. In mammalian cells, the three Akt family members, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ are encoded by three different genes [3], [4]. They are ubiquitously expressed, although their levels are variable, depending upon the tissue type and pathological/physiological state. Increased expression or activation of Akt has been described as a frequent phenomena in human cancer [1], [5], [6]. Akt has been demonstrated to phosphorylate a number of proteins involved in apoptotic signaling cascades, including the Bcl-2 family member BAD [7], pro-caspase 9 [4], the forkhead transcription factors, FKHR and FKHRL1 [8], [9], and p21 cipWAF1. Phosphorylation of these proteins prevents apoptosis through several mechanisms [10]. Apoptosis, or programmed cell death, is an evolutionarily conserved mechanism of elimination of unwanted cells [11]. Apoptosis is triggered via two principal signaling pathways [12]. The extrinsic pathway is activated by the engagement of death receptors on the cell surface [13]. The other pathway is triggered by various intracellular and extracellular stresses, such as growth–factor withdrawal, hypoxia, DNA damage, and anticancer therapy [13], [14]. Intrinsic-pathway induced-apoptosis is generally regulated by the fine balance of Bcl-2 family proteins in a cell- and tissue-specific manner [11]. Apoptosis is believed to be the major mechanism responsible for chemotherapy-induced cell death in cancer. However, tumor cells often retain the ability to evade drug-induced death signals because of the activation of anti-apoptotic mechanisms [15]–[17]. Understanding these evading mechanisms is a first step needed for the design of rational anticancer therapy. Therefore, we decided to address the role of Akt in apoptosis resistance in human cancer by finding new partners involved in resistance to cell death. To this end, we performed a two hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the possible interactors of Akt, we decided to focus on Bcl-w, a member of the Bcl-2 family. Biochemical experiments confirmed the interaction of Akt with Bcl-w. Further, we demonstrate that Akt modulates the half-life of Bcl-w. We also found that Bcl-w is a substrate of Akt and, more importantly, that Akt regulates its anti-apoptotic activity and interaction with some of the pro-apoptotic members of the Bcl-2 family. Methods Materials Media, sera, and antibiotics for cell culture were from Life Technologies, Inc. (Grand Island, NY, USA). Protein electrophoresis reagents were from Bio-Rad (Richmond, VA, USA), and Western blotting and ECL reagents were from GE Healthcare. All other chemicals were from Sigma (St. Louis, MO, USA). Plasmids Plasmids pEF FLAG(hs) Bcl-w , pEF EE Bax, pEF EE Bik, pEF EE Bad cDNAs were kindly provided by Elisabeth Cory and David Huang laboratories (Victoria, Australia). Akt wild type (HA-Akt, cDNA), Akt E40 K (constitutively active Akt cDNA, HA-Akt-D+) and Akt K179M (dominant negative Akt cDNA, HA-Akt-D-) were a kind gift of Prof. G.L. Condorelli (University of Rome “La Sapienza”). Cell culture Human HeLa and HEK-293 cell lines were grown in DMEM containing 10% heat-inactivated FBS and with 2 mM L-glutamine and 100 U/ml penicillin-streptomycin. Yeast Two-hybrid System All experiments were performed in the yeast reporter MaV203. The cDNA library was synthesized from rat FRTL-5 cell poly(A)+ RNA plasmid by Life Technologies and cloned into the pPC86GAL4AD vector, and was kindly provided by Prof. Roberto Di Lauro (Naples, Italy). Screening of the library was performed essentially following instructions for the ProQuest two-hybrid system (Life Technologies) and has been previously described [18]. The GAL4 DNA-binding domain/hAkt fusion was obtained from Dr. Alfonso Bellacosa (Fox Chase Cancer Centre, Philadelphia, Pennsylvania, USA). Subsequently, yeast pLEx4-Akt plasmid was transformed with the pPC86AD-cDNA library and plated onto plates lacking histidine in the presence of 3AT (aminotriazole; 10 mM). Approximately 1.2×106 individual clones were plated, and about 200 grew on the selective medium. Resistant colonies were grown on a master plate and then replica-plated onto selection plates to determine their ability to induce three independent reporters (HIS3, URA3, and lacZ). Eighty independent clones were isolated after this first screening. DNA was isolated from each positive clone and sequenced to identify the inserts. Independent pPC86AD clones were retransformed into yeast and tested for interaction with a fresh Akt clone. Generation of Bcl-w deletion mutants We generated by PCR two deletion mutants of Bcl-w cDNA, using as template the plasmid pEF FLAG Bcl-w: the following primers were used for the bclw-BH4 mutant, which included only the N-terminal BH4 domain (45 aa): BH4-For-HINDIII:cccaagcttatggactacaaagacgatgacgataaag and BH4-Rev-Xba1: gctctagaggcttggtgcagcgggtc; the following primers were used for CT-Bcl-w, which included the remaining coding sequence of 97aa: CT-For-HINDIII: cccaagcttcccagcagctgacccgct and CT-Rev-Xba1: gctctagatcacttgctagcaaaaaaggccc. Temperature cycles were the following: 95°C 1 minute; 95°C 50 seconds, 60°C 50 seconds, 68°C 7 minutes for 35 cycles; 68°C 2 minutes. The amplified sequences were cloned in p3X-Flag-CMV previously linearized with the restriction enzymes HINDIII and XbaI. Generation of stable transfectants HeLa cells were transfected with 4 µg of Flag-Bcl-w cDNA using lipofectamine 2000 according to the manufacturer's protocol (InVitrogen, Carlsbad, CA). After 48 hr of transfection, cells were selected using a medium containing 10% FBS, 2 mMol L-glutammine, 100 U/ml pen/strep, and 3.75 µg/ml of puromicine. After 15 days the clones were isolated and maintained in culture with 2.5 µg/ml of puromicine. Twenty colonies were isolated and tested through western blot to verify the expression of the construct. Western blotting Total protein from HeLa and HEK 293 cells was extracted with RIPA buffer (0.15 mM NaCl, 0.05 mM Tris-HCl, pH 7.5, 1% Triton, 0.1% SDS, 0.1% sodium deoxycolate and 1% Nonidet P40). Fifty µg of sample extract were resolved on 7.5–12% SDS-polyacrylamide gels using a mini-gel apparatus (Bio-Rad Laboratories, Richmond, CA) and transferred to Hybond-C extra nitrocellulose. Membranes were blocked for 1 hr with 5% non-fat dry milk in TBS containing 0.05% Tween-20, incubated over night with primary antibody, washed and incubated with secondary antibody, and visualized by chemiluminescence. The following primary antibodies were used: Anti Flag M2 and anti-β-actin antibody from Sigma (St. Louis, MO, USA), anti HA and anti EE from Covance (Berkeley,CA USA); anti Bcl-w from Abcam (Cambridge, MA); anti-Akt, -Phospho Akt substrate, -phospho ser473 Akt from Cell signalling (Danvers, MA USA); anti-Bcl2, - BAD, -BIK and -BAX from Santa Cruz, Inc (Santa Cruz, CA USA), caspase -9 and -3 from Cell Signaling (Danvers, MA USA), and PARP antibodies from Santa Cruz (Santa Cruz, CA USA). Phosphorylation experiments In order to study Bcl-w phosphorylation in intact cells, 293 cells were transiently transfected with different Akt cDNAs constructs as indicated. After 24 h, the cells were rinsed with 150 mM NaCl and incubated in serum-free culture medium for 16 h at 37°C. Insulin (final concentration, 100 nM) or 20% serum was then added, and the cells were rapidly rinsed with ice-cold saline followed by solubilization with 0.5 ml of RIPA buffer per dish for 1 hr at 4°C. Lysates were centrifuged at 5,000×g for 20 min, and solubilized proteins were precipitated with the indicated antibodies, separated by SDS-PAGE, and revealed by western blot with the anti-Akt substrate antibody that recognizes all the phosphorylated Akt substrates (Cell Signaling, Danvers, MA USA). Phospho-(Ser/Thr) Akt Substrate Antibody preferentially recognizes peptides and proteins containing phospho-Ser/Thr preceded by Lys/Arg at positions −5 and −3. Some cross-reactivity has been described for peptides that contain phospho-Ser/Thr preceded by Arg/Lys at positions −3 and −2, thus recognizing also a low-stringency Akt kinase motif. Immunoprecipitation Cells were cultured at a final concentration of 90% in p100 plates. The cells were harvested with RIPA Buffer on a shaker for 30 minutes. 1 mg of total extract was immunoprecipitated using the indicated antibodies (5 µg/ml Anti-FLAG, 2 µg/ml Anti-HA, 3 µg/ml anti-Akt, 5 µg/ml anti-Bcl-w, 3 µg/ml anti-EE), for 16 hrs on shaker. Then, A/G beads (Santa Cruz, Santa Cruz, CA USA) were added for two hrs. The beads were washed for three times with washing buffer (50 mM Tris Hcl pH 7.5, 150 mM NaCl, 0.1% Triton, 10% glycerol), and then 20 µl of sample buffer was added; the samples were boiled at 100°C for 5 minutes and then the supernatants resolved by SDS-PAGE. Cytosol/mitochondria separation Cells were grown in p100 plates and the mitochondrial and cytoplasmic fractions isolated using the Mitochondria/Cytosol Fractionation Kit (Biovision San Francisco, CA USA) according to the manufacturer's protocol. Akt Kinase Assay Akt activity was assayed in vitro as previously reported [19]. Briefly, HEK-293 cells were transfected with 4 µg of Flag-Bcl-w cDNA. 1 mg of total cell extract was immunoprecipitated using an anti-FLAG antibody (Sigma) and A/G beads (SantaCruz, Santa Cruz, CA USA) for 18 hr. The beads were incubated in a kinase reaction mixture containing 20 mM HEPES [pH 7.2], 1 mM MgCl2, 10 mM MnCl2,1 mM dithiothreitol, 5 mM ATP, 0.2 mM EGTA, 1 mM protein kinase inhibitor, 10 µCi of [γ–32P]ATP, and 2 µg of rAkt (Cell signaling, Danvers, MA USA) for 30 minutes at room temperature. The samples were boiled at 100°C for 5 minutes, centrifuged and the supernatant loaded on a 12.5% maxi protean gel (BioRad, Richmond VA, USA). The gel was run overnight and then visualized by autoradiography. Cell death and cell proliferation quantification Cells were plated in 96-well plates in triplicate and incubated at 37°C in a 5%CO2 incubator. Different chemotherapics (30 mg/ml cisplatin, 10 mg/ml epirubicin) were added for 24 hrs to the medium. Cell viability was evaluated with the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI), according to the manufacturer's protocol. Metabolically active cells were detected by adding 20 µl of MTT to each well. After 2 h of incubation, the plates were analyzed in a Multilabel Counter (Bio-Rad, Richmond, VA, USA). Apoptosis was assessed using PI (propidium iodide)-FITC staining followed by flow cytometric analysis. Cells were seeded at 1.8×106 cells per 100 mm dish, grown overnight in 10% FBS/DMEM, washed with PBS, then treated for 24 hours with chemotherapics. Following incubation, cells were washed with cold PBS and removed from the plates by mild trypsinization. The resuspended cells were washed with cold PBS and stained with PI-FITC staining according to the instructions provided by the manufacturer (Roche Applied Science, Indianapolis, IN). Cells (50,000 per sample) were then subjected to flow cytometric analysis. Flow cytometry analysis were done as described [20]. The percentage of apoptosis indicated was corrected for background levels found in the corresponding untreated controls. siRNA transfection HeLa cells were cultured to 80% confluence and transiently transfected using LIPOFECTAMINE 2000 with 100 nM anti-Akt siRNA (Dharmacon, Lafayette, CO USA), a pool of 4 target-specific 20–25 nt siRNAs, or 150 nM anti-Bcl-w si-RNA (InVitrogen, Carlsbad, CA) with 6 µl transfection reagent, as described in the manufacturer's protocol. Results Akt interacts with Bcl-w To find new interactors of Akt, we performed a yeast two-hybrid screening with human full-length Akt c-DNA sequence as bait and a murine c-DNA thyroid library as prey. Among the 100 clones obtained, two were identified as Bcl-w, covering its full coding sequence. To confirm the interaction between Akt and Bcl-w, we immunoprecipitated proteins from untreated, Akt-transfected, and Bcl-w-transfected cells with an anti-Bcl-w antibody. We found that Akt co-immunoprecipitates with Bcl-w in extracts from untransfected and transfected cells (Figure 1A and 1B). The extent of Akt binding with Bcl-w was comparable to that with its substrate, Bad (Figure 1B). 10.1371/journal.pone.0004070.g001Figure 1 Akt interacts with Bcl-w. (A) Co-immunoprecipitation of endogenous Akt with Bcl-w. Wt HeLa cells were lysed and 1 mg of protein extract immunoprecipitated using an anti-Bcl-w antibody. Immunoprecipitates and total lysates (50 µg) were separated on 12%SDS polyacrilamide gel and blotted with an anti-Akt antibody. As negative control, proteins were incubated with beads without antibody (B) Co-immunoprecipitation of transfected Akt with FLAG-Bcl-w or EE-BAD. HEK-293 cells were tansfected with 2 µg of HA-Akt and 2 µg of FLAG-Bcl-w or EE-BAD cDNAs, as indicated. After 48 hr, cells were lysed, and 1 mg of protein extract was immunoprecipitated using an anti-HA antibody. Immunoprecipitates were subsequently blotted with anti-HA, anti-Flag or anti-EE antibodies, as indicated. (C) HEK-293 cells were transfected with 2 µg of either wt-Bcl-w cDNA or the deletion mutants, Bcl-w/BH4 or Bcl-w/CT, as indicated. Protein extracts were immunoprecipitated using an anti-Akt antibody. Immunoprecipitates and total lysates were resolved on 12%SDS-PAGE and transferred to Hybond-C nitrocellulose. Membranes were incubated with an anti-FLAG antibody. Both deletion mutants, Bclw/BH4 and Bclw/CT, immunoprecipitated with Akt. Bcl-w contains four Bcl-2 homology (BH) domains and a transmembrane (TM) fragment at the C-terminal region, important for its insertion into the mitochondrial outer membrane. We verified whether these regions are important for the interaction with Akt. For this, HA-Akt cDNA was transfected together with one of two Bcl-w domain-region cDNAs obtained by PCR and fused to the FLAG epitope: these were the BH4 domain (45 aa) of Bcl-w, located at the N-terminus, and the remaining portion of the protein (97aa). Extracts were immunoprecipitated with an anti-Flag antibody and blotted with an anti-HA antibody. We found that Akt interacts with both Bcl-w deletion mutants, indicating that Akt may interact with Bcl-w at multiple sites (Figure 1C). Role of Akt activation on Akt/Bcl-w interaction To find whether the activity of Akt influences its interaction with Bcl-w, HeLa cells were transfected either with wild type Akt (Akt wt) cDNA or with one of two mutants: an HA-tagged kinase dead-Akt construct (Akt D−) with dominant negative functions, and a constitutively active Akt construct (Akt D+). Protein extracts were immunoprecipitated with a monoclonal anti-HA antibody and then blotted with an anti-FLAG antibody. We found that Bcl-w interacts with wild type Akt and more efficiently with the activated kinase, but not with the kinase-dead Akt (Figure 2A). 10.1371/journal.pone.0004070.g002Figure 2 Akt activity regulates Bcl-w expression. (A) HeLa cells were transfected with 2 µg of HA-Akt wt, Akt D+, or HA-Akt D− cDNA and 2 µg Flag-Bcl-w for 48 hrs. Protein extracts were immunoprecipitated with an anti-HA monoclonal antibody. Immunoprecipitates were resolved on 12% SDS-PAGE and transferred to Hybond-C nitrocellulose. Membranes were incubated with anti-Flag antibody (0.2 µg/ml). 50 µg of total sample extracts were also analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (B) HeLa cells were transfected with 4 µg of HA-Akt wt, HA-Akt D+, or HA-Akt D− cDNA for 48 hrs. Protein extracts were blotted with anti-Bcl-w antibody in order to detect endogenous levels of Bcl-w. Loading control was obtained with anti-β actin antibody. (C) Cells were transfected with 100 nM of siAkt-RNA for 48 hrs. Cellular proteins were solubilized and analyzed by western blot using the indicated antibodies. (D) HeLa cells were treated with 10, 20 or 40 µM of LY294002 for 24 hrs. Protein extracts were analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (E) Bcl-w HeLa cells were treated with 10 µM of MG-132 for 8 hrs. 40 µg of protein extracts were analyzed by western blot with anti-Bcl-w antibodies. Loading control was obtained using anti-β actin antibody. Akt regulates Bcl-w expression When we transfected cells with Akt D−, we noticed a fall in the expression of Bcl-w (Figure 2A). Therefore, lack of interaction between Bcl-w and the kinase-dead Akt could have been due to reduced expression of Bcl-w rather than to poor interaction with Akt D−. To address this issue, we inhibited Akt in three different ways: by interfering with its endogenous function; by treating cells with Akt-siRNA; and by inhibiting the PI3K/Akt pathway with a specific drug. In order to interfere with endogenous Akt activity, we transfected cells with the previously described Akt mutant cDNAs (Akt wt, Akt D+, and Akt D−). We found that Bcl-w was reduced after transfection with inactive Akt, whereas Bcl-w expression increased upon transfection with Akt D+ (Figure 2B). In order to knock down endogenous Akt, HeLa cells were transfected with a pool of Akt siRNAs. We found that endogenous Akt expression, analyzed by Western blot, was reduced by >80% after 48 hrs. This reduction in Akt expression was followed by a drastic reduction in the level of Bcl-w. Moreover, the expression of the anti-apoptotic protein, Bcl-2, but not of the pro-apoptotic protein, Bax, was also reduced (Figure 2C). Finally, incubation of HeLa cells with 10, 20 or 40 µM of LY294002, a specific inhibitor of the PI3K pathway, resulted in reduced amount of Bcl-w protein (Figure 2D). All these results provide evidence that the kinase activity of Akt regulates the expression of Bcl-w. To gain insight on the mechanism of Akt-mediated Bcl-w regulation, we treated Bcl-w/HeLa cells with the proteasome inhibitor, MG-132, for 8 hrs and then analyzed Bcl-w levels by western blot (Figure 2E). The inhibition of the proteasome did not result in an increase in Bcl-w expression, suggesting that the ubiquitin pathway is not directly involved in the regulation of Bcl-w by Akt. Role of Akt in Bcl-w subcellular localization Bcl-w is an anti-apoptotic protein weakly linked to the outer mitochondrial membrane [21]. To verify its intracellular localization, extracts of HeLa cells were fractionated to isolate mitochondria from the cytosol. We found that Bcl-w is present mainly in mitochondrial protein extracts (Figure 3A). To clarify the role of Akt in determining Bcl-w cellular localization, HeLa cells were transfected with Akt wt, Akt D+, or Akt D− cDNAs before fractional separation. We found that the presence of the kinase-dead Akt mutant reduced the amount of Bcl-w linked to the mitochondrial fraction and induced only a slight increase in the cytosolic one (Figure 3B). Similar results were obtained in cells transfected with Akt siRNA (Figure 3C). Thus, Akt acts mainly on Bcl-w expression. 10.1371/journal.pone.0004070.g003Figure 3 Akt controls Bcl-w localization. (A) HeLa cells were subjected to fractionated separation of mitochondrial/cytosolic proteins using a mitochondria/cytosol fractionation kit (Biovision). Protein extracts were loaded onto 15% SDS polyacrilamide gel, and analyzed by western blot by anti-Bcl-w antibody. As a control of the mitochondrial fraction, an anti-cox4 antibody was used. (B) HeLa cells were transfected with 2 µg of HA-Akt WT, D+, or D− for 48 hrs. Cells were subjected to mitochondria/cytosol separation as above. Protein extracts were analyzed by western blot using anti-Bcl-w, anti-Akt, or anti-cox4 antibodies. (C) Cells were transfected with 100 nM of siAkt-RNA for 48 hrs. Cytosol and mitochondria were isolated as described in the methods and analyzed by western blot using the indicated antibodies. Akt phosphorylates Bcl-w Akt is a serine threonine kinase that phosphorylates different pro- and anti-apoptotic proteins. Thus, in vitro and in vivo phosphorylation assays were performed to uncover whether Bcl-w is a substrate of Akt. For in vitro assays, cells were transfected with Flag-Bcl-w and the extracts obtained immunoprecipitated using a monoclonal anti-Flag antibody. Immunoprecipitates were incubated with a constitutively active Akt recombinant protein in the presence of γP32ATP. We found that Akt phosphorylates Bcl-w in vitro, although not with the same efficiency as histone H2B (Figure 4A). 10.1371/journal.pone.0004070.g004Figure 4 Akt phosphorylates Bcl-w in vitro and in vivo. (A) HeLa cells were transfected with 2 µg of DNA of Flag Bcl-w, solubilized, and 1 mg of protein extract was immunoprecipitated with an anti-M2 Flag antibody. Immunoprecipitates were incubated with recombinant constitutive active Akt (rAkt), and in vitro kinase assay was conducted as described in the methods. Samples were loaded onto 2.5% SDS-PAGE and analyzed by autoradiography. As positive control we used Histone2B (H2B). (B) HeLa Bcl-w stable expressing clones were serum starved for 18 hrs and then stimulated with 100 nM insulin or with 20% serum for 15 min as indicated. Cells were solubilized and immunoprecipitated with an anti-M2 Flag antibody. Immunoprecipitates were loaded onto SDS-PAGE and blotted with an anti-phospho Akt substrate antibody that recognizes all the phosphorylated Akt substrates. Total extracts were analyzed by western blot using the indicated antibodies. (C) HeLa cells were transfected with 2 µg of pcDNA3 empty vector or 2 µg of HA-GSK3β, and 2 µg of Flag-Bcl-w for 48 hrs. Cells were stimulated with 100 nM insulin for 15 min, solubilized, immunoprecipitated using an anti-HA antibody, and analyzed by western blot using an anti-phospho-Gsk3 antibody. Total extracts were analyzed by western blot using the indicated antibodies. Bcl-w overexpression does not affect Akt activity. To study the effects of Akt kinase activity on Bcl-w phosphorylation in intact cells, we generated HeLa cells that stably expressed Flag-Bcl-w (HeLa/Bcl-w). HeLa/Bcl-w cells were stimulated with insulin or 10% serum for 15 min, and protein extracts then immunoprecipitated using an anti-Flag antibody and blotted with an anti-phospho (Ser/Thr) Akt substrate antibody that recognizes the Akt substrate motif. We found that the phosphorylated band corresponding to Bcl-w immunoprecipitates upon stimulation with serum or insulin. These results taken together provide evidence that Bcl-w may be a substrate of Akt both in vitro and in intact cells (Figure 4B). In turn, to investigate whether Bcl-w overexpression regulates Akt kinase activity, HEK293 cells were co-transfected with Flag-Bcl-w and HA-tagged Gsk3β, one of the main Akt substrates. 48 hours after transfection, the cells were stimulated with insulin for 10 min, cellular extracts immunoprecipitated with an anti-HA antibody, and then immunoblotted with an antibody that recognizes the phosphorylated form of Gsk3β. We did not find a change in the extent of Gsk3β phosphorylation by overexpressing Bcl-w (Figure 4C). Therefore, Bcl-w binds to Akt and is a direct substrate of Akt; however, this binding does not alter the activity of Akt on other substrates. Role of Bcl-w/Akt interaction on cell death Given that Bcl-w is an anti-apoptotic member of the Bcl-2-family, we investigated the role of Akt activity on this function. We first analyzed the effect of Bcl-w overexpression in preventing apoptosis induced by two different chemotherapics, i.e. cisplatin and epirubicin, in HeLa/Bcl-w compared to parental untransfected HeLa cells. Cells were treated with 30 µg/ml cisplatin or with 10 µg/ml epirubicin for 24 hr. Cell death was assessed with a cell viability assay, with propidium iodide staining followed by FACS analysis, or by caspase 9, −3, and PARP activation. We found that HeLa/Bcl-w cells were 80–90% resistant to cell death induced by the chemotherapics. This was confirmed by analysis of the activation state of the intrinsic apoptotic pathway (caspase 9, −3, and PARP) (Figure 5A). 10.1371/journal.pone.0004070.g005Figure 5 Akt regulates the anti-apoptotic function of Bcl-w. (A) HeLa control cells and HeLa cells stably expressing Flag-Bcl-w were plated in 96 well plates in triplicate and treated with 30 µg/ml of cisplatin or 10 µg/ml of epirubicin for 24 hr. Apoptosis was analyzed by Cell Vitality assay, by propidium iodide staining and FACS analysis, or by western blot for caspase cascade activation with anti-caspase-3, -9, and PARP antibodies. Loading control was obtained with anti β-actin. (B) HeLa-Flag Bcl-w cells were transfected with 4 µg of HA-Akt D− cDNA or with 100 nM of siAkt-RNA for 48 hrs and then treated with 30 µg/ml of cisplatin for 24 hr. Cell death was then analyzed as described above. Total lysates were analyzed by western blot using an anti-PARP antibody. Loading control was obtained with an anti-β-actin antibody. Inactivation of Akt activity resulted in a reduction in the protective effect of Bcl-w on cell death. To test the role of Akt activity on the antiapoptotic function of Bcl-w, we repeated the above experiments in Bcl-w/HeLa cells transfected for 48 hr either with Akt D− cDNA or with Akt siRNAs. We found that the inhibition of Akt kinase activity or protein quantity resulted in a strong activation of the downstream effector PARP (Figure 5 b, left panel), that is partially reflected as reduction of pro-survival effect of Bcl-w (∼20%) (Figure 5B). Thus, Akt activity mediates the anti-apoptotic function at least in part by regulating the intracellular levels of Bcl-w. Given that inhibiting Akt results in a reduction of Bcl-w levels, these results suggest that Akt may contribute to Bcl-w protective effects mainly by regulating its intracellular levels. To further confirm this, we down-regulated Bcl-w expression with two specific Bcl-w-siRNAs, and analyzed the effects of Bcl-w down-regulation on chemotherapy-induced cell death. We found that 72 hrs of incubation with Bcl-w siRNAs drastically reduced Bcl-w protein (Figure 6 A) although to different extents (siRNA61 was more effective than siRNA62). The assessment of cell viability (Figure 6B) and of apoptotic cells (Figure 6C) provided evidence that the effect of cell death induced by chemotherapics was proportional to the expression of Bcl-w. Furthermore, by reducing Bcl-w level, we obtained the same ∼20% increase in cell death that we observed in HeLa cells treated with Akt siRNA. Thus, the reduction in Bcl-w expression secondary to Akt inactivation contributes to the resistance of cancer cells to chemotherapy-induced cell death. 10.1371/journal.pone.0004070.g006Figure 6 Effects of Bcl-w si RNA on cell death. (A) Cells were transfected with 150 nM of siBcl-w-RNAs for 72 hrs. Total lysates were analyzed by western blot using anti-Bcl-w antibodies. Loading control was obtained with an anti-β-actin antibody. (B, C) Cells were transfected with 150 nM of siBcl-w-RNAs for 48 hrs. Then, the cells were splitted into 96 wells and then treated with 30 µg/ml of cisplatin for 24 hr. Cell death was then analyzed with MTT (B) or propidium iodide staining and FACS analysis (C). Bcl-w down-regulation induces an increase of cell death. Akt regulates Bcl-w interaction with Bcl-2 family members The intrinsic apoptotic pathway is regulated by the net interactions of pro- and anti-apoptotic Bcl-2 members [22]. To evaluate the effect of Akt activity on the interaction of Bcl-w with the pro-apoptotic Bcl-2 members, we set up co-immunoprecipitation experiments with Bcl-w and Bad, Bik, or Bax in cells overexpressing the dominant negative Akt cDNA. We found that Akt inactivation resulted in a drastic reduction of Bcl-w interaction with the pro-apoptotic proteins (Figure 7). This further confirms the stimulatory role of Akt activity on Bcl-w anti-apoptotic function. 10.1371/journal.pone.0004070.g007Figure 7 (A) Akt activity regulates Bcl-w interaction with Bcl-2 family members. Flag-Bcl-w/HeLa cells were transfected with 2 µg of either HA-Akt D+ or HA-Akt D− cDNA, and 2 µg of EE-Bax cDNA for 48 hr. Cells were harvested and 1 mg of total lysate immunoprecipitated using an anti-Flag antibody. The immunoprecipitates were then blotted with an anti-EE antibody. Total protein was normalized using anti-EE, -HA or -β-actin antibodies, as indicated. (B) Flag Bcl-w/HeLa cells were transfected with 2 µg of HA-Akt D+ or HA-Akt D− cDNA, and 2 µg of either EE-Bad or EE-Bik cDNA, as indicated, for 48 hr. Cells were harvested and 1 mg of total lysate immunoprecipitated using anti-Flag antibody. The immunoprecipitates were then blotted with an anti-EE antibody. Total protein was normalized using anti-EE, -HA or -β-actin antibodies, as indicated. Inactivation of Akt induced a reduction of Bcl-w interaction with the pro-apoptotic Bcl-2 members. Discussion Apoptosis is believed to be the major mechanism of chemotherapy-induced cell death in cancer [23], [24]. Unfortunately, many tumour cells evade drug-induced death signals [25]. Akt is an important survival-signaling molecule, whose function is frequently found altered in human cancer [5], [26]. Therefore, we decided to address the role of Akt in apoptosis resistance in human cancer by finding new partners of Akt by two hybrid screening in yeast. Among the interactors of Akt that we found, we focused on Bcl-w, a pro-survival member of the Bcl-2 protein family [27], [28] that has received less attention compared to its other family members. By genetic and biochemical methods, we have demonstrated here that Akt interacts with the N- and C-terminal sequences of the Bcl-w protein, and phosphorylates Bcl-w both in vitro and in the intact cell. The analysis of the Bcl-w sequence did not reveal a canonical Akt phosphorylation motif [29]. However, there is evidence that Akt may phosphorylate cellular substrates at the level of a partially conserved sequence motif [29]. Bcl-w posses at least 6 serine/threonines that are included in “degenerated” Akt phosphorylation sites. By site-directed mutagenesis, we mutated two of these sites (ser 62 and ser 83) substituting the serine with an alanine (data not shown). These mutations did not result in a change of Bcl-w phosphorylation state, so the hypothetical Akt phosphorylation site must be located elsewhere. We are now addressing this issue. We have also demonstrated here that interfering with the activity or amount of Akt reduces the quantity of Bcl-w protein; oppositely, transfection of a dominant active Akt mutant increased the content of Bcl-w in cells. Akt-mediated Bcl-w down-regulation was observed to occur also in glioma (data not shown). Thus, Akt affects Bcl-w function in various cell types at least in part by regulating its expression. The mechanisms underlying this are not clear, but the regulation of Bcl-w protein levels is unlikely mediated by the ubiquitin-proteasome pathway, as evidenced by the negative result obtained with a proteasome inhibitor. Furthermore, Akt inhibition did not produce an effect on Bcl-w mRNA, as evaluated by Real Time PCR (data not shown). Other possible Akt-mediated regulatory effects on RNA or protein stability are under investigation in our laboratory. Several studies have suggested that Akt may regulate the balance between pro- and anti-apoptotic signals, at least in part by regulating the cellular localization of Bcl-2 family members [30], [31]. Thus, in this study we have analyzed the effect of Akt activation on the subcellular localization of Bcl-w. We found Bcl-w predominantly associated with the mitochondrial fraction, as previously described also by O'Reilly et al. [32]. The presence of the kinase-dead Akt mutant reduced the amount of Bcl-w linked to this fraction, but it did not increase Bcl-w in the cytosol; we obtained similar results with cells transfected with Akt siRNA. Thus, via binding and phosphorylating Bcl-w, Akt may control Bcl-w activity mainly through the regulation of Bcl-w protein expression. We are conducting experiments with Bcl-w phosphorylation mutants to formally prove this conclusion. Moreover, with the intent to clarify the role of Akt-mediated regulation of Bcl-w on its anti-apoptotic functions, we established a Bcl-w overexpressing cell line. These cells exhibit a significant decrease of chemotherapy-mediated cell death. When we evaluated the effects of decreasing Akt activity on survival in Bcl-w/HeLa cells, we found a ∼20% increase in cell death. However, when we analyzed cell death by western blot of PARP activation, the active PARP fragment was present exclusively in Bcl-w/HeLa cells incubated with Akt D− cDNA. Thus, even though the differences that we observe with FACS analysis and cell vitality are of small entity, the end point, that is cell death evaluated as PARP activation, is reached only in Bcl-w cells where Akt has been inactivated. On the other hand, our data provide evidence that Bcl-w is not the only defense mechanism of the cell toward chemotherapy-induced apoptosis, and many other Bcl-2 family members may mediate anti-apoptotic signals. Therefore, downregulation of Akt may result in a pronounced efficacy in cancer cells were Bcl-w predominates over the other Bcl-2 family members [33]–[35]. When appropriate stimuli are present, homodimerization of pro-apoptotic members of the Bcl-2 family activates the intrinsic apoptotic cascade. Bcl-w interacts with pro-apoptotic members of the Bcl-2 family, such as Bad, Bax, and Bik, blocking the formation of the homodimers and, thus, the activation of the apoptotic cascade. Events that inhibit the formation of these Bcl-w/pro-apoptotic Bcl-2 member complexes may lead to the activation of apoptosis [36]. We show here that Bcl-w/Bax, Bcl-w/Bad, and Bcl-w/Bik interactions were drastically reduced in cells overexpressing dominant-inactive Akt cDNA, indicating that Akt activity is necessary for these interactions. Therefore, Akt may regulate the anti-apoptotic function of Bcl-w, reducing its amount in the cell and, thus, impairing the balance of homo- and heterodimer formation upon apoptotic stimuli. Bcl-w can be up-regulated in tumors such as gastric and colorectal cancer [33]–[35]. Interestingly, the PI3k-Akt pathway is involved in the progression and chemoresistance of these types of cancer [37]–[39]. Therefore, increased Akt activity can be speculated to promote survival and anti-apoptotic signaling in cancer cells at least in part through increasing Bcl-w levels. Recently, Bcl-w was reported to promote gastric cancer cell invasion, by inducing matrix metalloproteinase-2 expression [34]. Bcl-w is up-regulated also through pathways besides the Akt one: Tran et al. demonstrated that Bcl-w can be up-regulated via the NFkB pathway activated by TWEAK (tumor necrosis factor-like weak inducer of apoptosis) through stimulation of its receptor, Fn14; moreover, the TWEAK-Fn14 pathway can induce survival of glioma cells, at least in part by up-regulating the quantity of Bcl-w protein [40]. In addition, Yao et al. reported that up-regulation of Bcl-w protein mediates the neuroprotective effect of estrogens [41]. Therefore, Bcl-w participates in a number of different systems that regulate survival and anti-apoptotic pathways. The results that we have presented here provide the first evidence that Akt interacts with, and regulates the levels of, Bcl-w, moving the balance of the Bcl-2 family toward anti-apoptotic members. Enhancement of this Akt/Bcl-w anti-apoptotic pathway can be speculated as one mechanism responsible for the reduced sensitivity to apoptosis of cancer cells that are resistant to chemotherapy-induced cell death. This finding may be of importance in optimizing a strategy for the treatment of cancers, such as gastric and colon adenocarcinoma, in which Bcl-w has been found to be increased. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was partially supported by funds from: Associazione Italiana Ricerca sul Cancro, AIRC (G.C.), MIUR-FIRB (RBIN04J4J7), EU grant EMIL (European Molecular Imaging Laboratories Network) contract No 503569. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. C.Q. is a recipient of Clinica Mediterranea- Federico II FiXO Training program. G.R. is recipient of Fondazione SDN fellowship. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1913210108-PONE-RA-04841R310.1371/journal.pone.0004168Research ArticleMathematicsPublic Health and Epidemiology/Global HealthPublic Health and Epidemiology/Health Services Research and EconomicsPublic Health and Epidemiology/ImmunizationPublic Health and Epidemiology/Infectious DiseasesPredicting the Herd Immunity Threshold during an Outbreak: A Recursive Approach Herd Immunity PredictionGeorgette Nathan T. * Science Department, Allen D. Nease High School, Ponte Vedra, Florida, United States of America von Elm Erik EditorUniversity of Bern, Switzerland* E-mail: [email protected] and designed the experiments: NTG. Performed the experiments: NTG. Analyzed the data: NTG. Contributed reagents/materials/analysis tools: NTG. Wrote the paper: NTG. 2009 9 1 2009 4 1 e416826 5 2008 8 12 2008 Georgette.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background The objective was to develop a novel algorithm that can predict, based on field survey data, the minimum vaccination coverage required to reduce the mean number of infections per infectious individual to less than one (the Outbreak Response Immunization Threshold or ORIT) from up to six days in the advance. Methodology/Principal Findings First, the relationship between the rate of immunization and the ORIT was analyzed to establish a link. This relationship served as the basis for the development of a recursive algorithm that predicts the ORIT using survey data from two consecutive days. The algorithm was tested using data from two actual measles outbreaks. The prediction day difference (PDD) was defined as the number of days between the second day of data input and the day of the prediction. The effects of different PDDs on the prediction error were analyzed, and it was found that a PDD of 5 minimized the error in the prediction. In addition, I developed a model demonstrating the relationship between changes in the vaccination coverage and changes in the individual reproduction number. Conclusions/Significance The predictive algorithm for the ORIT generates a viable prediction of the minimum number of vaccines required to stop an outbreak in real time. With this knowledge, the outbreak control agency may plan to expend the lowest amount of funds required stop an outbreak, allowing the diversion of the funds saved to other areas of medical need. ==== Body Introduction The World Health Organization (WHO) has emphasized case management over outbreak response immunization (ORI) based on cost-effectiveness per mortality avoided [1], [2], demonstrating that cost is a significant issue with regard to ORI. However, studies have demonstrated the effectiveness of ORI at limiting the spread of a VPD and thus reducing the number of resulting morbidities and mortalities [1]–[3]. Vaccination has two purposes: 1) individual protection, and 2) population or “herd” protection [4]. This research focuses on determining the minimum number of vaccines required to achieve the population protection. In other words, the objective is to predict the minimum vaccination coverage required to reduce the mean number of secondary infections per infectious individual to less than unity. Poorer nations may choose to focus on achieving population protection rather than on ensuring individual protection for as many as possible to conserve funds in resource poor settings. The method proposed in this paper demonstrates a manner through which impoverished countries may predict the number of vaccines necessary to achieve protection of the population and thus preserve funds. Key Definitions As two terms with similar meanings are used throughout this paper, I will clarify the distinctions: Herd Immunity Threshold: The overall fraction of a population that must be vaccinated (regardless of when these vaccinations occur) to reduce the mean number of secondary infections per infectious individual to less than one for a VPD [5]. This threshold level reduces the probability of infection of unimmunized persons, thus essentially granting indirect protection to those without immunity and thereby gradually stopping the outbreak [5]. An alternative definition has been offered [6], which would characterize the above approach as “herd effect” rather than the herd immunity threshold. The noun “threshold” is used to reflect the quantitative nature of the phenomenon. Herd Immunity Threshold = Pre-Outbreak Vaccine Coverage+ORIT. Outbreak Response Immunization Threshold (ORIT): The fraction of a population that must be vaccinated during the outbreak to reduce the mean number of secondary infections per infectious individual to less than one, thus causing the cessation of the outbreak. It should be noted that achieving the ORIT also results in the achievement of the herd immunity threshold. By attaining the ORIT, the outbreak control agency may reduce the probability of infection among the unimmunized to a sufficient degree, thus gradually stopping the outbreak [5]. The ORIT may be guaranteed, at a rather high financial cost, by immunizing as much of the population as possible. This high expenditure is problematic since measles, along with most VPDs, primarily affects the poorest nations around the world [7]. In addition, implementing an ORI program of the scale required to end an outbreak requires a large expenditure of resources in the form of vaccines and personnel [8]. Therefore, immunizing the minimum number of persons required to achieve the ORIT can preserve critical resources in a poor nation. These resources may be diverted to other areas of dire medical concern. By immunizing only the minimum number of persons required to gradually stop an outbreak the outbreak control agency can save vaccines for a later date. For example, unused MMR vaccines, with an average shelf life of 2 years according to the Centers for Disease Control and Prevention [9], may be employed at a later date to help stop a future outbreak. To immunize the minimum number of persons to stop an outbreak, the outbreak control agency must have advance knowledge of an approximation for the ORIT. This prediction would also grant an outbreak control agency several days to prepare, plan for, and coordinate a massive immunization drive according to this target value, thus improving the overall efficiency of outbreak control. Other approaches The herd immunity threshold is most often presented as being dependent on the mean number of infections caused by a single infectious individual during his/her duration of infectiousness [10], [11], which is defined as the individual reproduction number [12] in this paper. Several time-based models for a variety of reproduction numbers have been developed previously, with some recent examples including [9], [12]–[15], through which the individual reproduction number (synonymous with the effective reproduction number), and by extension the ORIT, may be predicted. With the individual reproduction number, the ORIT may be approximated by solving for the vaccination coverage that satisfies Ri<1 where Ri is the individual reproduction number [4]. In other words, when the ORIT is achieved, each infectious person infects less than one other person on average, resulting in the ending of an outbreak [5]. Other methods for determining the minimum vaccination deployment required to end an outbreak have been developed. One notable perspective involves the “firefighter problem,” first introduced in [16], in which the minimum number of firefighters required to control a fire spreading through a grid is calculated. In the epidemiological application of this problem, the fire is a VPD and the firefighters are vaccines, protecting the vertices at which they are located. In other words, the minimum number of vaccines needed to contain the outbreak may be solved for through an iterative algorithm solution to the firefighter problem. Significant advances have been made on this front, including the examination of grids with three or more dimensions [17], and even generalization for grids of several dimensions [18]. Purpose This paper proposes a simple and readily applicable recursive predictive algorithm based on a tractable model I previously developed (hereinafter the “Threshold Model”) for the ORIT in [19]. This research builds on that previous work. The outbreak control agency may use this predictive algorithm to obtain an accurate prediction of the ORIT from up to six days in advance based on survey data input from two prior days. The paper then goes on to analyze the effects of a variety of factors on the accuracy of the prediction. In addition, I develop a novel formulation for determining the effects of additional vaccination on changes in the individual reproduction number. Methods Underlying model The Threshold Model proposed earlier [19] is as follows: (1) In the Threshold Model, P is the initial fraction of a population susceptible, I is the fraction of the population infectious, R is the fraction of a population recovered, is the recovery rate (fraction of the infectious persons that recover per day), S is the fraction of a population susceptible, N is the initial fraction of the population infectious, and is the immunization rate, where the value for may be calculated with the following equation: (2) In (1), the VT term is the most critical aspect. It represents the fraction of the population that must be immunized during the outbreak to achieve the herd immunity threshold, and is defined as the Outbreak Response Immunization Threshold (ORIT) in this paper. As most populations have high pre-outbreak immunity rates (often around 0.80 [20], [21]), the actual herd immunity threshold is calculated as: Pre-Outbreak Immunization Coverage+VT. Thus, VT values in this paper will be significantly lower than the largely accepted value of the herd immunity threshold for measles, which is around 0.90 [1]. In many cases, a significant portion of the population has been immunized during the outbreak before the ORIT is calculated using the Threshold Model in (1). The Threshold Model calculates the total fraction of the population that must be immunized during the outbreak to achieve the herd immunity threshold, not the fraction remaining. In other words, the fraction of the population that must be immunized from the point of calculation onwards is equal to: ORIT–fraction already immunized during the outbreak. In addition, (1) is based on a system of differential equations (which itself is based on the classic SIR model) that assumes homogenous mixing of the population and the mass action principle [19]. It also does not incorporate household mixing, outbreak response protocols (except vaccination), or the fraction of the population exposed but not infectious [19]. While these assumptions limit the accuracy of the Threshold Model, they simultaneously allow for greater simplicity and real time applicability. Based on the planned recursive nature of the predictive algorithm and the lack of data available for its testing (the number of infections and immunizations per day) a specific plan is established. The first stage, development, includes the analysis of the 2003 Republic of Marshall Islands (RMI) measles outbreak [20]. The second stage, testing, includes the application of the predictive algorithm to the 2003 RMI outbreak (also used in development) and a new test data set for the 2006 Fiji measles outbreak [21]. Data from both the 2003 RMI outbreak [20] and the 2006 Fiji measles outbreak [21] also was used to test the Threshold Model [19]. Development of ORIT algorithm To develop the recursive predictive algorithm, I first graph the Threshold Model's estimations for the ORIT over the duration of the 2003 Republic of Marshall Islands (RMI) measles outbreak [20]. Measles is chosen due to its high impact on poor nations and its high infectiousness [7]. This graph is presented in Figure 1 . 10.1371/journal.pone.0004168.g001Figure 1 To begin to understand the trends in the changes in the Outbreak Response Immunization Threshold (ORIT), I calculate the ORIT, using the Threshold Model, at several points during the 2003 RMI measles outbreak. I also graph the fraction of the population immunized during the outbreak. A strong relationship between the changes in the ORIT and the rate of immunization is noticeable. With this in mind, I posit the following inverse, direct, and constant relationships: (3.1) (3.2) (3.3) Where k is an arbitrary constant of proportionality and t is time, in days. As ρ is a fraction, the 4th root is taken to increase its magnitude. This higher value reflects the high impact of ρ on changes in the ORIT. I first calculate the change in the ORIT between two consecutive days for several instances during the 2003 RMI measles outbreak and find significant variation in this slope among the tested instances. This variability in the slope of the ORIT line (an approximation for ) eliminates the possibility that is constant, thus eliminating (3.3). A comparison of the slopes at each of these points with their respective immunization rates () demonstrates that the slope and immunization rate are close to inversely proportional. I conclude that the inverse relationship (3.1) is the most reasonable possibility and find that (3.1) only applies when . To generate a recursive model based on this function, the value of k must constantly update in a recursive fashion. Based on this necessity, I apply a modified form of Euler's method with secant, rather than tangent, lines. First, the slope of the secant line (defined as m) over the two previous days is found to be: (4) With (4), the value for k may be calculated based on the relationship between m and in the following equation: (5) I set as an approximation for based on the previous constant of proportionality calculated in (5). The definition of is shown below: (6) Therefore, the basic recursive formula for the value of is generated as follows based on the modified version of Euler's method discussed above: (7) Inserting (4) into (5), then (5) into (6), and finally (6) into (7) yields the final recursive definition with which the ORIT may be predicted: (8) The final recursive definition described in (8) predicts VT several days in advance by first predicting VT for the very next day. Using this prediction, the threshold for the following day is predicted and this procedure is repeated until the final value is determined. To expedite the application of the recursive predictor, I develop an algorithm for the prediction of the ORIT using the recursive formula in (8) for the Java programming language, which I call the Recursive Prediction of the ORIT Algorithm (RPORITA). The application of the RPORITA consists of the following steps: Enter the background information of the outbreak, including total population, vaccination coverage at beginning of outbreak, and recovery rate (defined as 1/8 for measles [7] in this paper). Enter the survey data for two consecutive days, including number of persons infectious and recovered for each day and total number of persons immunized during the outbreak for each day. Select the day number for which the herd immunity threshold should be predicted and enter the number of immunizations that will occur on each day until the day of prediction. The RPORITA then returns the predicted ORIT for the selected day of prediction. Development of an individual reproduction number model The ability to determine the effects of additional vaccination on the individual reproduction number would provide insight into outbreak response and improving cost effectiveness. For this reason, I seek to develop a model for , the change in the individual reproduction number, as a function of , the change in the vaccination coverage. To approximate this relationship, I solve for dRi/dV. The model for the individual reproduction number I developed concurrently with the Threshold Model in [19] is as follows: (9) By introducing variables into equation (2) from above, the formulation for the immunization rate becomes: (10) With V = the fraction of the population immunized during the outbreak, c = the current day number, and b = the day number on which vaccination began during the outbreak. The vaccinations that cause the are assumed to occur instantaneously for the purposes of this model. Therefore, both c and I are held constant. However, S would decrease due to the increase in V caused by the vaccinations, yielding the relationship below: (11) To simplify the Ri function, I decompose it into functions of its numerator and denominator, or y and z, respectively. (12) (13) Taking the derivatives of the numerator and denominator individually with respect to V yields: (14) (15) By the quotient rule for differentiation: (16) Inserting (12,13,14,15) into (16) results in: (17) To determine the effects of changes in V on Ri at extremely low incremental values, the following approximation is established: (18) By (18), can be determined with the function below: (19) Results Measles outbreak application I predict the ORIT for an approximate 20 day period with an average of 5 day intervals for both datasets [20], [21]. The interval of the highest rate of immunization was chosen for the test period. The numerical results of these applications are presented in Tables 1 and 2 , and the graphical depictions can be seen in Figure 2 . 10.1371/journal.pone.0004168.g002Figure 2 This figure presents a graphical depiction of the comparison between the Recursive Prediction of the Outbreak Response Immunization Threshold Algorithm (RPORITA) prediction for the Outbreak Response Immunization Threshold (ORIT) and the Threshold Model estimation based on direct data input. I apply the RPORITA to both the 2006 Fiji measles outbreak (A) and the 2003 RMI measles outbreak (B). I show the RPORITA prediction generated with each prediction day difference (PDD). 10.1371/journal.pone.0004168.t001Table 1 Comparison of the RPORITA-generated predictions for the ORIT and ORIT approximations based on direct input of survey data for the 2006 Fiji measles outbreak. Date (DSI) Threshold Model estimated ORIT RPORITA prediction average Prediction error based on average RPORITA predicted ORIT Prediction error PDD 3/29 (9) 0.1528 0.1454 −0.0074 0.1469 −0.0059 4 0.1452 −0.0076 5 0.1440 −0.0088 6 4/03 (14) 0.1326 0.1329 −0.0003 0.1330 0.0004 4 0.1358 0.0032 5 0.1299 −0.0027 6 4/08 (19) 0.1135 0.1122 −0.0013 0.1131 −0.0004 4 0.1113 −0.0022 5 0.1122 −0.0013 6 4/14 (25) 0.1045 0.1027 −0.0018 0.0969 −0.0076 4 0.1081 0.0036 5 0.1032 −0.0013 6 4/18 (29) 0.0919 0.0926 0.0007 0.0975 0.0056 4 0.0956 0.0037 5 0.0846 −0.0073 6 The RPORITA signifies the Recursive Prediction of the Outbreak Response Immunization Threshold Algorithm, which I use to produce a comparison of the RPORITA prediction and the Threshold Model approximation for the ORIT, or Outbreak Response Immunization Threshold, based on direct data input. The prediction day difference (PDD) is the difference between the second day of direct input on which the prediction is based and the day of the prediction. The Days since Start of Immunization (DSI) is the number of days between the day of prediction and the start of the ORI. The prediction error is defined as follows: prediction error = RPORITA prediction−Threshold Model approximation. 10.1371/journal.pone.0004168.t002Table 2 Comparison of RPORITA-generated predictions for the ORIT and ORIT approximations based on direct input of survey data for the 2003 RMI measles outbreak. Date (DSI) Threshold Model estimated ORIT RPORITA prediction average Prediction error based on average RPORITA predicted ORIT Prediction error PDD 8/20 (19) 0.1292 0.1319 0.0027 0.1348 0.0056 4 0.1300 0.0008 5 0.1308 0.0016 6 8/25 (24) 0.1212 0.1200 −0.0012 0.1158 −0.0054 4 0.1169 −0.0043 5 0.1272 0.0060 6 8/30 (29) 0.1166 0.1182 0.0016 0.1156 −0.0010 4 0.1198 0.0032 5 0.1192 0.0026 6 9/05 (35) 0.1074 0.1106 0.0032 0.1090 0.0016 4 0.1105 0.0031 5 0.1123 0.0049 6 9/10 (40) 0.1016 0.0995 0.0021 0.0992 −0.0024 4 0.0993 −0.0023 5 0.0999 −0.0017 6 The RPORITA signifies the Recursive Prediction of the Outbreak Response Immunization Threshold Algorithm, which I use to produce a comparison of the RPORITA prediction and the Threshold Model approximation for the ORIT, or Outbreak Response Immunization Threshold based on direct data input. The prediction day difference (PDD) is the difference between the second day of direct input on which the prediction is based and the day of the prediction. The Days since Start of Immunization (DSI) is the number of days between the day of prediction and the start of the ORI. The prediction error is defined as follows: prediction error = RPORITA prediction−Threshold Model approximation. Error Analysis The number of days between the second day of direct data input and the day of the prediction can be defined as the Prediction Day Difference (PDD). I define the difference between the day of prediction and the beginning of ORI as Days since Start of Immunization (DSI). To better understand the specific factors affecting the level of prediction error of the RPORITA, two relationships were examined: 1) between DSI and prediction error, and 2) between PDD and prediction error. These relationships are examined using data from both outbreaks, allowing several conclusions to be drawn. Tables 1 and 2 illustrate several key aspects of the RPORITA. First, all of the prediction errors are within 0.009 of the Threshold Model-based approximation using direct input of data. It is important to note, however, that only four of the thirty predictions (4/30 = 13.3%) have a prediction error of greater than 0.006. In addition, all of the mean prediction errors are within 0.005. Given the multitude of factors that affect the ORIT and the randomness inherent to all biological action, the low error values provide supporting evidence for the accuracy of the RPORITA. The most striking prediction errors occur on 29 March in the 2006 Fiji measles outbreak, the day of the most extreme error value of −0.0088 for the prediction with a PDD of 6. At this point, the \|prediction error\| mean is 0.0074, a much greater value than any of the other means. This extreme nature may be attributed to the relatively low DSI. This relationship is most likely caused by the high infectiousness, and therefore high variability, of the disease dynamics at such an early point in the ORI. Based on this fact, it appears that the RPORITA is best applied with a DSI of at least 10 days. Based on Tables 3 and 4 , the most accurate method for applying the RPORITA may be determined. An overarching trend cannot be established between either 1) the PDD and prediction error, or 2) DSI and prediction error. However, in both outbreaks, the prediction error mean is the highest when six days separate the day of data input and the day for prediction (PDD = 6). Overall, based on the numerical data presented in Tables 3 and 4 , the two most accurate methods for applying the RPORITA may be established: Predict the ORIT five days in advance (with overall mean \|prediction error\| of 0.00340). Or, more accurately, Predict the ORIT four days in advance by averaging the predictions generated with PDDs of 4, 5, and 6 (with overall mean \|prediction error\| of 0.00223). 10.1371/journal.pone.0004168.t003Table 3 Data demonstrating the two key relationships concerning RPORITA prediction error for the 2006 Fiji measles outbreak. DSI \|Prediction error\| mean PDD \|Prediction error\| mean 9 0.0074 4 0.00398 14 0.0021 5 0.00406 19 0.0013 6 0.00428 25 0.0042 29 0.0055 The prediction day difference (PDD) is the difference between the second day of direct input on which the prediction is based and the day of the prediction. The Days since Start of Immunization (DSI) is the number of days between the day of prediction and the start of the Outbreak Response Immunization. This data presents a numerical depiction of the two critical relationships with regard to Recursive Prediction of the Outbreak Response Immunization Threshold Algorithm (RPORITA) error: 1) between DSI and prediction error, and 2) between PDD and prediction error. 10.1371/journal.pone.0004168.t004Table 4 Data demonstrating the two key relationships concerning RPORITA prediction error for the 2003 RMI measles outbreak. DSI \|Prediction error\| mean PDD \|Prediction error\| mean 19 0.0027 4 0.00320 24 0.0052 5 0.00274 29 0.0023 6 0.00336 35 0.0032 40 0.0021 The prediction day difference (PDD) is the difference between the second day of direct input on which the prediction is based and the day of the prediction. The Days since Start of Immunization (DSI) is the number of days between the day of prediction and the start of the Outbreak Response Immunization. This data presents a numerical depiction of the two critical relationships with regard to the Recursive Prediction of the Outbreak Response Immunization Threshold Algorithm (RPORITA) error: 1) between DSI and prediction error, and 2) between PDD and prediction error. Discussion In this study, I developed a recursive algorithm (the RPORITA) for predicting the vaccination coverage required to reduce the individual reproduction number to less than one. I then tested the RPORITA against data from two actual measles outbreaks. In addition, I developed a model to demonstrate how changes in vaccination coverage affect changes in the individual reproduction number. Strengths The ability to predict the ORIT would allow an outbreak control agency to better coordinate a future immunization drive intended to stop the outbreak. Several methods for predicting the threshold are available, most of which involve time based functions for the individual reproduction number, a crucial aspect in the calculation of the threshold [10]. With respect to other approaches for analyzing the individual reproduction number and the ORIT, the RPORITA developed herein differs primarily in its fundamental nature. The RPORITA, in contrast with other time-based approaches, allows each outbreak to essentially define its own dynamics. The RPORITA bases the prediction of the threshold on the previous threshold values within the specific outbreak, allowing for a more widespread application. Another possible method for predicting the ORIT through the Threshold Model would involve the prediction of each of the necessary variables: S, I, and R. However, this approach requires three predictions, while the RPORITA requires only one, thereby reducing the number of opportunities for error to affect the results. In addition, the RPORITA provides a simpler method for the prediction of the ORIT, involving only a single recursive definition that may be quickly applied with readily available survey data. The only data input requirement of the RPORITA, besides the survey data from two consecutive days, is the number of vaccinations that will occur on each day until the prediction, the value of which is under the control of the outbreak control agency. The individual reproduction number and herd immunity threshold have a complex relationship [10]. Perhaps the most basic distinction is that the individual reproduction number is a largely abstracted and theoretical value intended more to inform policy (whether or not certain outbreak control strategies are effective) than to quantify the amount of resources needed for immunization programs [15]. To determine the ORIT, the vaccination coverage that results in an individual reproduction number of less than one must be solved for [9], involving intermediary steps. In contrast, the predicted value for the ORIT generated using the RPORITA may be immediately applied to ORI without any of these interceding steps. Therefore, the direct prediction of the ORIT sidesteps any additional predictions or mathematical methods required by the reproduction number prediction. Limitations I applied the RPORITA to only two outbreaks due to the paucity of available data necessary for its implementation (specifically the day-by-day breakdown of rash onsets and immunizations). However, the RPORITA development is based solely on the dynamics of the 2003 RMI outbreak, given the assumption that the recursive nature of the algorithm would allow for its wide applicability. I then tested the RPORITA on both the data used for the development (the 2003 RMI outbreak) and new test data (the 2006 Fiji outbreak). Although this data is not generally collected in databases, it would be readily available in the field based on real time survey data during an outbreak. As more data becomes available, the RPORITA must be applied to several additional outbreaks, those of both measles and other VPDs, to obtain a greater understanding of its accuracy. Another key aspect of the RPORITA is that equation (8) only applies when the slope of the ORIT curve is less than zero. However, during the significant majority of the ORI program, the ORIT curve has a negative slope. At a time for which the ORIT curve has a positive slope, the outbreak control agency would still be identifying and formulating a response to the outbreak. Therefore, it is highly unlikely that this factor would interfere with the successful application of the RPORITA. Other limitations of the RPORITA result from the Threshold Model on which it is based. This model assumes homogenous mixing of the population and the mass action principle and does not take into account the exposed portion of the population [19]. All of these aspects limit its accuracy. In this respect, several aspects of the RPORITA may be improved. The RPORITA must be applied to several more outbreaks, especially those of other VPDs, before being deemed fully ready for field application. In addition, the incorporation of more variables into the RPORITA, such as vaccine efficacy, quarantine, school closings, and heterogeneity of the population, would improve its accuracy. However, the simultaneous difficulty in determining these variables in real time for use in the RPORITA would limit their practical application. Another limiting aspect involves the difficulty inherent to identifying those who could benefit from immunization, in other words: susceptible persons. For the RPORITA to accurately predict the ORIT, the vaccines must be used to immunize susceptible persons against the disease, although this limitation is by no means a fatal flaw of the RPORITA or the Threshold Model. All previously vaccinated persons and those who have presented or are presenting symptoms of the VPD may be legitimately excluded from the estimated susceptible pool for the purposes of the ORI. Those symptoms for measles include runny nose, red eyes, cough, small white spots, and a rash [7]. Once enough susceptible persons have been identified, the outbreak control agency may apply the RPORITA prediction by simply vaccinated the stated fraction of the population and ensuring, based on available data, that those vaccinated persons are susceptible. Conclusions The immunization strategy discussed in this paper is not the only option for outbreak control agencies. In fact, there are three possible general courses of action with regard to ORI. The outbreak can run its course, maximizing the number of infections at the lowest cost. Or, the outbreak control agency can immunize as many persons as possible, thus minimizing the number of infections and maximizing cost. However, there is middle road, the one facilitated by the RPORITA developed in this research: the outbreak control agency can immunize the minimum number of persons required to achieve the ORIT at that point in the outbreak. This approach strikes a balance between these inversely related objectives by limiting both infections and cost, resulting in an optimal strategy for impoverished nations seeking to preserve limited funds. It is duly noted that higher vaccination coverage will reduce the number of infections caused by the outbreak, as it would further reduce the individual reproduction number. However, once the individual reproduction number has become less than unity, the outbreak will eventually cease regardless of its exact value within the range from 0 to 1. The specific individual reproduction number determines the number of infections. The model for the individual reproduction number in equation (19) can be applied to outbreak control strategy to determine the effects of vaccination on the ability of the VPD to spread through the population at different points in an outbreak. This equation incorporates three critical factors affecting the ability of vaccination to reduce the individual reproduction number: the point in the outbreak at which these vaccinations would occur (involves variables c, S, and I), the vaccination coverage before the new vaccinations (which determines V), and the number of additional vaccinations (). With this tool the time at which vaccination has the optimum impact on the individual reproduction number may be determined. Also, equation (19) may help answer the question: are these vaccinations worth their financial cost at this point in the outbreak? Overall, the primary purpose of the RPORITA is to predict, several days in advance, the minimum number of vaccines required to achieve ORIT and thereby gradually stop an outbreak. This prediction capability would allow poorer nations to plan and coordinate an immunization drive that implements the minimum amount of resources needed to guarantee the end to a VPD outbreak at that point. Despite the moderate prediction error, considering the multitude of variables and factors that affect the ORIT during an outbreak, the RPORITA proves to be accurate in its prediction of the ORIT. Overall, the RPORITA strikes a delicate balance between real-time applicability (through simplicity) and accuracy, thus achieving the overall goal of this research. I would like to thank Diane Mutolo for her assistance locating sources of raw data and her comments on my manuscript. I also appreciate the helpful input of the reviewers, as these comments markedly improved this paper. Competing Interests: The author has declared that no competing interests exist. Funding: The author has no support or funding to report. ==== Refs References 1 Sniadack D Moscoso B Aguilar R Heath J Bellini W 1999 Measles epidemiology and outbreak response immunization in a rural community in Peru. B World Health Organ 77(7) 545 552 2 Grais R Conlan A Ferrari M Djibo A Le Menach A 2007 Time is of the essence: exploring a measles outbreak response vaccination in Niamey, Niger. J R Soc Interface 5(18) 67 74 3 White C Koplan J Orenstein W 1985 Benefits, Risks, and Costs of Immunization for Measles, Mumps, and Rubella. Am J Public Health 75(7) 739 744 3923849 4 Farrington C Whitaker H 2003 Estimation of effective reproduction numbers for infectious diseases using serological survey data. Biostatistics 4(4) 621 632 14557115 5 Fine PE 1993 Herd Immunity: History, Theory, Practice. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1917219008-PONE-RA-07216R110.1371/journal.pone.0004282Research ArticleCell Biology/Neuronal Signaling MechanismsNeuroscience/Neuronal Signaling MechanismsAnesthesiology and Pain Management/Basic Science of Pain ManagementNaloxone's Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptor–Gs Coupling and CREB Activation of Acute Morphine Filamin A and Opioid SignalingWang Hoau-Yan 1 Burns Lindsay H. 2 * 1 Department of Physiology & Pharmacology, City University of New York Medical School, New York, New York, United States of America 2 Pain Therapeutics, Inc., San Mateo, California, United States of America Karl-Wilhelm Koch EditorUniversity of Oldenburg, Germany* E-mail: [email protected] and designed the experiments: HYW. Performed the experiments: HYW. Analyzed the data: HYW. Wrote the paper: LB. 2009 27 1 2009 4 1 e42826 11 2008 22 12 2008 Wang et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Gαs and its Gβγ dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 µM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA2561–2565), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR–FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S133. Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS133CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR–Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR–Gs coupling by 100 pM NLX/NTX or 10 µM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential. ==== Body Introduction Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia, minimize opioid tolerance and dependence [1], [2] and attenuate the addictive properties of opioids [3], [4]. Early electrophysiology data suggested that ultra-low-dose opioid antagonists block excitatory signaling of opioid receptors [1]. Chronic opioid-induced excitatory signaling is mediated by a switch in G protein coupling by MOR from Gi/o to Gs proteins [5], 6 and by stimulation of adenylyl cyclase II and IV by the Gβγ dimer [5], [7] originating from the MOR-associated Gs protein [8]. Ultra-low-dose NLX co-treatment suppresses opioid tolerance and dependence by preventing these MOR signaling alterations [5], and we recently identified the NLX binding site that mediates its protective effects as a pentapeptide segment in c-terminal FLNA [9]. A scaffolding protein best known for its actin-binding and cell motility function, FLNA also regulates cell signaling by interacting with a variety of receptors and signaling molecules [10], [11]. Onoprishvili et al. [12] first showed FLNA to interact with MOR and suggested a role in MOR downregulation and desensitization. Also implicating FLNA in desensitization, our recent organotypic striatal slice culture data showed that potentially disrupting the MOR–FLNA interaction via NLX's high-affinity binding to FLNA blocks the chronic morphine-induced G protein coupling switch by MOR [9]. Specifically, FLNA peptide fragments containing the NLX binding site blocked the protective effect of NLX on both the MOR–Gs coupling and downstream cAMP accumulation induced by chronic morphine (twice daily 1-hr exposures for 7 days), presumably by interfering with NLX's binding to FLNA in the tissues. Again using organotypic striatal slice cultures, we show here that acute morphine causes a dose-dependent and transient MOR–Gs coupling that resolves by 1 hr. This is the first indication that MOR–Gs coupling occurs acutely and dynamically and is not only a consequence of chronic opioid treatment. To assess the involvement of FLNA in this acute opioid-induced Gs coupling, we co-treated slices with NLX, naltrexone (NTX), or FLNA2561–2565, their pentapeptide binding site, as a decoy for MOR. We also examined the effects of these treatments on cAMP production and downstream phosphorylation of the cAMP-response-element-binding protein (CREB) at S133 as a marker of addictive processes, since ultra-low-dose NTX attenuates the acute rewarding effects of opioids [3], [4]. CREB is activated by phosphorylation at S133 predominantly by protein kinase A (PKA) through binding of cAMP. Hence, increasing levels of cAMP from activation of adenylyl cyclase following MOR–Gs coupling could contribute to the acute rewarding or addictive properties of opiates. A partial mediation of the acute rewarding effects of opiates by Gs signaling could explain the apparent discrepancy that ultra-low-dose NLX or NTX can enhance opioid analgesia while also attenuating the addictive properties of opioids. Methods Animals Male Sprague Dawley rats (200 to 250 g) purchased from Taconic (Germantown, NY) were housed two per cage and maintained on a regular 12-hr light/dark cycle in a climate-controlled room with food and water available ad libitum. For organotypic brain slice cultures, rats were sacrificed by rapid decapitation and striata were removed on ice and treated in vitro as described below. All procedures in this protocol are in compliance with the City College of New York IACUC on the use and care of animals. Organotypic striatal slice cultures Rat brain slice organotypic culture methods were modified from those published previously [13], [14]. Striatal slices (200 µm thick) were prepared using a McIlwain tissue chopper (The Mickle Laboratory Engineering Co., Surrey, UK) at 4°C. Slices were immediately transferred to sterile, porous culture inserts (0.4 µm, Millicell-CM), using the rear end of a glass Pasteur pipette. Each culture insert unit contained 2 slices and was placed into one well of the 12-well culture tray. Each well contained 1.5 ml of culture medium composed of 50% MEM with Earl's salts, 2 mM L-glutamine, 25% Earl's balanced salt solution, 6.5 g/l D-glucose, 20% fetal bovine serum (FBS), 5% horse serum, 25 mM HEPES buffer and 50 mg/ml streptomycin and 50 mg/ml penicillin. The pH was adjusted to 7.2 with HEPES buffer. Cultures were first incubated for 2 days to minimize the impact of injury from slice preparation. Incubator settings were 36°C with 5% CO2. Determination of MOR–G protein coupling To determine the dose-response of acute morphine on MOR–G protein coupling, slices were exposed to 0.1, 1, 10 or 100 µM morphine for 15 min. To assess the effect of ultra-low-dose NLX or NTX on the signaling switch induced by 100 µM morphine, some slices were exposed to 100 µM morphine plus 100 pM NLX or NTX. To determine whether NLX's effect may be mimicked by the site on FLNA that interacts with MOR, brain slices were incubated with morphine plus 10 µM VAKGL (FLNA2561–2565) or control pentapeptide, VAAGL. Peptides were purchased from Sigma-Genosys (The Woodlands, TX). A separate experiment was performed to assess the effect of 100 pM (+)NLX, the stereoisomer of NLX that is inactive at MOR. Slices were exposed to (+)NLX or (+)NLX plus 100 µM morphine for 15 and 60 min. Prior to treatments, culture medium was removed and the culture insert containing the slices was gently rinsed twice with warm (37°C) PBS (pH7.2) before incubation in 0.1% FBS-containing culture medium for 2 hr. Slices were transferred to fresh 0.1% FBS-containing culture medium with indicated agents. Tissues were harvested 5, 15 or 60 min after drug exposures by centrifugation. To determine of MOR–G protein coupling, slices were homogenated to generate synaptic membranes and cytosolic fractions. Synaptic membranes (400 µg) were solubilized with 0.5% digitonin/0.2% sodium cholate/0.5% NP-40 in 250 µl of immunoprecipitation buffer (25 mM HEPES, pH7.5; 200 mM NaCl, 1 mM EDTA, 50 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml soybean trypsin inhibitor, 0.04 mM PMSF and a mixture of protein phosphatase inhibitors). Following centrifugation, striatal membrane lysates were immunoprecipitated with immobilized anti-Gαs/olf or -Gαo antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated with immobilized protein A-agarose beads (Pierce-Endogen). The level of MOR in anti-Gαs/olf or -Gαo immunoprecipitates was determined by Western blotting using specific anti-MOR antibodies (Santa Cruz Biotechnology). Determination of cAMP accumulation To measure the MOR-mediated increase in cAMP production, brain slices were incubated with Kreb's-Ringer (basal), 100 µM morphine and/or co-treatment with 100 pM NLX, NTX or 10 µM VAKGL (FLNA2561–2565 ) for 5, 15 and 60 min in the presence of 100 µM phosphodiesterase inhibitor, IBMX. For the 60-min timepoint, IBMX was added 15 min after morphine exposure. For the experiment that assesses cAMP production by Go-coupled MOR, slices were incubated with morphine, morphine plus 10 µM VAKGL, or 10 µM VAKGL for 5 or 60 min prior to incubation for 5 min with 10 µM forskolin. The reaction was terminated by placing slices in ice-cold Ca2+-free Kreb's-Ringer containing 0.1 mM EDTA and centrifugation. Tissues were homogenized by sonication and protein precipitated with 1M TCA and the supernatant obtained after centrifugation was neutralized using 500 mM Tris, pH9.0. The level of cAMP in brain lysates was measured by a cAMP assay kit (PerkinElmer Life Science, Boston) according to manufacturer's instructions. Determination of CREB activation To assess whether acute high-dose morphine induces CREB activation and whether co-treatment with 100 pM NLX, NTX or the FLNA pentapeptide alter it, the level of protein kinase A-phosphorylated CREB, pS133-CREB, was assessed in cytosolic and nucleus fractions generated as described above from striatal slices. CREB in the solubilized cytosol and nucleus was immunoprecipitated with immobilized anti-CREB antibodies (Santa Cruz Biotechnology) and the level of pS133-CREB determined by Western blotting using a specific antibody directed against pS133-CREB (Santa Cruz Biotechnology). Since CREB phosphorylation at the S133 can also be induced in response to elevated intracellular Ca2+, experiments were designed to assess the contribution of Ca2+ by including cell permeable intracellular Ca2+ chelator, BAPTA-AM. In this study, striatal slice cultures were treated with 100 µM BAPTA-AM for 30 min in serum-free medium prior to incubation with 100 µM morphine with or without 100 pM NLX, 100 pM NTX or 10 µM VAKGL pentapeptide. pS133-CREB levels in the solubilized cytosol and nucleus were measured as described above. Assessment of VAKGL peptide binding to MOR and FLNA proteins To elucidate whether the pentapeptide FLNA fragment VAKGL functions as a MOR decoy by binding to MOR or interacts with full-length FLNA, we utilized a cell-free system. VAKGL biotinated at either n- or c-terminus (0.5 µg/well) was coated onto streptavidin-coated plates (Reacti-BindTM NeutrAvidinTM High binding capacity coated 96-well plate, Pierce). Each condition had 12 replicates. After 3 washes with 200 µl PBS, immunoaffinity-purified FLNA (0.5 µg) from rat brain and A7 cells or MOR (0.1 µg) from rat brain or SK-N-MC cells were added into designated wells and incubation was carried out for 1 hr at 25°C with constant shaking. The plate was washed with PBS three times and the bound FLNA and MOR detected using anti-FLNA and -MOR antibodies followed by FITC-conjugated anti-mouse and -rabbit IgG, respectively. Following three washes with PBS, The FLNA and MOR signals were detected using a multi-mode plate reader (DTX-880, Beckman). The background FITC signal from matched wells, defined by the signal produced by unconjugated secondary antibodies at 100-fold higher dilution, was subtracted from the total signal. This background FITC signal was <15% of total signal in test wells. Data Analysis All data are presented as mean±standard error of the mean. Treatment effects were evaluated by two-way ANOVA followed by Newman-Keuls test for multiple comparisons. Two-tailed Student's t test was used for post hoc pairwise comparisons. The threshold for significance was p<0.05. Results Acute morphine induces dose-dependent MOR–Gs coupling After 15-min incubation with 0.1, 1, 10 or 100 µM morphine, some MOR–Gs coupling was noted in response to 0.1 µM morphine. With increasing dose, the Gs coupling progressively increased as the level of Go coupling decreased (Fig. 1). While a true comparison of the levels of Gs vs. Go coupling cannot be made since the antibodies detecting Gαs vs. Gαo may have different affinities, the progressive switch from Go to Gs coupling at 15 min of exposure to increasing doses of morphine is clearly visible. Additionally, although the anti-Gαs/olf antibody does not distinguish between these functionally similar Gs family members, the Gα protein detected here in striatum may be predominantly Golf[15]. 10.1371/journal.pone.0004282.g001Figure 1 Acute morphine causes a dose-dependent MOR – G protein coupling switch. Densitometric quantitation (B) of blots (A) shows that the level of MOR–Gs coupling after 15-min morphine exposure increases progressively as dose increases from 0.1 to 100 µM concentrations. In parallel, the level of MOR–Go coupling decreases with increasing morphine concentration. n = 4. p<0.01 compared to respective Go or Gs/olf coupling of vehicle condition. Acute morphine-induced G protein coupling switch blocked by 100 pM NLX/NTX or 10 µM FLNA pentapeptide An acute morphine-induced Go-to-Gs coupling switch in striatal slice cultures was blocked by co-exposure to 100 pM NLX or NTX or 10 µM concentration of their pentapeptide binding site on FLNA (Fig. 2). Morphine (100 µM) caused a complete Go-to-Gs coupling switch by MOR at 5 and 15 min of exposure. After 60 min of morphine exposure, the switch had resolved and no Gs coupling was detected. Co-treatment with 100 pM NLX or NTX prevented this acute G protein coupling switch by MOR (Fig. 2B). Similarly, pre-treatment with the pentapeptide binding site of NLX or NTX on FLNA (VAKGL; FLNA2561–2565) at 10 µM concentration also completely prevented this acute morphine-induced coupling switch by MOR (Fig. 2B). The control peptide, FLNA2561–2565 with one mid-point lysine-to-alanine substitution (VAAGL), did not prevent the MOR–Gs coupling, but mildly preserved Go coupling at 15 minutes. Exposure to NLX, NTX or either peptide alone without morphine had no effect on MOR coupling. In sum, the only significant differences compared to the vehicle condition for both Go and Gs coupling were the 5 and 15 minute timepoints for both morphine and morphine + the control peptide (VAAGL) (p<0.01 for each). 10.1371/journal.pone.0004282.g002Figure 2 The Go-to-Gs coupling switch is transient and blocked by FLNA pentapeptide or 100 pM NLX/NTX. After 5, 15 or 60 min of drug treatment, striatal membranes were solubilized and immunoprecipitated with immobilized anti-Gα prior to detection with anti-MOR by Western blotting. Densitometric quantitation (B) of blots (A) shows the acute morphine-induced Go-to-Gs/olf coupling switch at 5 and 15 min timepoints that is prevented by co-treatment with 100 pM NLX or NTX or pre-treatment with the FLNA2561–2565 pentapeptide (VAKGL), but not the control pentapeptide (VAAGL). n = 4. p<0.01 compared to respective Go or Gs/olf coupling of vehicle condition. (+)NLX also blocks acute morphine-induced MOR–Gs coupling The naloxone isomer that is inactive as an opioid antagonist, (+)NLX, was used to confirm that the blockade of MOR–Gs coupling occurs by a mechanism other than antagonism of MOR. Similar to the blockade by a 100 pM concentration of the active isomer of NLX seen in Fig. 2, 100 pM (+)NLX blocked the Gs coupling induced by 15 min of 100 µM morphine (Fig. 3). 10.1371/journal.pone.0004282.g003Figure 3 (+)NLX also blocked the transient Go-to-Gs coupling switch induced by acute high-dose morphine. After 15 or 60 min of drug treatment, striatal membranes were solubilized and immunoprecipitated with immobilized anti-Gα prior to detection with anti-MOR by Western blotting. Densitometric quantitation (B) of blots (A) shows that the inactive isomer of NLX, (+)NLX, blocks the acute morphine-induced Go-to-Gs coupling switch at 100 pM similar to NLX or NTX or pre-treatment with the VAKGL pentapeptide in fig. 2. n = 4. p<0.01 compared to respective Go or Gs coupling of vehicle condition. NLX/NTX or the FLNA pentapeptide block acute morphine-induced cAMP production and CREB activation Since 100 µM morphine induced a complete Go-to-Gs switch (Figs 1 and 2), we determined whether cAMP production is elevated in response. As expected, acute 100 µM morphine significantly increased cAMP levels at 5 and 15 min (Fig. 4A). This acute morphine effect was blocked by co-treatment with 100 pM NLX or NTX (Fig. 4A). Similarly, VAKGL but not the control peptide VAAGL at 10 µM concentration also completely prevented this acute morphine-induced cAMP elevation (Fig. 4A). Exposure to NLX, NTX or either peptide alone without morphine had no effect on cAMP levels. As with the morphine-induced Go-to-Gs switch, cAMP differed from vehicle control only for morphine and morphine+the control peptide (VAAGL) at the 5- and 15-min timepoints (p<0.01 for each). To demonstrate that MOR returns to Go coupling and inhibition of cAMP accumulation at 60 min, we performed a separate experiment using forskolin-stimulated cAMP production. With 10 µM forskolin added to each condition for 5 min, the inhibition of cAMP after 60 min of morphine exposure was visible (Fig. 4B), illustrating that the increased cAMP is not merely a result of desensitization of MOR with this high dose of morphine. 10.1371/journal.pone.0004282.g004Figure 4 VAKGL or 100 pM NLX/NTX block an increased cAMP accumulation that parallels the coupling switch. A: Without stimulation by forskolin, the morphine-induced increase in cAMP accumulation was greatest in striatal slice cultures treated with morphine for 5 or 15 min but was still visible at 60 min. This increase was blocked by all co-treatments except the control peptide VAAGL. B: With forskolin stimulation, morphine increased cAMP accumulation at 15 min but decreased it at 60 min, indicating a reversion back to Go coupling. The VAKGL peptide again blocked the morphine-induced increase in cAMP accumulation. n = 4. p<0.05; p<0.01 compared to Kreb's-Ringer at the respective timepoint. #p<0.01 comparing VAKGL+morphine to morphine. To further investigate this Gs-mediated activation of adenylyl cyclase by acute high-dose morphine (100 µM), we measured the potential downstream activation of CREB by its phosphorylation at serine-133. Morphine increased pS133CREB with a timecourse that paralleled that of the Gs coupling and cAMP accumulation (Fig. 5). A robust level of pS133CREB was detected in striatal tissue from slice cultures after 5 and 15 min of morphine exposure, while only a residual level was detected at 1 hr. Co-treatment with 100 pM NLX or NTX, or pre-treatment with 10 µM of the VAKGL pentapeptide, the binding site of NLX or NTX on FLNA (FLNA2561–2565), virtually abolished this signal. In contrast, pre-treatment with the control peptide VAAGL did not significantly diminish pS133CREB levels seen in the morphine only condition. Treatment alone with NLX, NTX or either peptide did not result in pS133CREB detection. Again, only morphine and morphine+the control peptide produced significant differences compared to the vehicle condition (p<0.01 for 5- and 15-min timepoints and p<0.05 for 60 min). 10.1371/journal.pone.0004282.g005Figure 5 VAKGL or 100 pM NLX/NTX blocked the CREB activation induced by acute morphine. Densitometric quantitation (B) of Western blots (A) shows the activation of CREB, measured by pS133CREB detection, at 5 and 15 min of morphine exposure and its blockade by co-treatment with 100 pM NLX or NTX or pre-treatment with the VAKGL but not the VAAGL pentapeptide. n = 4. p<0.01 and p<0.05 compared to vehicle. Because phosphorylation of CREB at S133 can also be achieved through elevation of Ca2+, we assessed the contribution of cAMP and Ca2+ to acute high-dose morphine-induced pS133CREB by inclusion of the cell permeable Ca2+ chelator, BAPTA-AM. Removal of Ca2+ did not affect the ability of morphine to increase pS133CREB or the blockade of this effect by NLX, NTX or VAKGL (Fig. 6). These data suggest that acute high-dose morphine increases pS133CREB mainly through the cAMP and protein kinase A pathway. 10.1371/journal.pone.0004282.g006Figure 6 The calcium chelator BAPTA-AM did not notably diminish morphine-induced CREB activation or alter co-treatment effects. Densitometric quantitation (B) of Western blots (A) shows the activation of CREB, measured by pS133CREB detection, at 5 and 15 min of 100 µM morphine exposure and its blockade by co-treatment with 100 pM NLX or NTX or pre-treatment with the VAKGL but not the VAAGL pentapeptide. n = 4. p<0.01 and **p<0.05 compared to vehicle. VAKGL binding to MOR or FLNA proteins We last investigated whether the VAKGL pentapeptide is binding to MOR or full-length FLNA to prevent, in a manner similar to ultra-low-dose NLX, MOR's Gs coupling, the increased cAMP accumulation and the CREB activation following acute high-dose morphine. FLNA and MOR purified from two different sources each were tested for binding to VAKGL-coated plates and detected with anti-MOR or anti-FLNA antibodies followed by FITC-labelled secondary antibodies. Both MOR and FLNA bound VAKGL-coated plates (Fig. 7), suggesting that the VAKGL pentapeptide may disrupt the MOR–FLNA interaction by binding to MOR as a decoy for FLNA and/or by binding to FLNA itself. 10.1371/journal.pone.0004282.g007Figure 7 The VAKGL pentapeptide binds both MOR and FLNA proteins. To assess the mechanism whereby FLNA2561–2565 pentapeptide (VAKGL) disrupts the MOR–Gs coupling, cAMP accumulation and CREB activation in a manner similar to 100 pM NLX or NTX, purified MOR and FLNA proteins were tested for binding to streptavidin-coated plates coated with biotinated VAKGL peptide. Both proteins, purified from two different sources, showed marked binding to the peptide, regardless of the peptide's orientation on the plate. The bound proteins were visualized with specific antibodies followed by FITC-conjugated secondary antibodies, and the background FITC signal from matched wells was subtracted from the total signal. n = 12. Discussion Preferentially coupling to pertussis toxin-sensitive G proteins Gi and Go to inhibit the adenylyl cyclase/cAMP pathway (Laugwitz et al., 1993;Connor and Christie, 1999), MOR switches to Gs coupling after chronic opioid administration, resulting in excitatory signaling by both Gαs and Gβγ subunits [5], [6], [8]. Ultra-low-dose NLX co-treatment prevents this chronic opioid-induced G protein coupling switch and the instatement of Gβγ interacting with adenylyl cyclase, as well as the associated opioid tolerance and dependence [5]. Although the molecular pharmacology demonstrations of MOR–Gs coupling have used morphine, it is reasonable to assume that a variety of opioid agonists may similarly induce MOR–Gs coupling since ultra-low-dose NLX or NTX have been shown to enhance and prolong analgesia and prevent tolerance and dependence [1], [2], [16], [17], and attenuate addictive properties [3], [4] of oxycodone as well as morphine. While some level of Gs coupling has been previously detected in opioid-naïve or acute morphine exposed CHO cells [6], the present work in organotypic striatal slice cultures is the first to demonstrate that acute exposure to a high concentration of morphine causes predominant but transient MOR–Gs coupling similar to the persistent change following chronic morphine. This transient and dose-dependent G protein coupling switch of MORs, evident at 5 and 15 minutes of morphine exposure, is unlikely to represent an acute tolerance/dependence effect since MOR had completely reverted to its native Go coupling by 1 hour of morphine exposure. Instead we propose that with even this brief period of continued stimulation following high-dose morphine, MOR initially couples to Gs instead of Gi/o but is able to recover. With the repeated or prolonged MOR stimulation of chronic opiate exposure, however, this dynamic cycling from Gs back to Go evidently becomes impaired, leading to a persistent Gs coupling, behaviorally manifest as opioid analgesic tolerance and dependence. Similar to ultra-low-dose NLX's suppression of the chronic morphine-induced coupling switch, the present data show that this acute morphine-induced coupling switch by MOR can be completely prevented by co-treatment with 100 pM NLX or NTX or 10 µM concentration of their pentapeptide binding site on FLNA. In addition, the equal efficacy in blocking this Gs coupling of 100 pM (+)NLX, the isomer that is inactive as an opioid antagonist, further confirms a mechanism of action other than antagonism of MOR. We previously discerned the precise binding site of NLX as FLNA2561-2565 using overlapping peptides and then blocked the protective effects of NLX on MOR–Gs coupling and cAMP accumulation in organotypic striatal slice cultures by co-incubation with decapeptides containing this binding site [9]. In this prior study, FLNA peptides presumably bound to NLX, blocking its prevention of the morphine-induced MOR–Gs coupling by preventing it from binding full-length FLNA in the tissues. These data led us to postulate that the interaction between MOR and FLNA, first demonstrated by Onoprishvili et al. (2003), was critical to the G protein coupling switch. We theorized that a particular FLNA–MOR interaction enables MOR to release from the signaling complex to couple to Gs upon subsequent receptor stimulation. In addition to FLNA, MOR has recently been shown to interact with another scaffolding protein, spinophilin (Charlton et al., 2008). While blocking the MOR–FLNA association, at least via ultra-low-dose NLX/NTX, diminishes tolerance, dependence and the rewarding effects of opiates, preserving or enhancing MOR's association with spinophilin appears to similarly modify these opioid effects. In the present study, we showed the pentapeptide FLNA2561–2565 itself may act as a decoy for MOR to prevent the MOR–FLNA interaction and subsequent Gs coupling by MOR. This VAKGL pentapeptide was as effective as NLX or NTX as all three completely blocked the transient switch to Gs coupling by MOR during acute, high-dose morphine exposure. NLX and NTX presumably prevent the MOR–FLNA interaction by binding to their VAKGL binding site on FLNA, and this binding site on FLNA does appear be the approximate site on this protein that interacts with MOR. The demonstration that both purified MOR and FLNA proteins bind to VAKGL suggests that the VAKGL pentapeptide can similarly prevent the MOR–FLNA interaction but by binding to either protein. It is also possible that NLX and NTX block MOR–Gs coupling by changing the conformation of FLNA upon binding rather than by direct interference, and that by binding to “itself” in the full-length protein, VAKGL similarly prevents the changed FLNA conformation. Finally, since the VAKGL peptide can both prevent NLX's protective effects as in our prior study (Wang et al., 2008) as well as mimic NLX's protective effects when used alone in the current study, we theorize that this pentapeptide preferentially binds NLX but in its absence will interact with MOR or full-length FLNA. Further study is needed to elucidate the precise VAKGL interaction site on FLNA and MOR. Our finding that all three co-treatments, NLX, NTX or the decoy pentapeptide also prevented morphine-induced CREB activation (as indicated by ser133 phosphorylation) in these striatal slice cultures may offer a mechanistic explanation for the attenuation of opioid reward and addictive processes by ultra-low-dose NTX. Found in all cells of the brain, CREB is a transcription factor implicated in addiction as well as learning and memory and several other experience-dependent, adaptive (or maladaptive) behaviors [18]. In general, CREB is inhibited by acute opioid treatment, an effect that is completely attenuated by chronic opioid treatment, and activated during opioid withdrawal [19]. However, a regional mapping study showed that opioid withdrawal activates CREB in locus coeruleus, nucleus accumbens and amygdala but inhibits CREB in lateral ventral tegemental area and dorsal raphe nucleus [20]. In the striatum, CREB activation has been viewed as a homeostatic adaptation, attenuating the acute rewarding effects of drugs [21], [22]. This view is supported by nucleus accumbens overexpression of CREB or a dominant-negative CREB mutant respectively reducing or increasing the rewarding effects of opioids in the conditioned place preference test [23]. In conflict, however, reducing nucleus accumbens CREB via antisense attenuated cocaine reinforcement as assessed in self-administration [24]. Clearly, CREB activation is implicated in addiction, but whether it directly contributes to the acute rewarding effects of drugs or initiates a homeostatic regulation thereof appears less clear. Again, it is possible that the several-fold increase in pS133CREB observed here following acute, high-dose morphine might indicate acute dependence rather than acute rewarding effects; however, the transient nature of the MOR–Gs coupling and the return to inhibition of cAMP at 1 hr suggests otherwise. The correlation of pS133CREB with the MOR–Gs coupling and cAMP production following acute high-dose morphine exposure, as well as the similar treatment effects on all, suggest that this alternative signaling mode of MOR may contribute to the acute rewarding or addictive effects of opioids. This counterintuitive notion may explain the apparent paradox that ultra-low-dose NTX, while enhancing the analgesic effects of opioids, decreases the acute rewarding or addictive properties of morphine or oxycodone as measured in conditioned place preference or self-administration and reinstatement paradigms [3], [4]. If one considers analgesic tolerance, opioid dependence, and opioid addiction together as adaptive regulations to continued opioid exposure, a treatment that prevents MOR's signaling adaptation of switching its G protein partner may logically attenuate these seemingly divergent behavioral consequences of chronic opioid exposure. However, the acute rewarding effects of opioids are not completely blocked by ultra-low-dose opioid antagonists, suggesting that a MOR–Gs coupling may only partially contribute to the addictive or rewarding effects. Although ultra-low-dose NTX blocks the conditioned place preference to oxycodone or morphine [3], its co-self-administration only reduces the rewarding potency of these opioids but does not abolish self-administration outright [4]. Nevertheless, it is tempting to theorize that a direct stimulatory effect on VTA neurons, as opposed to the proposed disinhibition via inhibition of GABA interneurons [25], may play some role in opioid reward. Finally, a MOR–Gs coupling mediation of reward, increasing with increasing drug exposure, is in keeping with current theories that the escalation of drug use signifying drug dependence may not indicate a “tolerance” to rewarding effects but instead a sensitization to rewarding effects [26]. In summary, the present work has demonstrated that acute, high-dose morphine causes an immediate but transient switch in G protein coupling by MOR from Go to Gs similar to the persistent switch caused by chronic morphine. Ultra-low doses of NLX, NTX or even (+)NLX prevent this switch, just as ultra-low-dose NLX has previously been shown to attenuate the chronic morphine-induced coupling switch by MOR. The transient nature of this acute altered coupling suggests that in recycling, the receptor eventually recovers and couples to its native G protein. We hypothesize that with chronic opioid exposure, the receptor loses the ability to recover and continues to couple to Gs, activating the adenylyl cyclase/cAMP pathway, upregulating protein kinase A, and phosphorylating CREB as one downstream effector example. The persistently elevated phosphorylated CREB may then shape the expression of responsive genes including those closely related to drug addiction or withdrawal, such as brain-derived neurotrophic factor (BDNF)[27], and tolerance. Importantly, the equivalent blockade of Gs coupling and pS133CREB by the NLX and NTX pentapeptide binding site on FLNA further elucidates the mechanism of action of ultra-low-dose NLX and NTX in their varied effects. These data strengthen our hypothesis that a particular MOR–FLNA interaction allows MOR to couple to Gs and that disrupting this interaction, either by NLX/NTX binding to FLNA or via a FLNA peptide binding to MOR and/or FLNA, can prevent the altered coupling and attenuate tolerance, dependence and addictive properties associated with opioid drugs. Competing Interests: This study was funded by Pain Therapeutics, Inc., and LHB is an employee of this company. Funding: This study was funded by Pain Therapeutics, Inc., and LHB is an employee of this company. The funders had no role in study design, data collection and analysis. 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==== Front BMC NeurolBMC Neurology1471-2377BioMed Central 1471-2377-8-521910275910.1186/1471-2377-8-52Research ArticleIn-hospital cerebrovascular complications following orthotopic liver transplantation: A retrospective study Ling Li [email protected] Xiaoshun [email protected] Jinsheng [email protected] Zhijian [email protected] Department of Neurology and Stroke Centre, First Affiliated Hospital, Sun Yat-Sen University, No. 58 Zhongshan Road 2, Guangzhou, 510080, PR China2 Department of Organ Transplantation, First Affiliated Hospital, Sun Yat-Sen University, No. 58 Zhongshan Road 2, Guangzhou, 510080, PR China3 Department of Neurology and Stroke Centre, First Affiliated Hospital, Guangxi Medical University, No. 23, Shuangyong Road, Nanning, 530021, PR China2008 22 12 2008 8 52 52 26 7 2008 22 12 2008 Copyright © 2008 Ling et al; licensee BioMed Central Ltd.2008Ling et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Cerebrovascular complications are severe events following orthotopic liver transplantation (OLT). This study aimed to observe the clinical and neuroimaging features and possible risk factors of in-hospital cerebrovascular complications in the patients who underwent OLT. Patients and methods We retrospectively reviewed 337 consecutive patients who underwent 358 OLTs. Cerebrovascular complications were determined by clinical and neuroimaging manifestations, and the possible risk factors were analyzed in the patients with intracranial hemorrhage. Results Ten of 337 (3.0%) patients developed in-hospital cerebrovascular complications (8 cases experienced intracranial hemorrhage and 2 cases had cerebral infarction), and 6 of them died. The clinical presentations were similar to common stroke, but with rapid deterioration at early stage. The hematomas on brain CT scan were massive, irregular, multifocal and diffuse, and most of them were located at brain lobes and might enlarge or rebleed. Infarcts presented lacunar and multifocal lesions in basal gangliar but with possible hemorrhagic transformation. The patients with intracranial hemorrhage had older age and a more frequency of systemic infection than non-intracranial hemorrhage patients. (P = 0.011 and 0.029, respectively). Conclusion Posttransplant cerebrovascular complications have severe impact on outcome of the patients who received OLT. Older age and systemic infection may be the possible risk factors of in-hospital intracranial hemorrhage following OLT. ==== Body Background With the rapid development of transplant technique and immunosuppressive therapy, orthotopic liver transplantation (OLT) has been carried out all over the world and accepted as one of the most effective treatments for the patients with end-stage liver diseases. However, postoperative complications are still the most important causes resulting in death of patients undergoing OLT. The incidences of posttransplant cerebrovascular complications or intracranial hemorrhage were 2.2%–3.9% in United States [1-3], 3.3% in United Kingdom[4], 3.7% in Chile[5], 6% in Spain[6], 6.5% in Hong Kong[7] clinically, and 32.7% in post-mortem patients after OLT in United States[8]. But cerebrovascular complications following OLT in patients of mainland China have not been reported. In the present study, we reported the incidence, mortality and clinical and neuroimaging features, then analyzed the possible risk factors of in-hospital cerebrovascular complications in patients following OLT at a center in southern China. Methods Patients We retrospectively reviewed 337 consecutive patients who underwent 358 OLTs in which 19 patients had a second transplant, and one had a third transplant, at an organ transplant center in southern China from Janurary 1st,1996 to June 30th, 2005. The mean age was 47 ± 11.5 years (3 months to 75 years), and 288 of 337 patients were males. The primary liver diseases before OLT are listed in Table 1. Table 1 The primary liver diseases before orthotopic liver transplantation The primary liver diseases Cases (%) Primary hepatic carcinoma 198 (58.75%) Cirrhosis 94 (27.89%) Hepatitis B 21 (8.01%) Secondary hepatic carcinoma 6 (1.78%) Polycystic liver disease 4 (1.19%) Giant hemangioma of the liver 3 (0.89%) Primary sclerosing cholangitis 3 (0.89%) Drug-induced hepatitis 2 (0.59%) Congenital biliary atresia 2 (0.59%) Hepatitis C 1 (0. 30%) Congenital hepatic fibrosis 1 (0. 30%) Budd-Chiari syndrome 1 (0. 30%) Acute fatty liver and liver function failure of pregnancy 1 (0. 30%) Total 337 (100%) Management All patients underwent OLT using Piggyback technique and were managed in an intensive care unit before being transferred to a general ward following surgery. FK506, cyclosporine A or corticosteroids were used as immunosuppressives and their serum level was measured daily postoperativly. Blood tests such as blood platelet counts, prothrombin time (PT), activated partial prothrombin time (APPT), the function of the liver and kidney and other necessary tests were measured routinely. Brain computed tomography (CT) or magnetic resonance imaging (MRI) was performed in patients when neurological symptoms occurred after OLT. We focused on the clinical and neuroimaging features of the patients with in-hospital cerebrovascular complications following OLT, and analyzed the possible risk factors in the patients with intracranial hemorrhage. The research protocol was approved by the local ethical committee for clinical research and all procedures involving the participant were conducted according to institutional guidelines in compliance with the regulations. Both oral and written informed consents were obtained from the patients or their families. Statistical analysis All statistical calculations were performed on microcomputer using SPSS13.0 (SPSS Inc). Continuous variables were compared using two tails student's t-test, and categorical variables analyzed by a chisquare analysis or Fisher's exact test. A p value less than 0.05 was considered significantly. Results In all 10 patients (6 males, 4 females) aged 56 ± 8.4 years (40 to 67 years) developed in-hospital cerebrovascular complications following OLT, resulting in an incidence of 3.0% in 337 patients. The mean in-hospital time were 39 ± 21.7 days (8 to 70 days). All patients with cerebrovascular complications had brain CT scan. Among them, eight patients (8/337, 2.4%) experienced intracranial hemorrhage, including 5 with lobe hematomas, 2 with subdural hematomas, and 1 with lobe and subarachnoid hemorrhage, and 7 of 8 intracranial hemorrhages occurred within posttransplant 1 month. The clinical presentations, such as unconsciousness, headache, aphasia, hemiparesis, seizures, were similar to common hemorrhagic stroke, but with rapid deterioration at early stage. Five of 8 patients with intracranial hemorrhage deteriorated clinically and then died within two weeks after onset, including two patients with enlargement of hematoma and intraventricular mass, and a marked midline shift on repeated CT. Two of 337 (0.6%) patients had lacunar cerebral infarctions on the 6th and the 58th postoperative day respectively. One patient's lesions located in basal ganglia and cerebellum, the other patient with generalized seizures at the oneset had a lacunar infarction at left basal ganglia. Although the seizure stopped soon after antiepileptic therapy, the aptient became apathia and reactiveless, then persistent coma. Neurological examination showed bilateral unclear papilla opticas with left papilla optica retinal vein bleeding and stiff neck. It's a pity that the repeat CT scan was not done due to the severe conditions. The patient died of multiple organ failure on the 11th days after OLT. All clinical and laboratory data, as well as brain CT findings of patients with cerebrovascular complications following OLT are shown in an Additional Table (see additional file 1). Among the 8 patients with intracranial hemorrhage, two of them received decompressive craniectomy and evacuation of intracranial hematoma, but both died of brain herniation soon after the surgery. Other 6 patients received medical treatement, and 3 of them survived. One of 2 patients with cerebral infarction died. Altogether, six of 10 patients with in-hospital cerebrovascular complications died following OLT. To analyze the possible risk factors and outcome of intracranial hemorrhage following OLT, we compared the clinical and laboratory data between the patients with and without intracranial hemorrhage (Table 2). In our series, the patients with intracranial hemorrhage had older age and a more frequency of systemic infection than non-intracranial hemorrhage patients (56.3 ± 8.7 vs 46.7 ± 11.7 years old,p = 0.011; 7/8 vs152/329, p = 0.029, respectively). The patients over 55 years were prone to have intracranial hemorrhage than those less than 55 years (5/75 vs 3/262, p = 0.015). Additionly, seven of 8 patients with systemic bacterial or fungal infection experienced intracranial hemorrhage, which had a more frequency than those without infection (7/155 vs 1/182, p = 0.026). The patients over 55 years with systemic infection had more frequency of intracranial hemorrhage than those less than 55 years and without systemic infection (5/37 vs 1/52,p = 0.043). Seven of 8 patients with intracranial hemorrhage had infection, including 4 patients experienced bacterial pneumonia, one had an aspergillus pneumonia, an aspergillus combinded Candida tropicalis pneumonia and a bacterial pneumonia combinded urinary tract infection respectively. The patients with intracranial hemorrhage had a higher mortality rate than those without intracranial hemorrhage (5/8 vs 74/329, p = 0.019). Table 2 Comparison of clincal features between the patients with and without intracranial hemorrhage after orthotopic liver transplantation Features intracranial hemorrhage (n = 8) non-intracranial hemorrhage (n = 329) age (years) 56.3 ± 8.7 (40–67) 46.7 ± 11.7 (0.3–75)* >55 years 5 (62.5%) 70 (21.3%) ≤55 years 3 (37.5%) 259 (78.7%) operative time (h) 6.3 ± 0.5 (5.5–10.0) 7.2 ± 1.9 (4–15) previous abdominal surgery 4 (50%) 113 (34.3%) retranplatation 0 (0) 20 (6.1%) introperative blood loss volume (ml) 4312.5 ± 4566.5 (1500–15000) 3900.0 ± 4389.7 (100–38000) thrombocytopenia 1 (12.5%) 66 (20.1%) PT(s) 16.3 ± 5.2 (9.8–22.6) 18.8 ± 6.4 (9.7–46.3) APTT(s) 35.7 ± 11.4 (26.1–58.6) 39.2 ± 13.6 (22.9–180) preoperative hypertension 0 (0) 5 (1.5%) introperative hypotension 5 (62.5%) 110 (33.4%) postoperative hypertension 2 (25%) 23 (7.0%) bacterial or fungal infections 7 (87.5%) 148 (45.0%)* sepsis 1 (12.5%) 29 (8.8%) bleeding at extracerebral sites 1 (12.5%) 31 (9.4%) death 5 (62.5%) 74 (22.5%)* *: vs intracranial hemorrhage, p < 0.05. PT: prothrombin time; APTT: activated partial prothrombin time. Discussion Incidence and mortality rate In the past 20 years, more and more patients with various end-stage diseases have benefited from organ transplantation. But cerebrovascular events, especially intracranial hemorrhage, have been concerned as severe neurological complications after transplantation. The reported clinical incidences of cerebrovascular complications were 1.7%–6.5% [1-7], and the mortality rate of cerebrovascular complications or intracranial hemorrhage were 57%–100% following OLT[1,3,7]. Moreover, the incidence was 32.7% in autopsy cases of patients who underwent OLT[8]. Cerebrovascular complications are also common in other transplantation patients. It has been reported that the incidence of ischemic and hemorrhagic strokes was 6.8% after kidney transplantation[9], 2.9% after bone marrow transplantation[10], 0.9% after reduced-intensity stem cell transplantation[11] and 2% in pediatric patients after cardiac transplantation[12]. Our study showed that the incidence of cerebrovascular complications in patients of southern China who underwent OLT was 3.0%, and 6 of 10 died, which was similar to the previous international reports [1-7]. These data indicate cerebrovascular events, especially intracranial hemorrhage, are severe posttransplant complications which deserve more attention for the neurologists, neuro-intensivists and surgeons for organ transplantion, and more endeavours should be done prospectively to identify or aviod such complications in clinical practice. Clinical and neuroimaging features In our study, the intracranial hemorrhage patients presented with unconsciousness, headache, aphasia, hemiparesis, seizures, which was consistent with the findings of previous report[2]. These presentations were similar to common stroke, but with more urgent onset, progressing clinical course and rapid deterioration at early stage. In our experience, sudden conscious disturbance (or loss of consciousness) after OLT was strongly associated with intracranial hemorrhage. In the present of coagulopathy, metabolic disturbance or multiple organ failure in early postoperative time, liver transplantation recipients were prone to developed massive intracranial hematomas. Moreover, these hematomas were not prone to cease spontaneously and might enlarge gradually or rebleed, even with a tendency to form cerebral herniation, which results in the death. It is important to examine the consciousness and pupils in patients after OLT. Brain CT scan shoud be performed promptly to identify whether intracranial hemorrhage occurs once the patient has sudden conscious disturbance or loss of consciousness following OLT. Additionally, liver transplantation recipients may occurr ischemic infarction due to the haemodynamic change and coagulopathy, and then develop hemorrhagic infarction, then deteriorated rapidly. Possible risk factors Most liver transplantation recipients are in the end-stage of liver diseases, preoperative liver dysfunction even liver failure results in thrombocytopenia and absence of coagulation factors. Moreover, liver function may not return to normal level in the postoperative early stage. All these could result in coagulopathy, which can trigger intracranial hemorrhage after OLT[2]. But in our series, no significant differences were found in the incidence of thrombocytopenia, PT, or APPT between the patients with and without intracranial hemorrhage, which suggests that the causes of intracranial hemorrhage after OLT are multiple and complex, except for coagulopathy, there may be other factors responsible for this complication. In our study, we found that intracranial hemorrhage patients were older than non-intracranial hemorrhage patients, moreover, the patients over 55 years had a more frequency of intracranial hemorrhage than those less than 55 years old. Additionly, the patients with systemic infection had a more frequency of intracranial hemorrhage than those without infection. The results indicate that older age and systemic infection might be important risk factors of intracranial hemorrhage following OLT. Cox and colleagues[13] reported that an 11-year-old boy who had cystic fibrosis died of an intraventricular and intracerebral hemorrhage caused by an aspergillus brain abscess on the 48th day after OLT. Wijdicks and colleagues[2] reported that in the group of 8 patients with intracranial hemorrhage after OLT, one had a Candida-associated mycotic aneurysm demonstrated by autopsy and another had disseminated aspergillosis. Generally, it is presumed that under the condition of systemic infection related to immunosuppresion following OLT, aspergillus, toxoplasma and any virulent bacterial organism may lead to inflammation of the arterial wall and formation of an aneurysm[2,14-16]. In our series, seven of the 8 patients with intracranial hemorrhage had systemic bacterial or fungal infection, and all that them had pneumonia, including 4 with bacterial pneumonia, one with aspergillus pneumonia, one with aspergillus combinded Candida tropicalis pneumonia and one with bacterial pneumonia combinded urinary tract infection respectively. But it was very pitiful that no evidence to support bacterial or fungal infection is the direct cause of intracranial hemorrhage due to no vascular image or post-mortem exams in the present study. A published study showed that patients with introperative hypotension were prone to develop cerebral infarction after OLT[7]. In the present study, one patient over 60 years old with cerebral infarction indeed had introperative hypotension, indicating introperative hypotension may be a potential risk factor of cerebral infarction after OLT. Conclusion Although posttransplant cerebrovascular complications are not common, they have severe impact on outcome of the patients who received OLT. Age and systemic infection may be the possible risk factors of in-hospital intracranial hemorrhage following OLT. More effctive measures should be taken to prevent posttransplant infection, such as improvement of patient's systemic condition, bacteriologic surveillance and infection control measures. It is urgent to early diagnosis and take more prompt systemic antibiotic/antifungal therapy once infection occurs, especially in old patients. Further prospective study is necessary to explore the risk factors and optimize the preventive and therapeutic regimen of intracranial hemorrhage following OLT. Competing interests The authors declare that they have no competing interests. Authors' contributions LL collected the data and wrote the primary manuscript. HX participated in the study. ZJ designed the study, interpreted the results and critically revised the manuscript. LZ assisted with the statistical analysis. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 All clinical and laboratory data, as well as brain CT findings of patients with cerebrovascular complications following orthotopic liver transplantation are shown this additional table. Click here for file Acknowledgements This study was supported by the grants from the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of the Ministry of Education, China (2002), the China Medical Board of New York Inc. (CMB 00-730, No.06837), the Fund for Priority Subjects in Clinical Medicine, Chinese Ministry of Health (2004), the Key and Scientific Project of the Natural Science Foundation of Guangdong Province, China (Nos. 2003B30303, and 2003D30301). ==== Refs Bronster DJ Emre S Boccagni P Sheiner PA Schwartz ME Miller CM Central nervous system complications in liver transplant recipients- incidence, timing, and long-term follow-up Clin Transplant 2000 14 1 7 10693627 10.1034/j.1399-0012.2000.140101.x Wijdicks EF de Groen PC Wiesner RH Krom RA Intracerebral hemorrhage in liver transplant recipients Mayo Clin Proc 1995 70 443 446 7731253 Saner F Gu Y Minouchehr S Ilker K Fruhauf NR Paul A Neurological complications after cadaveric and living donor liver transplantation J Neurol 2006 253 612 617 16511638 10.1007/s00415-006-0069-3 Lewis MB Howdle PD Neurologic complications of liver transplantation in adults Neurology 2003 61 1174 1178 14610116 Uribe M Buckel E Ferrario M Godoy J Blanco A Hunter B Epidemiology and results of liver transplantation for acute liver failure in Chile Transplant Proc 2003 35 2511 2512 14611998 10.1016/j.transproceed.2003.09.025 Pujol A Graus F Rimola A Beltran J Garcia-Valdecasas JC Navasa M Predictive factors of in-hospital CNS complications following liver transplantation Neurology 1994 44 1226 1230 8035920 Wang WL Yang ZF Lo CM Liu CL Fan ST Intracerebral hemorrhage after liver transplantation Liver Transpl 2000 6 345 348 10827237 Estol CJ Pessin MS Martinez AJ Cerebrovascular complications after orthotopic liver transplantation: A clinicopathologic study Neurology 1991 41 815 819 2046922 Lentine KL Rey LA Kolli S Bacchi G Schnitzler MA Abbott KC Variations in the risk for cerebrovascular events after kidney transplant compared with experience on the waiting list and after graft failure Clin J Am Soc Nephrol 2008 3 1090 1101 18385393 10.2215/CJN.03080707 Coplin WM Cochran MS Levine SR Crawford SW Stroke after bone marrow transplantation: frequency, aetiology and outcome Brain 2001 124 1043 1051 11335706 10.1093/brain/124.5.1043 Kishi Y Miyakoshi S Kami M Ikeda M Katayama Y Murashige N Early central nervous system complications after reduced-intensity stem cell transplantation Biol Blood Marrow Transplant 2004 10 561 568 15282534 10.1016/j.bbmt.2004.05.004 Groetzner J Reichart B Roemer U Reichel S Kozlik-Feldmann R Tiete A Cardiac transplantation in pediatric patients: fifteen-year experience of a single center Ann Thorac Surg 2005 79 53 60 15620914 10.1016/j.athoracsur.2003.12.075 Cox KL Ward RE Furgiuele TL Cannon RA Sanders KD Kurland G Orthotopic liver transplantation in patients with cystic fibrosis Pediatrics 1987 80 571 574 3309864 Duchini A Redfield DC McHutchison JG Brunson ME Pockros PJ Aspergillosis in liver transplant recipients: successful treatment and improved survival using a multistep approach South Med J 2002 95 897 899 12190228 Shimazu M Kitajima M Living donor liver transplantation with special reference to ABO-incompatible grafts and small-for-size grafts World J Surg 2004 28 2 7 14639495 10.1007/s00268-003-7263-6 Stracciari A Guarino M Neurological complications of liver transplantation Metab Brain Dis 2001 16 3 11 11726086 10.1023/A:1011698526025	19102759	PMC2636841	CC BY	2021-01-04 17:26:35	yes	BMC Neurol. 2008 Dec 22; 8:52
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1923459508-PONE-RA-06271R210.1371/journal.pone.0004567Research ArticleDiabetes and EndocrinologyGeriatricsPhysiologyEvolutionary Biology/Animal GeneticsDiabetes and Endocrinology/EndocrinologyDiabetes and Endocrinology/Neuroendocrinology and PituitaryDiabetes and Endocrinology/ObesityDiabetes and Endocrinology/Type 2 DiabetesDisruption of Growth Hormone Receptor Prevents Calorie Restriction from Improving Insulin Action and Longevity CR, GH, Insulin & LifespanBonkowski Michael S. 1 2 Dominici Fernando P. 3 Arum Oge 1 Rocha Juliana S. 1 4 Al Regaiey Khalid A. 1 5 Westbrook Reyhan 1 Spong Adam 1 Panici Jacob 1 Masternak Michal M. 1 Kopchick John J. 6 Bartke Andrzej 1 2 * 1 Department of Internal Medicine – Geriatrics Research, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America 2 Department of Pharmacology, and Physiology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America 3 Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina 4 Department of Morphology, Laboratory of Cellular Biology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil 5 Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia 6 Department of Biomedical Sciences, Edison Biotechnology Institute, Ohio University, Athens, Ohio, United States of America Maedler Kathrin EditorUniversity of Bremen, Germany* E-mail: [email protected] and designed the experiments: MSB FPD AB. Performed the experiments: MSB FPD OA JSR KAR RW AS JP. Analyzed the data: MSB. Contributed reagents/materials/analysis tools: MSB KAR MMM JJK. Wrote the paper: MSB FPD AB. 2009 23 2 2009 4 2 e45678 9 2008 9 12 2008 Bonkowski et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Most mutations that delay aging and prolong lifespan in the mouse are related to somatotropic and/or insulin signaling. Calorie restriction (CR) is the only intervention that reliably increases mouse longevity. There is considerable phenotypic overlap between long-lived mutant mice and normal mice on chronic CR. Therefore, we investigated the interactive effects of CR and targeted disruption or knock out of the growth hormone receptor (GHRKO) in mice on longevity and the insulin signaling cascade. Every other day feeding corresponds to a mild (i.e. 15%) CR which increased median lifespan in normal mice but not in GHRKO mice corroborating our previous findings on the effects of moderate (30%) CR on the longevity of these animals. To determine why insulin sensitivity improves in normal but not GHRKO mice in response to 30% CR, we conducted insulin stimulation experiments after one year of CR. In normal mice, CR increased the insulin stimulated activation of the insulin signaling cascade (IR/IRS/PI3K/AKT) in liver and muscle. Livers of GHRKO mice responded to insulin by increased activation of the early steps of insulin signaling, which was dissipated by altered PI3K subunit abundance which putatively inhibited AKT activation. In the muscle of GHRKO mice, there was elevated downstream activation of the insulin signaling cascade (IRS/PI3K/AKT) in the absence of elevated IR activation. Further, we found a major reduction of inhibitory Ser phosphorylation of IRS-1 seen exclusively in GHRKO muscle which may underpin their elevated insulin sensitivity. Chronic CR failed to further modify the alterations in insulin signaling in GHRKO mice as compared to normal mice, likely explaining or contributing to the absence of CR effects on insulin sensitivity and longevity in these long-lived mice. ==== Body Introduction A vast majority of the mutations that delay aging and prolong lifespan in the mouse (Mus musculus) either directly or indirectly alter somatotropic and/or insulin signaling [1], [2]. There is considerable intra- and extra-cellular crosstalk between growth hormone (GH), insulin-like growth factor 1 (IGF-1), and insulin in mediating growth and metabolism in mammals [3]. These signaling pathways are highly conserved across phyla; and mutations affecting homologous IGF-1/insulin-like signaling and downstream gene expression increase longevity in yeast (Saccharomyces cerevisiae), worms (Caenorhabditis elegans), and flies (Drosophila melanogaster) [4], [5]. Taken together, the above-mentioned findings posit GH/IGF-1/insulin (and homologous) signaling as one of the key mediators of longevity. Calorie restriction (CR) is the only environmental treatment known to consistently increase average and maximal lifespan and delay aging in organisms ranging from yeast to mammals [6], [7]. In addition to extended longevity and reduced cancer incidence, the most consistent responses to CR in mammals include reductions in peripheral (i.e. blood) insulin, GH, IGF-1, and glucose levels [6], [8]. These biomarkers of a “CR response” are reported in species ranging from mice to humans [4]. Interestingly, there is considerable phenotypic overlap between mice on CR and many long-lived mutant mice. These similarities include reductions in body weight, body size, neoplastic disease incidence, peripheral GH/IGF1, insulin, and glucose, relative to their respective controls [9]–[11]. In addition, most of the long-lived mutant mice and mice on long-term CR show improvements in insulin sensitivity, feed efficiency and health-span. These similarities suggest that studying interactions between the life-extending mutations and CR may reveal what pathways and mechanisms are utilized by CR to alter aging. GH receptor/binding protein knockout (GHRKO) mice were developed by targeted disruption of the GHR/GHBP gene [12]. This insertion mutation renders the GH receptor nonfunctional and causes severe GH resistance, leading to suppression of peripheral IGF-1 and insulin levels. In addition, GHRKO mice are markedly insulin-sensitive, tend to be hypoglycemic, have reduced body weight and size, reduced incidence of neoplastic disease and a pronounced increase in all lifespan parameters, including increased mortality rate doubling time (MRDT), as well as median, average and maximal lifespan [10], [13], [14]. We previously reported that in contrast to hypopituitary, GH-deficient Ames dwarf mice [15], GHRKO mice did not receive a CR benefit in insulin sensitivity and most measures of longevity [16]. From a genetic perspective, these findings suggest that CR may work partially via GH signaling to mediate improvements in insulin sensitivity and extended lifespan, especially since it is well known that GH can induce insulin resistance [17]–[19]. One possible caveat is that when using a single level of CR, it is difficult to conclude whether the mutation ablates the effects of CR at all “doses” (i.e., flat-lines the dose-response curve) or if the mutation altered (or shifted) the CR dose response curve [20]. While both outcomes are important, the former would implicate GHR-based signaling as the paramount pathway controlling the effects of CR on aging and lifespan in mice and thus potentially one of the pathways that may be exploited to induce CR-like effects in humans. We report here two long-term studies, of the interaction between GHR disruption and CR in male mice that were designed to address the following questions: 1) Does the GHRKO mutation modulate or completely ablate the benefits of CR on lifespan? 2) How does CR improve insulin sensitivity and longevity in normal mice? 3) Why does CR fail to improve insulin sensitivity and longevity in GHRKO mice? Results and Discussion Longevity characteristics of normal and GHRKO mice on mild CR To determine whether the previously documented differential responses of GHRKO and normal (N) mice to 30% CR may have been limited to this level of dietary restriction, we conducted an additional longevity study using a milder degree of CR in both mice. Every other day (EOD) feeding resulted in approximately a 10–15% reduction in the average daily food intake compared to ad libitum (AL) control mice (data not shown) [21], [22]. In normal male mice, EOD feeding led to a 16% increase in median lifespan (N AL = 851 days vs. N EOD = 1010 days, hazard ratio (HR) 2.62 confidence interval (CI) 1.31–6.03). In contrast, median lifespan of GHRKO mice was not extended (KO AL = 1178 days vs. KO EOD = 1158 days, HR 1.063, CI 0.50–2.29). Compared to N AL mice, EOD feeding increased overall lifespan in normal mice as evaluated by log-rank analysis (P<0.008); however, there were no significant diet effects in GHRKO mice (P = 0.6727). Thus, a milder and different form of CR that increased longevity in normal control mice failed to further increase the lifespan of long-lived GHRKO mice, corroborating our previous findings using a more severe (30%) traditional CR regimen [16]. Taken together, these studies indicate that GH resistance ablates rather than modulates (or shifts) the benefits of CR on longevity in male GHRKO mice. Effects of long-term 30% CR on metabolic characteristics of normal and GHRKO mice To search for potential mechanisms for the differential responses of GHRKO and normal mice to CR and to further investigate the interaction between CR, GH resistance and insulin sensitivity, 30% CR was initiated at 2 months of age and maintained for one year in normal and GHRKO male mice. In comparison to animals fed AL, CR produced the expected decrease in body weight trajectory in both normal and GHRKO mice (Figure 1A). Furthermore, we fasted mice in all groups overnight to determine body weight in the non-fed state. Compared to AL controls, one year of 30% CR led to reduced fasted body weight in both normal and GHRKO mice (Figure 1B). There were no significant differences in the fasted peripheral glucose levels in any of the groups tested (Figure 1C). In normal mice, CR decreased the level of fasted insulin (P<0.001), while both AL and CR GHRKO mice had reduced insulin levels compared to normal mice on either diet (P<0.001 for each comparison). Calorie restriction did not further decrease fasted peripheral insulin levels in GHRKO mice (Figure 1D). 10.1371/journal.pone.0004567.g001Figure 1 Metabolic parameters of male GHRKO (KO) and normal (N) mice fed ad libitum (AL) or subjected to 30% calorie restriction (CR) for one year. Panel (A) shows time-course changes in body weight over a one year period. After one year of CR, all groups were fasted overnight and bodyweight (B), peripheral glucose (C), insulin (D), and calculated homeostatic model of assessment (HOMA, E) were determined. Values with unlike superscripts/letters are significantly different (P<0.05). The relationship between peripheral glucose and insulin levels is an important indicator of the overall insulin sensitivity of the animal. One model that utilizes fasted glucose and insulin levels to represent the whole organism's insulin sensitivity is the Homeostatic Model of Assessment (HOMA), with a relatively lower score representing improved insulin sensitivity. Figure 1E shows that in normal mice, one year of CR led to a reduction in the HOMA score compared to AL controls (P<0.01), while CR did not alter the already improved HOMA score in GHRKO mice. To summarize, CR reduced body weight in both phenotypes and improved fasting insulin sensitivity in normal mice, but failed to further improve the already insulin-sensitive status of GHRKO mice, thus uncoupling the effects of CR on body weight/growth from its effects on insulin sensitivity and lifespan. To further explore the interaction between GH resistance, CR, and insulin sensitivity, we investigated the activation state of the insulin signaling cascade in these groups in response to an exogenous insulin challenge. Effects of CR on the response of normal and GHRKO mice to insulin stimulation The actions of insulin are critical for the regulation of the processes involved in glucose homeostasis. The maintenance of carbohydrate homeostasis involves multiple enzymatic processes, in addition to cellular signaling processes, with liver and muscle playing different roles. To determine the molecular mechanism of differential impact of CR on insulin sensitivity in normal mice and GHRKO mice, we evaluated insulin signal transduction through the IR/IRS/PI3K/AKT1/AKT2 pathway in liver and skeletal muscle in normal and GHRKO mice fed AL or subjected to CR for one year in response to an insulin challenge (10 IU/kg insulin versus saline). Liver responses to insulin stimulation Calorie restriction increased total amount of insulin receptor (IR) protein in normal mice (P<0.03), but did not increase the already-elevated levels of IR in GHRKO mice on both AL and CR diets (Figure 2A). The heightened insulin induced upstream events in GHRKO mice corresponds with previous studies in these insulin sensitive mutants [23], [24]. In response to acute insulin stimulation, normal mice on CR had a 2-fold greater increase in IR activation (as estimated by measuring IR phosphorylation at tyrosine (Tyr) residue 1158 [pY1158] as a proxy for activation) than the normal AL mice, while CR did not increase the already elevated insulin-induced IR activation in GHRKO mice (Figure 2B). 10.1371/journal.pone.0004567.g002Figure 2 Liver insulin signaling cascade total proteins and phospho-proteins in response to insulin stimulation (10 IU/kg) versus saline treated controls in GHRKO (KO) and normal (N) mice fed ad libitum (AL) or 30% calorie restricted (CR) for one year. ELISA with antibodies directed towards total insulin receptor (IR; A), IR pY1158 (B), AKT1 (D), and AKT1 pS473 (E) were performed. Liver homogenates were immunoprecipitated (IP) with anti-p85, separated using SDS-PAGE and the level of non-specific Tyr phosphorylation was determined at approximately 180 kDa using anti-pY99 (C). Total AKT2 protein (F) and AKT2 pS473/474 (G) was determined from liver protein homogenates first subjected to IP using anti-AKT2. Panel (H) summarizes key insulin-stimulated phospho-proteins compared to insulin-stimulated N AL mice. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05). Next, we determined the levels of activation of a major intracellular signaling intermediate in the insulin signaling cascade, namely phosphatidylinositol-3 kinase (PI3K). In normal mice, CR significantly increased insulin-induced p85/IRS pY99 phosphorylation from 1.8-fold (N AL) to 2.2-fold in (N CR). However, this parameter was not affected by CR in GHRKO mice (Figure 2C). This finding suggests that while the enhanced amplitude of the insulin signal from the hepatic IR was maintained at the level of PI3K in normal mice on CR, GHRKO mice on either diet did not show the level of activation (as presumed by phosphorylation) that corresponded to the upstream activation of the insulin receptor (Figure 2B). An important downstream target of the insulin/PI3K signaling cascade is protein kinase B (AKT1). Total AKT1 protein in liver did not differ in any of the groups tested (Figure 2D). In normal mice, CR increased the insulin-induced phosphorylation of AKT1 at serine (Ser) residue 473 (pS473, a marker of activation of this enzyme) compared to values measured in insulin-stimulated N AL mice (P<0.03, Figure 2E). Insulin-induced pS473 levels of AKT in KO AL and KO CR groups did not significantly differ from the corresponding values in N AL or N CR mice. In the liver of normal mice, CR increased AKT2 total protein compared to AL controls (P<0.03, Figure 2F). GHRKO mice had comparable AKT2 total protein levels when compared to N AL mice. Acute insulin stimulation increased the levels of Ser phosphorylated AKT2 (pS474) in all groups (P<0.001). When compared to phenotype/treatment saline controls within groups, insulin increased the phosphorylation in N AL by 92%, while N CR, KO AL, and KO CR groups had more than a 2 fold increase in insulin stimulated phosphorylation (N CR 2.1 fold, KO AL 2.05 fold, and KO CR 2.1 fold). Hepatic insulin signaling cascade activation in response to insulin stimulation in normal mice on CR and in GHRKO mice on both diets is compared to N AL controls and summarized in Figure 2H. These data suggest that CR increases the levels of proteins involved in insulin signaling transduction and the activation of these proteins in response to insulin in normal mice, but has no effects in GHRKO mice. Curiously, the enhanced activation of the IR in response to insulin stimulation is not propagated downstream to the AKT proteins in GHRKO mice, whereas it is in the normal mice. In an attempt to understand this discrepancy, we explored the regulatory subunits of PI3K in normal and GHRKO mice fed AL or subjected to 30% CR. Determination of the PI3K regulatory subunits p85/p55/p50 To determine the relative protein abundance of the regulatory subunits of PI3K in the liver, we targeted the p85 subunit of PI3K for immunoprecipitation in liver homogenates and subsequently immunoblotted for the p85, p55 and p50 subunits of PI3K (Figure 3). In the liver of normal mice, CR did not alter p85 (Figure 3A), p55 (Figure 3B) or p50 (Figure 3C) total protein concentration. Similar to these findings, CR did not alter the level of these subunits in GHRKO mice, with the exception of a significant reduction in the p55 subunit compared to GHRKO AL controls (P<0.05). It has been shown that an alteration in a single regulatory subunit of PI3K may act to inhibit or bind up free IRS proteins and alter signal transduction [25], [26]. This could explain the partial decrease in signal amplification seen between the activation of the IR and PI3K phosphorylation in mice from the normal CR group. Irrespective of the diet, GHRKO mice had reduced p85, p55, and p50 regulatory subunits compared to normal mice. CR did reduce the p55α subunit in GHRKO mice. We believe this reduction of the regulatory subunits of PI3K in GHRKO mice may underpin the dampened downstream activation (compared to activation of IR) of the PI3K/IRS, AKT1 and AKT2 proteins seen in the insulin stimulation experiments (Figures 2 and 3). 10.1371/journal.pone.0004567.g003Figure 3 Liver PI3K subunit abundance in GHRKO (KO) and normal (N) mice fed ad libitum (AL) or 30% calorie restriction (CR) for one year. Liver protein isolates were immunoprecipitated (IP) with anti-pan-p85. Total p85α (A), 55α (B) and 50α (C) subunits were separated using SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-pan-p85. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05). Effects of insulin stimulation in the skeletal muscle Skeletal muscle is the major site of insulin-stimulated glucose uptake in the body, as demonstrated by various tissue-specific knockouts of the glucose transporter GLUT4 [27]. In normal mice, CR increased total IR from 6.92±0.64 to 8.87±0.47 units/100ug (P<0.02, Figure 4A). In contrast, GHRKO AL mice had elevated muscle IR protein that was reduced by CR (9.01±0.90 to 6.72±0.59 units/100ug; P<0.01). Insulin-stimulated phosphorylation of the IR in normal CR mice was increased approximately 3-fold compared to AL controls (P<0.001), while IR phosphorylation in GHRKO mice was unaffected by the diet, and values obtained were comparable to normal AL mice (Figure 4B). This finding was also reported previously [24], and this does not correspond with their heightened insulin stimulated glucose uptake [16] so we therefore further investigated downstream insulin signaling events associated with insulin mediated glucose uptake. 10.1371/journal.pone.0004567.g004Figure 4 Muscle insulin signaling cascade total proteins and phospho-proteins in response to insulin stimulation (10 IU/kg) versus saline treated controls in GHRKO (KO) and normal (N) mice fed ad libitum (AL) or 30% calorie restriction (CR) for one year. Levels of total insulin receptor (IR; A), IR pY1158 (B), AKT1 (D), and AKT1 pS473 (E) were determined using ELISA. Liver homogenates were immunoprecipitated (IP) with anti-p85, separated using SDS-PAGE and the level of non-specific Tyr phosphorylation was determined at approximately 180 kDa (corresponding to IRS proteins) using anti-pY99 (C). Total AKT2 protein (F) and AKT2 pS473/474 (G) was determined from liver protein homogenates first subjected to IP using anti-AKT2. Total GLUT4 protein (H) was determined in muscle homogenates using anti-GLUT4. Panel (I) summarizes key insulin-stimulated phospho-proteins compared to insulin-stimulated N AL mice. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05). Calorie restriction produced a significant increase in the PI3K/IRS phosphorylation of IRS associated with PI3K in the muscle of normal mice, yet did not further elevate the already heightened phospho-Tyr levels in the GHRKO mice (Figure 4C). This finding did not coincide with IR activation, where GHRKO mice did not show elevations in IR phosphoylation, yet there was an increase in the level of IRS phosphorylation at Tyr 99. To further probe this finding, we assessed the total protein and phosphor-Ser levels of the downstream targets AKT1, AKT2, and mTOR, as well as the total protein content of GLUT4. AKT proteins in skeletal muscle are critically involved in insulin-stimulated glucose uptake through the trafficking of GLUT4 to the cell surface. In normal mice, CR increased total AKT1 protein (N AL 11.2±0.94 versus N CR 16.4±2.0, P<0.01, Figure 4D). In contrast, CR did not increase the already-elevated level of AKT1 total protein in GHRKO mice. Calorie restriction increased insulin-induced AKT1 phosphorylation in normal mice (P<0.01), but it did not further increase the already-elevated AKT1 response to insulin in GHRKO mice (Figure 4E). This data coincide with the increases of p85/IRS activation in muscle homogenates shown in Figure 4C. As mentioned previously, AKT2 has been shown to mediate the stimulatory effects of CR on improved glucose uptake in skeletal muscle [27]. Calorie restriction did not alter muscle AKT2 protein abundance in either phenotype, but total AKT2 protein was elevated in GHRKO AL mice compared to normal AL controls (P<0.05, Figure 4F). It should be noted that normal CR mice had numerically elevated AKT2 values (compared to normal AL mice) that were not significantly different from the elevated values in GHRKO groups. Calorie restriction increased AKT2 phosphorylation in response to insulin stimulation in normal mice, compared to insulin-stimulated AL controls (P<0.01), while GHRKO mice had elevated phosphor-Ser 474 AKT2 levels (P<0.01 for both, Figure 4G), with no further changes in response to CR. The similarity of activated AKT1 and AKT2 between normal mice on CR and GHRKO mice on either diet coincides with the improved insulin sensitivity in these groups compared to normal AL controls, and with our previous results concerning CR effects on longevity [16]. Since the AKT proteins have been shown to regulate insulin-stimulated GLUT4 trafficking, we next determined the level of GLUT4 protein in skeletal muscle whole cell homogenates. Muscle GLUT4 total protein levels were significantly increased by CR in normal mice (P<0.003, Figure 4H). However, CR did not alter total protein levels in GHRKO mice. Both KO AL and KO CR mice had elevated muscle GLUT4 total protein levels compared to N AL controls (P<0.03 and P<0.04, respectively). These findings indicate that normal mice on CR and GHRKO mice on both diets have increased pools of GLUT4 available for recruitment. Figure 4I illustrates/summarizes the insulin signaling activation state of normal mice on CR and GHRKO mice (on both diets) compared to normal AL controls. GHRKO mice did not have elevated IR activation in comparison to their normal littermates, yet downstream PI3K/IRS-1, AKT1, AKT2 and GLUT4 levels or activity were elevated. While CR increased the activation of all proteins tested in normal animals, when compared to AL controls, the effects of CR in GHRKO mice were absent or opposite to those found in normal mice on CR. To further investigate this incongruity, we more closely examined the level of inhibition between the IR and PI3K signaling molecules. Calorie restriction and/or GH resistance decreases mTOR signaling and phosphoSer inhibition of IRS-1 in skeletal muscle A number of recent reports show that Ser/Thr kinases mTOR, JNK, and PKCζ mediate a negative feedback loop in the insulin signaling cascade by phosphorylating IRS-1 at Ser position 307 [28], [29]. While IRS proteins are normally Tyr-phosphorylated upon IR activation, Ser phosphorylation prevents binding to the beta subunit of the IR and subsequent Tyr phosphorylation, thereby preventing the progression of insulin signal transduction. Since we have previously reported a reduction in JNK activation and in PKCζ in the skeletal muscle of GHRKO mice [30], we investigated the levels of IRS-1 total protein and phospho-Ser307 IRS-1 in normal and GHRKO mice on both dietary regimens. Total IRS-1 protein levels were not different in any of the groups tested (Figure 5A). Compared to normal mice, GHRKO mice had more than a 50% reduction in the phosphorylation of IRS-1 at Ser 307 (Figure 5B). We believe that the decrease in IRS-1 phosphorylation at this inhibitory site (Ser 307) in GHRKO mice underpins the insulin-stimulated elevations in the activation of downstream PI3K/IRS, AKT1 and AKT2 in the absence of increased IR activation upstream. 10.1371/journal.pone.0004567.g005Figure 5 Muscle total IRS-1 protein (A) and IRS-1 pS307 inhibitory phosphorylation (B) in GHRKO (KO) and normal (N) mice fed ad libitum (AL) or 30% calorie restriction (CR) for one year was determined using ELISA. Bars represent 6–8 animals per phenotype/diet group. Values with unlike superscripts/letters are significantly different (P<0.05). It was recently reported in another long-lived, insulin-sensitive mouse, the Ames dwarf, that mTOR signaling was reduced in skeletal muscle [30]. Since mTOR has been directly implicated in mediating IRS-1 inhibition through phosphorylation at Ser 307, we determined the level of mTOR total protein and its phosphorylated isoform in normal and GHRKO mice on both diets. In normal mice, CR reduced total mTOR protein compared to AL controls (P<0.01), while CR did not further reduce the already low levels of mTOR in GHRKO mice (Figure 6A). The activation of mTOR through phosphorylation at Ser 2448 followed the same trends as total content of mTOR protein, where CR reduced basal activation (pS2448) of mTOR in normal mice but did not alter the already low levels of activation in GHRKO mice (Figure 6B). We believe that differences in mTOR signaling implied by these results together with reduced JNK and PKCζ activation previously reported by our laboratory [31] importantly contribute to, and perhaps account for, the decrease in skeletal muscle inhibitory IRS-1 Ser 307 phosphorylation and improved muscle and whole-animal insulin sensitivity in GHRKO mice. 10.1371/journal.pone.0004567.g006Figure 6 Muscle total mTOR protein (A) and mTOR pS2448 (B) in GHRKO (KO) and normal (N) mice fed ad libitum (AL) or 30% calorie restriction (CR) for one year were determined using western blotting. Bars represent 6–8 animals per phenotype/diet group. Conclusion In the present study, we addressed three major research questions: 1) Does the GHRKO mutation modulate, or ablate, the effects of different intensities of CR on longevity? 2) How does CR improve insulin sensitivity and longevity in normal mice? and 3)Why does CR fail to improve insulin sensitivity and longevity in GHRKO mice? In regard to the first question, we showed that while longevity of normal mice increased as expected in response to various intensities of CR, GHRKO mice did not derive longevity benefits from identical dietary interventions. This “flattening” of the median lifespan response in GHRKO mice on various levels of food intake shows that, in the absence of a functional GH receptor, CR does not affect longevity of male GHRKO mice. In other words, GH resistance ablates, rather than modulates, the key benefit of CR in these animals. In yeast and invertebrates the interactions between CR (or dietary restriction) and mutations that extend longevity have been explored [reviewed in 32]. While there seems to be homologous insulin signaling pathway mutations that ablate the CR response in yeast [i.e. Sch9, an AKT homologous protein and TOR, 33], mutations in chico (IRS-1 homologous proteins) modulate, rather than block, the CR response in flies [34]. These findings in less complex organisms are confounded by two major complications. First, yeast and invertebrates have not evolved GH signaling pathways. This fact makes it difficult to interpret findings across species. Second, the method by which dietary restriction, or CR is delivered in yeast and invertebrates is quite different than the traditional mammalian model, further complicating interaction studies [32]. In normal mice on CR, there is a major amplification of the insulin signaling cascade, with increases in protein concentration and/or (insulin-stimulated) activation of almost all the cellular mediators of insulin action tested in the present study (summarized in Figure 7). These increases in the insulin signaling cascade in liver and muscle show that the previously documented improvements in whole-animal insulin sensitivity in response to CR reflect heightened insulin cascade action. We previously postulated that improved whole-animal insulin sensitivity is tightly coupled to extended longevity [16]. 10.1371/journal.pone.0004567.g007Figure 7 Summarizes the effects of CR, or lack thereof, in wildtype and GHRKO mice under insulin-stimulated conditions. Solid grey proteins represent stimulatory activation, while solid black proteins are inhibitory. The present results suggest two different—but not necessarily mutually exclusive—answers to our third question: “Why does CR fail to increase insulin sensitivity and longevity in GHRKO mice?” Since skeletal muscle is the major mediator of post-prandial, insulin-stimulated glucose uptake, we believe our data showing heightened muscle insulin action in both normal mice on CR and GHRKO mice regardless of diet underpin their improved insulin sensitivity (summarized in Figure 7). This convergence between normal mice exposed to long-term CR and innately insulin-sensitive GHRKO mice could explain why CR fails to further improve insulin sensitivity of GHRKO mutants or further extend their already-long lives. Another conclusion can be drawn from the differential effects of CR on the responses to an insulin challenge in the liver's insulin signaling cascade. While normal mice on CR showed improved downstream insulin action through increases in total protein and/or insulin-stimulated activation of several key elements of the insulin signaling cascade in liver, CR failed to produce corresponding changes in GHRKO mice. Additionally, both KO AL and CR groups had reduced downstream insulin-induced activation. This tissue-specific phenotypic dimorphism may have contributed to the absence of CR effects on whole-animal insulin sensitivity and lifespan in GHRKO mice. Collectively, the present study identifies molecular steps of insulin signaling in the liver and in the muscle that are similarly affected by two conditions that extend life, CR in normal animals and deletion of the GH receptor, but are not altered by CR in GHRKO mice in which CR fails to increase insulin sensitivity and therefore longevity. This includes four parameters in liver and 8 in muscle (plus one altered by CR in the opposite directions in the two phenotypes). We suggest that these steps of insulin signaling are involved in the control of whole animal insulin sensitivity and mammalian longevity. Materials and Methods Animals GHRKO and normal mice were produced in the breeding colony at SIU-SOM Springfield, IL which was developed from mice kindly provided by J. J. Kopchick (Ohio University, Athens). Phenotypically normal siblings of GHRKO mice served as controls in these studies. Animals were weaned at 3 weeks of age and placed on Lab Diet Formula 5001 (Ralston Purina, St. Louis, MO). Animals were housed at 20–23°C on a 12/12 hour light/dark cycle. Sentinel animals were sent for serological testing every 3 months and the results were uniformly negative. These studies were approved by the Laboratory Animal Use and Care Committee of SIU-SOM. Longevity Study The every other day (EOD) feeding paradigm was conducted in normal and GHRKO mice starting at approximately 8 weeks of age. Mice were gradually shifted to EOD by placing them on every third day fast for the first week and then EOD feeding throughout the remainder of the study. The survival of male mice used in this study (approximately 15–20 per phenotype/dietary treatment) was evaluated once all four groups reached median lifespan. At the time of analysis, the remaining survivors from this still ongoing study were 6% of the N AL, 43% of the N CR, 41% of the KO AL and 46% of the KO CR mice. Log-rank analysis for partial survivorship was conducted. At the time of analysis, all female treatment groups did not yet reach median lifespan. Animals in the longevity study were checked daily for health and survival, and were handled only for cage changes and bi-weekly body measurements. Animals that appeared near death (listless, unable to walk, and cold to the touch) or had large bleeding neoplastic growth approaching 10% of their body weight were euthanized and date of euthanasia was considered date of death. Metabolic Parameters In the mice on the terminal insulin stimulation study, metabolic parameters were evaluated from blood collected from the orbital plexus after 10 months of 30% CR. Following a 12 hour fast, animals were anesthetized using isoflurane (Abbott Laboratories, Chicago, IL) and glucose levels were determined using a glucometer (Lifescan, Johnson & Johnson, New Brunswick, NJ.) and insulin levels were determined using an enzyme-linked immunosorbant assay (ELISA, Crystal Chem. Inc., Downers Grove, IL). Homeostatic Model of Assessment (HOMA) score was calculated as 22.5/(insulin [mU/L] X glucose [mmol/L]. Insulin Stimulation Study GHRKO mice and phenotypically normal male siblings were gradually calorie restricted starting at 8 weeks of age, by receiving 90% of the amount of food consumed by AL controls in the initial week, 80% the following week, and 70% for the remainder of the study. After one year of 30% CR, mice from all 4 groups (N AL, N CR, KO AL, and KO CR, n = 15–18 per group) were anesthetized [ketamine (100 mg/ml):xylazine (20 mg/ml), at a 0.1 ml per 10 grams of body weight] and injected in the inferior vena cava with either 10 IU/kg of porcine insulin (Sigma, St. Louis, MO) or with saline (control). Liver and hind limb muscle tissues were extracted and snap frozen at 1 and 3 minutes, respectively, after injection. Protein and Phosphoprotein Determination After storage at −80°C, liver and muscle tissues in T-Per (Pierce, Rockford, IL) with phosphatase inhibitor (Pierce, Rockford, IL) and protease inhibitors (Sigma, St. Louis, MO) at approximately 1 ml per 1 g of tissue. ELISA for insulin (CrystalChem, Downers Grove, IL), IR, IR pY1158, IRS-1, IRS-1 pS312, AKT1 and AKT1 pS473 were used for quantification of cellular proteins in accordance with manufacturer's instructions (Biosource Camarillo, CA). For immunoprecipitation (IP), equal amounts of solubilized protein were incubated at 4°C overnight with ∼4ug/ml of specific antibody. Polypeptide-Immuneglobin complexes were then collected by incubation with protein A-sepharose beads (Sigma, St. Louis, MO) washed solubilization buffer A, and boiled in Laemmli sample buffer (Biorad, Hercules, CA). Antibodies (Ab) against pan p85, AKT2, AKT (pS473), mTOR, mTOR (pS2448), and GLUT4 (Cell Signaling Technology, Inc. Danvers, MA) were used according to manufacturer's suggestions. Non-specific IRS Tyr phosphorylation of p85 was determined by detecting anti-pY99 (Santa Cruz Biotechnology, Santa Cruz, CA) signal in ∼180 kDa region in the anti-pan p85 immunoprecipitates. Immunoblotting was performed according to standard protocol. Protein content in tissue homogenates extracted with T-Per (described above) was determined by the BCA protein assay (Pierce, Rockford, IL). Protein lysates (40–60 ug) were subjected to SDS-PAGE using Criterion XT Precast Gels (BioRad, Hercules, CA), transferred to nitrocellulose membranes (BioRad, Hercules, CA) and even loading was verified using MemCode Reversible Protein Stain Kit (Pierce, Rockford, IL). Membranes were blocked for 1 hour at room temperature with either 5% dry skim milk or 3% BSA when determining phosphorylated proteins, in TBST (TBS+0.05% Tween-20). Blots were then washed with TBST and incubated with the primary Ab diluted in the appropriate Ab-specific blocking solution suggested by the manufacturer. ECL Plus chemiluminescent reagent was used for immunodetection (G.E. Healthcare Bio-sciences Corp, Piscataway, NJ). Digital images of blots were captured using a CCD camera (Hitachi Genetic Systems, Ameda, CA) and quantified using GeneTools software (SynGene, Cambridge, England). Statistical analysis Kaplan-Meier survival curves were used for survival analysis using log-rank test to evaluate significance of survivorship between groups. Median lifespan represents the age at which 50% of the population within groups remained alive and was reported with the corresponding hazard ratio (HR) and confidence interval (CI). Analysis of phenotype X diet was used to reveal interactions of characterization factors upon the phenotype under analysis, with subsequent independent Student's t-tests for within group comparisons. Analysis of basal and insulin-stimulated protein concentration across all groups was analyzed with three-way Analysis of Variance (ANOVA) followed by subsequent two-way ANOVA's and Students t-tests within groups when warranted. All statistics were conducted using SPSS version 13.0 (SPSS, Chicago, IL) with α = 0.05. All graphs were made using Prism 4.02 (GraphPad Software, San Diego CA.). Alpha is set to 0.05. With the exception of longevity data, all values are reported as mean±Standard Error of the Mean (SEM) throughout the figures and text. FP Dominici is a career investigator from CONICET of Argentina. The authors would like to thank Fieja Wang and Marty Wilson for laboratory assistance and Steve Sandstrom for editorial assistance. Competing Interests: The authors have declared that no competing interests exist. Funding: This project was supported by NIA, AG 19899 and u19 AG023122, by the Ellison Medical Foundation, and by the SIU Geriatrics Medicine Initiative. JJK is supported in part by the state of Ohio's Eminent Scholars Program that includes a gift from Milton and Lawrence Goll and by AG19899-05. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Tatar M Bartke A Antebi A 2003 The endocrine regulation of aging by insulin-like signals. 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==== Front Int Semin Surg OncolInternational Seminars in Surgical Oncology : ISSO1477-7800BioMed Central 1477-7800-6-41919647810.1186/1477-7800-6-4ReviewMale breast cancer: thirteen years experience of a single center Akbulut Sami [email protected] Ilker [email protected] Alper [email protected]ğmurdur Mahmut Can [email protected]ı Hamdi [email protected] Mehmet [email protected] Department of Surgery, University Hospital of Baskent, Ankara 06490, Turkey2 Department of Pathology, University Hospital of Baskent, Ankara 06490, Turkey2009 5 2 2009 6 4 4 17 10 2008 5 2 2009 Copyright © 2009 Akbulut et al; licensee BioMed Central Ltd.2009Akbulut et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background This retrospective study analysed the epidemiological, clinical, and therapeutic profiles of breast cancer in males. Methods We report our experience at the Hospital of the University of Baskent, where 20 cases of male breast cancer were observed and treated between 1995–2008. Results Median age at presentation was 66,7 ± 10,9 years. Average follow-up was 63 ± 18,5 months. The main presenting symptom was a mass in 65% of cases (13 patients). Ýnvasive ductal carcinoma was the most frequent pathologic type (70% of cases). Conclusion Male breast cancer patients have an incidence of prostate cancer higher than would be predicted in the general population. Cause of men have a higher rate of ER positivity the responses with hormonal agents are good. ==== Body Background Male breast cancer(MBC) is infrequent; it accounts for 0,8% of all breast cancers, less than 1% of all newly diagnosed male cancers, and 0,2% of male cancer deaths. The median age at diagnosis is 68 years, 5 years older than in women. [1-4]. The aim of this retrospective study was to present our results and analyse the epidemiological, clinical, and therapeutic profiles of this disease in 20 cases treated in our unit between1995 and 2008. Patients and methods Twenty male patients with breast cancer were treated at our University between 1995 – 2008. median age at presentation was 66,7 ± 10,9 years (range 56–90 years). Results Ýn two cases the disease was diagnosed incidentally after CT scan of the thorax for other conditions. In three cases, prostate cancer was conicident, renal cell cancer was present in one case. The disease developed after renal transplantation in one case, Kaposi sarcoma in one case. There was a family history of breast cancer in two cases. Fourteen patients (70%) had a left-sided breast cancer and six patients (30%) had a right-sided tumour. The median follow-up was 63 ± 18,5 months (range: 4–149 months). Three patients developed local recurrence (Chest wall in one patient, axillary lymph nodes in two patients). The presenting clinical symptom was a mass in 13 of cases. Pain was the second complaint in 4 of cases. The tumour associated with breast ulceration in 2 of cases and a bloody nipple discharge in one of cases (graphic 1). Upper outer quadrant masses were present in 80% of cases, a retroareolar mass in 10%, and inner quadrants mass in 10%. Bone pain was observed in one patient, related to presence of metastases. The diagnosis was confirmed by excisional biopsy in 75% cases and tru-cut biopsy in 25% cases. The type of surgical procedure and tumors size was showed in Figure 1 and 2. Figure 1 Distribution of patients according to the operation type. MRM: Modified radical mastectomy, Q+AND: Quadranectomy + axillary node dissection, L+AND: Lumpectomy + axillary node dissection, RM: Radical mastectomy. Figure 2 Dsitribution of patients according to the tumour size (T). Tis: carcinoma insitu. Pathogical analysis of the specimens revaled infiltrating ductal carcinoma (IDC) in 70% (14 cases), which one of has mucinous carcinoma togetherness, ductal carcinoma in situ (DCIS) in 25% (5 cases), and one case of coexistent apocrine carcinom and micropapillary carcinoma [5]. One of the IDC was associated with primary unkown adenocarcinoma. Pathological characteristics were showed in Figure 3 Figure 3 Invasive apocrine carcinoma with desmoplastic stroma (H&E, ×100). (A1), Estrogen receptor shows strong positivity (approximately %80 of the tumor cells) (A2), Progesteron receptor reveals the same features with ER (A3), Cytoplasm expression of HER2/neu, no membranous stainning (A4), Diffuse stainning for gross cystic disease fluid protein (GCDFP15) (A5), Invasive ductal component of the invasive mucinous carcinoma at the perifery of the tumor (H & E, ×40) (B1), H & E sections (×100) (B2), Approximately the whole of the tumor cells shows estrogen receptor positivity (B3), The same stainning features for progesteron (B4), There is no HER2/neu expression, whereas positive control reveals membranous staining (left corner) (B5), Tumor cells arranged in morula-like clusters, reffered to as an ''exfoliative appearance'' (H & E, ×100) (C1), There is no estrogen receptor positivity, whereas positive control reveals staining (left corner) (C2), Approximately 50% of the tumor cells show progesteron receptor positivity (moderate staining) (C3), There is no HER2/neu expression, whereas positive control reveals membranous staining (left corner) (C4), There is stainning for EMA (epithelial membran antigen) at the perifery (C5). Lymph node ınvolvement Axillary lymph nodes contained metastasis in 6 of the cases. Five of 6 cases had more than four lymph nodes involved. One of 6 cases had nipple areola complex invasion. Four of cases had positive lymph nodes with extra-capsular extension. Hormone receptors Fourteen cases had 30% to 100% positive ostrogene, progestrogene receptors. Three case had only positive ostregene receptor. In three patients examination of hormone receptore was not done Because the operations of these 3 patients was done between 1995–1998 and at that years receptor scanning was not been performed routinely. Examination of c-erb-2 oncoprotein was done in 15 patients. There was not any staining with c-erb b2 except one case who has apocrin carcinoma. Treatment Adjuvant radiation therapy with an average dose of 50 Gy was given to all of the patients who had axillary lymph node metastasis and to whom performed breast sparing surgery. In addition, tamoxifen therapy was also given if the hormone receptors were positive. Only hormonal therapy was given in one case who had an apocrine carcinoma, and had 80% positive hormone receptor and categorized as T2N0M0. Adjuvant radiation therapy, chemotherapy and hormonal therapy was given in patient(T2N0M0) who had IDC and mucinous carcinoma together and in patient who had a chest wall invasion. Outcome After the first therapy protocole the patients were taken to a 6 month follow-up. Only 2 cases, the cases with bone and lung metastasis, were lost. Of the cases with extracapsullary invasion two of them and n the patient with T4 tumour (in the chest wall) showed local recurrence. Radio- and chemotherapy were given to these cases with a difference of excision addition to the chest wall recurrence. At the end of the six month, local recurrences regressed totally. All of the alive 18 cases came to the control in the last year. The doses of agents placed in the therapy protocole of immunosuppressive patients, were reduced to minimum. None of them shows pathological evidence belong to metastasis or local invasion. Discussion MBC is an uncommon disease, which presents mostly in the latter decades of life. It represents less than 1% of all malignancies in men and is responsible for 0,1% of male cancer deaths [2]. It behaves similiar to female breast cancer in most cases [6]. In our clinic incidence ratio is 2, 9:100 which is more than literature [7]. Men have a higher rate of ER positivity, which accounts for good responses with hormonal agents as in our study. We used only tamoxifen in all ER positive, DCIS cases. We used tamoxifen combined with radiotherapy in ER positive, IDC cases. The prevalence of MBC increases with age, with a mean age of 60–65 years at presentation [8]. Risk factors include increasing age, radiation exposure, and factors related to abnormalities in estrogen and androgen balance, including testicular disease, infertility, obesity, and cirrhosis. Risk factors related to a genetic predisposition include Klinefelter's syndrome, family history, and BRCA gene mutations, particularly BRCA2 mutations. Gynecomastia is not a risk factor [9]. Male breast cancer patients have an incidence of prostate cancer higher than would be predicted in the general population; this risk factor has implications for prostate cancer screening. In a recent study of Lee UJ et all, 12 of 69(17%) patients with male breast cancer also had a diagnosis of prostate cancer [10]. In our study 3 of 20(15%) patients had breast cancer with prostate cancer. Breast cancer occurred in two patients with immunosuppressive disorders in our series. MBC is usually diagnosed at an advanced stage, with a typical subareolar mass associated with axillary lymph nodes and pain [11,12]. The male breast contains only ductal tissue, hence, most MBCs are of the ductal type. Histologically, 90% of male breast cancers are invasive ductal carcinomas. Approximately 80% are ER positive, 75% are PR positive, and 35% overexpress HER-2/neu. The remaining 10% are DCIS. Given the absence of terminal lobules in the normal male breast, lobular carcinoma, both invasive and in situ, is rarely seen [3,4]. The most common surgical treatment is modified radical mastectomy with axillary node dissection. Adjuvant radiotherapy has an important role in reducing the risk of local recurrence in large tumors, lymph node and muscle involvement [13]. Tamoxifen is the most widely used adjuvant therapy, because it improves survival. It may be associated with more limited side effects in men than in women, like decreased libido, weight gain, hot flushes, mood alterations, depression, insomnia and deep vein thrombosis. There is no evidence that chemotherapy improves long term survival. Chemoterapy may be useful in node positive and locally advanced disease. The use of adjuvant RT has not been conclusively proven to reduce local recurrence. Prognostic factors in male breast cancer are the same as in female breast cancer and include nodal involvement, tumor size, histologic grade, and hormone receptor status. When matched for age and stage, survival is similar to that in women. Poor prognosis is attributed to old age, axillary lymph node metastases(>4) and negative hormone receptors [13,14]. Competing interests We confirm, none of the authors listed in this manuscript have any financial or other conflicts of interest to disclose. Authors' contributions SA, MCY and HK contributed writing the article and review of the literature as well as undertaking a comprehensive literature search; SA and IA contributed design and manuscript preparation; AK provided the histopathologycal information. Acknowledgements This work was performed in University Hospital of Baskent, Ankara, Turkey. The authors wish to thank Prof Dr Mehmet Haberal (President of Baskent University) for supporting this work. ==== Refs English JC Middleton C Patterson JW Slingluff CL Cancer of the male breast J Dermatol 2000 39 881 6 El Omari-Alaoui H Lahdiri I Nejjar I Hadadi K Ahyoud F Hachi H Alhilal M Errihani H Benjaafar N Souadka A El Gueddari BK Male breast cancer. A report of 71 cases Cancer/Radiother 2002 6 349 351 10.1016/S1278-3218(02)00250-0 Lanitis S Rice AJ Vaughan A Cathcart P Filippakis G Mufti RA Hadjiminas DJ Diagnosis and management of Male Breast Cancer World Journal of Surgery 2008 32 2471 6 18787895 10.1007/s00268-008-9713-7 Winer EP Hudis C Burstein HJ Chlebowski RT Ingle JN Edge SB Mamounas EP Gralow J Goldstein LJ Pritchard KI Braun S Cobleigh MA Langer AS Perotti J Powles TJ Whelan TJ Browman GP American Society of Clinical Oncology technology assessment on the use of aromatase inhibitors as adjuvant therapy for women with hormone receptor-positive breast cancer: status report J Clin Oncol 2002 20 3317 27 12149306 10.1200/JCO.2002.06.020 Liberman L Bracero N Vuolo MA Dershaw DD Morris EA Abramson AF Rosen PP Percutaneous large-core biopsy of papillary breast lesions AJR Am J Roentgenol 1999 172 331 7 9930777 Contractor KB Kaur K Rodrigues GS Kulkarni DM Singhal H Male breast cancer: is the scenario changing World J Surg Oncol 2008 6 58 18558006 10.1186/1477-7819-6-58 El Hajjam M Khaiz D Benider A Lakhloufi A Abi F Kahlain A Bouzidi A Cancer of the breast in men. 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A multiinstitutional survey Cancer 1998 83 498 509 9690543 10.1002/(SICI)1097-0142(19980801)83:3<498::AID-CNCR19>3.0.CO;2-R Stranzl H Mayer R Quehenberger F Prettenhofer U Willfurth P Stöger H Hackl A Adjuvant radiotherapy in male breast cancer Radiotherapy and Oncology 1999 53 29 35 10624850 10.1016/S0167-8140(99)00122-X Vetto J Jun SY Padduch D Eppich H Shih R Stages at presentation, prognostic factors, and outcome of breast cancer in males Am J Surg 1999 177 379 83 10365874 10.1016/S0002-9610(99)00067-7	19196478	PMC2642849	CC BY	2021-01-04 17:27:29	yes	Int Semin Surg Oncol. 2009 Feb 5; 6:4
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1927073108-PONE-RA-05912R210.1371/journal.pone.0004730Research ArticleBiochemistryBiochemistry/Chemical Biology of the CellBiochemistry/Protein ChemistryBiochemistry/Protein ChemistryThe Cellular Distribution of Serotonin Transporter Is Impeded on Serotonin-Altered Vimentin Network 5HT Alters SERT TranslocationAhmed Billow A. 1 Bukhari Irfan A. 1 Jeffus Brandon C. 1 Harney Justin T. 1 Thyparambil Sheeno 1 Ziu Endrit 1 Fraer Mony 2 Rusch Nancy J. 3 Zimniak Piotr 3 Lupashin Vladimir 4 Tang Dale 5 Kilic Fusun 1 * 1 Department of Biochemistry and Molecular Biology, College of Medicine, The University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America 2 Department of Internal Medicine, College of Medicine, The University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America 3 Department of Pharmacology, College of Medicine, The University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America 4 Department of Physiology, College of Medicine, The University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America 5 Center for Cardiovascular Sciences, Albany Medical College, Albany, New York, United States of America Kreplak Laurent EditorDalhousie University, Canada* E-mail: [email protected] and designed the experiments: IB BJ FK. Performed the experiments: BA IB BJ JH ST VL FK. Analyzed the data: BA JH EZ MF NJR DT FK. Contributed reagents/materials/analysis tools: EZ NJR DT FK. Wrote the paper: EZ NJR PZ VL FK. 2009 9 3 2009 4 3 e473011 8 2008 15 1 2009 Ahmed et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments. Methodology/Principal Findings We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Δ14 and Δ20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S611, T613, and T616 arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter. Conclusions/Significance Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network. ==== Body Introduction The serotonin transporter (SERT) is a member of a larger family of Na+- dependent transporters in prokaryotes and animals, which is designated the SLC6 or NSS family. The biogenic amine transporter family shares about 60% amino acid identity overall [1]–[4]. SERT exists as a 630 amino acid plasma membrane bound glycoprotein in which both the amino (N) and carboxyl (C) termini are cytosolic. The termini domains of monoamine transporter proteins have garnered significant attention for their importance in transport function and localization. Several proteins have been identified in association with the C-terminus of SERT such as PICK1 [5]–[7], the actin cytoskeleton [8], neuronal nitric oxide synthase, Sec23A, Sec24C (5), fibrinogen, an activator of integrin αIIbβ3 [9]. Additionally, the interaction with MacMARCKS has been shown to modulate 5HT uptake, endocytosis, and phosphorylation of SERT via activating protein kinase C (PKC) [10] in a biphasic manner [11]. Studies have also shown that PKC-dependent modulation of SERT is correlated with extracellular 5HT levels [12], [13]. More specifically, it has been suggested that the final 20 amino acids of the C-terminal of SERT are critical for the functional expression of the transporter [14], [15]. Our recent findings explained the role of the C-terminus in the localization and trafficking of SERT via Rab4 a small GTPase, in a plasma 5HT-dependent manner. These studies demonstrated that elevated plasma 5HT “paralyzes” the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation [15]. In endogenous, platelet system, we have also observed the biphasic effect of plasma 5HT on platelet SERT [16]. More specifically, in the serum of prehypertensive subjects in which the plasma 5HT level was slightly higher than physiological levels, 5HT uptake rates and the density of SERT on the platelet plasma membrane were found significantly higher than those on platelets from normotensive states [16]. However, in plasma of hypertensive subjects in which 5HT concentration was further elevated, the 5HT uptake rates of SERT was low due to a decrease in the number of the transporters on the platelet plasma membrane [16]. Importantly, neither the mediators playing a role in 5HT-dependent regulation of SERT density on the plasma membrane nor the mechanism by which they are effective on SERT density as a factor of plasma 5HT-levels have fully been identified yet. In a series of previously reported experiments, it was found that 5HT-stimulation of cells activates p21 activating kinase (PAK), which in turn phophorylates vimentin on the serine residue at position 56 [17]. Following phosphorylation, the curved filamentous structure of vimentin undergoes reorganization and straightens [18]. Therefore, as reported here, we analyzed the vimentin-SERT association in platelets and then explored the role of plasma 5HT on this association, i.e., whether the disassembly and spatial reorganization of the vimentin network affects the translocation and, in turn, the cellular distribution of SERT molecules. Our biochemical and proteomic analysis of the proteins associated with the C-terminus of SERT identified vimentin, an intermediate filament in between many other platelet proteins. Based on our studies detailed here, we propose that phosphate modification of the SITPET sequence of SERT one at a time exposes the C-terminus domain of SERT for vimentin association. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced specifically on the plasma membrane which controls the cellular distribution of SERT on an altered vimentin network. Materials and Methods Plasmids, constructs, and cell line expression systems Human SERT (hSERT) tagged on its amino terminus with yellow fluorescent proteins (YFP) was studied for the specificities previously and no significant differences between the 5HT uptake efficiencies of tagged or wild-type hSERT were observed [15]. The mutant transporters were constructed utilizing a Stratagene Quickchange XL site-directed mutagenesis kit. The primer sequences are listed in Table S1. All synthetic constructs were verified via DNA sequencing. Cells were grown, and transfection was achieved, as described previously [19]. To test the impact of 5HT on cellular SERT system, transfected cells were pretreated with 5HT for 30-min at room temperature (RT) and then the assays were performed. Immunofluorescent (IF) analysis IF imaging cells grown on glass coverslips to 50–60% confluence in 35-mm dishes were transfected with the respective plasmids, fixed, stained and imaged after 24 h. We used the 63× oil 1.4 numerical aperture (NA) objective of a LSM510 Zeiss Laser inverted microscope outfitted with confocal optics for image acquisition. Subsequent scanning for each individual channel was performed. YFP was exited at 488 nm with Argon2 laser and emission was recorded through a 500–530-nm infrared band-pass filter. Texas Red fluorescence was excited at 543 nm with a helium-neon laser, and the emitted light was recorded through a 560-nm long-pass filter. Single z-sections were collected (0.8 μm thick) using Zeiss LSM 510 software (Release Version 4.0 SP1). Images were cropped with Adobe Photoshop 6.0 software. 5HT Uptake Assay Transport assays were performed in 24 well plates at RT. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.3) (PBS) containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS/CM). Transport was measured by incubating the cells in 250 μl of PBS/CM containing the radio-labeled substrate (1,2-3H(N))5HT (14.6 nM final concentration; specific activity) 20.5 Ci/mmol (New England Nuclear, Inc., Boston, MA; #NET-498) for 10 min at RT, an interval previously determined to include only the initial, linear phase of the transport. Each well was washed very quickly three times with ice-cold PBS. The cells were lysed in 100 ml of 1% SDS and each well's contents were transferred to a scintillation vial and counted in 3 ml of Scintisafe Econo1 (Fisher Scientific, Pittsburgh, PA) [19]. Background accumulation of (3H)-serotonin was measured in the same experiment using mock-transfected cells and subtracted from each experimental value. Maximum background accumulation was 0.01 pmol/mg protein/min. All determinations were performed at least in triplicate. Data were then plotted using OriginLab 7.5 (Northampton, MA), and statistical analyses were conducted using the NCSS software package (Number Cruncher Statistical Systems, Kaysville, UT). Analysis of variance (ANOVA) was used to determine whether mutations or deletions changed transport activity significantly relative to control or according to cell line. P-values were adjusted for multiple comparisons using either Dunnett's or Bonferroni correction procedures. Peptide-Affinity Chromatography and Mass Spec Analysis To identify platelet proteins that interact with the C terminus of SERT, we used a proteomic approach based on peptide affinity chromatography by using a synthetic peptide corresponding to the last 26 amino acids from the C-terminus (586–630) of SERT. Proteintech Group, Inc. (Chicago, IL) synthesized the peptide and conjugated to GST Sepharose beads. GST was used as an arm between peptide and Sepharose to increase the distance between peptide and matrix to facilitate the interaction between peptide and the cytosolic protein in the cell lysate. Our control column was 3 ml of GST-Sepharose without peptide. Once we set the peptide-affinity column, the platelets were isolated and the soluble proteins were prepared to run on column. Platelets were isolated from 20 ml blood samples and lysed. 1.0% TX-100 soluble lysates were loaded on control GST columns and GST-peptide columns. The proteins bound to the columns were eluted using 500 mM NaCl and fractions were collected. The peak fractions were pooled and concentrated on microfilterfuge tubes with a 10 kD cut off (Rainin Instrument Co., Oakland, CA). The concentrated samples were resolved by SDS-PAGE. Two major bands appeared in the peptide-GST Sepharose column but did not appear in our control, the GST-Sepharose column. The 115 and 60 kD bands were eluted and processed robotically using a ProGest instrument (Genomic Solutions), as previously described [20]. The resultant 50 ml peptide pools were analyzed using nano LC/MSMS on a LCQ Deca XP Plus ion trap mass spectrometer. Western Blot (W/B) Analysis Cells were solubilized in PBS containing 0.44% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor mixture (PIM). The PIM, which contained 5 μg/ml pepstatin, 50 μg/ml leupeptin, and 5 μg/ml aprotinin, was included with each lysis buffer [19]. Samples were analyzed by SDS-PAGE and transferred to a nitrocellulose membrane. SERT was detected by using SERT monoclonal antibody (MAb Technology, Stone Mountain, GA) (diluted 1:400) and HRP-conjugated anti-mouse IgG (diluted 1:5,000). The signal was visualized by using an enhanced chemiluminescence W/B detection system (Pierce, Rockford, IL). The polyclonal SERT antibody was purchased from Chemicon International (Temecula, CA; catalog number: AB1594P). In W/B analysis, PAK-phosphorylation of vimentin was detected with a specific vimentin Ser-56 (pS56) antibody (Ab) [17], [18]. pS56-Ab was prepared against a synthetic phosphopeptide (Ser-Leu-Tyr-Ala-Ser-phosphoSer56-Pro-Gly-Gly-Ala-Tyr-Cys) by SynPep Inc. (Dublin, CA). Using standard affinity purification procedures, pS56A Ab was purified on Protein A Sepharose column [21]. Vimentin-Ab (Research Diagnostics, Inc. Concord MA) reacts with vimentin and phosphovimentin dually; in contrast, phosphovimentin-(pS56)-Ab reacts only with phosphovimentin [17]. Cell surface biotinylation Cell surface expression of the transporters was detected after biotinylation with the membrane-impermeant biotinylation reagent sulfo-NHS-SS-biotin, as described previously [15], [19]. Briefly, cells were treated with 100 mM glycine to complete quenching of the unreacted NHS-SS-biotin and lysed in TBS containing 1% SDS, 1% TX100, and PIM/PMSF. The biotinylated proteins (500 μl) were recovered with an excess amount of streptavidin-agarose beads (400 μl) after overnight incubation. After biotinylated proteins were eluted in 100 μl sample buffer and separated on SDS-PAGE, they were transferred to nitrocellulose and were detected with anti-SERT antibody, as described [19]. Densitometric scanning of W/B was done on VersaDoc digital imaging system (BioRad). On each gel, samples were compared with the biotinylation procedure applied to the same amount of cells as determined by the BCA protein assay (Pierce, Rockford, IL). The experiments were performed within the linear range of densitometry reading of the SERT band as a function of the amount of protein applied according to control experiments with varying amounts of protein load per lane. Densitometry data were captured as total signal in the rectangular area encompassing the band of study corrected for background; the same rectangular area was used for estimates of the same band in other lanes of gel. Results from different scans were uniform. Results Vimentin and phosphovimentin associate with SERT in platelets A synthetic peptide corresponding to the last 26 amino acids from the C-terminus (586–630) of SERT was conjugated to GST Sepharose beads. As described in the Methods section, the GST was used as an arm between the peptide and Sepharose to increase the distance between the peptide and matrix to facilitate the interaction between the peptide and the cytosolic protein in the cell lysate. Detergent solubilized platelet lysate was run on the peptide-affinity column. The proteins bound to the columns were eluted and concentrated on microfilterfuge tubes with a 10 kD cut off. The concentrated samples were resolved by SDS-PAGE (data not presented). Two major 115 and 60 kD bands appearing in the peptide-GST but not control GST-column were eluted, processed, and analyzed using nano LC/MSMS on a LCQ Deca XP Plus ion trap mass spectrometer as previously described [20]. Our proteomic approach identified vimentin as one of many platelet proteins bound to the C-terminus of SERT. Furthermore, we analyzed these findings with biochemical techniques following the endogenous expression of vimentin and SERT in platelets with W/B assays (Fig. 1A). The co-IP assays agreed with our ESI-MS/MS mass spectrometry result showing the association between vimentin and SERT in platelet (Fig. 1B and C). 10.1371/journal.pone.0004730.g001Figure 1 SERT and vimentin interaction in human platelets. (A) Endogenous vimentin and SERT expression in platelets were analyzed with W/B analysis. The association between vimentin and SERT was determined by a co-IP in platelets stimulated with 0, 1, and 2 nM 5HT. Following 5HT-stimulation, platelet lysate was divided into two portions; the half portion of lysate was incubated in anti-vimentin monoclonal Ab (B), the other half portion in anti-SERT Ab coated protein A Sepharose beads (C). Next day, vimentin- or SERT-Ab pulled down proteins were eluted from sepharose beads; both IP eluents were analyzed either with a polyclonal SERT Ab, or with pS56-Ab, respectively. Nonspecific adsorption of Sepharose beads was not determined in the absence of antibodies. The association between endogenous vimentin and SERT was altered in a 5HT concentration-dependent manner. The highest amount of SERT was pulled down by vimentin-Ab in 2 nM 5HT-stimulated platelets. Additionally, vimentin associated with SERT after 2 nM-5HT stimulation was in phosphorylated form. (D) Expression of SERT and vimentin in total cell lysates was determined by W/B analysis as a loading control. All lanes contain protein recovered from the same number of platelets (1.5×108). Figures show representative images from 2 to 4 separate experiments. It is reported that stimulation of cells with 5HT induced phosphorylation of vimentin on serine at position 56, resulting in the reorganization of the vimentin network [17], [18]. Consequently, we investigated the impact of 5HT stimulation on vimentin-SERT association. Platelets in platelet poor plasma (PRP) were first stimulated with 1 or 2 nM 5HT, which represents the plasma levels of 5HT in normotensive and hypertensive patients, respectively, and thus a physiologically relevant stimulus [16]. Following a 30-min pretreatment with 5HT at RT, platelets were pelleted, lysed in IP-lysing buffer, and precleared [19]. The platelet lysate was divided into two half portions. The IP assay was performed on both portions using either a monoclonal vimentin-Ab or monoclonal SERT-Ab. The proteins precipitated on vimentin-Ab were subjected to immunoblot analysis using anti-SERT Ab (Fig. 1B). The level of SERT on vimentin-Ab was increased in a 5HT concentration-dependent manner. Stimulation of platelets with 2 nM 5HT enhanced SERT-vimentin association in whole platelet significantly (Fig. 1B). Since 5HT-stimulation of cells leads to the phosphorylation of vimentin on the Serine56 residue which alters the filamentous structure of this cytoskeletal protein [17], [18], the reorganization of the vimentin network should regulate the translocation of proteins that utilize the vimentin network [18]. Therefore, we next evaluated the impact of 5HT stimulation on SERT-phosphovimentin association in order to understand the involvement of phosphovimentin in the translocation process of SERT. We then analyzed the proteins precipitated on SERT-Ab by W/B using a polyclonal phosphovimentin-(pS56)-Ab, which reacts only with the phosphovimentin (Fig. 1C). In contrast, vimentin-Ab dually reacts with vimentin and phosphovimentin [17]. pS56-Ab identified a major band around 55 kD only in 2 nM 5HT pretreated platelet lysate. Thus, these data demonstrate the presence of an association between SERT and phosphovimentin (Fig. 1C). Therefore, the level of association between vimentin and SERT in Fig. 1B and C represents the total intracellular and plasma membrane. Overall, these findings show that vimentin associates with SERT in an endogenous system, the platelet. Their association was not due to 5HT-dependent stimulation of the platelet; even in the unstimulated form, vimentin-SERT association can be detected (Fig. 1B). However, when the level of 5HT was increased to 1 nM, the precipitated amount of vimentin on SERT was also elevated; therefore, 5HT enhances vimentin-SERT association (Fig. 1B). In the presence of 2 nM 5HT when vimentin is phosphorylated, a high affinity association between SERT and phosphovimentin was observed (Fig. 1C). Additionally, the levels of vimentin and SERT in the whole platelet lysate were not altered at different 5HT concentrations (Fig. 1D). Since the co-IP assays demonstrated that 5HT-stimulation enhanced the association between vimentin and SERT in platelets, we next addressed (i) whether their association was limited to intracellular locations or also occurred on the plasma membrane; (ii) whether 5HT-dependent elevation of phosphovimentin-SERT association also occurred on the plasma membrane. We performed surface biotinylation followed by W/B assays on platelets stimulated with different concentrations of 5HT. Vimentin and phosphovimentin associate with SERT on platelets plasma membrane To determine the involvement of phosphovimentin in the density of SERT on platelet plasma membrane, platelets in PRP were first pretreated with 5HT (0–2 nM) for 30 min at RT, then the pelleted platelets was biotinylated with membrane impermeable NHS-SS-biotin [16]. Biotinylated platelet plasma membrane proteins were retrieved on streptavidin beads and eluted from the beads. Half of each biotinylated sample was subjected to immunoblot analysis using anti-SERT Ab (Fig. 2A). The biphasic effect of plasma 5HT on the density of SERT on platelet plasma membrane was observed, as seen previously in hypertension model systems [16]. An intermediate level (1 nM) 5HT-stimulation increased the density of SERT on the platelet; however, at high level (2 nM), 5HT-stimulation lowered the surface density of SERT compared to untreated platelets [15], [16]. 10.1371/journal.pone.0004730.g002Figure 2 Effect of 5HT stimulation on SERT-vimentin association on platelet plasma membrane. 0–2 nM 5HT-stimulated platelets were biotinylated with NHS-SS-biotin [15], [16], [19], and biotinylated plasma membrane proteins were retrieved on streptavidin beads. Half of biotinylated platelet membrane proteins were analyzed for SERT (A), and the other half was analyzed for anti-phosphovimentin-Ab (pS56-Ab) (B) with W/B. Neither SERT not pS56-Ab detected recognizable proteins in mock biotinylated controls (carried through the biotinylation procedure without the addition of the biotinylation reagent); therefore, they are not presented in the blots. Plasma 5HT levels had a biphasic effect on the plasma membrane expression of platelet SERT [16]. To correlate SERT-phosphovimentin association on the surface density of platelet SERT, the biotinylated platelet membrane proteins were analyzed with pS56-Ab. Plasma 5HT, only at high level, showed an association between phosphovimentin-SERT. Intracellular SERT (C) and vimentin (D) were determined by W/B analysis of non-bound material and also served as a loading control. All lanes contain protein recovered from the same number of platelets (1.5×108). Figures show representative images from 3 separate experiments. Here, our data demonstrate that the association between SERT and vimentin is altered in a 5HT-dependent manner. Therefore, here we tested whether the cellular distribution of SERT is altered by 5HT-dependent phosphorylation of vimentin. The level of phosphovimentin on the plasma membrane 5HT-stimulated platelet was evaluated. The other half of the biotinylated platelet plasma membrane proteins was subjected to immunoblot analysis with pS56-Ab (Fig. 2B). Phosphovimentin appeared as one of the proteins associated with biotinylated plasma membrane-bound proteins in 5HT-stimulated platelets. SERT could be one of the other phosphovimentin-associated membrane proteins, but our co-IP data in 5HT-stimulated platelets also demonstrated an elevation in the association of SERT-phosphovimentin in whole platelet (Fig. 1C). Therefore, we tested SERT-phosphovimentin association in 5HT-stimulated platelets. The effects of 5HT-stimulation on the amount of intracellular SERT (flow through of the streptavidin beads) mirrored those of the cell surface SERT (Fig. 2C and D). Previously, it has been shown that 5HT-stimulation phosphorylates vimentin on the Serine56 residue, but the vimentin S56A mutant is not phosphorylated by 5HT-stimulation [18]. Therefore, to mechanistically determine how the vimentin-SERT association responds to 5HT for regulating the distribution of transporter molecules between plasma membrane and intracellular locations, the S56A mutant and the C-terminus truncated forms of SERT were studied in a CHO heterologous expression system. Obviously, not all aspects of 5HT biology in platelets can be recapitulated in CHO cells, but the CHO model system allows for the analysis of the association between vimentin and the C-terminus truncated forms of SERT and the nonphosphorylated mutant form of vimentin S56A. To ascertain the optimal 5HT concentration required to stimulate CHO cells expressing hSERT (CHO-hSERT), we measured the density of SERT proteins on the plasma membrane of CHO-hSERT cells and compared this finding to human platelet membranes using biotinylation [15]. In this previous study, we tried to model the effect of plasma 5HT on platelet SERT in a heterologous expression system. Simply stated, an equal amount of biotinylated membrane proteins from CHO-SERT cells and platelets were resolved and analyzed by W/B using SERT-Ab. These calculations together with the dose response analysis of CHO cells to 5HT-stimulation which was already conducted in our previous studies showed that the expression of SERT on the plasma membrane of CHO-SERT cells was 59-fold higher than on the platelet membrane [15]. This estimation indicated that the effect of plasma 5HT at a concentration of 1 nM on platelet SERT may correspond to exogenous 5HT at a concentration of ∼45 μM on CHO-SERT cells [15]. Co-localization of Vimentin and SERT with or without 5HT stimulation For the current study, we took a second approach for testing the association between phosphovimentin and SERT on the plasma membrane of 5HT-stimulated cells. The co-localization of the red vimentin and green YFP-SERT signals were captured in the overlaid images with YFP and Texas Red filter sets. If vimentin and SERT co-localized then the structures would appear light orange; otherwise, the distinct green and red signals would represent structures containing either one of the two proteins. Fifty cells were examined for colocalization of vimentin and SERT following 5HT stimulation. In all YFP-SERT transfected cells, a limited but consistent co-localization between vimentin and SERT on the plasma membrane was seen (Fig. 3). 10.1371/journal.pone.0004730.g003Figure 3 Vimentin-SERT co-localization and impact of 5HT-stimulation on their cellular distribution. CHO-YFP-SERT cells were pretreated with 5HT as indicated, labeled with vimentin monoclonal Ab, and stained with Texas Red conjugated rabbit IgG. In unstimulated cells, vimentin revealed curved filamentous structures; in 5HT-stimulated ones the vimentin filaments became straight as indicated with arrows. Cells were analyzed with a Zeiss LSM510 laser confocal microscope. To contrast the localization of SERT and vimentin, the overlaid images are presented with SERT signal pseudocolored in green. The bar (10 μm) indicates the magnification of the main figures; the insets are 2× the magnification of the main figures. Figures show representative images from at least 2 separate experiments. The colocalization of endogenously expressed vimentin and transiently expressed SERT was monitored in 5HT-stimulated CHO-(YFP-SERT) cells using IF microscopy. The cellular distribution of vimentin was significantly different in 5HT-stimulated cells than in control cells. To facilitate a comparison between the localization of SERT and vimentin, the SERT signal was pseudocolored in green in merged images. 5HT stimulation mostly located vimentin around the plasma membrane. Exposure of CHO-YFP-hSERT to 5-HT induced the spatial reorientation of vimentin filaments (Fig. 3). In control cells, vimentin exhibited a curved filamentous appearance (Fig. 3, control panel, insert). Vimentin filaments became more straight and bundled 30 min after stimulation with 100 μM 5HT (Fig. 3, 5HT-treated cells panel, insert). Vimentin binding domain on the C-terminus of SERT The C-terminus of the biogenic amine transporter plays a critical role in the regulation of transporter function and intracellular trafficking [5]–[15]. Our proteomic studies identified the C-terminus of SERT as a vimentin binding domain on SERT. To map the vimentin binding sequence on the C-terminus of SERT, we utilized the truncated form of transporters, Δ26, Δ20, Δ14, and Δ6 [15]. As we reported in a previous study, the 5HT uptake rates and the levels of surface expression of Δ6 and Δ14 of SERT were similar to the wild-type transporter [15]. These results were not due to altered protein translation as evident by W/B and densitometry analysis showing that the band densities of all constructs were similar [15]. Next, the association between endogenously expressed vimentin and transiently expressed truncated forms of transporters were tested in 5HT-stimulated CHO cells with IP analysis (Fig. 4). The cellular proteins on the vimentin-Ab coated protein A beads were eluted and separated on SDS-PAGE followed by immunoblotting with SERT-Ab (Fig. 4). The major band at 90 kD was detected in the CHO-SERT and -Δ6 cells (Fig. 4). 10.1371/journal.pone.0004730.g004Figure 4 Vimentin binding domain on the C-terminus of SERT. CHO cells expressing the indicated SERT constructs were lysed and prepared for IP. The cell lysate was incubated with monoclonal anti vimentin-Ab coated protein A sepharose beads. The presence of SERT truncations was detected with a polyclonal SERT Ab (Chemicon International). Since SERT-Δ6 bound to vimentin and the other truncations, we proposed that the amino acids 616–624 are at least one of the vimentin binding domains on the C-terminus of SERT. Nonspecific adsorption of Sepharose beads was not determined in the absence of vimentin-Ab and the data obtained from this set of experiment is not presented here since it can be found in Figure 1B. All lanes contain protein recovered from the same number of cells equivalent to 30% of one well from a confluent 24-well culture plate. Figure shows representative images from 3 separate experiments. Our recent study compared the distribution of these truncated YFP-SERT variants with that of Texas Red conjugated wheat germ agglutinin (WGA), a lectin marker for the plasma membrane [15]. The IF analysis and 5HT uptake rates showed a lack of colocalization between Δ26 and Δ20 and plasma membrane [15]. Although deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations [15], Δ14 like the other two mutants, Δ20 and Δ26, did not co-IP with vimentin (Fig. 4). These data identify the residues 616–624 in the SERT protein backbone as an essential domain for vimentin association. Characterization of SITPET sequence of SERT Inspection of the six amino acid difference between Δ20 and Δ14, the SITPET sequence, revealed 3 amino acids, S611, T613, and T616 as possible kinase action sites. We began to assess the effect of mutations of these 3 residues on uptake activity, whole cell, and surface expression. At each of the targeted locations, the original amino acid was changed to an alanine or aspartic acid (Fig. 5A). 10.1371/journal.pone.0004730.g005Figure 5 (A) 5HT uptake rates of SITPET mutants. Twenty-four hour post-transfection CHO cells were washed and assayed for analysis of 5HT uptake rate. Background accumulation of (3H)-serotonin was measured in the same experiment using mock-transfected cells and subtracted from each experimental value. Maximum background accumulation was 0.01 pmol/mg protein/min. Phospho-mimicking mutations in the C-terminal region caused mixed results. The S611D mutation resulted in the most significant change over its neutral counterpart, S611A. Another key point is the drastic reduction seen when all 3 sites were mutated to aspartic acid and when only the two threonines were mutated to aspartic acid. Bars represent means±SD of three or more independent experiments. ANOVA indicated that the effect of the construct was highly significant (p<0.001). Multiple post-hoc comparisons versus control (SERT) were made applying Dunnett's correction; values marked with (*) were significantly different from control at p<0.05. Expression of SITPET mutants. The trafficking of mutants was assessed by gel electrophoresis and W/B. The detergent extracts of total protein were analyzed to determine if the alteration in uptake ability was due to alterations in the amount of protein. All variations of the transporter demonstrated similar protein expression levels. All lanes contain protein recovered from the same number of cells equivalent to 30% of one well from a confluent 24 well dish. The loading control was performed with actin (inset in the figure). The immunoblots are representative of at least three independently performed experiments. (B) Colocalization of YFP-SERT mutants on the plasma membrane. Cells were stained with Texas Red-WGA to mark the plasma membrane. The images show that DD and DDD mutations were totally retained on intracellular structures; S611D was found partially on the intracellular compartments. However, AAA and T613D were predominantly on the plasma membrane (arrowheads), but a pool of them resided on intracellular organelles. A 1 μm bar is presented for the image set. A mutation to alanine is a relatively neutral change whereas a change to aspartic acid acts as a phospho-mimic at the site of mutation due to the charge and shape of the carboxylic acid functional group. 5HT uptake rates of each construct, S611A, T613A, T616A, and the triple mutation (AAA) retained 90, 74, 100 or 95% of the activity of wild-type transporter, respectively (Fig. 5A). However, the single mutation of S611D caused a dramatic decrease in the uptake activity of SERT, reducing transport capacity to approximately 38%, whereas T616D caused no noticeable change in uptake function (Fig. 5A). Furthermore, the triple mutation to aspartic acid (DDD) caused a 95% reduction in the 5HT uptake capacity of SERT (Fig. 5A), possibly indicating a synergistic relationship between these three positions. In all cases, changes in transport capacity were not the result of altered protein expression levels, as indicated by W/B and densitometry analysis of total protein blots (Table 1). 10.1371/journal.pone.0004730.t001Table 1 The whole cell expressions of all truncated and mutant forms of SERT in CHO cells were similar to those of the wild type transporter. SERT Δ26 Δ20 Δ14 Δ6 S611A S611D T613A T613D T616A T616D AA DD AAA % of the total protein on the cell surface 80.33±5.5 0 0 65.3±2.5 65.3±2.3 70.6±3.05 30.6±1.53 54.33±4.04 55±4 79.6±0.58 79.6±0.58 78.6±3.2 8.3±2.9 99.3±1.15 5HT Uptake rates of transporters % of wild-type SERT 100 0 0 88 82 90 38 74 70 100 100 92 17 95 The levels of transporter proteins on the plasma membrane were assessed by biotinylation of surface proteins followed by gel electrophoresis and W/B, and calculated as the percent of whole cell expression. The data presented in Table 1 are the average from three independent experiments, normalized as percentage of expression or 5HT uptake rates of wild-type transporter. Investigation into the role of S611 in the 5HT uptake capacity of SERT was analyzed using the S611A and S611D constructs originally produced, while DD and AA, i.e., the double mutation of T613 and T616, were constructed to investigate the role of the two threonine residues in the proposed mechanism. S611 appeared to exert the most influence on the 5HT uptake rate of SERT. Next, the expression of SITPET mutants on the plasma membrane was assessed by their colocalization with WGA, a plasma membrane marker (Fig. 5B). Mutants YFP-SERT variants were expressed in CHO cells and their distribution was compared with that of Texas Red conjugated wheat germ agglutinin, a lectin marker for the plasma membrane. The fluorescence data shown in Figure 5B indicate that the majority of DD and DDD were associated with intracellular structures. A significant part of AAA localized to the plasma membrane. Although a noteworthy pool of S611D appeared internally, some of S611D was observed on the plasma membrane (Fig. 5B). In summary, DDD and DD were predominantly found at intracellular compartments while S611D located on the plasma membrane partially and mostly at intracellular compartments. Thus, the S611D transporter apparently has difficulties in membrane trafficking. In contrast, AAA and T616D were found on the plasma membrane (Fig. 5B and Table 1). In an effort to explore the six amino acid difference between Δ20 and Δ14, the SITPET sequence, we began tested the association between endogenously expressed vimentin and transiently expressed mutant forms of transporters in 5HT-stimulated CHO cells with IP analysis (Fig. 6). The cellular proteins on the vimentin-Ab coated protein A beads were eluted and separated on SDS-PAGE followed by immunoblotting with SERT-Ab (Fig. 6). The major band at 90 kD was detected in the CHO-SERT, T613D, -T616D, and -AAA cells (Fig. 6). The 5HT uptake rates and the levels of surface expression of these forms were similar to the wild-type transporter (Fig. 5). Subsequently their levels of association with vimentin were high as well. On the other hand, the mutant that had a very minimal 5HT uptake rate and plasma membrane such as DDD did not associate with vimentin. Based on these findings, we hypothesize that the vimentin-binding ability of transporter is correlated with the density of transporter on the plasma membrane. In deed, S611D neither fully appeared on the plasma membrane, nor was pulled down by anti-vimentin antibody (Fig. 6). The 5HT uptake rate and the density of S611D on the plasma membrane (approximately 30% of the wild-type, Table 1) showed a similar pattern with its vimentin binding ability. The level of S611D precipitated on vimentin-Ab was 30% of the level of wild-type on vimentin-Ab. 10.1371/journal.pone.0004730.g006Figure 6 Vimentin binding ability of SITPET mutants. CHO cells expressing the indicated SERT constructs were lysed and prepared for IP. The cell lysate was incubated with anti vimentin-Ab coated protein A sepharose beads. The presence of SERT truncations was detected with a polyclonal SERT Ab (Chemicon International). Nonspecific adsorption of Sepharose beads was not determined in the absence of vimentin-Ab and the data obtained from this set of experiment is not presented here since it can be found in Figure 1B. All lanes contain protein recovered from the same number of cells equivalent to one of well from a confluent 24-well culture plate. The triple mutant AAA and two of the single mutants, T613D and T616D, bound to vimentin; S611D showed a low affinity for binding to vimentin. Figure shows representative images from 3 separate experiments. Vimentin-SERT Association on the plasma membrane Next, we evaluated the impact of 5HT stimulation on the density of truncated and mutant forms of transporters on the plasma membrane. CHO cells expressing Δ26, Δ20, Δ14, Δ6, S611D, T613D, T616D, AAA, and DDD were either directly (Fig. 7A, B ) or after stimulation with 100 μM 5HT (Fig. 7C, D) subjected to the cell surface biotinylation assay. Biotinylated membrane proteins were pulled down on streptavidin beads and eluted from the beads in SDS-PAGE sample buffer. 10.1371/journal.pone.0004730.g007Figure 7 Impact of 5HT stimulation on the plasma membrane density of truncated and mutant transporters. CHO cells expressing truncated or mutant forms of transporter were either directly (A and B) or after stimulated with 100 μM 5HT (C and D) subjected to the cell surface biotinylation assays. Biotinylated membrane proteins were pulled down on streptavidin beads and eluted from the beads in SDS-PAGE sample buffer. The biotinylated eluents were subjected to immunoblot analysis using either anti-SERT (A and C), or anti-vimentin (B), or pS56-Ab (D). All lanes contain protein recovered from the same number of cells equivalent to one of well from a confluent 24-well culture plate. In the surface protein experiments, we included two control experiments: mock biotinylated CHO cells transfected with SERT and mock transfected CHO cells. The first control, mock biotinylated CHO cells, were carried through the biotinylation procedure without the addition of the biotinylation reagent and neither of antibodies, SERT-, vimentin- nor pS56-Abs recognized proteins from these blots (data not presented). The next control, mock-transfected cells allowed us to observe (i) the nonspecific absorption of Sepharose beads and the proteins from the detergent soluble platelet lysate; (ii) if 5HT mediate any of these nonspecific interactions. Pretreatment with 100 μM 5HT increased the density of Δ14 and S611D, significantly or partially, respectively, and decreased the density of wild-type SERT and the truncated or mutant transporters which could to the plasma membrane. The transporters whose surface expressions were decreased with 5HT-stimulation, i.e., wild-type, Δ6, AAA, T616D, and T613D, were able to bind vimentin (B) and phosphovimentin (D). However, the truncated or mutant forms transporters which could not bind vimentin (B) and appeared on the plasma membrane at high levels in 5HT-stimulation (C), such as D14 and S611D, could not bind phosphovimentin (D), either. The total phosphovimentin (E) and vimentin (F) (as flow through of the streptavidin beads) did not differ in truncated and mutant forms of transporter expressing CHO cells. The levels of vimentin and phosphovimentin in cell lysate of 100 mM 5HT-stimulated cells show if the associations between these proteins with SERT or truncated/mutant forms are altered with the differences in their levels, and/or with 5HT stimulation (Figure 7E and F). All lanes contain protein recovered from the same number of cells equivalent to 30% of one well from a confluent 24-well culture plate. One half of the biotinylated membrane proteins were blotted with anti-SERT-Ab (Fig. 7A). Although there was some decrease in the densities of transporters on the plasma membrane of S611D transfected cells, Δ26, Δ20, and DDD were not located on the plasma membrane at all (Fig. 7A). To evaluate the association of vimentin and the truncated and mutant transporters on the plasma membrane, the second half of the same biotinylated samples were subjected to immunoblot analysis with vimentin-Ab (Fig. 7B). In untransfected cells our W/B analysis recognized the endogenous vimentin as one of the proteins pulled by the biotinylated plasma membrane-bound proteins (Fig. 7B, the lane labeled as NoDNA). Therefore, it is clear that vimentin had bound other plasma membrane proteins as well as SERT. In SERT transfected cells, the level of vimentin on the plasma membrane was much higher than the untransfected ones. This finding identifies SERT as one of the membrane-bound proteins that links vimentin to the plasma membrane. Similarly, the levels of vimentin on the plasma membrane of Δ26, Δ20, Δ14, and DDD transfected cells were the same as that on the plasma membrane of untransfected cells (Fig. 7B). This finding suggests a lack of association between vimentin and Δ26, Δ20, Δ14, or DDD on the plasma membrane. On the other hand, the vimentin-binding abilities of T616D and T613D on the plasma membrane were very similar to CHO-SERT and CHO-Δ6 cells. The levels of vimentin on the plasma membrane of S611D transfected cells was lower than that in wild-type transfected cells but higher than that in untransfected ones. These findings are in good agreement with the data in Figure 6. Collectively, they support our hypothesis that the density of transporter on the plasma membrane and the level of its vimentin binding are correlated. In summary, SERT is one of the proteins that link vimentin to the plasma membrane. Our co-IP studies in endogenous and heterologous expression systems, and IF analyses (Fig. 1, 3) demonstrate that in 5HT stimulated platelets the level of SERT precipitated on vimentin-Ab is higher than that in control platelets, which were altered by the extracellular level of 5HT. We [15], [16] and others [11], [12] reported that 5HT stimulation at high levels does not increase the 5HT uptake rates and the density of SERT on the plasma membrane. Thus, we attempted to determine: (i) whether extracellular 5HT at high levels facilitates the translocation of SERT from the plasma membrane via phosphovimentin; and (ii) modification of three C-terminus residues, S611D, T613D, T616D, are involved in phosphovimentin-SERT association (Fig. 7C and D). CHO cells expressing truncated and mutant forms of SERT were first stimulated with 100 μM 5HT, and then biotinylation assay was performed to separate the plasma membrane proteins and their partners. Biotinylated membrane proteins were pulled down on streptavidin beads and eluted from the beads in SDS-PAGE sample buffer. One half of the biotinylated membrane proteins were blotted with anti-SERT-Ab (Fig. 7C). In agreement with reported studies, a pretreatment with 100 μM 5HT did not elevated the densities of SERT, Δ6, and the mutant transporters that mimic the phosphorylated forms, T613D and T616D, as 10 μM 5HT-stimulation did [15]. However, 5HT-stimulation increased the density of Δ14 truncated transporter and did not change the density of S611D mutant transporters on the plasma membrane (Fig. 7C). In exploring the impact of phosphovimentin-SERT association on the plasma membrane density of SERT, the second half of the same biotinylated samples were subjected to immunoblot analysis with pS56, phosphovimentin-Ab (Fig. 7D). The data indicated that the association affinity between SERT and phosphovimentin on the plasma membrane was enhanced by 5HT stimulation. The mutants, AAA, T613D, T616D, and the truncated form of the transporter, Δ6, which show decreased densities on the plasma membrane in response to 5HT stimulation, associated with phosphovimentin with high affinity (Fig. 7D). 5HT-stimulation does not alter the cell surface expression of S611D significantly or its association with phosphovimentin. In these experiments, we included two control experiments: (i) mock biotinylated CHO cells transfected with SERT; and (ii) mock transfected CHO cells. The first control, mock biotinylated CHO cells, were carried through the biotinylation procedure without the addition of the biotinylation reagent and none of antibodies, SERT-, vimentin- nor pS56-Abs, recognized proteins from these blots (data not presented). The next control, mock-transfected cells, allowed us to observe (i) the nonspecific adsorption of Sepharose beads and the proteins from the detergent soluble platelet lysate; (ii) if 5HT mediate any of these nonspecific interactions. The total phosphovimentin and vimentin (as flow through of the streptavidin beads) was similar between truncated and mutant forms of transporter expressing CHO cells (Fig. 7E and F). The levels of vimentin and phosphovimentin in cell lysate of 100 mM 5HT-stimulated cells show if the associations between these proteins with SERT or truncated/mutant forms are altered with the differences in their levels, and/or with 5HT stimulation (Figure 7E and F). Overall, these data suggest that the 5HT-dependent decrease in the surface expression of SERT directly correlates with its binding to phosphovimentin. Therefore, we hypothesize that in cells stimulated with a high level of 5HT, the surface density of SERT is decreased due to an increase in its association with phosphovimentin. In exploring this hypothesis, we tested the 5HT uptake rates and level of SERT on the cell surface in CHO cells transfected with 5HT-dependent phosphorylation site mutant vimentin, S56A and SERT. The impact of 5HT-stimulation on plasma membrane density of transporter The amount of SERT on the plasma membrane is one of the important factors in determining the 5HT uptake rates of cells, which is controlled in a dynamic manner by the relative rates of transporter recycling from endosomes and internalization from the cell surface. In evaluating the role of phosphovimentin-SERT association on the surface expression of transporter, CHO cells co-expressing SERT and the vimentin S56A mutant were used in biotinylation assays followed by quantitative W/B either with SERT- or with pS56-Ab (Fig. 8A). 10.1371/journal.pone.0004730.g008Figure 8 Effect of SERT-vimentin interaction on the surface expression of SERT. (A) CHO-SERT cells were pretreated with 0, 10, or 100 μM 5HT and biotinylated with NHS-SS-biotin (15, 16, 19). Biotinylated plasma membrane proteins were retrieved on streptavidin beads and analyzed for SERT or phosphovimentin- (pS56-) Ab by W/B. All lanes contain protein recovered from the same number of cells equivalent to one well from a confluent 24-well culture plate. (B) Intracellular SERT, vimentin, and phosphovimentin were also determined by W/B analysis of non-bound material. Mock-transfected CHO cells served as a control. Vimentin served as a loading control. The results of W/B analysis are the summaries of combined data from three densitometric scans. represent differences from 0 μM 5HT-matched samples. All lanes contain protein recovered from the same number of cells equivalent to 30% of one well from a confluent 24-well culture plate. (C) CHO cells expressing SERT and phosphovimentin mutant S56A were first pretreated with (0–100 μM) 5HT, and then their 5HT uptake rates were measured in intact cells as described under “Materials and Methods.” Background accumulation of (3H)-serotonin was measured in the same experiment using mock-transfected cells and subtracted from each experimental value. Maximum background accumulation was 0.01 pmol/mg protein/min. Rate of uptake is expressed as the means and SD values of triplicate determinations from three independent experiments. ANOVA indicated that the interaction between 5HT level and cell line was highly significant (p<0.001) for both figures (B and C). Post-hoc comparisons were adjusted using a Bonferroni correction. Significant comparisons with the uptake rates of mock transfected CHO cells within 5HT level are indicated with *, and with 0 μM 5HT within cell line are indicated with # (all p≤0.001). The plasma membrane density of SERT in CHO-SERT cells stimulated with 100 μM 5HT appeared lower than in cells stimulated with 10 μM 5HT. The quantification of these data is summarized in Table 2. 10.1371/journal.pone.0004730.t002Table 2 Effect of 5HT pretreatment on SERT Expression. Percent change of SERT in 5HT pretreated cells compare to untreated cells CHO-SERT CHO-(SERT+S56A) CHO-SERT CHO-(SERT+S56A) SERT expression on cell membrane SERT expression in cell lysate 10 μM (5HT)ex 44.4% ↑ 47% ↑ 35% ↓ 35% ↓ 100 μM (5HT)ex 5.7% ↓ 40% ↑ 5% ↑ No change In CHO-SERT and CHO-(SERT+S56A) cells, the effect of 5HT pretreatment on the surface density of SERT proteins was tested at two different concentrations: at low (10 μM) and high (100 μM) 5HT. Twenty-four hour post-transfected cells were pretreated with 5HT and biotinylated with NHS-SS-biotin [18], [23], [26]. Intracellular SERT and biotinylated plasma membrane proteins were analyzed with W/B with SERT antibodies. The results of W/B analysis are the summary of combined data from three densitometric scans denoted as the percent change of SERT density in 5HT pretreated cells compared to untreated cells. Stimulation with 10 μM 5HT increased the density of SERT on the plasma membrane of CHO-SERT cells 44.4%, whereas 100 μM 5HT stimulation did not show this enhancement on the density of SERT on the plasma membrane compared to untreated CHO-SERT cells. At high concentrations, 5HT stimulation reduced the plasma membrane density of SERT and resulted in a loss of uptake function in platelet system (30%) that was more severe than that in the heterologous system (5.7%). Apparently, these differences are due to the factors involved in the translocation of SERT from/to the plasma membrane, which are either not found in endogenous and heterologous expression systems equally, or the expressions levels in both systems are not stochiometrically sufficient to play their roles correctly. Additionally, the immunoblots revealed that in cells stimulated with 100 μM 5HT, the transporters bound significant amounts of phosphovimentin on the plasma membrane (Fig. 8A). Next, we tested the impact of SERT-phosphovimentin association on the plasma membrane density of SERT. CHO cells were co-transfected with SERT and pS56A constructs, stimulated with 10 or 100 μM 5HT, and then subjected to cell surface biotinylation. Immunoblots of the biotinylated membrane proteins demonstrated that in the absence of phosphovimentin, 100 μM 5HT-stimulation kept the plasma membrane density of SERT at the level found in 10 μM 5HT-stimulated levels (Fig. 8A and B). We wanted to follow up these findings by correlating the biochemical characteristics of CHO-(SERT+pS56A) cells with their 5HT uptake measurement (Fig. 8C). Cells co-expressing the phosphorylation mutant form of vimentin, pS56A and transporter, did not reveal the wild-type phenotype of 5HT-downregulated 5HT uptake. In this respect, they behaved identically to the cells stimulated with 10 μM 5HT. Discussion The plasma membrane level of SERT is altered by the rate of the translocation transporter protein to/from the plasma membrane which is controlled through its interaction with other proteins in these pathways. It was well documented that the plasma level of 5HT plays a role in the density of SERT on the plasma membrane via PKC-mediated phosphorylation of SERT [12], [22]. Additionally, the C-terminus region of SERT is vital to the ability of these transporters to function [14]. Our studies here identify a novel pathway by correlating how plasma 5HT plays a role on the translocation of SERT from the plasma membrane via using the C-terminus region of transporter. The density of SERT on the plasma membrane is modulated by its interaction with other proteins such as an adaptor protein, Hic-5 plays a role in the internalization of SERT in platelets [13]. Also, the C-terminal region of SERT was identified as a domain of interaction with the actin cytoskeleton [8]. Relevant findings include that the C-terminal of SERT interacts with MacMARCKS, a substrate of PKC that binds to the actin cytoskeleton, and the fact that PKC modulators, such as β-PMA, modulate the activity of SERT [8], [10], [11], [23]. Our data further support this contention by demonstrating that C-terminal truncated forms of SERT show a loss of functional membrane trafficking. This loss of function may relate to the level of interact between SERT and cytoskeleton network. Our studies with SERT in transient transfection systems reveal that the truncation of various lengths of the C-terminus altered the 5HT uptake rate of SERT transporters. Truncation of the final 26 and 20 amino acid residues of SERT completely abolished uptake, whereas truncation of the final 14 and 6 residues resulted in a 12% to 18% loss in transport capacity as compared to full length SERT. These results agree with published reports for NET and SERT [14], [24, respectively], which demonstrate that truncation of the C-terminus abolished the uptake rates of these transporters. However, a single residue removal from the C-terminus of NET caused a 60% reduction in uptake capacity [24]. Here, we demonstrate that truncation of the final 14 residues of SERT resulted in a transporter that still retained approximately 90% of its 5HT uptake rate. Further analysis of the difference between Δ20 and Δ14 truncations of the SERT C-terminus, which retained 0% compared to 90% transport rates, respectively, revealed the sequence SITPET. Within this region, there are 3 potential phosphorylation sites at S611, T613, and T616. Several studies have demonstrated that PKC modulators, such as βPMA, reduce SERT localization on the plasma membrane and blunt 5HT uptake capacity [1], [11], [12], [22], [23], [25]–[28]. Additionally, these studies also established an interaction of PP2A, a component of the protein phosphatase complex, with SERT [29]–[31]. Based on these findings, we analyzed the effects of phosphorylation-mimicking amino acids on the 5HT uptake rate of SERT. Our results indicate that S611 may be a key site for phospho-regulation, since the single mutation of S611 to D caused a 61% decrease in 5HT uptake rate whereas the single mutation of S611 to A caused no reduction in 5HT uptake. T613 and T616 individually do not appear to be critical phospho-regulatory residues since neither mutation (A or D) of T613 and T616 showed a similar level of 5HT uptake rate. However, it is possible that these sites work in conjunction with each other to modulate the function of the transporter since our results indicate that the triple mutation DDD of 611, 613, and 616 retained only 5% of its 5HT uptake capacity as compared to control SERT. It is also important to note that the presence of such a large amount of negative charge on the end of the protein could cause alterations in protein folding or protein-protein associations that are important for protein function, resulting in the observed blunting of transport capacity. Next, we analyzed the impact of four truncations of the SERT C-terminus on the trafficking and expression of SERT on the plasma membrane using biotinylation and IF assays. Our data indicate that depending on the amount of truncation from the C-terminus of SERT, there was altered localization of the transporter. Therefore, we carried out a biotinylation analysis on some of the phosphorylation-mimicking mutations in an effort to determine the plasma membrane localization of these mutants, i.e., whether the mutation arrests them intracellularly or whether the mutants can still traffic to the plasma membrane. The data indicate that 3 possible phosphorylation sites do contribute to the 5HT uptake rates of transporters via inhibiting their proceedings toward the plasma membrane. The proteins involved in the membrane trafficking of SERT are still under investigation by many laboratories. Our biochemical and proteomic analysis of the platelet proteins associated with the C-terminus of SERT demonstrate an association between vimentin, an intermediate filament, and SERT in platelets. Association also was noted in a CHO heterologous expression system. Vimentin is the major type III intermediate filament expressed in cells of mesenchymal (e.g. endothelium, fibroblasts, megakaryocytes) and myogenic origin [32]. Vimentin, a minor component of the platelet cytoskeleton [33], is associated with the Triton X-100 insoluble fraction of human platelets [34], [35]. Studies have shown that vimentin forms a network of intermediate filaments that form a ring close to the cell membrane, as well as a network that activates PAK-dependent phosphorylation of vimentin, altering the filamentous structure of this cytoskeletal compound. Our studies demonstrate an association between vimentin and SERT in the cytosol and on the plasma membrane of platelet and CHO-expression system within 5HT-independent manner. Collectively, these findings point to vimentin as a possible candidate for facilitating the translocation of SERT between intracellular compartments and plasma membrane. When 5HT-stimulation dependent phosphorylation was eliminated, the surface expression and the 5HT uptake rates of SERT were restored in CHO cells treated with 100 μM 5HT. Of the several C-terminus mutant transporters, only Δ14 and S611D did not associate with vimentin although both could appear on the plasma membrane in active form. Additionally, neither the plasma membrane expressions of these two mutant transporters were decreased in 100 μM 5HT-treated cells, nor they were able to bind phosphovimentin. These data strengthen our hypothesis that the modification of the SITPET sequence differentially, one amino acid at a time, exposes the vimentin binding domain on the C-terminus of SERT. However, elevated plasma 5HT controls the cellular distribution of SERT on an “altered” vimentin network, the translocation of SERT from the plasma membrane is accelerated on the 5HT-altered vimentin network. Thus, in plasma of hypertensive subjects in which 5HT reaches a high level, the platelet SERT may continue to clear plasma 5HT with a lower Vmax, most likely until the plasma 5HT levels come back to the physiological level [16]. Therefore, to the best of our knowledge, this is the first study to identify a sequence on the C-terminus of SERT that regulates the rate of 5HT uptake by altering the density of SERT on the plasma membrane via differential phosphorylation of SITPET sequence, which facilitates the association of SERT with an intermediate filament, vimentin. Recent investigations indicate a system of phosphorylation for SERT that incorporates two phases of phosphorylation [11]. The first phase of phosphorylation is said to affect the serine residues, whereas the second phase involves the threonine residues. It is suggested that the first phase of phosphorylation causes the transporters to shut down, and the second phase of phosphorylation tags the proteins for internalization via the SERT recycling mechanism. According to the biphasic model, a S611D construct should shut down the uptake ability of the transporter while the DD construct should demonstrate a reduced or eliminated 5HT uptake capacity due to its intracellular localization. Indeed, our data agree with the study by Jayanthi et al. [11], who reported that S611D reduces transport capacity by ∼39%, whereas DD (T613+T616) demonstrates an uptake capacity of ∼16%. On the basis of our findings, we hypothesize that the blunted activity (∼39%) of S611D may be due to additional serine residues that play a role in reducing the uptake capacity of SERT. A finding that was not consistent with the biphasic theory was the localization of S611D, which is mainly found at intracellular locations. In summary, in an endogenous platelet system and in heterologous expression systems, our studies demonstrate that vimentin associates with SERT. The last 20 amino acids from the C-terminus of SERT are required and are at least one of the binding-domain(s) of vimentin. SERT becomes a bridge between vimentin and the plasma membrane. At physiological plasma 5HT levels, vimentin-SERT association was found at intracellular locations and on the plasma membrane (Fig. 7B). However, when plasma 5HT level was higher than physiological level, their association was enhanced and the level of SERT on the plasma membrane was decreased. Therefore, we hypothesize that SERT utilizes the vimentin network during translocation from the plasma membrane. Furthermore, the 5HT-dependent phosphorylation of vimentin on the S56 residue accelerates the translocation of SERT on the 5HT-altered vimentin network. Future analysis of these mutants in stable transfection systems, as well as continued experiments with the phospho-mimicking mutants presented here, will further reveal the mechanism of action that governs transporter C-terminal phosphorylation. These studies also will advance our understanding of the specific processes by which phosphorylation of the C-terminus plays a role. Supporting Information Table S1 (0.03 MB PDF) Click here for additional data file. We thank Ms. Shelly Lensing for critical review of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: Support provided by the National Science foundation (MCB-0234822; MCB-0645163 to V.L.) and the National Institute of Child Health and Human Development (HD053477 to FK) and American Heart Association (0660032Z to FK). 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==== Front PLoS GenetPLoS GenetplosplosgenPLoS Genetics1553-73901553-7404Public Library of Science San Francisco, USA 1930049008-PLGE-RA-0927R310.1371/journal.pgen.1000424Research ArticleGenetics and Genomics/Genetics of DiseaseGenetics and Genomics/Genetics of the Immune SystemRheumatology/Rheumatoid ArthritisAn African Ancestry-Specific Allele of CTLA4 Confers Protection against Rheumatoid Arthritis in African Americans CTLA4 SNP Associated with Protection from RAKelley James M. 1 Hughes Laura B. 1 Faggard Jeffrey D. 1 Danila Maria I. 1 Crawford Monica H. 1 Edberg Yuanqing 1 Padilla Miguel A. 1 Tiwari Hemant K. 1 Westfall Andrew O. 1 Alarcón Graciela S. 1 Conn Doyt L. 2 Jonas Beth L. 3 Callahan Leigh F. 3 Smith Edwin A. 4 Brasington Richard D. Jr 5 Allison David B. 1 Kimberly Robert P. 1 Moreland Larry W. 1 ¤ Edberg Jeffrey C. 1 Bridges S. Louis Jr 1 * 1 University of Alabama at Birmingham, Birmingham, Alabama, United States of America2 Emory University, Atlanta, Georgia, United States of America3 University of North Carolina, Chapel Hill, North Carolina, United States of America4 Medical University of South Carolina, Charleston, South Carolina, United States of America5 Washington University School of Medicine, St. Louis, Missouri, United States of AmericaMarchini Jonathan EditorUniversity of Oxford, United Kingdom* E-mail: [email protected]¤: Current address: University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America Conceived and designed the experiments: JMK LBH JCE SLB. Performed the experiments: JMK JDF YE. Analyzed the data: JMK LBH MID MAP HKT AOW DBA SLB. Contributed reagents/materials/analysis tools: GSA DLC BLJ LFC EAS RDB RPK LWM JCE SLB. Wrote the paper: JMK LBH MID MHC SLB. 3 2009 20 3 2009 5 3 e100042423 7 2008 17 2 2009 Kelley et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is ∼1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462×10−26, Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13–0.26, p = 2.4×10−28, Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF = 0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations. Author Summary Rheumatoid arthritis (RA) is a systemic autoimmune condition affecting the synovial membranes of diarthrodial joints. The etiology of RA is unclear but is thought to result from an environmental trigger in the context of genetic predisposition. We report that a single nucleotide polymorphism (SNP) (rs231778) in CTLA4, which encodes a negative regulator of T cell activation, is associated (p = 2.4×10−28) with protection from developing RA among African Americans. rs231778 is only polymorphic in populations of African ancestry. Protective alleles such as this one may contribute to the purported lower prevalence of RA in African Americans. Our finding appears to be independent from confounding by linkage with the HLA-DRB1 shared epitope or by genetic admixture. Furthermore, we did not replicate associations of CTLA4 SNPs with RA or other autoimmune diseases previously reported in Asians and Caucasians, such as rs3087243 (+60C/T) and rs231775 (+49A/G). The associations of different SNPs with RA susceptibility specific to different populations highlight the importance of CTLA4 in the pathogenesis of RA and demonstrate the ethnic-specific genetic background that contributes to its susceptibility. ==== Body Introduction Cytotoxic T-lymphocyte associated protein 4 (CTLA4, CD152) is a negative regulator of T-cell activation. As the T-cell activation signal propagates due to costimulatory B7 molecule (CD80, CD86) binding of CD28, cell surface expression of CTLA4 increases to compete with CD28 [1]. CTLA4 also prevents further clonal expansion of effector T-cells, including regulatory T cells (Treg) [2],[3], and can inhibit osteoclast formation [4]. Genetic variation in CTLA4 (Chromosome 2q33) could contribute to unchecked T cell or osteoclast activation with resultant onset of autoimmune disease such as rheumatoid arthritis (RA). CTLA4 was modestly associated with RA in a recent genome wide association study (GWAS) of RA in Caucasians [5]. CTLA4 single nucleotide polymorphisms (SNP), such as rs231775 (+49A/G), have been associated with multiple autoimmune conditions including RA, Addison's disease, autoimmune pancreatitis [6], autoimmune thyroid disease, celiac disease, chronic inflammatory arthritis [7]. multiple sclerosis [8], type I diabetes mellitus, Sjögren's syndrome [9], and systemic lupus erythematosus (SLE) [10]. An association with another SNP, rs3087243 (+60C/T), and RA was found in a Chinese Han population [11]; however, these results were not replicated in Irish [7], United States Caucasian [12], or, when corrected for multiple testing, British Caucasian [13] populations. Analysis of a much larger group of Caucasians from North America and Sweden associated this marker with RA [particularly with the anti-cyclic citrullinated peptide (anti-CCP) antibody positive RA subset] [14]. Given the association of CTLA4 with multiple diseases in various populations, we sought to characterize the genetic contribution of CTLA4 to RA in African Americans – a population not yet explored. RA is purported to be less prevalent in African Americans than in Caucasians based on clinical observation and data in black continental Africans [15]–[19]. African-specific protective alleles might explain the lower disease prevalence among persons of African ancestry and should be evaluated in genetic studies with this population. In this study, we genotyped CTLA4 haplotype tagging SNPs (htSNPs) in two groups totaling 505 African American patients with RA and 712 African American healthy controls. We found and replicated a novel protective association at an ethnic-specific intronic SNP, rs231778, in both independent groups. While this SNP is polymorphic only in the HapMap Yoruba population, we confirmed a lack of variation by genotyping 530 Caucasians. Importantly, we did not detect significant confounding for association of rs231778 when our patients were stratified by level of European admixture or by RA subclassification such as presence of the HLA-DRB1 shared epitope (SE) or anti-cyclic citrullinated peptide (anti-CCP) antibodies [20]. We also did not find association with two SNPs (rs3087243 and rs231775) previously reported to have disease associations with RA in European ancestry populations or with other autoimmune diseases. Our data reveal a protective African ancestry-specific allele that may contribute to the purportedly lower prevalence of RA in persons of African ancestry and provide suggestions for future research into the relationship between T cell regulation and RA pathogenesis. Methods The Consortium for the Longitudinal Evaluation of African Americans with Early Rheumatoid Arthritis (CLEAR) Registry enrolled self-identified African Americans with RA who met the American College of Rheumatology (ACR) 1987 diagnostic criteria [21]. Participants for CLEAR were recruited from the University of Alabama at Birmingham (UAB) [coordinating center]; Emory University/Grady Hospital (Atlanta, GA); University of North Carolina at Chapel Hill; Medical University of South Carolina (Charleston, SC); and Washington University (St. Louis, MO). Recruitment occurred in two phases: enrollment of patients with early RA (<2 year disease duration) followed longitudinally until 5 years disease duration, from 2000 to 2007 (CLEAR I); and enrollment of patients with RA of any duration from the same sites as part of a cross-sectional analysis from 2007 to present (CLEAR II). Comprehensive demographic, clinical, and radiographic data are being collected on all CLEAR participants, and serum and DNA samples are being stored [22]. These data allow for stratification of RA patients [20] by presence of the HLA-DRB1 SE and anti-CCP antibody positivity. We have also measured estimated global admixture using a panel of ancestry informative markers (AIMs), as previously reported [23]. A group of healthy African American controls, for the longitudinal arm of this study, with similar sex, age, and geographic location has been recruited, as previously described [23]. All participants were recruited with informed consent under the approval of each respective Institutional Review Board. Genomic DNA was isolated using standard methods and stored at −70°C. This study included 282 African American RA patients and 149 African American controls from the CLEAR longitudinal study (CLEAR I) and 223 African American RA patients from the CLEAR cross-sectional study (CLEAR II). We also obtained DNA samples from an additional 563 healthy African Americans from Alabama recruited for a case-control study of SLE [24] to use as controls for the CLEAR II RA patients. Demographics for CLEAR I and CLEAR II RA patients are presented in Table 1. Controls were younger than the RA patients (mean age: CLEAR I = 45±14 years, CLEAR II = 35±11 years). Similar to the patient groups, both of the control sets were predominantly female (percent female: CLEAR I = 82%, CLEAR II = 74%). In total, we analyzed 505 African American RA patients and 712 African American controls. We used RA patients and controls from CLEAR I as an initial test set and RA patients from the CLEAR II and additional Alabama controls as a replication group. 10.1371/journal.pgen.1000424.t001Table 1 Demographics of populations used in this study. CLEAR I (Longitudinal) [n = 282] CLEAR II (Cross-Sectional)[n = 223] P value Age at onset, average (±SD) 55.6 (±13.3) 57.5 (±11.2) NS % Female 82.2 86.3 NS Disease Duration (yrs), mean (±SD) 1.6 (±0.6) 5.9 (±1.6) <0.0001* Rheumatoid Factor Positive 72.8 80.3 NS Anti-CCP Antibody Positive 62.0 60.8 NS % HLA-DRB1 Shared Epitope Positive 42.7 39.2 NS Anti-CCP – anti-cyclic citrullinated peptide; SD – standard deviation; NS – not significant. * Inclusion criteria were <2 years disease duration for CLEAR I and any disease duration for CLEAR II. All SNPs within the CTLA4 region (±2 kb) that have a minor allele frequency (MAF) ≥0.05 in the Yoruba HapMap population (Phase II/Release 21) were genotyped: rs231775, rs231776, rs231777, rs231778, rs231779, and rs3087243. Data from the resequencing of CTLA4 in both African and European populations contracted to SeattleSNP (Dr. Debbie Nickerson, University of Washington) were kindly provided from the Population Genetics Study coordinated at UAB (Drs. Richard Kaslow and Robert Kimberly). CTLA4 SNPs detected by SeattleSNP that capture information on polymorphisms not present in HapMap for Africans with a MAF ≥0.05 were additionally genotyped: rs11571319, rs231772, rs231780, rs34031880, rs733618, and 5251. 5251 is not yet listed in dbSNP: its physical location is 54945227 in NCBI contig file NT_005403, and its surrounding sequence is ATGGTAGCCTTGCTTATTGT [G/T] GGTGGCAACCTTAATAGCAT. Genotyping was performed by the Illumina FastTrack GoldenGate BeadXpress genotyping service (San Diego, CA) for CLEAR I for SNPs from the International HapMap Consortium. All other genotyping was performed using Applied Biosystems TaqMan Allelic Discrimination Assays (Foster City, CA) on an ABI 7900HT Genetic Analyzer. Overall, between both platforms for all SNPs, our genotyping success rate was 99.4%. We successfully genotyped rs231778 among 74 samples using both platforms with 100% reproducibility. To confirm the monomorphic nature of rs231778, we genotyped this SNP in 530 Caucasian samples from the UAB Treatment of Early Aggressive Rheumatoid Arthritis (TEAR) study. Fisher's exact tests were performed on SAS 9.0 (Cary, NC) and exact logistic regression tests performed on LogXact 8.0 (Cambridge, MA). We controlled for potential confounding by HLA-DRB1 status, anti-CCP antibody positivity, and genetic admixture following the approach of Redden et al. [25]. Linkage Disequilibrium and haplotype analyses were performed with HaploView v3.31 [26]. All SNPs were in Hardy-Weinberg Equilibrium (tested with Chi squared tests), except rs231776 (HWE p = 0.0085), which was excluded from further analysis. Results Resequencing and Description of Genetic Variation in CTLA4 Data available from the International Haplotype Mapping Consortium (HapMap), as accessed in February 2008, appear incomplete with regard to coverage of CTLA4. Only SNPs present from the 5′ region through intron 1 (rs231775, rs231776, rs231777, rs231778, rs231779) are represented with detailed genotyping data. HapMap does not provide data for SNPs among the remaining exons and introns of CTLA4 but does present information for polymorphisms in the 3′ end of the gene, such as rs3807243. To select htSNPs that cover the remaining interior portions of this gene, we accessed resequencing data available from SeattleSNP that provided detailed genotypes on YRI and CEU populations. SeattleSNP routinely resequences only 500 basepairs into each end of a given intron. A portion of intron 1 (the longest intron) is the only region of CTLA4 not completely resequenced by SeattleSNP; however, intron 1 was completely covered by HapMap, allowing the combination of these two resources to provide the most detailed haplotype tagging strategy for this gene. See Figure 1. 10.1371/journal.pgen.1000424.g001Figure 1 Only SNPs genotyped in this study are listed by name. SNPs in linkage disequilibrium with SNPs genotyped in this study are shown in brown. SNPs in gray were not genotyped or haplotype tagged due to low minor allele frequency (MAF). SNP - Single Nucleotide Polymorphism. 5251* is not yet registered in dbSNP as described in the methods. Due to the limited public information on CTLA4 in African Americans, we used HaploView to calculate linkage disequilibrium (LD) across all genotyped SNPs. A plot representing the LD (r2 values) of SNPs is included as Figure 2. 10.1371/journal.pgen.1000424.g002Figure 2 This plot, generated from HaploView v3.31 (Broad Institute), shows the r2 values between SNPs genotyped in CTLA4. Darker boxes represent stronger r2 values. 5251* is not yet registered in dbSNP as described in the methods. Association of rs231778 with Rheumatoid Arthritis in African Americans We detected a protective effect for RA in African Americans with the G allele of rs231778 in both CLEAR study groups (longitudinal and cross-sectional) independently and together (CLEAR I and CLEAR II combined Fisher's exact p = 4.46×10−26). See Table 2. Because homozygotes for the G allele were rare, we compared the frequency of persons with genotypes GG and AG to those with genotype AA. From the odds ratios of the two groups combined, it can be seen that the presence of the G allele confers a protective effect (OR = 0.19, 95% CI: 0.13–0.26, p = 2.4×10−28, Fisher's exact). See Table 3. rs231778 is not in LD with any other SNP, which suggests any genetic effect it confers is likely independent. See Figure 2. 10.1371/journal.pgen.1000424.t002Table 2 Association with rheumatoid arthritis at rs231778 in African Americans. Genotype AA AG GG All Fisher's Exact P value Longitudinal RA 249 (0.90) 25 (0.09) 2 (0.01) 276 1.572×10−8 Controls 99 (0.67) 44 (0.30) 4 (0.03) 147 Cross-sectional RA 186 (0.89) 21 (0.10) 1 (0.01) 208 5.093×10−14 Controls 324 (0.59) 202 (0.37) 24 (0.04) 550 Combined RA 435 (0.90) 46 (0.10) 3 (0.01) 484 4.462×10−26 Controls 433 (0.62) 236 (0.34) 28 (0.04) 697 Values indicate number of samples with allele frequency in parentheses. Analysis compares number of patients and controls for each genotype (AA vs AG vs GG) in a 3×2 Fisher's exact test. 10.1371/journal.pgen.1000424.t003Table 3 Odds ratios for the protective effect of the rs231778 G allele in African Americans with RA. OR 95% CI Fisher's Exact P value Longitudinal 0.23 0.13–0.39 2.127×10−8 Cross-sectional 0.17 0.10–0.28 3.354×10−17 Combined 0.19 0.13–0.26 2.437×10−28 Analysis compares number of patients and controls for genotype AA vs those with either AG or GG in a 2×2 Fisher's exact test. OR – odds ratio; CI – confidence interval. The G allele of rs231778 is relatively specific for African populations as only the A allele is detected among Asians and Caucasians genotyped in the International HapMap Project and in Caucasians genotyped by SeattleSNP. Since variation at rs231778 was not found in the HapMap (n = 24) or Perlegen (n = 60) based European samples, we genotyped an additional 530 self-identified Caucasians to assess ethnic specific variation at this site. Among these 530 subjects, only 3 were heterozygous at rs231778, and none were homozygous for the G allele, which yields a MAF of 0.0028. In the 697 healthy African American individuals we genotyped, the MAF is 0.209 illustrating the ethnic specificity of this marker. Since the presence of the SE has been associated with susceptibility to RA in our population [23] and known to confound association with RA at other immunologically relevant loci such as PTPN22 [27], we evaluated our findings in CTLA4 for possible confounding by the HLA-DRB1 SE, the strongest known genetic risk factor for RA. We found that the MAF of rs231778 was not different within cases or controls when stratified for number of SE alleles present. Because only 4 control samples have two SE alleles, we cannot rule out any possible influence of the SE on the genetic contribution of CTLA4 in RA susceptibility, but it appears to be unlikely. See Table 4. 10.1371/journal.pgen.1000424.t004Table 4 Minor allele frequency (MAF) of rs231778 segregated by HLA-DRB1 shared epitope status. 0 SE Alleles 1 SE Alleles 2 SE Alleles Total Patients 0.05 (N = 12/274) 0.06 (N = 9/160) 0.06 (N = 2/34) 0.05 Controls 0.21 (N = 27/130) 0.21 (N = 8/38) 0.25 (N = 1/4) 0.21 This table combines samples from both CLEAR I and CLEAR II and only incorporates data where HLA-DRB1 shared epitope (SE) status has been determined. The ‘N’ listed below each MAF represents the number of ‘G’ alleles over the total number of alleles for each number of SE alleles present. SE – HLA-DRB1 shared epitope. Since our study focuses on African Americans, a group with known recent population admixture [28], we assessed percentage of European admixture as a confounding factor in the association of RA with rs231778. Data from a genome-wide admixture panel performed at the Broad Institute from our previously reported work [23] allowed calculations of global admixture estimates (percent European ancestry) for 282 cases and 94 controls (total N = 366). Of these 366 with admixture data, there was successful genotyping for the CTLA4-containing region of Chromosome 2 in 266 cases and 81 control samples (total N = 347). We show the mean percentage of European ancestry segregated by genotype for cases and controls in Table 5. The degree of admixture was not associated with rs231778 genotype (Fisher's exact p = 0.2367). We confirmed that admixture difference between cases and controls was not significant using the robust Welch test, which produced a value of 2.308 (degrees of freedom = 215.578, p = 0.130). 10.1371/journal.pgen.1000424.t005Table 5 Mean percentage of European ancestry segregated by genotype of rs231778. Genotype AA AG or GG AA, AG, or GG Case 0.172 (0.0997) N = 240 [246] 0.114 (0.062) N = 26 [27] 0.166 (0.098) N = 266 [273] Control 0.146 (0.052) N = 67 [75] 0.169 (0.078) N = 14 [18] 0.150 (0.057) N = 81 [93] Total 0.166 (0.092) N = 307 [321] 0.133 (0.072) N = 40 [45] 0.162 (0.090) N = 347 [366] This table provides data for the 347 samples with complete admixture and rs231778 genotyping data. Standard error is provided in parenthesis. Brackets indicate frequency counts used in determining association of the rs231778 G allele with rheumatoid arthritis among 366 samples, as described in the text. We did not find a significant association with RA of the G allele among the 347 samples with complete admixture data and CTLA4 genotypes (asymptotic p = 0.0674); we suspect that this is due to the reduced statistical power of analysis of a smaller number of subjects and controls. When we based calculations upon the 366 samples used in our previous admixture-based manuscript [23], this small increase in sample size regained statistical significance of association with RA (asymptotic p = 0.0183). To illustrate further the lack of significance among the 347 samples is due to lack of power, frequency counts of genotypes among the 366 samples are incorporated in Table 5 to demonstrate a similar pattern of genotype distributions with and without these additional samples. Lack of Genetic Associations with RA at Other CTLA4 Loci Nonsynonymous SNPs previously associated in other populations and autoimmune phenotypes (rs3087243 and rs231775) were not associated with RA in our study. See Table 6. We also found no association when we analyzed data based upon deduced haplotypes or at any individual SNP when stratified by RA subclassification (SE status, anti-CCP antibody status, or percent European ancestry) as has been observed with RA associations at other sites in the genome [23] and with CTLA4 SNPs in Caucasian populations [14]. See Table 6. 10.1371/journal.pgen.1000424.t006Table 6 Minor allele frequencies of CTLA4 SNPs for African American RA patients and controls. SNP Minor Allele Position CCP+ Patients CCP− Patients SE+ Patients SE− Patients All Patients Controls Public Database: African Public Database: European Fisher's Exact P values rs733168 C −1722 0.134 0.128 0.158 0.113 0.123 0.142 0.15 0.07 0.0113 rs231772 A −1113 0.030 0.031 0.034 0.033 0.028 0.053 0.09 0.00 0.0462 rs231775 G 48 0.424 0.392 0.442 0.381 0.413 0.372 0.42 0.39 0.2827 rs231776 A 184 0.023 0.031 0.034 0.026 0.027 0.038 0.10 0.00 0.0418 rs231777 T 922 0.240 0.232 0.252 0.238 0.244 0.220 0.13 0.27 0.7651 rs231778 G 1155 0.053 0.046 0.063 0.046 0.054 0.209 0.09 0.00 4.462×10−26 rs231779 T 1821 0.464 0.448 0.490 0.424 0.460 0.452 0.42 0.38 0.3718 5251* A 2107 0.060 0.041 0.034 0.066 0.045 0.040 0.08 0.00 0.8275 rs231780 G 4031 0.082 0.119 0.102 0.096 0.097 0.124 0.17 0.00 0.0667 rs34031880 C 6222 0.036 0.067 0.054 0.043 0.045 0.063 0.04 0.00 0.2222 rs3087243 A 6253 0.161 0.186 0.170 0.169 0.167 0.195 0.17 0.41 0.6343 rs11571319 A 6272 0.217 0.180 0.199 0.215 0.197 0.141 0.12 0.20 0.0731 This table represents combined data from CLEAR I and CLEAR II. rs3087243 (+60C/T) has been associated with RA in Caucasians, and rs231775 (+49A/G) has been associated with other autoimmune conditions. Position is the physical location of each polymorphism relative to the start codon (position = 0) in dbSNP build 129. CCP refers to anti-cyclic citrullinated antibody status. SE refers to presence of HLA-DRB1 shared epitope. Public databases include allele frequencies from HapMap or SeattleSNP. rs231776 was not in Hardy-Weinberg Equilibrium. 5251 is not currently listed in dbSNP as described in the methods. We found no significant association with the G allele of rs3087243, even when stratified for presence of anti-CCP antibody, as previously reported in Europeans with RA [14]; the distribution of genotypes and allele frequencies of this SNP were similar in anti-CCP antibody-positive and anti-CCP antibody-negative RA patients. Similarly, we found no significant differences in allele frequency between anti-CCP positive RA patients and anti-CCP negative RA patients at the SNP associated with RA in our study (rs231778). In the initial analysis of the CLEAR longitudinal arm, we found a protective effect (lower allele frequency in patients than controls) of the minor allele (G) of rs231780 allele (Fisher's Exact p = 0.0123). However, upon replication in the CLEAR cross-sectional arm, this difference in MAF between cases and controls was not significant in the cross-sectional arm or in both arms combined [Fisher's exact p = 0.0667; MAF 0.097 in patients, 0.124 in controls]. See Table 6. Of note, the rs231780 SNP appears to be African-ancestry specific as well, with a MAF of 0.17 in Africans and ∼0.00 in Europeans among HapMap subjects. It is possible that our lack of association at this marker is due to a true negative state or due to lack of power for detecting a positive association, as our the p value is bordering on significance (p = 0.07). Although rs231780 is also an ethnic-specific SNP, there is not significant LD between it and the strongly associated SNP rs231778 (r2 = 0.107, D′ = 0.445). The lack of association of the African-specific SNP, rs231780, with RA might be sufficient to rule out genetic admixture as the cause of the association at rs231778. We also found an association with rs231776 (Fisher's exact p = 0.0418) when both study groups were combined; however, this SNP was not in Hardy-Weinberg equilibrium (HWE p = 0.0085), complicating interpretation of these results. Discussion We detected a significant novel genetic association with RA in African Americans at the CTLA4 SNP rs231778. In this case-control study, African Americans with at least one minor (G) allele were 0.19 times as likely to have RA as those without a minor allele (95% CI 0.13 0.26, Fisher's Exact p = 2.437×10−28). This P value does not appear to be subject to the inaccuracy introduced by cancellation error by complementation [29]. Our study is limited in sample size due to its exclusive focus on a minority population, which may introduce influence by bias in sample collection, genotyping errors, and lack of power. However, due to our efforts in matching patients and controls and validating our genotyping results (100% reproducibility in 74 samples on different genotyping platforms), we believe such biases have been minimized. We believe that our study is sufficiently powered to detect associations as we found a statistically significant result in two separate arms of the study. The associated SNP, rs231778, is located in intron 1 and is not in LD with any genotyped SNP in CTLA4 such as the disease-associated rs3087243 and rs231775 markers. See Figures 1 and 2. It is possible, however, that LD could span farther than assessed in this study allowing the possibility that rs231778 is a surrogate marker for another associated polymorphism well outside of the gene boundaries of CTLA4. LD has been shown to span several megabases in African Americans, which supports this possibility [30]. Additional genotyping of 5–10 AIMs in this chromosomal region in a large number of African Americans may allow a better understanding of the long-range haplotype structure. Our study did include five African-specific SNPs (rs231772, rs231776, rs231780, rs34031880, 5251) and one AIM, defined as a difference in MAF >0.20 between populations, (rs3087243) that did not associate with RA. The association of the African-specific allele of rs231778 and RA and the lack of association at these ethnic-specific markers supports the idea that the association of rs231778 is independent from bias by genetic admixture. Interestingly, rs231778 is monomorphic in both Asian and Caucasian populations, according to genotype data from HapMap and SeattleSNP, and virtually absent in our genotyping of 530 Caucasians. Given the ethnic-specific status of this SNP, it is possible that our finding helps to explain the purported, but as yet unproven, observation of a lower prevalence of RA in African Americans compared to Caucasians. We would anticipate the association of such African-specific protective alleles with resistance to RA. Racial or ethnic differences have now been suggested in the association of RA with several genes, including PTPN22 [31], PADI4 [32], SLC22A4 and RUNX1 [33], and in CTLA4, particularly between Asian and Caucasian populations [10],[34]. These data highlight the need for additional research into the genetic background of RA in various populations such as African Americans to uncover additional ethnic-specific associations. Our study included 697 healthy African American controls that possessed a MAF of 0.209 at rs231778. This finding is surprising since public resources such as the International HapMap Consortium has a MAF of 0.09 in their panel of 60 Yorubans and 0.00 in 60 Caucasians. African Americans are considered to be an admixed population with an African background and contribution of approximately 20% European genetic ancestry. In fact, we calculated that European ancestry contributes 15±5% of the genetic composition of African Americans in the CLEAR study. Therefore, we would expect a MAF for rs231778 to be between 0.00 and 0.09. Given that our participants were collected at multiple centers across the Southeastern United States (with each center having similar MAFs), that we genotyped 74 samples with 100% reproducibility on dual platforms (TaqMan and Illumina), and that our study included a larger number of samples (n = 697) than public resources (n = 60), we believe our results are accurate. Such a difference from the expected MAF may be due to reduced power in HapMap compared to this work or due to population stratification (i.e. the MAF of 0.09 for Yorubans in Nigeria could be markedly lower than elsewhere on the continent from where ancestors of our participants may have lived). More work into the genetic population structure across Africa and in admixed populations such as African Americans is needed to appreciate such differences. Population-based differences in susceptibility to RA are observed through previous reports that show an association between RA and rs3087243 (+60C/T), a polymorphism known to affect the expression levels of soluble CTLA4 protein [35], in Swedish and North-American populations [14] or a lack of association at this locus in studies based in Massachusetts or Northern Ireland [7],[13]. We failed to find an association of rs3087243 in RA among African Americans. Even when stratifying for a clinical subclassification more strongly associated with CTLA4 [14] (anti-CCP positivity), we could not reproduce these results in African Americans. This non-replication finding may be due to genuine population-specific differences in allele frequency or different patterns of LD among African and European ancestry individuals, but our relatively small sample size precludes definitive conclusions. For example, to detect a small genetic effect [OR = 1.08 (95% CI: 1.01–1.17)] in a meta-analysis of genotypes, Plenge et al. analyzed ∼4,000 Caucasian RA samples [14], a much higher number of subjects than is available for our analysis. We also failed to find an association with the nonsynonymous SNP, rs231775 (+49A/G), which has been implicated in multiple autoimmune diseases, again possibly due to small sample size. CTLA4 is an important molecule in preventing an inappropriate immune response and in dampening osteoclast formation [4], both of which may have implications for the pathogenesis of RA. CTLA4 stimulation functions in regulatory T cell development including proliferation and frequency [2],[3],[36], providing another possible mechanism for this protein to influence RA pathogenesis. While we do not address possible functional consequences of this polymorphism, future work may reveal a relationship between rs231778 and T cell/osteoclast development or linkage disequilibrium with a SNP outside of the CTLA4 gene boundaries that influences expression or function. In conclusion, our results suggest a need for greater understanding of CTLA4 function and of the ethnic-specific genetic contributions to RA including relationship to disease pathogenesis. We gratefully acknowledge the following physicians who enrolled patients: Jacob Aelion, MD, Jackson, TN; Charles Bell, Birmingham, AL; Sohrab Fallahi, MD, Montgomery, AL; Richard Jones, PhD, MD, Tuscaloosa, AL; Maura Kennedy, MD, Birmingham, AL; Adahli Estrada Massey, MD, Auburn, AL; John Morgan, MD, Birmingham, AL; Donna Paul, MD, Montgomery, AL; Runas Powers, MD, Alexander City, AL; William Shergy, MD, Huntsville, AL; Cornelius Thomas, MD, Birmingham, AL; Ben Wang, MD, Memphis, TN. We gratefully acknowledge staff and coordinators at the following sites: University of Alabama at Birmingham: Stephanie Ledbetter, MS; Zenoria Causey, MS; Selena Luckett, RN, CRNC; Laticia Woodruff, RN, MSN; Candice Miller; Emory University: Joyce Carlone, RN, FNP-BC; Karla Caylor, BSN, RN; Sharon Henderson, RN; University of North Carolina: Diane Bresch, BSN; Medical University of South Carolina: Trisha Sturgill. The CLEAR Registry is a national resource, with clinical data, DNA, and other biological samples available. For details, see the following website: http://www.dom.uab.edu/rheum/CLEAR%20home.htm. The authors have declared that no competing interests exist. This work was supported by NIH N01 AI40068, N01 AR02247, P01 AR40894, R01 AR51394, and NIH GCRC/CTSA grants M01 RR00032, U54 RR025777 (University of Alabama at Birmingham) and M01 RR 000046 (University of North Carolina). Funding bodies had no role in study design; collection, analysis, and interpretation of data; writing of the paper; and decision to submit it for publication. ==== Refs References 1 Noel PJ Boise LH Thompson CB 1996 Regulation of T cell activation by CD28 and CTLA4. Adv Exp Med Biol 406 209 217 8910687 2 Tang AL Teijaro JR Njau MN Chandran SS Azimzadeh A 2008 CTLA4 expression is an indicator and regulator of steady-state CD4+ FoxP3+ T cell homeostasis. 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==== Front BMC MicrobiolBMC Microbiology1471-2180BioMed Central 1471-2180-9-361921674810.1186/1471-2180-9-36Research articleNF-κB activation by Helicobacter pylori requires Akt-mediated phosphorylation of p65 Takeshima Eriko [email protected] Koh [email protected] Hirochika [email protected] Chie [email protected] Shigeki [email protected] Mariko [email protected] Masachika [email protected] Fukunori [email protected] Hitomi [email protected] Chihiro [email protected] Jiro [email protected] Naoki [email protected] Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan2 Division of Control and Prevention of Infectious Diseases, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan3 Division of Child Health and Welfare, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan4 The Japanese Society for the Promotion of Science (JSPS), Japan5 Division of Oral and Maxillofacial Functional Rehabilitation, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan6 Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan7 Department of Endoscopy, University Hospital, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan8 Department of Microbiology and Immunology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan2009 12 2 2009 9 36 36 13 9 2008 12 2 2009 Copyright ©2009 Takeshima et al; licensee BioMed Central Ltd.2009Takeshima et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background The inflammatory response in Helicobacter pylori-infected gastric tissue is mediated by cag pathogenicity island (PAI)-dependent activation of nuclear factor-κB (NF-κB). Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is known to play a role in NF-κB activation, but little information is available on the relationship between H. pylori and PI3K/Akt signaling in gastric epithelial cells. We examined whether H. pylori activates Akt in gastric epithelial cells, the role of cag PAI in this process and the role of Akt in regulating H. pylori-induced NF-κB activation. Results Phosphorylated Akt was detected in epithelial cells of H. pylori-positive gastric tissues. Although Akt was activated in MKN45 and AGS cells by coculture with cag PAI-positive H. pylori strains, a cag PAI-negative mutant showed no activation of Akt. H. pylori also induced p65 phosphorylation. PI3K inhibitor suppressed H. pylori-induced p65 phosphorylation and NF-κB transactivation, as well as interleukin-8 expression. Furthermore, transfection with a dominant-negative Akt inhibited H. pylori-induced NF-κB transactivation. Transfection with small interference RNAs for p65 and Akt also inhibited H. pylori-induced interleukin-8 expression. Conclusion The results suggest that cag PAI-positive H. pylori activates Akt in gastric epithelial cells and this may contribute to H. pylori-mediated NF-κB activation associated with mucosal inflammation and carcinogenesis. ==== Body Background Helicobacter pylori causes various human gastric diseases. In 10 to 20% of infected individuals, H. pylori-induced chronic gastric inflammation progresses to gastroduodenal ulcers, gastric cancer or gastric mucosa-associated lymphoid tissue lymphoma [1,2]. Bacterial, environmental and host genetic factors may affect the progress and outcome of gastric disease in these individuals. Virulence of individual H. pylori strains is one such factor responsible for severe disease, and several virulence factors have been described such as the presence of a cag pathogenicity island (PAI) and vacuolating cytotoxin (VacA) [3-6]. The presence of cag PAI genes correlates strongly with the development of ulcer diseases and gastric cancer [7-9]. Nuclear factor-κB (NF-κB) is a crucial regulator of many cellular processes, including immune response, inflammation and apoptosis [10]. It has been established that inflammation plays an important role in cancer development [11]. The five known mammalian Rel genes encode seven Rel-related proteins: RelA/p65; p105 and its processing product, p50; p100 and its processing product, p52; c-Rel; and RelB. Each contains an N-terminal Rel homology domain that mediates DNA binding, dimerization and interaction with the IκB family of NF-κB inhibitors. p65, c-Rel and RelB contain C-terminal transactivation domains, but p50 and p52 do not. The prototypical NF-κB complex is a p50–p65 heterodimer. In resting cells, NF-κB is complexed to cytoplasmic NF-κB inhibitors. IκBα is the best characterized of these inhibitors. NF-κB activation requires phosphorylation of two conserved serine residues within the N-terminal domain of IκBα (serines 32 and 36) [12]. Phosphorylation leads to ubiquitination and 26S proteasome-mediated degradation of IκBα, thereby releasing NF-κB from the complex where it translocates to the nucleus and activates various genes. Stimulation such as cytokines results in the activation of specific intracellular signaling pathways with subsequent activation of the IκB kinase (IKK) complex. This complex comprises two catalytic subunits (IKKα and IKKβ) and the regulatory subunit (IKKγ), and can phosphorylate IκBα [12]. Only H. pylori strains containing the cag PAI (cag PAI+) can direct signaling in gastric epithelial cells to activate the IKK complex and thus NF-κB, leading to the release of chemoattractants such as interleukin (IL)-8 [13]. However, the exact mechanism by which cag PAI+ H. pylori strains induce activation of NF-κB in gastric epithelial cells is not clear yet. The cag PAI encodes a bacterial type IV secretion capable of translocating effector molecules [14]. Based on the observations that mutants of CagA, the only type IV secretion system effector protein, often induce a considerable amount of IL-8, early studies reported that CagA did not activate NF-κB or IL-8 secretion in infected cells [15,16]. However, CagA was recently reported to induce IL-8 release through NF-κB activation in time- and strain-dependent manners [17]. Protein kinases are also required for optimal NF-κB activation by targeting functional domains of NF-κB protein itself. Phosphorylation of the p65 subunit plays a key role in determining both the strength and duration of the NF-κB-mediated transcriptional response [18,19]. Sites of phosphorylation reported to date are serines 276 and 311, in the Rel-homology domain, and serines 468, 529 and 536, three phosphoacceptor sites located in the transactivation domain. Importantly, phosphorylation at serine 536 reduced the ability of p65 to bind IκBα [20] and facilitated the recruitment of TAFII31, a component of the basal transcriptional machinery [21]. Phosphorylation at serine 536 is also responsible for recruiting coactivators such as p300 [22]. The above data emphasize the importance of p65 phosphorylation at serine 536 in the function of NF-κB. In contrast, p50 phosphorylation does not regulate NF-κB activation, because p50 lacks a transactivation domain. Akt is a downstream effector of phosphatidylinositol 3-kinase (PI3K) that has been implicated in phosphorylation of serine 536 on the p65 subunit [18,19]. Akt activation also mediates multiple biological activities including increased survival, proliferation and growth of tumor cells. The present study investigated whether Akt regulates NF-κB activation in response to H. pylori infection. Results Immunohistochemical studies H. pylori-positive gastritis biopsies of 10 patients were immunostained for phosphorylated Akt. Staining was limited to mucosal epithelial cells in all 10 patients (Figure 1A and Figure 1B), whereas no such staining was observed in the normal mucosa of all three healthy volunteers (Figure 1C and Figure 1D). Epithelial cells from three patients showed strong staining, while those of six patients showed moderate staining, and the remainder demonstrated weak staining. Figure 1 Phosphorylation of Akt in H. pylori-infected gastric mucosa. Immunohistochemical detection of phosphorylated Akt in tissues of patients with H. pylori-positive gastritis. Serial sections of gastric biopsy specimens were stained with monoclonal antibody against phospho-Akt (serine 473). (A and B) Representative examples of mucosa from patients with H. pylori-positive gastritis. (C and D) Representative examples of normal mucosa. Note the positive staining for phospho-Akt in the mucosal epithelial cells of patients with H. pylori-positive gastritis. Original magnification, ×200. Cag PAI is required for H. pylori-mediated IL-8 induction in gastric epithelial cells The cag PAI is a 40-kbp cluster of approximately 27 genes and encodes a type IV secretary apparatus which injects the CagA protein, and possibly other unknown proteins, into eukaryotic cells [14]. virD4 is one of seven genes in cag PAI that are virulent (vir) gene homologues [23]. In H. pylori, virD4 is thought to act as an adapter protein for the transfer of CagA protein and possibly other yet unknown proteins into the transfer channel formed by other Vir proteins in cag PAI [24]. The virD4 mutant cannot translocate CagA [24]. IL-8 cytokine is chemotactic for neutrophils and lymphocytes, and is induced in response to H. pylori infection. Many of the cis-elements that regulate IL-8 expression have been identified, including binding sites for NF-κB [25]. H. pylori-induced IL-8 expression is NF-κB dependent [26]. To examine the role of virulence factors in H. pylori-mediated NF-κB activation, we compared IL-8 induction in gastric epithelial cells infected with Δcag PAI, ΔVacA, ΔvirD4 or wild-type H. pylori strain. Infection with wild-type strain 26695 induced IL-8 mRNA expression in MKN45 cells, while the isogenic mutant that lacked cag PAI expression did not induce IL-8 mRNA expression (Figure 2A). Wild-type H. pylori strain but not Δcag PAI strain induced IL-8 mRNA expression in AGS cells (Figure 2B). In contrast, VacA and virD4 null mutants induced IL-8 mRNA expression similar to the parental strain (Figure 2A). Our study on isogenic mutants derived from the 26695 strain suggests that H. pylori cag PAI plays an important role in the induction of IL-8 mRNA expression. Figure 2 cag PAI products of H. pylori are required for induction of IL-8 mRNA expression. Total RNA was extracted from MKN45 (A) or AGS cells (B) infected with the wild-type strain 26695 (WT) or isogenic mutant ΔVacA, ΔvirD4 or Δcag PAI (Δcag) for the indicated times and used for RT-PCR. Lane M contains markers. Representative results of three similar experiments. H. pylori activates Akt and induces phosphorylation of the NF-κB p65 subunit in gastric epithelial cells We next examined whether coculture of gastric epithelial MKN45 cells with H. pylori results in activation of Akt, using Western blot analysis. As shown in Figure 3A (rows 2 and 3), phosphorylated Akt levels increased after only 30 min of coculture and this phosphorylation persisted for 3 h. There was no significant change in total Akt protein level in H. pylori-infected MKN45 cells (row 1). In vitro Akt kinase activity also increased 30 min after the addition of H. pylori to MKN45 cells (Figure 3A, bottom row). Since Akt is an upstream kinase implicated in p65 phosphorylation [27], we then assessed p65 phosphorylation with an antibody specific for p65 phosphorylated on serine 536. p65 phosphorylation was induced after 1 h of stimulation with H. pylori (Figure 3A, row 5). H. pylori infection also induced phosphorylated IκBα (Figure 3A, row 7). Kinetic analysis of H. pylori-induced degradation and resynthesis of IκBα in MKN45 cells revealed gradual increase in IκBα levels (Figure 3A, row 6). These results indicate that H. pylori-induced phosphorylation of IκBα leads to proteasome-mediated degradation of IκBα, thereby releasing NF-κB from the complex followed by its translocation to the nucleus to activate genes. This signal is terminated through cytoplasmic resequestration of NF-κB, which depends on IκBα synthesis, a process requiring NF-κB transcriptional activity [12]. Similar results were obtained in AGS cells (Figure 3A). Figure 3 H. pylori activates Akt and induces p65 phosphorylation. (A) MKN45 or AGS cells were infected with H. pylori (ATCC 49503) for the indicated times. Cells were harvested, lysed and subjected to immunoblotting with the indicated antibodies. Akt in vitro kinase assay was performed after immunoprecipitation of Akt, with GSK-3 fusion protein serving as the exogenous substrate for Akt. Kinase reactions were analyzed by immunoblotting with monoclonal antibody for phospho-GSK-3 (serines 21 and 9). (B) The cag PAI of H. pylori is required for induction of Akt phosphorylation. MKN45 or AGS cells were infected with either the wild-type H. pylori strain 26695 (WT) or its isogenic cag PAI-lacking mutant strain (Δcag) for 1 h. Cells were harvested, lysed and subjected to immunoblotting with the indicated antibodies. Representative results of three similar experiments in each panel. We next examined whether the observed Akt activation was specific to the cag PAI domain, based on the above results indicating the importance of cag PAI expression for IL-8 induction in gastric epithelial cells in vitro (Figure 2). We used a wild-type H. pylori strain (26695) and an isogenic cag PAI mutant (Δcag PAI). Stimulation with the wild-type strain induced Akt phosphorylation in MKN45 and AGS cells, while the isogenic mutant that lacked the expression of cag PAI did not (Figure 3B). These results suggest the important role of H. pylori cag PAI in the phosphorylation of Akt. H. pylori-induced p65 phosphorylation is PI3K-dependent Akt is a substrate for PI3K, and thus we investigated the role of this kinase in H. pylori-induced Akt activation and p65 phosphorylation. As expected, inhibition of PI3K with LY294002 inhibited H. pylori-induced Akt activation (Figure 4A, top row), but interestingly, also abrogated H. pylori-induced p65 phosphorylation (Figure 4A, row 2). Despite being mutually dependent, the nuclear translocation, DNA binding and transcriptional activity of NF-κB may rely on independent regulatory elements. We investigated the role of PI3K in each of these processes by using the LY294002 inhibitor. MKN45 cells were infected with H. pylori and NF-κB DNA binding was assessed by electrophoretic mobility shift assay (EMSA). As shown in Figure 4B, a complex was induced in these cells within 10 min after infection with H. pylori. This binding activity was reduced by the addition of either cold probe or a typical NF-κB sequence derived from the CCL20 gene but not by an oligonucleotide containing the AP-1 binding site (Figure 4C, lanes 2–4). Furthermore, an NF-κB DNA complex composed of p50 and p65 was induced in MKN45 cells within 10 min after infection with H. pylori, but pretreatment of MKN45 cells with LY294002 did not inhibit H. pylori-mediated NF-κB DNA binding activity (Figure 4B and Figure 4C). Figure 4 Involvement of PI3K in H. pylori-mediated Akt activation and p65 phosphorylation. (A) MKN45 cells were pretreated for 60 min with LY294002 (20 μM) or medium alone, and infected with H. pylori (ATCC 49503) for the indicated times (30–180 min). Cells were harvested, lysed and subjected to immunoblotting with the indicated antibodies. Akt in vitro kinase assays were performed as shown in Figure 3A. (B) LY294002 had no effect on the H. pylori-stimulated DNA binding activity of NF-κB. MKN45 cells were pretreated for 60 min with LY294002 (20 μM) or medium alone, and infected with H. pylori (ATCC 49503) for the indicated times for EMSA (10–60 min). (C) H. pylori stimulated the formation of a p65–p50 heterodimer in MKN45 cells infected with H. pylori (ATCC 49503) for 60 min. The cells were lysed and the competition and supershift assays were performed with the competitor oligonucleotides and the indicated antibodies (Ab), respectively. H. pylori-stimulated NF-κB transcriptional activity is dependent on PI3K/Akt Next, to assess whether H. pylori-induced PI3K activity affected NF-κB transcriptional activity, we transfected MKN45 cells with an NF-κB reporter construct (κB-LUC). In contrast to the effect of LY294002 on the DNA-binding activity of NF-κB, LY294002 pretreatment caused 65% decline in H. pylori-stimulated luciferase expression from κB-LUC (Figure 5A). Overexpression of the dominant-negative Akt mutant also suppressed the ability of H. pylori to stimulate κB-LUC in a dose-dependent manner (Figure 5B). The above findings indicate that the transcriptional activity but not the DNA binding activity of NF-κB is sensitive to inhibition of Akt and PI3K. Figure 5 NF-κB-mediated transactivation induced by H. pylori is inhibited by either LY294002 or transfection of a dominant-negative Akt mutant. (A) MKN45 cells were transfected with κB-LUC and phRL-TK for 24 h. Where indicated, the cells were preincubated with LY294002 (20 μM) for 60 min prior to infection with H. pylori (ATCC 49503). They were infected subsequently with H. pylori for 24 h. Luciferase activity was assayed for each sample. Readings were normalized for each sample as expressed κB-LUC over constitutively expressed phRL-TK and plotted as -fold stimulation. (B) Dominant-negative Akt blocked H. pylori signaling to an NF-κB-dependent promoter. MKN45 cells were cotransfected with κB-LUC and phRL-TK, together with either a vector or a construct expressing a dominant-negative Akt (Akt K179A/T308A/S473A). The cells were infected with H. pylori (ATCC 49503) 24 h later. Data are mean ± SD of three independent experiments. PI3K inhibition or transfection with small interference RNAs for p65 and Akt suppresses H. pylori-induced IL-8 expression Finally, we investigated the effect of inhibition of H. pylori-induced PI3K activity on IL-8 expression. Pretreatment of MKN45 cells with LY294002 reduced H. pylori-stimulated IL-8 mRNA expression as determined by reverse transcription-polymerase chain reaction (RT-PCR) (Figure 6A). Inhibition of PI3K also significantly decreased the amount of IL-8 secreted by MKN45 cells stimulated with H. pylori in a dose-dependent manner (Figure 6B). Figure 6 LY294002 inhibits H. pylori-induced IL-8 expression and production. (A) MKN45 cells were preincubated with LY294002 (20 μM) for 60 min prior to infection with H. pylori (ATCC 49503), harvested at the indicated time points and assayed for IL-8 mRNA expression by RT-PCR. Lane M contains markers. (B) LY294002 inhibits H. pylori-induced IL-8 production. MKN45 cells were preincubated with the indicated concentrations of LY294002 for 60 min prior to infection with H. pylori (ATCC 49503). For IL-8 protein determination, supernatants were collected 24 h after infection and assessed for IL-8 production by ELISA. Data are mean ± SD of three experiments. LY294002 is a chemical inhibitor, and thus its target specificity may be questionable. Thus, small interference RNAs (siRNAs) for p65 and Akt were used to examine the role of p65 and Akt activation in the signal transduction pathway leading to IL-8 expression by H. pylori infection. Each siRNA specifically inhibited the expression of p65 and Akt (Figure 7). Figure 7 also shows that H. pylori-induced IL-8 mRNA expression was inhibited by siRNAs for p65 and Akt, confirming that p65 and Akt are important in H. pylori-induced IL-8 expression. Figure 7 Transfection of siRNAs for p65 and Akt inhibits H. pylori-induced IL-8 expression. MKN45 cells were transfected with siRNAs for p65 and Akt, followed by stimulation with H. pylori (ATCC 49503) for 6 h. The RNA was subjected to RT-PCR for IL-8 and p65 mRNAs. Lane M contains markers. Discussion NF-κB activation is known to regulate various cellular responses, including apoptosis, and is required for the induction of inflammatory and tissue-repair genes [10]. As reported previously [26,28], we demonstrated that H. pylori modulates the NF-κB system in gastric epithelial cells by inducing IκBα phosphorylation and degradation, NF-κB DNA binding activity, and NF-κB transcriptional activity. Although this investigation was a preliminary in nature in a small number of patients, we also showed that H. pylori infection activated Akt in epithelial cells, both in vivo and in vitro, and that this is dependent on an intact cag PAI in vitro. Interestingly, H. pylori also stimulated endogenous p65 phosphorylation on serine 536. Phosphorylation of p65 at serine 536 in the transactivation domain enhances the transcriptional activity of NF-κB [19]. Although previous studies using pathogenic strains containing the cag PAI showed NF-κB activation and cytokine expression in gastric epithelial cells [13,28], ours is the first demonstration that cag PAI+ H. pylori strains induce gene expression through p65 phosphorylation, and that H. pylori-induced p65 phosphorylation is PI3K/Akt-dependent. The role of PI3K/Akt signaling cascades in the regulation of NF-κB transactivation remains controversial. The present study agrees with previous investigators in demonstrating that activation of PI3K/Akt promotes the activation of p65 [29], while some others found that inhibition of the PI3K/Akt pathway augmented p65 activation [30]. We also analyzed how H. pylori-stimulated PI3K activation leads to the activation of NF-κB, and identified a pathway initiated by the PI3K activation that is distinct from NF-κB DNA binding. In contrast to the lack of effect of inhibition of PI3K on NF-κB DNA binding, pretreatment of MKN45 cells with LY294002 resulted in marked inhibition of H. pylori-stimulated p65 phosphorylation and the ability of H. pylori to activate NF-κB-dependent transcription. Furthermore, a dominant-negative derivative of Akt blocked the ability of H. pylori to activate an NF-κB-dependent promoter. Therefore, the results established a clear role of PI3K and its downstream effector Akt in modulating the transactivation potential of p65. However, the kinases and signaling pathway responsible for H. pylori-induced p65 phosphorylation remain unknown. Our data demonstrated for the first time that PI3K and Akt participate in H. pylori-mediated NF-κB transcriptional activity. Further studies are required to define the exact signaling cascade involved in bacteria-induced p65 phosphorylation and NF-κB activity. Conclusion Our data demonstrated the role of PI3K/Akt in H. pylori-induced NF-κB transcriptional activity and subsequent IL-8 production in gastric epithelial cells. We also demonstrated an important role of PI3K/Akt in the regulation of gastric responses to H. pylori infection, thereby elucidating a novel mechanism that controls both transcription and gene expression in bacterial pathogenesis. Methods Antibodies and reagents Polyclonal antibodies to Akt, phospho-Akt (threonine 308), phospho-Akt (serine 473), p65 and phospho-p65 (serine 536), as well as monoclonal antibodies to phospho-Akt (serine 473) and phospho-IκBα (serines 32 and 36) were purchased from Cell Signaling Technology (Beverly, MA, USA). Polyclonal antibodies to IκBα and NF-κB subunits p50, p65, c-Rel, p52 and RelB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibody to actin was purchased from NeoMarkers (Fremont, CA, USA). PI3K inhibitor LY294002 was obtained from Calbiochem (La Jolla, CA, USA). Bacterial strains H. pylori ATCC 49503 (American Type Culture Collection, Rockville, MD, USA) was used. Isogenic H. pylori mutants lacking the cag PAI [31], VacA and virD4 were also studied together with their parental wild-type strain (26695). Isogenic null mutants derived from 26695 were constructed by insertional mutagenesis, using aphA (conferring kanamycin resistance). H. pylori strains were plated on blood agar plates and incubated at 37°C for 2 days under microaerophilic conditions. Using inoculating needles, bacteria harvested from the plates were suspended in 50 ml of brucella broth containing 5% fetal bovine serum (FBS) and then cultured in a liquid medium at 37°C for 1 day in a controlled microaerophilic environment. Bacteria were harvested from the broth culture by centrifugation and then resuspended at the concentrations indicated below in antibiotic-free medium. All procedures were approved by the appropriate institutional biosafety review committees and were conducted in compliance with biohazard guidelines. Cell culture The human gastric epithelial cell lines MKN45 and AGS were maintained in RPMI 1640 containing 10% FBS and antibiotics. On the day of the experiment, cells were plated on fresh serum- and antibiotic-free medium and cocultured with H. pylori at a final concentration of 107 colony forming unit/ml for the times indicated below. Tissue samples We examined stomach biopsy specimens from 10 patients with H. pylori gastritis and three histopathologically-normal stomach biopsies. We analyzed the phosphorylation status of Akt at serine 473 and the presence of H. pylori infection by culture, serological analysis (with anti-H. pylori IgG antibody), rapid urease test and histological visualization with Giemsa staining. Patients with H. pylori gastritis showed polymorphonuclear neutrophil infiltration in the gastric epithelium in conjunction with bacteria consistent with H. pylori. All subjects provided informed consent before obtaining the biopsy samples. RT-PCR Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using an RNA PCR kit (Takara Bio, Otsu, Japan). Thereafter, cDNA was amplified using 25 cycles for IL-8, 35 cycles for p65 and Akt, and 28 cycles for β-actin. The specific primers used are listed in Table 1. The thermocycling conditions for the targets were as follows: 94°C for 30 s (for IL-8 and β-actin) or for 60 s (for p65 and Akt), 60°C for 30 s (for IL-8 and β-actin) or for 60 s (for p65), or 55°C for 60 s (for Akt) and 72°C for 90 s (for IL-8 and β-actin) or for 60 s (for p65 and Akt). The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining. Table 1 Specific primers used in RT-PCR Primer Sequence Product size (bp) IL-8 sense 5'-ATGACTTCCAAGCTGGCCGTG-3' 302 antisense 5'-TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC-3' p65 sense 5'-GCGGCCAAGCTTAAGATCTGCCGAGTAAAC-3' 150 antisense 5'-GCGTGCTCTAGAGAACACAATGGCCACTTGCCG-3' Akt sense 5'-ATGAGCGACGTGGCTATTGTGAAG-3' 330 antisense 5'-GAGGCCGTCAGCCACAGTCTGGATG-3' β-actin sense 5'-GTGGGGCGCCCCAGGCACCA-3' 548 antisense 5'-CTCCTTAATGTCACGCACGATTTC-3' Plasmids The Akt dominant-negative mutant plasmid (pCMV5-K169A, T308A, S473A-Akt) encodes lysine169 (the ATP-binding site), threonine 308 and serine 473 (the phosphorylation sites) to alanine mutations. Reporter plasmid κB-LUC is a luciferase expression plasmid controlled by five tandem repeats of the NF-κB-binding sequences of the IL-2 receptor (IL-2R) α chain gene. Transfection and luciferase assay MKN45 cells were transfected with 1 μg of the appropriate reporter plasmid and 5 μg of effector plasmid using Lipofectamine (Invitrogen). After 24 h, H. pylori was added at a ratio of bacteria to cells of 20:1 and incubated for another 24 h. Luciferase activities were measured using the dual luciferase assay system (Promega, Madison, WI, USA) and normalized by the renilla luciferase activity from phRL-TK. Preparation of nuclear extracts and EMSA Cell pellets were swirled to a loose suspension and treated with lysis buffer (0.2 ml, containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM AEBSF and 1 mM DTT) with gentle mixing at 4°C. After 10 min, NP40 was added to a final concentration of 0.8% and the solution was immediately centrifuged for 5 min at 700 rpm at 4°C. The supernatant was removed carefully and the nuclei diluted immediately by the addition of lysis buffer without NP40 (1 ml). The nuclei were then recovered by centrifugation for 5 min at 700 rpm at 4°C. Finally, the remaining pellet was suspended on ice in the following extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 2 mM AEBSF, 33 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml E-64 and 10 μg/ml pepstatin A) for 30 min to obtain the nuclear fraction. All fractions were cleared by centrifugation for 15 min at 15,000 rpm. NF-κB binding activity with the NF-κB element was examined by EMSA as described previously [32]. In brief, 5 μg of nuclear extracts were preincubated in a binding buffer containing 1 μg poly(dI-dC)·poly(dI-dC) (Amersham Biosciences, Piscataway, NJ, USA), followed by the addition of a radiolabeled oligonucleotide probe containing NF-κB element from the IL-2R α chain gene (approximately 50,000 cpm). The radiolabeled oligonucleotide was prepared by filling in the overhang with the Klenow fragment of DNA polymerase I in the presence of 32P-dCTP and 32P-dATP. These mixtures were incubated for 15 min at room temperature. The DNA protein complexes were separated on a 4% polyacrylamide gel and visualized by autoradiography. For competition experiments, the cold oligonucleotide probe or competitors were used, and supershift analysis was performed using antibodies against p50, p65, c-Rel, p52 or RelB. The probe or competitors used were prepared by annealing the sense and antisense synthetic oligonucleotides as follows: for the NF-κB element of the IL-2R α chain gene, 5'-GATCCGGCAGGGGAATCTCCCTCTC-3'; for the NF-κB element of the CCL20 gene, 5'-GATCGATCAATGGGGAAAACCCCATGTG-3'; and for the AP-1 element of the IL-8 gene, 5'-GATCGTGATGACTCAGGTT-3'. The above underlined sequences are the NF-κB and AP-1 binding sites. Western blot analysis Cells were lysed in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 6% 2-mercaptoethanol and 0.01% bromophenol blue. Equal amounts of protein (20 μg) were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, followed by transfer to a polyvinylidene difluoride membrane and sequential probing with the specific antibodies. The enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK) was used for detection. The membranes were stripped in stripping buffer for probing with a different antibody. Actin served as an internal control in the Western blot procedure. Akt kinase assay A non-radioactivity-based Akt kinase assay kit was purchased from Cell Signaling Technology. After immunoprecipitation of Akt, the kinase reaction was performed using the instructions provided by the manufacturer with glycogen synthase kinase (GSK)-3 fusion protein as an exogenous substrate. The kinase reaction was analyzed by immunoblotting, using an anti-phospho-GSK-3 antibody (serines 21 and 9). Measurement of IL-8 production MKN45 cells were cultured in RPMI 1640 supplemented with 10% FBS in 24-well plates. Subconfluent monolayers of cells were cocultured with H. pylori for 24 h. The supernatants were collected and stored at -80°C. IL-8 was measured by ELISA (BioSource, Camarillo, CA, USA). RNA interference The siGENOME mixtures for p65 and Akt were obtained from Dharmacon (Chicago, IL, USA). All siRNA transfections were performed using a MicroPorator (Digital Bio, Seoul, Korea), pulsed once at 1,100 V for 20 ms. The siGENOME non-targeting siRNA served as controls. Immunohistochemical analysis Serial sections were deparaffinized in xylene and dehydrated using graded ethanol solutions. For better detection, sections were pretreated with ready-to-use proteinase K (Dako, Carpentaria, CA, USA) for 10 min at 37°C. This procedure increased the number of antigenic sites available for binding by the antibody. In the next step, the tissues were placed in 3% hydrogen peroxide and absolute methanol for 5 min to reduce endogenous peroxidase activity, followed by washing in PBS. Primary antibody incubations included anti-phospho Akt (serine 473) monoclonal antibody or a control IgG. After washing with PBS, the sections were covered with EnVision plus (Dako) for 40 min at 37°C and washed in PBS. Antigenic sites bound by the antibody were identified by reacting the sections with a mixture of 0.05% 3,3'-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl buffer and 0.01% hydrogen peroxide. Sections were counterstained with methyl green. Authors' contributions ET carried out the experiments and drafted the manuscript. KT and HK collected and assembled the data. CI, SS and MT contributed to the experimental concept and design and provided technical support. MS performed immunohistochemical staining. HM and CS provided bacterial strains. FK and JF participated in the discussion on the study design. NM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors thank T. Hirayama for providing H. pylori strain (ATCC 49503); J. Fujisawa for providing reporter plasmid κB-LUC; and D. R. Alessi for providing the dominant negative mutant of Akt. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1939917209-PONE-RA-08185R110.1371/journal.pone.0005373Research ArticleImmunology/Immune ResponseImmunology/ImmunomodulationImmunology/Innate ImmunityIndirect Recruitment of a CD40 Signaling Pathway in Dendritic Cells by B7-DC Cross-Linking Antibody Modulates T Cell Functions CD40 Mediated Treg ConversionRadhakrishnan Suresh Cabrera Rosalyn Bruns Kristina M. Van Keulen Virginia P. Hansen Michael J. Felts Sara J. Pease Larry R. * Department of Immunology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America Unutmaz Derya EditorNew York University School of Medicine, United States of America* E-mail: [email protected] and designed the experiments: SR LRP. Performed the experiments: SR RAC KMB VPVK MJH. Analyzed the data: SR RAC KMB LRP. Wrote the paper: SR SJF LRP. 2009 28 4 2009 4 4 e537314 1 2009 1 4 2009 Radhakrishnan et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40−/− DCs treated with B7-DC XAb (DCXAb) activated DAP12, but failed to activate NFκB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D3. CD40−/− DCXAb also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DCXAb to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant. ==== Body Introduction Generation of a vigorous T cell response is dependent on the activation of the T cells by professional antigen presenting cells and requires stable pMHC∶TCR interaction (signal 1), co-stimulation through CD28 and other membrane molecules (signal 2), and secretion of T cell growth factors (signal 3). B7-DC XAb is an IgM antibody from the serum of a patient with Waldenstrom's macroglobulinemia that binds to the surface of mouse and human DCs [1]. This binding leads to activation of the DCs and an augmentation of a number of phenotypic functions that are distinct from those elicited in DCs activated by TLR ligands or CD40L [2]. The pentameric structure of the IgM antibody is mandatory for the antibody to execute its effect on DCs. Monomers fail to bind and activate the DC and can prevent DC binding by the pentamer [1]. A candidate gene approach revealed that antibody binding is dependent on the expression of the co-stimulatory molecule B7-DC/ PD-L2 [1]. Therefore, we refer to this antibody as B7-DC cross-linking antibody (B7-DC XAb). Importantly, B7-DC XAb also activates human DC [3] and has actions in several experimental models of cancer in which results from the use of TLR ligands or TNF family members have been less impressive [4], [5]. The B7-DC molecule has a short cytoplasmic tail (5 amino acids), does not have charged amino acids, and by itself cannot convey signals from the membrane to the cytoplasm. We have recently shown that treatment of DCs with B7-DC XAb (DCXAb) induces multiple membrane proteins to become organized into a cell surface cap [3]. These molecules include B7-1 (CD80), B7-2 (CD86), class-II, TREM-2 and CD11c. Stimulation of antigen accumulation by DCXAb requires TREM-2 and is mediated by the activation of DAP12 and Syk. TREM-2 is also required for B7-DC XAb-mediated tumor protection in mice. However, in subsequent experiments, we found that TREM-2 was dispensable for the activation of an NFκB pathway necessary for DC treated with B7-DC XAb to remain viable under stress [6]. CD40 activation has been shown to increase the viability of a number of cell types including dendritic cells, to activate NFκB, and to increase secretion of a number of cytokines such as IL-1, IL-6 and TNFα [7]–[9]. CD40 activation mechanisms can also lead to tumor immunity [10]–[12]. Thus, we hypothesized that CD40 may play a role in some of the phenotype responses observed in DCXAb. In this report we show that CD40 is indeed present in a B7-DC XAb-induced cell surface complex and is required for activation of NFκB, protection of DCXAb from cell death signals, and for the secretion of IL-6. CD40 expression is also required for DCXAb to reprogram T regulatory cells to IL-17+ effectors, an outcome of B7-DC XAb shown previously to break tolerance in an antigen-specific manner [5]. Finally, presence of CD40 on the DCs in vitro and in vivo is required for the generation of tumor-specific cytolytic effector cells and to protect mice from tumors. Thus B7-DC XAb modulation of DC phenotypes is through its ability to bind a cell-specific molecule to simultaneously recruit and activate multiple signaling cascades in a combinatorial manner to directly regulate DC responses and indirectly regulate T cell functions. Results B7-DC XAb induces CD40 recruitment into the multi-molecular cap on DCs Previous dissection of the mechanism of the B7-DC XAb action showed that its binding to DC caused a reorganization of cell surface molecules (class II, CD80, CD86) into a cap-like cluster that also contained the signaling molecule TREM-2 [3]. While DCs from TREM-2 knockout mice were not able to accumulate antigen in response to B7-DC XAb, they were still able to activate NFκB, a mediator of DC viability following B7-DC crosslinking. Since CD40 is a known activator of NFκB, we used FRET analysis to determine whether CD40 was among the cell surface molecules recruited into the B7-DC XAb-induced cap on DC. A FRET signal between the PE-labeled anti-CD40 and APC labeled anti-Class II was detected as early as 5 minutes in DCs stimulated with B7-DC XAb (Fig. 1A, unfilled histograms). Thus, B7-DC XAb cross-linked CD40 to a previously identified constituent of the cell surface cap. The signal was maintained for as long as 30 minutes, but no FRET signal was detected upon stimulation with control antibody (filled histograms). To directly demonstrate that CD40 molecules were present in a multi-molecular complex, we performed Western blots on MHC class II immunoprecipitates. CD40 was detected in MHC class II complexes isolated from the DCs stimulated with B7-DC XAb but not from DCs stimulated with control antibody (Fig. 1B). Taken together, these data suggest that CD40 is recruited into the multimolecular cap that forms on DCs upon treatment with B7-DC XAb. 10.1371/journal.pone.0005373.g001Figure 1 CD40 is rapidly recruited into complexes containing MHC MHC class II on dendritic cells treated with B7-DC XAb. (A) DCs were pre-stained with APC-labeled antibody against MHC class II and PE-labeled antibody against CD40 for 15 min. An aliquot of cells was analyzed by flow cytometry (0 min time point). The remaining cells were then treated with 10 µg/ml IgM control antibody (filled histograms) or B7-DC XAb (open histograms). Cells were sampled at the indicated time points and analyzed for a FRET (FL3 channel). (B) Lysates were prepared from untreated DC (0′) or DC treated with control antibody or B7-DC XAb for the indicated times and subjected to immunoprecipitation using an antibody against MHC class II. The resultant complexes were resolved by SDS-PAGE, transferred to PVDF membrane, and probed for CD40. IgH serves as a loading control. The results shown are representative of 2 experiments. CD40 is required for NFκB activation in DC treated with B7-DC XAb Activation of CD40 culminates in stimulation of NFκB in multiple cell types [8], [13], [14]. Since CD40 was found to complex with class II, we asked whether CD40 mediated NFκB activation in DCXAb. Bone marrow derived DCs from wild type, CD40−/−, or TREM2−/− mice were stimulated with control antibody or B7-DC XAb for 15 minutes. In addition, parallel CD40−/− DC were stimulated with anti-CD40 or the TLR4 agonist, LPS. Cells were permeabilized and stained with an antibody that recognizes only the activated NFκB complex [6] and visualized by confocal microscopy. Abundant nuclear fluorescence (FITC-anti-p65 alone, upper left panels; merged with DAPI, lower panels) shows that NFκB was activated in wild type and TREM2−/− DCs stimulated with B7-DC XAb but not in CD40−/− (KO) DC stimulated with B7-DC XAb, or anti-CD40 antibody (Fig. 2A). However, CD40−/− DC still activated NFκB when stimulated through TLR4. Control antibody treatment failed to activate NFκB in all groups of DCs. The CD40−/− DCs activated DAP12 and Syk kinase upon B7-DC crosslinking (Fig. 2B), underscoring these cells were only non-responsive to CD40-dependent signals. 10.1371/journal.pone.0005373.g002Figure 2 DCs require CD40 for B7-DC XAb mediated NFκB activation and protection from cell death. (A) NFκB activation in wild type (WT), CD40−/− or TREM2−/− (KO) DC was determined after 15 min stimulation with control antibody, B7-DC XAb, anti-CD40, or LPS (TLR4 ligand). Cells were fixed, stained and imaged using a confocal microscope. Upper left panels show FITC-anti-p65 staining, upper right panels show DAPI staining of nuclei, lower panels show merged images of the upper panels. (B) Wild-type or CD40−/− (KO) DC were treated with control antibody or B7-DC XAb for 5 min. DAP12- (left) or Syk- (middle) specific immunoprecipitates or whole cell lysate for ERK (right) were resolved and blotted using a phosphotyrosine-specific antibody. IgH or total ERK serves as a loading control. (C) Cell death was induced in wild type (filled bars) or CD40−/− (open bars) DC by treatment with vitamin D3. All cultures were subsequently incubated with Alamar Blue Dye. Cell viability was measured using a fluorescence plate reader after 24 hours and is expressed as relative fluorescence units (RFU). (D) Similar to (C) except cell death was induced by cytokine withdrawal. Wild type (filled bars) or CD40−/− (open bars) DC were cultured in the presence or absence of GM-CSF and IL-4 (+ or − Cytokine) for 24 h in the presence of 10 µg/ml control antibody, B7-DC XAb, anti-MHC class II IgM antibody 25-9-3, or the combination of 1 µg/ml B7-DC XAb and 0.1 µg/ml RANKL as indicated. The results shown are representative of 2 experiments. CD40 is important in promotion of B7-DC cross-linking Ab-induced DC survival under adverse conditions CD40-CD40L interaction has been shown to promote survival of a number of cell types [15], [16]. Since CD40 was shown to be present in the cell surface complex that forms after cross-linking B7-DC on DCs, we tested whether CD40 was required for the enhanced survival previously observed in DCXAb [6]. Bone marrow derived DCs from wild type or CD40−/− mice were matured with CpG then treated with the cell death inducer Vitamin D3 [17]. Cells were then mixed with Alamar Blue dye to determine viability (expressed as mean relative fluorescence). Wild type DCs that received B7-DC XAb remained viable in the presence of Vitamin D3 while control antibody failed to protect wildtype DC (Fig. 2C). The effect of B7-DC XAb was dependent on presence of CD40, as DCs that lacked CD40 were not protected from this cell death pathway (Fig. 2C). Wild type or CD40−/− DC were also tested for B7-DC XAb-induced survival when deprived of GM-CSF/IL-4 cytokines for 24 hours. Only wild type DCs treated with B7-DC cross-linking Ab were able to survive cytokine withdrawal, and B7-DC XAb treatment was unable to promote cell survival in CD40−/− DCs. However, CD40−/− DC were still protected cells from cytokine withdrawal if stimulated with RANK ligand, another known activator of NFκB and downstream pro-survival signals (Fig. 2D). CD40 ligation has been demonstrated to activate ERK, and activation of ERK promotes cell survival. Therefore, we tested the requirement for CD40 in ERK activation in response to B7-DC XAb. Wild-type DCs, but not CD40−/− DCs, exhibited ERK activation in response to B7-DC XAb cross-linking, (Figure 2B). Both groups of DCs activated ERK in response to TLR4 ligation. These findings demonstrate that CD40 is an essential upstream mediator of ERK and NFκB activation in DC treated with B7-DC XAb and link CD40 to the enhanced survival phenotype observed in DCXAb. Moreover, this pathway is distinct from the TREM2 pathway used by DCXAb to accumulate antigen. CD40 is necessary for IL-6 secretion by DCXAb and reprogramming of Tregs to IL-17+ effectors Treatment of DCs with B7-DC XAb leads to the production of IL-6 and IL-6-dependent conversion of T regulatory cells to IL-17+ effectors [5]. Blockade of NF-κB activation also prevents the secretion of cytokines including IL-6 from DCXAb [6]. Thus, we sought to determine if these two responses of DC to B7-DC XAb were linked through a requirement for CD40. Wild type or CD40−/− DCs were stimulated with control antibody or B7-DC XAb for 48 hours and the amount of IL-6 in the culture supernatants was determined by ELISA. Parallel cultures were stimulated with CD40 or TLR agonists. Wild type DCs secreted IL-6 when stimulated with B7-DC XAb or control stimuli targeting CD40 or toll-like receptors. Neither DC type secreted IL-6 when treated with control antibody. Importantly, CD40−/− DCs did not secrete IL-6 when treated with anti-CD40 or B7-DC XAb antibodies, but were still able to respond to TLR stimulation (Fig. 3A). 10.1371/journal.pone.0005373.g003Figure 3 DCs require CD40 in order to secrete IL-6 and convert Tregs to Th17 cells in vitro. (A) IL-6 was measured in culture supernatants of wild type or CD40−/− (KO) DC stimulated for 48 h with control antibody, B7-DC XAb, anti-CD40 or TLR agonists as indicated. Statistical analysis was performed using one-way ANOVA. IL-6 secreted by WT DCs stimulated with B7-DC XAb was significantly higher than IL-6 secreted by WT DCs stimulated with control antibody; IL-6 secreted by wild type or CD40−/− DC in response to TLR ligands was significantly higher than IL-6 induced in WT DC by B7-DC XAb (p<0.01). Error bars represent the standard deviation from the mean of triplicate cultures. (B) Enriched populations of DO11.10 non-Tregs or Tregs (>95% pure) were cultured with OVA-pulsed and control- or B7-DC XAb-treated DC isolated from wildtype, (C and E) CD40−/− mice and (D) IL-6−/− mice. After 48 h, expression profiles for FoxP3 and IL-17A were determined by intracellular staining and flow cytometry. All experiments were conducted at least twice. As a final test for the role of a CD40-NFκB-IL-6 pathway in the conversion of Tregs to T effector cells [5], wild type or CD40−/− DC were pulsed with ovalbumin and treated with the control antibody or B7-DC XAb. These DCs were then used in vitro as stimulators of cultures enriched for either CD4+ CD25+ Tregs or CD4+ CD25− non-Tregs isolated from DO11.10 TCR transgenic mice. After 48 hours, cells from each group were subjected to intracellular staining for the Treg specific transcription factor FoxP3 and the Th17 specific effector cytokine IL-17A. Non-Tregs that were stimulated with antigen-pulsed wildtype or CD40-deficient DCcntrl or DCXAb did not express FoxP3 or IL-17A (Fig. 3B). Tregs stimulated with antigen-pulsed wild type DCXAb downregulated FoxP3 and expressed IL-17A. CD40−/− DCXAbs did not mediate the conversion of T regulatory cells to IL-17+ effector cells (Fig. 3C) suggesting the necessity of IL-6 in this process. Wild type DCcntrl and CD40−/− DCcntrl also failed to cause T regulatory conversion to T effector cells. However, it is possible that IL-6 alone is not sufficient for inducing DCXAb mediated T regulatory cell conversion into effector cells. To test this, DCcntrl, DCXAb, CD40−/−cntrl, CD40−/−DCXAb, IL-6−/−cntrl and IL-6−/−DCXAb were analysed for their potential to induce T regulatory cell conversion by exogenous addition of IL-6. As shown in panels 3D and E, under culture conditions where the DCs are defective in making IL-6 (both IL-6−/− DCs and CD40−/−DCs), provision of IL-6 restored the induction of Th17 cells, whereas the CD40 deficient and IL-6 deficient DCcntrl supplemented with IL-6 failed to induce IL-17A+ cells. This data indicate IL-6 is necessary, but not sufficient to cause T regulatory cell conversion to Th17 cells, highlighting the importance of other factors induced by B7-DC cross-linking in these regulatory events. We have previously shown that DCXAb-mediated conversion of Tregs into IL-17+ effectors occurs in the absence of any significant cell proliferation [5]. Since the data in Fig. 3B show that non-Tregs did not express IL-17A after stimulation with DCcntrl or DCXAb, we conclude that the IL-17A expressing cells originated from Tregs and were not a product of outgrowth of contaminating non-Tregs. These data show that CD40 is necessary for DCs to not only respond to B7-DC XAb by secreting IL-6, but also to induce the concomitant conversion of T regulatory cells into effector cells. CD40 is important for the generation of anti-tumor T cell responses induced by B7-DC XAb A major response in mice receiving B7-DC XAb is the induction of a potent CTL response that drives the protection of those mice from a lethal melanoma challenge [18], [19]. To test the ability of the B7-DC XAb to confer protection against other tumor types, we injected cells derived from kidney, thymus, or breast tumors or leukemia or melanoma cells into genetically-matched hosts. Mice receiving each tumor type also received either control antibody or B7-DC XAb treatment. Nearly all of the mice (97%) that were treated with the control antibody succumbed to the tumor burden irrespective of the origin of the tumor or the tumor-host combination. Anti-tumor immunity was produced by B7-DC XAb treatment in either strain of mice tested and was also independent of tumor type (Table 1). 10.1371/journal.pone.0005373.t001Table 1 B7-DC XAb Induces Protective Immunity Against a Variety of Different Tumor Grafts. Tumor Number of Cells Mouse Strain Number of Mice Tumor Free/Total (Control Ab) Number of Mice Tumor Free/Total (B7-DC XAb) p Value B16 5×105 C57BL/6 0/5 23/23 <0.001 RENCA 106 C57BL/6 0/9 9/9 <0.001 EL4 5×105 C57BL/6 0/8 8/8 <0.001 WEHI-3 5×103 BALB/c 1/5 6/6 0.015 TUBO 5×105 BALB/c 0/5 10/10 <0.001 Mice received B7-DC XAb or control antibody (i.v.) at the time they were grafted with tumor cells (s.c.). Mice were monitored regularly for tumor growth and were euthanized if tumors reached 17×17 mm. Mice remaining tumor-free were monitored regularly for up to 30 days; TUBO mice were monitored up to 300 days. The scores indicate the number of mice in the indicated treatment group remaining free of tumor. CD40 has been shown to play a major role in cross-presentation of antigens to stimulate CD8 T cells [20], and anti-CD40 antibody treatment has been used to induce protection against tumors [10], [21]. Therefore, we analyzed the role of CD40 in the anti-tumor immunity elicited by B7-DC XAb in two tumor models. Wild type or CD40−/− mice were implanted with B16 melanoma or WEHI-3 leukemia cells and treated systemically with the control antibody or B7-DCXAb. On day 7, lymph node cells were isolated from some of the treated mice and used as effectors against 51Cr-labeled B16 or WEHI-3 targets. EL-4 or P815 cells served as MHC-matched negative controls. In the CD40−/− mice, potent effectors were not induced in response to B7-DC XAb treatment (Fig. 4). Moreover, when the remaining mice were followed for up to 90 days, we found that all the groups of mice succumbed to the tumor except the wild type mice receiving B7-DC XAb (Table 2). These data suggest that CD40 is necessary for the generation of the potent anti-tumor immunity induced by B7-DC XAb. 10.1371/journal.pone.0005373.g004Figure 4 CD40 is required for the DCXAb-induced generation of anti-tumor CTL responses in vivo. Wild type or CD40−/− mice were engrafted with B16 melanoma (A) or WEHI-3 leukemia (B) and treated intravenously with 30 µg B7-DC XAb or control antibody. On day 7, cells from draining lymph nodes were used as effectors in CTL assays. (A) CTL against 51Cr-labeled B16 tumor targets (Left) or unrelated EL-4 controls (Right). Filled squares show CTL for wild-type mice receiving B7-DC XAb. (B) CTL against 51Cr-labeled WEHI-3 tumor targets (Left) or unrelated, MHC-matched P815 controls (Right). Filled triangles show the CTL response from wild type mice receiving B7-DC XAb. Symbols and lines for all other treatment groups are nearly superimposable. The results shown are representative of 2 experiments. 10.1371/journal.pone.0005373.t002Table 2 B7-DC XAb-induced Tumor Protection is Dependent on CD40. Tumor Mouse Strain Number of Mice Tumor Free/Total (Control Ab) Number of Mice Tumor Free/Total (B7-DC XAb) p Value B16 B6 WT 0/8 8/8 <0.001 B16 CD40−/− 0/8 0/8 n.s. WEHI BALB/cWT 0/3 5/5 <0.001 WEHI CD40−/− 0/7 0/8 n.s. Wild type or CD40−/− mice were implanted with B16 or WEHI tumor (5×105 cells) and received control antibody or B7-DC XAb by intravenous administration. The mice were monitored for tumor growth and were euthanized if tumor size reached 17×17 mm. Tumor-free B16-implanted mice were monitored regularly for more than 90 days; tumor-free WEHI-implanted mice were monitored regularly for more than 60 days. The scores indicate the number of mice in the indicated treatment group remaining free of tumor. n.s. = not significant. Since CD40 is expressed on multiple cell types, we asked whether the expression of CD40 on DC alone was critical for B7-DC XAb-induced anti-tumor immunity. To test this, bone marrow-derived wild type or CD40−/− DCs were pulsed with B16 melanoma tumor cell lysate and treated with control antibody or B7-DC XAb. After overnight incubation, the DCs were adoptively transferred into normal mice as a vaccine. Mice were then implanted with live B16 tumor. The animals were followed for 90 days for the development of tumor. Whereas mice receiving wild-type DCXAb vaccine were protected from melanoma challenge, animals receiving the CD40−/− DCXAb succumbed to the tumor (Table 3). Taken together, these data show that CD40 on DC is critical for B7-DC XAb to confer tumor immunity in vivo. 10.1371/journal.pone.0005373.t003Table 3 DCXAb Vaccination Requires CD40 for Tumor Immunity. Tumor Mouse Strain Number of Mice Tumor Free/Total (Control Ab) Number of Mice Tumor Free/Total (B7-DC XAb) p Value B16 WT 0/4 4/4 0.029 B16 CD40−/− 0/4 0/4 n.s. Normal mice received 2×106 wild-type or CD40−/− DC pulsed with B16 tumor lysate and control antibody or B7-DC XAb as indicated. On the same day, 5×105 B16 tumor cells were implanted on the right flank. The animals were monitored for tumor growth for at least 90 days and were euthanized if the tumor reached 17×17 mm. The scores indicate the number of mice in the indicated treatment group remaining free of tumor. n.s. = not significant. Discussion Cross-linking dendritic cells with B7-DC XAb results in a number of phenotypic changes, including enhanced survival, increased ability to accumulate antigen, enhanced ability to activate naïve T cells, increased efficiency of seeding draining lymph nodes, and the up-regulation of key immunomodulatory cytokines [2], [22]–[24]. B7-DC has no known ability on its own to transduce an intra-cellular signal. Therefore, we have focused on delineating the events that occur on the surface of the DCs upon binding of B7-DC XAb and to determine what membrane and intracellular molecules mediate the various DC phenotypic responses. Previously, we found that cross-linking B7-DC induced a substantial reorganization of cell surface molecules, resulting in the clustering of MHC, CD80, CD86, and CD11c molecules into a cap [3]. In this report we document that CD40 is also present within this cell surface complex on DCXAb. The recruitment of CD40 is necessary for the B7-DC XAb-induced activation of NFκB and protection of DC from cell death signals, a response also linked to the secretion of cytokines such as IL-6. Moreover, CD40 expression is required for DCXAb-induced conversion of T regulatory cells to IL-17-producing effector cells in vitro and the induction of CTLs capable of conferring tumor-protective immunity in mice. However, under culture conditions where the DCs are compromised in their ability to secrete IL-6 in response to B7-DC XAb treatment, addition of exogenous IL-6 resulted in generation of IL-17 cells, albeit at a lesser frequency. Antigen presentation by DCcntrl in the presence of exogenously added IL-6 fail to induce Treg conversion. This implies that apart from IL-6, other unknown factors from DCXAb are necessary to cause complete conversion of T regulatory cells. Taken within the context of our previous finding that TREM-2 recruitment on DCXAb mediates the induction of antigen accumulation by mature DC [3], we conclude that B7-DC XAb has its many unique biologic effects due to its ability to specifically target the surface of DC, recruit, and activate common signaling pathways in DC that, in turn, modulate multiple T cell functions. Dendritic cells treated with B7-DC XAb exhibit some similarities to DC activated by other means, but there are key differences. Typically, stimulation through TNFR, CD40, or TLR as well as through TREM-2 results in a maturation response in DC, which is associated with the up-regulation of CD80, CD86, and MHC class II and a decrease in antigen uptake [25], [26]. However, DCXAb do not exhibit this classic “mature” phenotype. In fact, B7-DC XAb causes matured DC to regain the ability take up antigen [2], and it stimulates antigen presentation by immature DC [27]. B7-DC cross-linking has little effect on the levels of class II, CD80, and CD86, but upregulates CCR7 expression. CD40 ligation typically down-regulates TREM-2 [28], but our studies show that B7-DC XAb co-opts both CD40 and TREM-2 signals. Many TLR and TNFR family members activate NFκB through MyD88 [29] whereas B7-DC XAb activation of NFκB is MyD88-independent [6]. Other cell surface receptors present on DC (TLRs, CD80, CD86, and TREM-1) can activate NFκB [29], but experiments with CD40−/− DC suggest that CD40 is the one required by B7-DC XAb. TREM-2 binds DAP12 to activate Syk. Stimulation of DC via TREM-2 also induces a maturation response and activates ERK [25]. B7-DC XAb activates ERK, but ERK is not involved in the TREM-2 mediated response of DC to B7-DC XAb [3]. However, ERK activation was dependent on the expression of CD40 on the dendritic cells activated with B7-DCXAb. These findings suggest that the combined signals from CD40 and TREM-2 upon B7-DC cross-linking are different than when either signal is generated through traditional means, or that each of these signals (and perhaps other signals not yet delineated) are only partially “on” in DCXAb. Thus, fundamental to the mechanism of action of B7-DC XAb is its ability to activate CD40 and TREM2 pathways in an indirect, but DC-specific manner. Cell-specific activation of multiple pathways is potentially advantageous from a therapeutic perspective, especially when it can be induced with a single reagent. Furthermore, B7-DC XAb is effective as a modulator of T cell functions in animal models, even in the absence of ex vivo manipulations. Teasing out additional details in the signals emanating from B7-DC XAb will be critical to furthering our understanding of how of this novel immune modulator works. For example, various TNF receptor associated factors (TRAFs) mediate a diverse array of events downstream of CD40 and related receptors [30]. A role for TRAFs in DCXAb remains to be tested. Whether B7-DC XAb also activates other kinases that regulate NFκB (e.g., pim1 dependent kinase, [31]) is not known. Whether B7-DC XAb affects cIAP, Bcl molecules, or additional survival-promoting pathways is also not known. The net effect of using this reagent in animal models is an antigen-specific modulation of immune responses resulting in the alleviation of allergic responses, the clearance of tumors, and the induction of specific autoimmunity [4], [5], [18], [19], [32]. Studies with B7-DC XAb have provided new insights into the plasticity of T cell lineages and have expanded the potential of immune-based therapies to impact disease. In this regard, we have shown that B7-DC XAb also activates human DC [6], allowing for results in mouse models to be rapidly translated into the clinic. Generation of such highly antigen specific immunity in the absence of autoimmunity with B7-DC XAb [5] suggests a hypothesis that IgM-based therapeutic antibodies provide a way to induce the integration of complex signals in a cell-specific manner. Testing of this hypothesis and translation of B7-DC XAb to treat human cancers is now underway. Materials and Methods Ethics Statement All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the Institutional Animal Care and Use Committee of Mayo Clinic (protocol numbers A10306, A207, and A33403). Mice and reagents C57BL/6J mice, BALB/CJ mice, DO11.10 mice, CD40−/− mice, and IL-6−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Male mice were used, and all experiments were conducted with IACUC oversight. The B16 melanoma cell line was kindly provided by Dr. Richard Vile (Mayo Clinic, Rochester, MN) and the RENCA cell line was kindly provided by Dr. Eugene Kwon (Mayo Clinic, Rochester, MN). EL-4 and WEHI-3 lines were purchased from American Type Culture Collection (ATCC, Vanassas, VA). The TUBO cell line, derived from a spontaneous breast carcinoma in a Balb-neuT mouse [33], was a gift from Dr. Esteban Celis (Moffitt Cancer Center, Tampa, FL). All tumor cell lines were maintained in DMEM (Life Technologies Invitrogen) containing 10% cosmic calf serum (Hyclone). Anti-mouse CD4-PE (RM4-5), MHC class II specific IgM (25-9-3), and purified anti-mouse MHC class II(M5/114.15.2) were purchased from BD Biosciences (San Jose, CA). APC-coupled FoxP3 antibody (FJK-16s), anti-mouse IL-17A-PE (eBio17B7) and purified and PE-coupled anti-mouse CD40 (IC10) and APC-coupled anti-mouse MHC class II antibody (M5/114.15.2) were purchased from eBioscience (San Diego, CA). Antibody against Syk kinase (4D10) and ERK (C.14) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine (4G10), anti phospho ERK (9101) and Goat anti-mouse antibodies were obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). Rabbit antibody against DAP12 (MC457) was developed by Dr. Paul Leibson (Mayo Clinic, Rochester, MN). Protein A-Sepharose was purchased from Pierce Biotechnology (Rockford, IL). For analysis of co-precipitating signaling molecules, affinity purified antibody against mouse Class II (I-Ab) (KH74) (BD Biosciences, San Jose, CA) was used. Vitamin D3 (1α, 25-dihydroxyvitamin D3) was a gift from Dr. Matthew Griffin (Mayo Clinic, Rochester, MN). Receptor activator of the NF-κB (RANK) ligand was purchased from Chemicon International. The TLR ligands were used at 10 µg/ml and were as follows: TLR2 ligand, Pam3CSK4 (InVivogen); TLR3 ligand, polyI∶C (Calbiochem); TLR 4 ligand, LPS (Sigma); TLR7/8 ligand, Gardiquimod (InVivogen); TLR9 ligand, CpG (synthesized in Mayo core facility). B7-DC XAb The human monoclonal IgM antibodies B7-DC XAb (sHIgM12) and isotype-matched control (sHIgM39) were identified in a screen for mouse DC-binding antibodies present in a bank of serum samples from patients with monoclonal gammopathies. The DC-binding sHIgM12 antibody and the non-binding sHIgM39 control antibody and were purified as described [1]. Because of the dependence on B7-DC for its biologic properties, the requirement for the pentameric form, and the observed signals in DC elicited by antibody binding, we refer to sHIgM12 as B7-DC cross-linking antibody (B7-DC XAb). Flow Cytometry and Fluorescence Resonance Energy Transfer (FRET) Flow cytometry was used to quantify close molecular interactions on the cell surface as described previously [34]. Briefly, mouse DC were stained with anti-Class II-APC (M5/114.15.2) and anti-CD40-PE (IC10). All staining was for 15 minutes. Cells were stimulated with 10 µg/ml control antibody or B7-DC XAb or purified anti-mouse MHC class IIIgM (25-9-3) and aliquots from different groups were taken at different time points. The cells were washed and fixed in 2% paraformaldehyde prior to analysis by FACS performed by the Mayo Flow Cytometry Core Facility using a FACSCaliber (BD Biosciences, Franklin Lakes, NJ). FRET (upon excitation of PE at 488 nm and emission of APC at 660 nm) was visualized in FL3 channel (650–670 nm LP). Data collected as log10 fluorescence were analyzed using CellQuest (BD Biosciences). MFI = Mean Fluorescence Intensity. Isolation of Tregs and non-Tregs Splenocytes were isolated from pooled spleens harvested from three mice. Tregs were isolated by positive selection using Mouse Treg Isolation kit from Miltenyi Biotec (Auburn, CA), as per the manufacturer's protocol [5]. Briefly, splenocytes were incubated with anti-CD25 antibody coupled to magnetic beads for 15 min prior to binding to the MACS column. Cells that were not bound were washed three times with RPMI/10% FBS and used as non-Tregs. Adherent cells (Tregs) were eluted and washed prior to use. In vitro activation of Tregs and non-Tregs Bone marrow derived WT or CD40−/− immature DCs (2×106) were pulsed with antigen and treated with control antibody or B7-DC XAb overnight then used to stimulate naïve DO11.10 Tregs in culture (together in 24-well plates) at a 1∶1 ratio in vitro for 48 hours. After 48 hours, cells were removed, pooled, and prepared for analysis by intracellular staining for the expression of FoxP3 and IL-17A. In some experiments, 20 ng/ml of IL-6 (R&D Systems, Minneapolis) was added at the start of the culture. Briefly, cells were permeabilized with CytoFix/CytoPerm kit (BD Biosciences, San Diego, CA) and incubated with the appropriate conjugated antibody at 4°C according to the manufacturers' suggestions prior to analysis by flow cytometry as described [5]. Generation of bone marrow DCs DCs were generated from the mouse bone marrow [35]. Bone marrow cells were plated (1×106/ml) in RPMI /10% serum containing 10 µg/ml of murine GM-CSF and 1 ng/ml of murine IL-4 (PeproTech, Rocky Hill, NJ). The culture medium was changed on day 2. DCs were used on day 6 unless otherwise indicated. For adoptive transfer experiments, day 6 DC were pulsed with antigen (1 mg/ml) and isotype control antibody or B7-DC XAb (10 µg/ml) overnight and injected into mice the following day. Confocal microscopy Wild type or CD40−/− DC were stimulated with 10 µg/ml of Ab or TLR ligand for 15 minutes, fixed and permeablized using Cytofix/Cytoperm kit (BD Pharmingen). Subsequently, Ab against a C-terminal peptide of NFκB was added followed by anti-rabbit FITC. Nuclei were stained with DAPI (Sigma-Aldrich) before being observed with a LSM510 laser scanning confocal microscope (Carl Zeiss) at 40× magnification and LSM510 software was used for the analysis. Cell viability assay Wild type or CD40−/− DCs were tested for their viability in a cytokine-deprived environment as previously described [6]. In brief, day 5 DCs were plated in triplicate into 96-well plates (2×104 cells/well) in RPMI 1640 without serum, GM-CSF, or IL-4 and in presence of B7-DC XAb, isotype control Ab, or RANK ligand (10 µg/ml). For experiments using vitamin D3 to induce cell death [17], DCs were matured with 200 ng/ml CpG plus 10 nM vitamin D3. All cultures were maintained overnight then Alamar Blue (Biosource International) was added to a final concentration of 10% (v/v). Readings were taken after additional 24 h of culture using a Spectra Max M2 Multi Detection Reader (Molecular Devices) set to an excitation wavelength of 520 nm and an emission reading of 590 nm. Tumor experiments All in vivo tumor experiments were carried out as previously described [19]. Briefly, mice (wild-type or CD40−/−) were injected with the indicated number of tumor cells in the right flank, and received control antibody or B7-DC XAb (30 µg) by intravenous administration. The mice were monitored regularly for tumor growth. To assess CTL responses, draining lymph node cells (from 2 mice in each group) were harvested on day 7, pooled, and used as effectors against the 51Cr-labeled B16 melanoma or WEHI-3 target and EL4 or P815 control cells. The remaining mice were monitored for tumor growth for the times indicated. For experiments using adoptive transfer of DC, mice received B16 melanoma lysate-pulsed wild type or CD40−/− dendritic cells (2×106, intraperitoneally) pretreated overnight with 10 µg/ml control antibody or B7-DC XAb. In all experiments, mice bearing tumors of size 17×17 mm were euthanized as per the Institutional Animal Care and Use Committee recommendations. Fisher exact probability test analyses were conducted using SigmaPlot software. IL-6 ELISA Bone marrow-derived DC from wild type or CD40−/− mice (1×106 DC/well) were stimulated for 48 h with 10 µg/ml B7-DC XAb, control antibody, anti-CD40 antibody, or TLR ligand. Murine IL-6 was detected in culture supernatants using Ready-Set-Go ELISA kits from e-bioscience (San Diego, CA, USA) as per the manufacturer's instructions. Samples were run in triplicate and values are recorded as mean±standard error of the mean. Statistical analysis was performed by one-way ANOVA using Sigma Stat software. Competing Interests: Potential conflict of interest is declared as Mayo Clinic holds intellectual property rights to the immune modulator B7-DC XAb, and authors LRP, SR, and VV could receive monetary compensation at some future time. However, no actual financial conflict is declared and potential conflicts are being managed by our institutional conflict of interest committee. Funding: This work was supported by National Institutes of Health (www.nih.gov/) grants CA104996-05 and HL077296-3 to LRP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscript. ==== Refs References 1 Radhakrishnan S Nguyen LT Ciric B Ure DR Zhou B 2003 Naturally occurring human IgM antibody that binds B7-DC and potentiates T cell stimulation by dendritic cells. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1939918809-PONE-RA-08477R110.1371/journal.pone.0005365Research ArticleOncology/Breast CancerOncology/Prostate CancerOncology/Skin CancersA Novel Triterpenoid Isolated from the Root Bark of Ailanthus excelsa Roxb (Tree of Heaven), AECHL-1 as a Potential Anti-Cancer Agent Anticancer Activity of AECHL-1Lavhale Manish S. 1 Kumar Santosh 2 Mishra Shri Hari 1 Sitasawad Sandhya L. 2 * 1 Pharmacy Department, Faculty of Technology and Engineering, The M. S. University of Baroda, Vadodara, Gujarat, India 2 National Centre for Cell Science, NCCS Complex, University of Pune Campus, Ganeshkhind, Pune, Maharashtra, India Bauer Joseph Alan EditorCleveland Clinic, United States of America* E-mail: [email protected] and designed the experiments: SK SLS. Performed the experiments: MSL SK. Analyzed the data: MSL SK SHM SLS. Contributed reagents/materials/analysis tools: SHM SLS. Wrote the paper: SK SLS. 2009 28 4 2009 4 4 e53653 2 2009 11 3 2009 Lavhale et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background We report here the isolation and characterization of a new compound Ailanthus excelsa chloroform extract-1 (AECHL-1) (C29H36O10; molecular weight 543.8) from the root bark of Ailanthus excelsa Roxb. The compound possesses anti-cancer activity against a variety of cancer cell lines of different origin. Principal Findings AECHL-1 treatment for 12 to 48 hr inhibited cell proliferation and induced death in B16F10, MDA-MB-231, MCF-7, and PC3 cells with minimum growth inhibition in normal HEK 293. The antitumor effect of AECHL-1 was comparable with that of the conventional antitumor drugs paclitaxel and cisplatin. AECHL-1-induced growth inhibition was associated with S/G2-M arrests in MDA-MB-231, MCF-7, and PC3 cells and a G1 arrest in B16F10 cells. We observed microtubule disruption in MCF-7 cells treated with AECHL-1 in vitro. Compared with control, subcutaneous injection of AECHL-1 to the sites of tumor of mouse melanoma B16F10 implanted in C57BL/6 mice and human breast cancer MCF-7 cells in athymic nude mice resulted in significant decrease in tumor volume. In B16F10 tumors, AECHL-1 at 50 µg/mouse/day dose for 15 days resulted in increased expression of tumor suppressor proteins P53/p21, reduction in the expression of the oncogene c-Myc, and downregulation of cyclin D1 and cdk4. Additionally, AECHL-1 treatment resulted in the phosphorylation of p53 at serine 15 in B16F10 tumors, which seems to exhibit p53-dependent growth inhibitory responses. Conclusions The present data demonstrate the activity of a triterpenoid AECHL-1 which possess a broad spectrum of activity against cancer cells. We propose here that AECHL-1 is a futuristic anti-cancer drug whose therapeutic potential needs to be widely explored for chemotherapy against cancer. ==== Body Introduction According to the World Health Organization based on morbidity, mortality, economic burden, and emotional hardship, cancer may be considered the most onerous health problem afflicting people worldwide [1]. Currently, over 22.4 million people in the world are suffering from cancer. Approximately 10.1 million new cases are diagnosed with cancer annually, and more than 6.2 million die of the disease in the year 2000 [2]. This represents an increase of around 19% in incidence and 18% in mortality since 1990. An important aim of cancer research is to find therapeutic compounds having high specificity for cancerous cells/tumor and fewer side effects than the presently used cytostatic/cytotoxic agents. Numerous plant-derived compounds used in cancer chemotherapy include vinblastine, vincristine, camptothecin derivatives, etoposide derived from epipodophyllotoxin, and paclitaxel (taxol®) [3]. However most of these compounds exhibit cell toxicity and can induce genotoxic, carcinogenic and teratogenic effects in non-tumor cells, and some of them failed in earlier clinical studies [4], [5]. Another most widely used metal-based drug at present against selected types of cancers is cisplatin [6], but use of cisplatin in curative therapy was associated with some serious clinical problems, such as severe normal tissue toxicity and resistance to the treatment [7]. These side effects limit their use as chemotherapeutic agents despite their high efficacy in treating target malignant cells. Consequently, new therapies and treatment strategies for this disease are necessary for treating patients with this disease. Therefore, the search for alternative drugs that are both effective in the treatment of cancers as well as non-toxic to normal tissue is an important research line [8]. Terpenoids are used extensively for their aromatic qualities. They play a role in traditional herbal remedies and are under investigation for antibacterial, antineoplastic, and other pharmaceutical functions. Natural triterpenoids, such as oleanolic acid and ursolic acid, are compounds with anti-tumorigenic and anti-inflammatory properties [9]. Synthetic triterpenoid derivatives such as 2-Cyano-3, 13 dioxooleana-1,9(11)-dien-28-oic acid (CDDO) [10] and its derivative 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) [11] also have anti-tumor activity. Root bark of Ailanthus excelsa Roxb (Tree of Heaven), a tree belonging to family Simaroubaceae is widely used in Ayurveda as evidenced by phytotherapy [12]. Other species from this family are well known for their anti-cancer activities [13]. Chemical constituents of A. excelsa include some triterpenes and alkaloids [14]. In the present study we have evaluated the in vitro and in vivo anti-cancer activity of a novel triterpenoid, AECHL-1 isolated from the root bark of the plant and found to be highly effective in cancer cells of different lineage. Materials and Methods Isolation and characterization of AECHL-1 The root bark of A. excelsa was botanically verified by Professor Shrihari Mishra (one of the authors in the present manuscript) and the extraction and fractionation of air-dried powdered root bark was done using chloroform. Isolation of AECHL-1 was done using silica gel column chromatography and characterized by ultra violet (Shimadzu 1700), infra red (Perkin Elmer Spectrum RX1), nuclear magnetic resonance (Bruker Avance I NMR Spectrometer) and mass spectroscopy (by Jeol SX 102 mass spectrometer). The purity of the AECHL-1 was assessed by HPLC on a RP C-18 Phenomenex column using methanol-water (90∶10, volume for volume) as the mobile phase. The purified compound, AECHL-1 was dissolved in DMSO as stock solutions. Cell lines Normal human embryonic kidney cell line (HEK 293), mouse melanoma B16F10 cells (B16F10), human breast carcinoma (MDA-MB-231), human breast adeno-carcinoma (MCF-7) and human prostate (PC3) cells were obtained from ATCC (Manassas, VA). HEK 293, MCF-7 and B16F10 cells were cultured in Dulbecco's modified Eagle's medium and PC3 in Ham's F-12 media (Gibco) at 37°C under 5% CO2. MDA-MB-231 cells were cultured in Leibovitz's L-15 (Gibco) supplemented with 10% FCS (Gibco), 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere at 37°C. Cell viability assay Direct interference between different concentrations of AECHL-1 (0–200 µM) and MTT in a cell-free system was not observed, therefore, MTT assay was used to test cell viability in the current system. HEK 293, B16F10, PC3, MCF 7 and MDA-MB-231 cells (4×103/well) were cultured in 96-well plates and after 24 h treated with different concentrations of AECHL-1 (0–200 µM), cisplatin (0–100 µM) or paclitaxel (0–50 µM) for 12, 24, and 48 hr at 37°C. Cell viability was assessed by MTT (0.5 mg/ml) conversion as described previously [15]. Cell proliferation assay Proliferation of MCF-7 cells was determined by measuring (3H) thymidine incorporation. Briefly, aliquots of complete medium containing 4×103 cells were distributed into 96-well tissue culture plates. After 24 hr, the media were replaced with various concentrations of the AECHL-1 (0–100 µM), cisplatin (0–100 µM) or paclitaxel (0–50 µM). Six hours after the treatment 1 µCi/well (3H) thymidine (Board of Radiation and Isotope Technology, Mumbai, India) was added and the cultures were incubated further for 42 hr at 37°C. Cells were rinsed and collected in scintillation mixture, and radioactivity incorporated into the DNA was determined with a liquid scintillation counter (Canberra Packard). Annexin V-FITC binding assay B16F10, MDA-MB-231 and MCF-7 cells (3×105/ml) were treated with various concentrations of AECHL-1 (0–40 µM) for 24 hr at 37°C. Cells were harvested after 24 hr, apoptosis was detected by using Annexin V-FITC apoptosis detection Kit (Calbiochem, USA) with flow cytometry (FACS Vantage–BD Sciences, USA). The data was analyzed using Cell Quest software for determining the percent of apoptotic cells. Cell cycle analysis B16F10, PC3, MDA-MB-231 and MCF-7 cells (3×105/ml) were treated with various concentrations of AECHL-1 (0–100 µM), or paclitaxel (0–10 µM) for 24 hr. Cell cycle analysis was performed as described earlier [16], with flow cytometry (FACS Vantage–BD Sciences, USA). The data was analyzed using Cell Quest software. Immunocytochemistry MCF-7 cells were fixed with 3.7% paraformaldehyde, and then incubated with anti-α-tubulin antibodies (1∶10000; Sigma, St. Louis, MO). After the antibodies were washed off, the cells were incubated with alexa-conjugated secondary antibodies (1∶200; Sigma, St. Louis, MO). Images were captured with a confocal laser scanning microscope (Zeiss LSM510). Animal tumor models Male C57BL/6 (6–8 weeks of age) and female athymic nude mice, NIH, nu/nu Swiss (10 weeks) were maintained in accordance with the Central Animal Ethical Committee procedures and guidelines. B16F10 melanoma cells were harvested, suspended in PBS, and subcutaneously injected into the right flank (2×106 cells/flank) of C57BL/6 mice and MCF-7 cells (5×106 cells/flank) into female athymic nude mice. Each athymic mouse was implanted subcutaneous with a 0.72-mg of 17-β-estradiol pellets, 2 weeks before inoculation of MCF-7 cells [17], [18]. Tumor size was measured every 3–4 days by a caliper and tumor volumes determined by the length (L) and the width (W): V = (LW 2)/2 [19]. After two weeks, AECHL-1 (50 µg), AECHL-1 (100 µg), cisplatin (100 µg) and PBS as vehicle control were injected subcutaneously to the site of tumor for 15 days in C57BL/6 mice (n = 6) and AECHL-1 (5 µg), AECHL-1 (10 µg), paclitaxel (20 µg) and PBS as vehicle control were injected subcutaneously to the site of tumor per day for 10 days in female athymic nude mice (n = 6). Tumor volume was measured at regular interval during the study. At the end of the experiment tumor and other organs were dissected out for histological analyses and western blots. Immunohistochemistry Tissues and organs of C57BL/6 and nude mice were fixed in alcohol formalin for 24 hr and embedded in paraffin as previously described [20]. Tissue sections (5 µm) were stained with hematoxylin and eosin (H & E), visualized and photographed with an inverted microscope (Nikon, ECLIPSE, TE2000-U, Japan). Immunoblotting Tumor tissue was homogenized in RIPA buffer (20 mM Tris–HCl pH 7.5, 120 mM NaCl, 1.0% Triton ×100, 0.1% SDS, 1% sodium deoxycholate, 10% glycerol, 1 mM EDTA and 1× protease inhibitor cocktail, Roche) proteins were isolated in solubilized form and concentrations were measured by Bradford assay (Bio-Rad protein assay kit). Solubilized protein (60 µg) was denatured in 2× SDS-PAGE sample buffer (sigma), resolved in 10% SDS–PAGE and transferred to nitrocellulose membrane followed by blocking of membrane with 5% nonfat milk powder (w/v) in TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween 20). The membranes were incubated with rabbit polyclonal anti-p21 and anti-pp53 antibodies (1∶1000; Santa Cruz, CA), mouse monoclonal anti-CDK4, anti-Cyclin D1 antibodies (1∶1000; Cell Signaling Technology, Beverly, MA), mouse monoclonal anti-c-Myc antibody and mouse monoclonal anti-p53 antibody (1∶1000; Abcam, USA), followed by HRP-conjugated appropriate secondary antibodies and visualized by an enhanced chemiluminescence (Pierce) detection system. Membranes were stripped and re-probed with β-actin primary antibody (1∶10000; MP Biomedicals, Ohio, USA) as a protein loading control. Statistics The data reported for tumor volumes are expressed as mean±SEM. Statistical differences were determined by ANOVA and post test applied was Tukey-Kramer multiple comparison Test. Results Chemistry: Ultraviolet, infra red, nuclear magnetic resonance, and mass characterization of AECHL-1 IR (KBr): 3425, 3419 (hydroxyl group), 2972, 2966, 2923, 2873 (alkyl C-H stretch), 1733 (δ lactone), 1718 (Bi acetyl), 1680 (C = O conjugation with alkene), 1652 (-C = C stretching), 1600 (aromatic), 1492, 1454, 1394 (methyl stretching), 1222 (δ lactone), 1184, 1110, 1051, 1031 (acetals), 1018 nm (alkanes). 1H-NMR (DMSO, 400 Hz) δ: 0.95 (3H, t, 4′-CH3), δ:1.15 (3H, d, H-24), δ:1.235 (3H, d, 5′-CH3), δ: 1.5 (2H, ddd, 5′-CH2), δ: 1.73 (3H, ddd, H-21), δ: 1.83 (1H, s, H-9), δ: 1.87 (1H, s, H-14), δ: 1.9 (2H, s, H-18), δ: 2.16 (3H, s, H-18), δ: 2.3 (3H, d, H-19) δ: 2.71 (2H, s, H-20), δ: 3.45 (2H, dd, H-23), δ:3.65 (2H, d, H-22), δ: 3.95 (1 H, t, H-12), δ: 4.05 (2H, s, H-22), δ: 5.30 (1H, s, H-15), δ: 5.46 (1H, s, OH-2), δ: 5.73 (1H, d,OH-2′), δ: 6.89 (1H, s, H-3), δ: 8.82 (1H, s, OH-11). Fast atom bombardment mass spectroscopy: m/z: 1068 due to dimmer formation. The actual (M+) was considered to be 543.8, 463.3 (M-C4H1O2), 461.4 (M-C4H2O2), 459.4 (M-C4H4O2), 361.2 (M-C9H11O4) (Figure 1B) and Mass Spectra (Figure S1). AECHL-1 is a solid, mp. 248–250°C possessed a molecular formula of C29 H36O10 as indicated by EI and ES mass spectra. The IR spectrum showed the presence of hydroxyl (s) (3425 nm, 3419 nm), δ lactone (1733 nm), and aromatic moiety (1600 nm). The UV spectrum gave a characteristic absorption maximum at 235 nm, indicating the presence of auxochromic groups like hydroxyl and ketone. The 1H-NMR spectrum of AECHL-1 revealed the presence of an aromatic proton δ 6.89 and a singlet at δ 5.30 which is characteristic of the ester function at C-15. H-22 appeared as an AB system as a singlet at δ 4.05 and doublet at δ 3.65 and H-12 appeared as a triplet at δ 3.95. The methyl group H-19 on the aromatic ring appeared as singlet at δ 2.3. A doublet at δ 1.235 for six protons is assigned at H-5′. H-4′ appeared as a triplet at δ 0.95. The methyl group, H-18 appeared as a singlet at δ 2.16 (Figure 2). 10.1371/journal.pone.0005365.g001Figure 1 Purity of AECHL-1 as assessed by HPLC. Single peak indicated that the preparation was >99% pure. 10.1371/journal.pone.0005365.g002Figure 2 Structure of AECHL-1 with its mass fragments by NMR spectroscopy. Inhibition of cell viability, proliferation, and apoptosis by AECHL-1 Effect of AECHL-1 on the viability of B16F10, PC3, MDA-MB-231 and MCF-7 cells was assessed. AECHL-1 inhibited cell growth of MCF-7 cells in a concentration- and time-dependent manner by MTT assay (Figure 3A). AECHL-1 inhibited cell growth in different cancer cell lines with a minimum growth inhibition in HEK 293 at 48 hr (Figure 3B). HEK 293 treated with 200 µM AECHL-1 exhibited high survival rate (>90%) as compared to cancer cells. AECHL-1 was found to be more effective on MCF-7 in comparison with B16F10, PC3 and MDA-MB-231 in cell proliferation inhibition as observed by the (3H) thymidine uptake after 48 hr (Figure 3C). Moreover, AECHL-1 was found to be more potent than paclitaxel or cisplatin in cell proliferation inhibition in MCF-7 cells after 48 hr (Figure 3D). 10.1371/journal.pone.0005365.g003Figure 3 Growth inhibition and cell proliferation of different tumor cell lines by AECHL-1 in vitro. (A) Cell growth by MTT assay in MCF-7 cells were treated with different concentrations of AECHL-1 (10, 20, 40 and 100 µM) for 12, 24 and 48 hr and cell viability was determined by MTT assay; (B) Cell growth by MTT assay in B16F10, PC3, MDA-MB-231 MCF-7 and HEK-293 cells. Cells were treated with different concentrations of AECHL-1 (10, 20, 40 100 and 200 µM) for 48 hr, and cell viability was determined by MTT assay; (C) Cell proliferation by (3H) thymidine incorporation in B16F10, PC3, MDA-MB-231, and MCF-7 cells. Cells were treated with different concentrations of AECHL-1 (10, 20, 40 and 100 µM) for 48 hr, and cell proliferation was determined by (3H) thymidine incorporation; (D) Comparison of AECHL-1 with other chemotherapeutic drugs. MCF-7 cells were treated with different concentrations (5, 10, 20, and 50 µM) of paclitaxel, cisplatin and AECHL-1 for 48 hr, and cell proliferation was determined by (3H) thymidine incorporation. Data are means±SEM of three independent experiments. Annexin V-conjugated FITC and propidium iodide (PI) stain was used to analyze the total percentage of apoptotic cells induced by AECHL-1. The investigator to identify early apoptotic cells (Annexin V-FITC positive, PI negative), cells that are in late apoptosis (Annexin V-FITC and PI positive), the necrotic cells (PI positive only) and cells that are viable (Annexin V-FITC and PI negative). Total percentage of apoptotic cells increased up to 36.25% and 37.18% at 20 µM in B16F10, MDA-MB-231 cells respectively and 60.66% at 5 µM in MCF-7 cells (Figure 4). 10.1371/journal.pone.0005365.g004Figure 4 Effect of AECHL-1 on apoptosis of tumor cells. Detection of apoptosis was done by the Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions and then analyzed by flow cytometry: UR indicates the percentage of late apoptotic cells (Annexin V and PI positive cells), and LR indicates the percentage of early apoptotic cells (Annexin V positive cells) The data are presented in dot blots depicting annexin/fluorescein isothiocyanate (x axis) vs. PI staining (y axis). The percentage of cells in each quadrant is shown. The results are representative of three independent experiments. AECHL-1 induced cell cycle arrest in cancer cells To determine the phase of the cell cycle at which AECHL-1 exerts its growth-inhibitory effect, exponentially growing B16F10, PC3, MDA-MB-231 and MCF-7 cells were treated with different concentrations of AECHL-1 for 24 hr and analyzed by flow cytometry (Table 1). We observed that B16F10 cells treated with AECHL-1 showed an increase in the population in G1 phase (52.18–72.08 %) with a concomitant decrease in the percentage of cells in S-G2/M phase (47.98–26.16%), suggesting a G1 arrest. In contrast, the number of PC3, MDA-MB-231 and MCF-7 cells in S-G2/M phase increased from 42.91% to 57.62%, 49.40% to 77.16% and 45.13% to 70.97% respectively in response to treatment with AECHL-1 and a decreased in G1 phase from 55.65% to 39.02%, 49.54% to 22.82%, 53.67% to 27.85% respectively suggesting a growth arrest in S-G2/M phase in PC3, MDA-MB-231 and MCF-7 cells. Paclitaxel treatment showed an increase in the population of MCF-7 cells in G2/M phase (29.30% to 72.55%) with a decrease in the percentage of cells in G1 phase (48.30% to 4.62%) suggesting a growth arrest in G2/M phase (Table 1). These results suggest that inhibition of cell cycle progression could be one of the molecular events associated with selective anti-cancer efficacy of AECHL-1 in cancer cells. 10.1371/journal.pone.0005365.t001Table 1 Cell cycle analysis of AECHL-1–treated cells. Cell line Compound Conc. (µM) Phase of cell cycle (% of cells) Sub G0 G1 S G2/M B16F10 AECHL-1 0 0.24 52.18 21.39 26.59 10 0.39 56.55 20.46 23.02 20 0.65 57.67 18.4 23.6 40 2.05 72.08 11.43 14.73 100 7.82 64.04 15.41 13.23 PC3 AECHL-1 0 1.44 55.65 15.21 27.7 10 1.66 50.74 16.21 30.94 20 1.79 48.96 13.99 34.99 40 4.14 44.26 17.71 33.82 100 3.36 39.02 20.25 37.37 MDA-231 AECHL-1 0 1.48 49.54 25.79 23.61 10 1.03 43.03 24.06 32.21 20 0.95 33.18 28.63 38.01 40 0.54 26.66 28.12 45.15 100 0.58 22.82 30.99 46.17 MCF-7 AECHL-1 0 1.64 53.67 19.03 26.1 4 1.61 35.04 27.2 36.65 10 1.99 27.85 34.19 36.78 20 1.81 29.68 37.44 31.98 40 2.55 36.26 32.5 29.42 MCF-7 Paclitaxel 0 3.17 48.3 20.42 29.3 1 3.45 4.62 18.65 72.55 2 5.21 7.41 22.89 63.71 5 3.98 5.34 18.35 72.86 10 3.47 4.86 18.96 69.36 Effect of AECHL-1 on cell cycle progression in B16F10, PC3, MDA-231, MCF-7 and paclitaxel in MCF-7 cells in 24 hr of treatment. Cell cycles were analyzed using propidium iodide. DNA content was analyzed using FACS to determine the cell cycle distribution. Effect of AECHL-1 on cellular microtubules Microtubule staining in control and cells treated with AECHL-1 and paclitaxel, showed that both AECHL-1 and paclitaxel resulted in microtubule disruption with an increase in the density of cellular microtubules and formation of thick microtubule bundles surrounding the nucleus in comparison to the untreated control cells (Figure 5). 10.1371/journal.pone.0005365.g005Figure 5 Effect of AECHL-1 on microtubules. MCF-7 cells were treated with the vehicle as a control, AECHL-1 (5 µM) and paclitaxel (5 µM) as a positive control for 24 h, and microtubules (red) were visualized by indirect immunofluorescence. DAPI was used to stain the cell nuclei (blue). Representative of 25–30 cells each in 3 separate experiments. Effect of AECHL-1 on primary tumor volume in allograft and xenograft We also examined the effects of AECHL-1 on the in vivo growth of primary tumors. Our preliminary studies showed that, of the various doses of AECHL-1 (0.5 to 5 mg/kg) injected intraperitoneal in C57BL/6 mice, the maximum tolerated dose was a single dose of 0.5 mg/kg that showed no obvious sign of toxicity when observed for one month. On this basis, the dose that was chosen was 50 and 100 µg/kg/day (a dose that was 10–20% of this maximum tolerated dose). On day 18 significant increase in tumor volume in control group (p<0.001) and a regression in tumor volume was evident in mice treated with 50 µg AECHL-1 (44.303±5.20 % (p<0.001)) and with 100 µg AECHL-1 (51.014±1.27% (p<0.001)). Tumors treated with 100 µg cisplatin showed a reduction of tumor volume (93.13±0.539% (p<0.001)). However, AECHL-1 (50 µg) vs. AECHL-1 (100 µg) was found to be non Significant (P>0.05). On day 24 control, AECHL-1 (50 µg) and AECHL-1 (100 µg) treated mice showed further increase in tumor volume (p<0.001) but mice treated with cisplatin showed reduction in tumor volume (p<0.001). However, although cisplatin showed further reduction in tumor volume, the damage caused to other organs was more than that in the AECHL-1 (50 & 100 µg) treated group in C57BL/6 mice (Figure 6A and 6B). 10.1371/journal.pone.0005365.g006Figure 6 Effect of AECHL-1 on primary tumor volume in allograft and xenograft. (A) Photographs of C57BL/6 mice showing 4-week-old allograft tumor growth by B16F10 cells; below, excised tumors with respective mice; (B) Tumor volume was determined at timed intervals as described in “Materials and Methods”. Tumor volume of experimental animals after treatment with 50, 100 µg AECHL-1 and 100 µg cisplatin was compared with the tumor volume of control animals; (C) Photographs of athymic nude mice showing 4-week-old xenograft tumor growth by MCF-7 cells; below, excised tumors with respective mice; (D) Tumor volume of experimental animals after treatment with 5, 10 µg AECHL-1 and 20 µg paclitaxel was compared with the tumor volume of control animals. Results represent the mean±SE of six starting animals in each group. Significant differences between Intra group at each time point are represented as: ns p>0.05, p<0.05, P<0.01, *P<0.001 and #Inter group at different doses are represented as ns P>0.05, #<0.05, ##P<0.01, ###P<0.001. Since the cytotoxic doses of AECHL-1 for MCF-7 cells in vitro were very low, the doses selected for tumor xenografts in female athymic nude mice injected with MCF-7 cells were 5 and 10 µg. These doses showed regression in tumor volume as: 35.72±0.05% for 5 µg (p<0.001) and 28.55±0.06% for 10 µg (p<0.001) whereas tumors treated with 20 µg paclitaxel showed a regression in tumor volume (14.19±0.32% (p<0.05)), which was less than the AECHL-1 treated group (Figure 6C and 6D). Effects of AECHL-1 treatment on tumor suppressor and cell cycle regulatory proteins in Tumor allograft of C57BL/6 Mice We evaluated the effect of AECHL-1 treatment on the expression of the tumor suppressor protein p53, the cell cycle regulatory protein Cyclin D and cdk4 and the oncogene c-Myc. As shown in Figure 4E, AECHL-1 at 50 µg/mouse/day administered to B16F10-implanted tumors in C57BL/6 mice resulted in an increase in the expression of wild-type p53 protein and then decreased at a higher concentration (100 µg/mouse/day). The level of the p53 was greater in the AECHL-1-treated group than in the cisplatin-treated group, indicating that the antitumor action of AECHL-1 was different from cisplatin. Since phosphorylation at the Ser-15 residue of p53 is critical for p53-dependent activation of cell cycle regulatory proteins for G1 arrest, we determined the phosphorylation status of p53 and cyclin D1 and cdk4. AECHL-1 treatment resulted in an increase in phosphorylation of p53 at serine 15 residue in tumors at 50 µg/mouse/day with a concomitant increase in the level of p21 and decreased at 100 µg/mouse/day. Western blot analysis revealed that treatment with 50 and 100 µg/mouse/day AECHL-1 caused a significant reduction in the cycle-regulatory proteins cyclin D1 and cdk4. Treatment with 50 and 100 µg/mouse/day AECHL-1 also caused a significant reduction in the oncogene c-Myc thus indicating that inhibition of the cell cycle may be responsible for antitumor effects of AECHL-1 (Figure 7). 10.1371/journal.pone.0005365.g007Figure 7 Effect of AECHL-1 on cell cycle regulatory proteins. Tumor tissue lysates were subjected to SDS-PAGE followed by Western immunoblotting. Membranes were probed with anti-p53, pp53, p21, c-myc, cyclin D1, cdk4, and β-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by enhanced chemiluminescence detection system. The experiments were repeated thrice with similar results and a representative blot is shown for each protein. Histological analysis of tumor tissue and other organs in C57BL/6 mice Histological examination of tumor in C57BL/6 control mice showed well developed blood vessels, increased neovascularization, cell density and presence of hemorrhagic areas with probable signs of angiogenesis with increased possibility of metastasis (Figure 8.1A). Treatment of tumors with 50 µg AECHL-1 did not show much influence on the tumor vascularization but showed less occurrence of hemorrhagic areas, decrease in tumor cell density and occurrence of picnotic/necrotic cells in the center of the tumor (Figure 8.1B). Treatment with 100 µg AECHL-1 showed increase in necrotic cells, disappearance of neovascularization, hemorrhagic areas and low cell density compared to control (Figure 8.1C), thus indicating that AECHL-1 prevented the progression of angiogenesis and risk of metastasis by blocking neovascularization. Cisplatin treated group showed significant increase in necrotic cells, decrease in tumor cell density and volume (Figure 8.1D). 10.1371/journal.pone.0005365.g008Figure 8 Histological analysis of tumor tissue and other organs in C57BL/6 and nude mice. (1) Representative H&E-stained sections from B16F10 allograft tumors and the characteristics of these tumors were analyzed (1A–1D). Morphological characteristics of heart (1E–1H), kidney (1I–1L), liver (1M–1P) and spleen (1Q–1T), six mice were used in each set of experiments. (2) Representative H&E-stained sections from MCF-7 xenograft tumors and the characteristics of these tumors were analyzed (2A–2D). Morphological characteristics of heart (2E–2H), kidney (2I–2L) and liver (2M–2P), spleen (2Q–2T), three mice were used in each set of experiments. Compared with control, the heart tissue of the mice treated with 50 µg AECHL-1 showed normal structure while 100 µg showed extensive myocardial fiber necrosis and contraction bands. The fragmentation and smudging of the muscle fibers characteristic of coagulative necrosis was seen (Figure 8.1E–8.1G). Cisplatin treated mice also showed necrosis of myocardial fiber, slight lymphocytic infiltration and also fragmentation and smudging of the muscle fibers (Figure 8.1H). Compared with control, the kidney of the mice treated with 50 µg AECHL-1 showed slight tubular vacuolization and tubular dilation with hemorrhagic areas with normal glomeruli appearing at the lower part. Treatment with 100 µg AECHL-1 showed tubular vacuolization, tubular dilation, hemorrhagic condition and scattered chronic inflammatory cell infiltrates (Figure 8.1I–8.1K). Cisplatin treated mice showed scattered lymphocytes in and around the vessel. Many neutrophils were also seen in the tubules and interstitium i.e. pyelonephritis (Figure 8.1L). Compared with control, the liver of the mice treated with 50 µg AECHL-1 did not affect the normal architect. Mice treated with 100 µg AECHL-1 retained the normal architect of the liver (Figure 8.1M–8.1O). In cisplatin treated mice however, extensive necrosis of hepatocytes were seen. The arrow at the right side shows dead hepatocytes and this pattern can be seen with a variety of hepatotoxins, where focal hepatocytes necrosis with lymphocytic infiltration occurs. In these tissues, lesions look similar to that of Tyzzer's disease characterised by necrosis with varying degrees of inflammation in response to the necrosis. Acute hepatic lesions consist of necrotic foci surrounded by minimal, primarily neutrophilic, inflammation (Figure 8.1P). Representative spleen sections from Control and mice treated with 50 µg AECHL-1 showed normal spleen architect and mice treated with 50 µg AECHL-1. Control and mice treated with 100 µg AECHL-1 and cisplatin showed hyperplasia of the white pulp, especially in the marginal zone (Ψ). Histology showed increased number of granulocytes in the marginal zones (Figure 8.1Q–8.1T). Histological examination of tumor tissue and other organs in nude mice Tumors from control mice showed pronounced neovascularization throughout the section surrounded by highly dense cells and absence of necrotic cells (Figure 8.2A). AECHL-1 at 5 µg dose showed decreased tumor cell density and lacunae throughout the tumor area. It also showed loss of neovasulization and absence of hemorrhagic areas (Figure 8.2B). AECHL-1 at 10 µg showed many empty spaces, occurrence of hemorrhagic areas was seen but reduction in the vasculization was not seen (Figure 8.2C). Treatment with Paclitaxel lowered the tumor cell density with occurrence of many empty spaces and necrotic areas in the section (Figure 8.2D). Treatment with 5 µg AECHL-1 did not show any change in the normal myocardium, while 10 µg AECHL-1 and Paclitaxel showed necrosis of myocardial fiber. Paclitaxel showed extensive myocardial fiber necrosis with fragmentation and smudging of the myocardium (Figure 8.2E–H). No significant change was observed in kidney structure from AECHL-1 treated groups, while paclitaxel treatment showed signs of tubular vacuolization dilation with hemorrhagic areas (Figure 8.2I–8.1L). Both AECHL-1 and Paclitaxel did not show any change in the normal architecture of liver (Figure 8.2M–8.2P) and Spleen sections (Figure 8.2Q–8.2T). Discussion In the present study, we report a new anti-cancer compound AECHL-1, isolated from root bark of the plant Ailanthus excelsa. AECHL-1 was characterized by UV, IR, NMR and mass spectroscopy and the purity was conformed by HPLC. It is a triterpenoid with high polarity and a molecular weight 453.8 (Figure 1 and Figure 2). The tumor-suppressor gene p53 plays a vital role in the development of various types of cancers. It is estimated that 50% of all cancers develop due to mutations in p53 [21]–[23]. Therefore, we first tested the effect of AECHL-1 cytotoxicity and proliferation in four different cancerous cell lines with different tissue origin that contain either wild-type or mutant p53, as well as p53 null. B16F10 and MCF-7 cells contain wild type p53, MDA-MB-231 cells contain mutant p53 and PC3 cells are p53 null. Cisplatin, a highly DNA damaging agent and paclitaxel, a tubulin based anti-mitotic agent were used as positive controls. We found that AECHL-1 inhibited the growth of MCF-7 cells in a concentration dependent manner at 12, 24 and 48 hr (Figure 3A). Cytotoxicity was also observed in the other cancer cells at 48 hr to a varying degree with a minimum growth inhibition of a normal human embryonic kidney cell line, HEK-293 (Figure 3B). The degree of cytotoxicity was MCF-7>B16F10>PC-3>MDA-MB-231>HEK-293 (Figure 3B) and inhibition of cell proliferation was MCF-7>B16F10>MDA-MB-231>PC-3 (Figure 3C). Compared with paclitaxel and cisplatin, AECHL-1 showed greater potency in MCF-7 (Figure 3D), B16F10 and MDA-MB-231 cell proliferation inhibition at 24 and 48 hr (data not shown). These results indicate that in MCF-7, B16F10 and MDA-MB-231 cell line AECHL-1 is more effective in inhibition of cell proliferation than cisplatin or paclitaxel. However, in PC-3 cells, paclitaxel is more effective than cisplatin and AECHL-1. In B16F10 cells, AECHL-1 was found to significantly induce cell cycle arrest in G1 phase, while in MCF-7, MDA-MB-231 and PC3 cells it showed arrest in S-G2/M phase in MCF-7 cells (Table 1). The cell cycle arrest in AECHL-1 treated MCF-7 cells was followed by concentration dependent apoptosis, but the percentage of cell death was dependent on the types of cell lines. Compared to B16F10 and MDA-MB-231 cells, AECHL-1 was highly effective in MCF-7 cells at both high and low concentrations (Figure 4). Therapeutic interference with the mitotic spindle apparatus is a widely used rationale for the treatment of tumors. The microtubule network required for mitosis and cell proliferation has been shown to be disrupted by the diterpenoid paclitaxel [24]. It has also been shown that microtubule disruption elevates p53 protein levels [25]. Our immunofluorescence staining of tubulin showed that similar to paclitaxel, AECHL-1 inhibited microtubule assembly (Figure 5). Our in vitro results demonstrate that AECHL-1 can act as a new class of microtubule damaging agent arresting cell cycle progression at mitotic phase and inducing apoptosis. AECHL-1 was tested in vivo in C57BL/6 mice allograft with melanoma, B16F10 and nude mice xenograft with human breast cancer cells, MCF-7. Injections of AECHL-1 to the tumor sites were found to inhibit tumor growth in both models. In case of B16F10 melanoma model, a daily dose of 50 µg and 100 µg showed a significant antitumor effect, leading to regression of established tumors (Figure 6A and 6B) however; cisplatin was more effective than AECHL-1 but AECHL-1 showed less toxicity to kidney and heart while cisplatin showed greater damage to kidney, heart, liver and spleen (Figure 8.1). In the MCF-7 breast cancer model, a daily dose of 5 and 10 µg showed a significant antitumor effect, leading to regression of established tumors (Figure 6C and 6D). In order to understand the major in vivo pathways through which AECHL-1 may induce tumor suppression, we studied the expression of the tumor suppressor, cell cycle regulatory proteins and oncogene in B16F10 melanoma. Our in vitro cell cycle analysis on B16F10 had showed a strong G1 arrest as a result of AECHL-1 treatment. Furthermore, mechanistic investigation in vivo in B16F10 tumor showed that both 50 µg of AECHL-1 and cisplatin up regulated the expression of p53 (Figure 7). However, AECHL-1 induced hyper phosphorylation of p53 at ser-15. Phosphorylation of p53 at ser15 help in strongly binding p53 to DNA for the up regulation of cell cycle regulatory proteins that helps in suppression of the growth of tumor [26]. Cisplatin also up regulated p53, but phosphorylation at ser-15 was not observed (Figure 7). This may be due to the fact that cisplatin suppress the growth of tumor cells by DNA damage and apoptosis [27]. The observation that ser-15 phosphorylation is required for p21 induction prompted us to investigate its role in G1 arrest [26], [28]. We found an increase in the expression of p21 in the AECHL-1 treated tumors. p21 forms a complex with CDK2/CDK4/CDK6 and inhibit the CDK-cyclin kinase activity phase [28], [29] and arrest the cells in G1 phase. c-Myc is an oncogene that is up regulated in cancer cells and help in the tumor growth [30]. We observed that treatment of AECHL-1 resulted in a marked decrease in c-Myc, CDK-4 and cyclin D1 levels that is known to arrest the cell in G1 [31]. Decreases level of c-Myc is known to be involved in the down regulation of cyclin D1 and CDK4 [32] (Figure 7). This also decreases the kinase activity and arrest the cell in G1 phase. In conclusion, our data clearly show that AECHL-1 is less toxic, more selective, and more effective in the treatment of cancer in comparison to plant derived anti-cancer compound paclitaxel and metal-based compound cisplatin. It is efficacious in inhibiting the proliferation of a broad range of cancer cells as well as solid tumors. The novel compound AECHL-1 is found to interact directly with tubulin, arrest the cell cycle, and induce apoptosis of tumor cells. The antitumor effect of AECHL-1 was comparable with or even superior to the conventional chemotherapeutic drugs tested. The positive outcomes of such an in vitro and in vivo study could form a strong basis for the development of AECHL-1 as a novel agent for human cancer prevention and/or intervention. Supporting Information Figure S1 (154 KB DOC) Click here for additional data file. We thank Dr. G. C. Mishra, Director, National Centre for Cell Science (Pune, India) for encouragement and support, Dr. M.K. Bhat for suggestion and technical support, Ms. A. N. Atre for assistance in capturing images on the confocal microscope, Mr. Swapnil for FACS analysis, and the staff of the experimental animal facility at National Centre for Cell Science. Competing Interests: The authors have declared that no competing interests exist. Funding: This research was support by the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi. Santosh Kumar received a research fellowship from the Indian Council of Medical Research and Manish Lavhale from the University Grants Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 American Cancer Society 2002 Cancer Facts & Figures 2 Ferlay J Bray F Parkin DM Pisani P 2001 Globocan 2000: Cancer Incidence and Mortality Worldwide. IARC Cancer Bases No. 5 Lyon IARCPress 3 Cragg GM Newman DJ 2004 Plants as a source of anti-cancer agents. Elisabetsky E Etkin NL Ethnopharmacology. In Encyclopedia of Life Support Systems (EOLSS). Developed under the Auspices of the UNESCO Oxford, UK Eolss Publishers 4 Philip PA 2005 Experience with docetaxel in the treatment of gastric cancer. 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==== Front BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central 1471-2474-10-381937144610.1186/1471-2474-10-38Research ArticleLow back pain status in elite and semi-elite Australian football codes: a cross-sectional survey of football (soccer), Australian rules, rugby league, rugby union and non-athletic controls Hoskins Wayne [email protected] Henry [email protected] Chris [email protected] Andrew [email protected] Peter [email protected] Andrew [email protected] Kate [email protected] George [email protected] Division of Environmental and Life Sciences, Department of Health and Chiropractic, Macquarie University, NSW 2109, Australia2 Norwest Orthopaedic and Sports Physiotherapy, Norwest, NSW 2153, Australia3 Enhance Chiropractic and Massage Sports Injury Centre, Ngunnawal, ACT 2913, Australia2009 17 4 2009 10 38 38 14 10 2008 17 4 2009 Copyright © 2009 Hoskins et al; licensee BioMed Central Ltd.2009Hoskins et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Our understanding of the effects of football code participation on low back pain (LBP) is limited. It is unclear whether LBP is more prevalent in athletic populations or differs between levels of competition. Thus it was the aim of this study to document and compare the prevalence, intensity, quality and frequency of LBP between elite and semi-elite male Australian football code participants and a non-athletic group. Methods A cross-sectional survey of elite and semi-elite male Australian football code participants and a non-athletic group was performed. Participants completed a self-reported questionnaire incorporating the Quadruple Visual Analogue Scale (QVAS) and McGill Pain Questionnaire (short form) (MPQ-SF), along with additional questions adapted from an Australian epidemiological study. Respondents were 271 elite players (mean age 23.3, range 17–39), 360 semi-elite players (mean age 23.8, range 16–46) and 148 non-athletic controls (mean age 23.9, range 18–39). Results Groups were matched for age (p = 0.42) and experienced the same age of first onset LBP (p = 0.40). A significant linear increase in LBP from the non-athletic group, to the semi-elite and elite groups for the QVAS and the MPQ-SF was evident (p < 0.001). Elite subjects were more likely to experience more frequent (daily or weekly OR 1.77, 95% CI 1.29–2.42) and severe LBP (discomforting and greater OR 1.75, 95% CI 1.29–2.38). Conclusion Foolers in Australia have significantly more severe and frequent LBP than a non-athletic group and this escalates with level of competition. ==== Body Background The prevalence of low back pain (LBP) in the general population has been well described [1,2]. Despite most LBP being low-intensity and low-disability [3], figures documentetime prevalence have been as high as 84% with point-prevalence between 12% and 33% [2]. Less interest has been afforded to investigating LBP in athletic populations. In particular there are very few studies assessing LBP amongst active competing athletes, especially at the elite level of competition. Studies have documented that LBP prevalence and severity can vary between sports, with an increase in pain noted in those with significant low back demands [4,5]. Noteworthy is the reported lack of significant difference in low back injury rates between contact and non-contact sports [6]. It is not clear whether competing athletes are at a risk of a higher prevalence or increased intensity of LBP compared with the non-athletic population. It also has not been investigated whether LBP prevalence or intensity varies at different levels of athletic competition. Bahr et al [4]. analyzed LBP prevalence between elite athletes competing in endurance based sports: cross-country skiing (n = 257), rowing (n = 199), orienteering (n = 278) as well as a non-athletic group (n = 197). LBP lifetime (51–65%), year (48–63%) and 7 day prevalence (20–25%) was similar between groups although lower in non-athletes. No other large study has used homogeneity in study design and methodology to make direct comparisons between active athletes and non-athletes. Evidence suggests that sporting participation in the general population, regardless of activity, contributes to less frequent LBP [7]. However, once LBP is established, participation in sporting activities may indirectly contribute to increased severity of LBP [7]. Despite this, much of the current sporting literature has tended to focus on sports with specific low back demands such as rowing [4,8,9], skiing [4,10-12], gymnastics [13,14], wrestling [15], golf [16] and American football [17]. Less interest has been afforded to running based power sports. The Australian football codes: football (soccer), Australian-Rules, rugby league and rugby union have established professional competitions. Each code has similar training and competition requirements, necessitating an all-round athletic ability with an emphasis on rapid acceleration running, endurance, agility, physical strength and code specific skills. Matches are played continuously over 90–120 minutes duration, typically weekly for the duration of the season. The extent of LBP in elite Australian football code participants has not been fully elucidated. This could partly be the result of injury surveillance in Australian professional sport typically adopting an injury definition involving missed participation from a match [18-20]. Subsequently, the knowledge surrounding the prevalence and magnitude of LBP remains largely unknown. It is uncommon that LBP is severe enough to prevent a professional footballer from competing or from relinquishing his place in team selection. This is particularly true when medical management frequently incorporates epidural steroid injections [21] and local anaesthetic [22], considered 'part of the game' in professional football [23]. Despite this, injury surveillances have documented that low back injury if present can be severe and have high recurrence rates. In elite soccer, LBP is the most common overuse injury reported [24]. In elite rugby league, 'back injuries' have been shown to have the highest rates of recurrence for all injuries [25], whilst in retired elites chronic LBP is the third most common complaint, reported by 39% [26]. In elite Australian-Rules, 5% of players will miss a match each season with a 'lumbar or thoracic spine' injury, causing them to miss 4 weeks/matches [20]. In amateurs, 27% report a long term or recurrent back problem [27]. In school children playing rugby union, LBP afflicts over 40% of participants [28]. Our understanding of the effects of elite football code participation on LBP is limited. Thus, it was the objective of this study to determine the prevalence, intensity, quality and frequency of LBP in Australian football code participants. It was also our objective to compare this data between elite and semi-elite participants and with that of a non-athletic group. Methods The study was designed as a cross-sectional survey of male Australian football code participants competing at the highest-level national or international elite competition (classified as elite), state based semi-elite competitions (classified as semi-elite) and a non-athletic control group (classified as non-athletic). The study was approved by the Macquarie University Human Ethics Committee. Elite clubs were pragmatically selected to participate until an approximate equal percentage representation of total elite players from each code was achieved. For semi-elite participants, clubs were chosen from the various competitions within Australia until an approximate equal number of total players for each code was achieved. At both of levels of play, if clubs consented to participate it was required that they provide access to their entire player rosters to ensure 100% compliance which would assist in providing an accurate reflection of the status of LBP in the participating clubs. The survey commenced in early 2005. An attempt was made to standardise the time of delivery of the questionnaires. However, the scheduling of seasons of the codes and the different levels of play do not directly overlap. As such the questionnaires were completed at different times of the respective seasons but a feature was that all surveys were completed whilst in a period including either competition or pre-season matches. All athletes on the playing lists of the selected clubs were invited to participate and complete the survey with responses remaining confidential. Non-athletic controls were drawn from a convenience sample of age matched male University students and office workers, with the specification that they not participate in a football code at the elite or semi-elite level. It should be noted that elite players are professional or 'full time' with their football commitments and this provides their sole income unless income is also received indirectly through their football requirements (e.g. endorsements). Semi-elite players receive financial payment but this is not enough to make them professional and they typically perform university study and/or work along with their football commitments to supplement their income. The was presented to the clubs and players as a low back pain survey. Questionnaires were either administered by an author of the study or by an official representing the participating club, at the preference of the club. In the case of the club wishing to administer the survey, the questionnaires were mailed out along with consent forms and instructions describing the purposes and procedures of the study and how the instruments were to be administered. This was followed by a telephone call to confirm that all procedures would be correctly administered, to ensure players completed every question and to make certain the club officials were qualified to answer questions of the players. None of the assessors were involved in the analysis of the data. Analysis was provided by a person independent to each of the group allocations. The questionnaire was developed using the validated and reliable Quadruple Visual Analogue Scale (QVAS) [29], the McGill Pain Questionnaire (short-form) (MPQ-SF) [30,31], along with a series of LBP questions either adapted from an Australian LBP epidemiological study with permission of the author [3] or that the authors developed themselves and thoroughly pilot tested. The additional questions were: • How old were you when you had your first ever attack or episode of LBP? • If applicable, was this a result of your sporting commitments and activities or not related to this? • When did your current episode of LBP commence? • If applicable, was this a result of your sporting commitments and activities or not related to this? • How often do you experience LBP? To assist with answering the questions a diagram of a mannequin that defined the anatomical boundaries of the low back as a shaded area between the last ribs and the gluteal folds was provided (Figure 1). For the purposes of this survey the shaded area represented the low back and subjects were told to focus only on LBP and not other sources of pain. This area was found to be the most commonly used in a review of methodologically sound LBP prevalence studies [2]. Figure 1 The anatomical boundaries representing the low back. The forms were manually entered using Microsoft Excel® and analyzed using an Excel add-in (EcStat). The subject characteristics are reported as means, standard deviations and age range. One-way ANOVA's and Pearson's Chi squared analysis were used to determine differences between groups. Pearson's chi-squared statistic was used to test independence between categorical outcome and predictor variables. When this hypothesis was rejected, odds ratios were used to assess the strength and directions of the association. For non-binary variables the odds ratios provided are computed by comparing the odds of each specified outcome level for each given predictor level with reference categories obtained by aggregating those for all other levels. Odds ratios are reported with 95% confidence limits. Results The subject characteristics of the different groups show groups were matched for age (Table 1). At the elite level 4 of the 14 (28.6%) Australian rugby league clubs (n = 93), 1 of the 2 (50.0%) rugby union clubs (n = 19), 3 of the 16 (18.8%) Australian-Rules clubs (n = 112) and 2 of the 6 (33.3%) soccer clubs (n = 47) participated. At the semi-elite level, 2 rugby league clubs participated (n = 52), 6 rugby union clubs (n = 139), 4 Australian-Rules clubs (n = 99) and 4 soccer clubs (n = 70). It appears that there was an almost linear trend of increasing LBP severity from the non-athletes, to the semi-elite and elite groups (Table 1). The elite athletes had significantly increased levels of the sensory, affective and total pain score when compared with both the semi-elite and non-athletic groups (Table 1). Elite athletes were approximately twice as likely to experience discomforting or greater LBP, less likely to have no LBP, whilst semi-elite athletes were less likely to experience discomforting or greater LBP and non-athletes more likely to experience no LBP (Chi squared χ2 18.67, p < 0.001) (Table 2). Using the results of the question "what describes your overall LBP" from table 2, in terms of prevalence, the elite group has a figure of 77.9%, semi-elite 66.7%, and non-athletic 62.2%. When solely looking at those with discomforting or worse pain the figure for the elite group is 42.1%, semi-elite 29.7% and non-athletic 28.4%. Table 1 Subject characteristics and results of the QVAS and MPQ-SF questionnaires by group (n = 779) Elite Semi-elite Non-athletes P value Number 271 360 148 Mean age (SD) 23.3 (4.0) 23.6 (4.0) 23.9 (4.5) 0.42 Age range 17–39 16–46 18–39 LBP nowa, b (SD) 25.4 (22.6) 17.7 (21.5) 11.2 (15.3) <0.001 LBP averagea (SD) 24.9 (19.8) 19.4 (19.0) 11.2 (12.3) <0.001 LBP besta (SD) 8.5 (11.9) 5.2 (9.1) 2.3 (4.9) <0.001 LBP worsta (SD) 56.2 (27.6) 48.1 (28.6) 39.8 (25.3) <0.001 Sensory pain scoreb (SD) 16.5 (14.0) 12.8 (12.4) 9.8 (9.2) <0.001 Affective pain scoreb (SD) 8.4 (13.2) 6.9 (11.6) 5.6 (8.2) 0.047 Total pain scoreb (SD) 14.3 (13.0) 12.2 (13.6) 9.3 (9.9) <0.001 a Questions from the QVAS "LBP now" refers to the question: How much LBP do you have right now? "LBP average" refers to the question: What is your typical or average pain? "LBP best" refers to the question: What is your pain level at its best (How close to "0" does your pain get at its best)? "LBP worst" refers to the question: What is your pain level at its worst (How close to "10" does your pain get at its worst)? b Questions from the MPQ-SF Table 2 Self reported present LBP intensity by category (none, mild, discomforting) by group (n = 779) Level Overall paina Odds ratio 95% CI Elite (n = 271) None (n = 60, 22.1%) 0.54 0.38–0.75 Mild (n = 97, 35.8%) 0.99 0.73–1.35 Discomforting + (n = 114, 42.1%) 1.75 1.29–2.38 Semi-elite (n = 360) None (n = 120, 33.3%) 1.31 0.96–1.77 Mild (n = 133, 36.9%) 1.08 0.81–1.45 Discomforting + (n = 107, 29.7%) 0.71 0.53–0.96 Non-athletic (n = 148) None (n = 56, 37.8%) 1.53 1.05–2.22 Mild (n = 50, 33.8%) 0.89 0.61–1.30 Discomforting + (n = 42, 28.4%) 0.74 0.50–1.09 a Question from MPQ-SF Breakdown of discomforting, distressing and excruciating pain for the groups were elite (n = 88, 24, 2), semi-elite (n = 93, 8, 6) and non-athletic (n = 39, 2, 1) respectively. These levels of pain have been combined and are described as "discomforting +" The results of the age of first ever episode of LBP were non-significant between groups (p = 0.40), with the means (SD): elites 17.9 (4.0), semi-elites 17.8 (4.0) and non-athletes 17.2 (4.1). Responders were 164 elites (60.5%), 220 semi-elites (61.1%) and 84 non-athletes (56.8%). Elite athletes were 2–5 times more likely to attribute their initial LBP to a sporting activity and less likely to be due to other factors (Table 3). Non-athletes experienced the opposite pattern whilst semi-elite athletes were also less likely to attribute sporting activity to initiate their first onset LBP (Chi squared χ2 55.10, p < 0.001). Elite athletes were more likely to have a recent onset of LBP and less likely to not experience a current episode (χ2 11.91, p = 0.018) (Table 3). Elite athletes were 2–4 times more likely to attribute sporting activity to have initiated their current LBP and less likely for other factors to be involved, whilst non-athletes report the opposite pattern, being 2–5 times more likely to attribute non-sporting factors to cause their LBP (χ2 73.05, p < 0.001) (Table 3). Elite athletes were approximately twice as likely to report daily or weekly LBP and less likely to experience less-frequent LBP, whilst non-athletes report the opposite pattern, being approximately 2–3 times more likely to report fortnightly or less frequent LBP (χ2 28.47, p < 0.001) (Table 3). Table 3 LBP profile (pain association, onset and frequency) by group (n = 779) Level Initial LBP due to sporta Odds ratio 95% CI Elite (n = 271) N/A (n = 13) 0.39 0.21–0.73 Yes (n = 229) 3.40 2.34–4.94 No (n = 29) 0.32 0.21–0.50 Semi-elite (n = 360) N/A (n = 42) 1.78 1.08–2.92 Yes (n = 235) 0.69 0.50–0.93 No (n = 83) 1.21 0.86–1.71 Non-athletic (n = 148) N/A (n = 16) 1.27 0.71–2.29 Yes (n = 78) 0.40 0.28–0.58 No (n = 54) 2.66 1.80–3.94 Start of current LBP episodeb Elite (n = 271) N/A (n = 85) 0.58 0.43–0.80 0–3 months (n = 110) 1.43 1.06–1.95 >3 months (n = 76) 1.25 0.89–1.74 Semi-elite (n = 360) N/A (n = 156) 1.34 1.01–1.79 0–3 months (n = 117) 0.80 0.60–1.08 >3 months (n = 87) 0.90 0.65–1.24 Non-athletic (n = 148) N/A (n = 67) 1.34 0.93–1.92 0–3 months (n = 47) 0.83 0.57–1.21 >3 months (n = 34) 0.86 0.56–1.31 Current LBP due to sportc Elite (n = 271) N/A (n = 85) 0.61 0.45–0.83 Yes (n = 173) 2.79 2.05–3.78 No (n = 13) 0.22 0.12–0.41 Semi-elite (n = 360) N/A (n = 151) 1.27 0.95–1.69 Yes (n = 157) 0.75 0.56–0.99 No (n = 52) 1.14 0.76–1.72 Non-athletic (n = 148) N/A (n = 67) 1.38 0.96–1.99 Yes (n = 40) 0.34 0.23–0.50 No (n = 41) 3.34 2.14–5.19 Frequency of LBPd Elite (n = 271) N/A (n = 51) 0.84 0.58–1.22 Daily or weekly (n = 107) 1.77 1.29–2.42 Fortnightly + (n = 113) 0.68 0.50–0.91 Semi-elite (n = 360) N/A (n = 84) 1.35 0.95–1.91 Daily or weekly (n = 111) 0.96 0.71–1.30 Fortnightly + (n = 165) 0.85 0.64–1.13 Non-athletic (n = 148) N/A (n = 26) 0.78 0.49–1.25 Daily or weekly (n = 26) 0.40 0.26–0.64 Fortnightly + (n = 96) 2.34 1.62–3.40 a Question: if applicable, was your first ever attack or episode of LBP a result of your sporting commitments and activities or not related to this? N/A = I have not had a first ever attack or episode of LBP b Question: when did your current episode of LBP commence? N/A = I do not have a current episode of LBP c Question: if applicable, is your current episode of LBP a result of your sporting commitments and activities or not related to this? N/A = I do not have a current episode of LBP d Question: how often do you experience LBP? N/A = I do not experience LBP "Fortnightly +" relates to experiencing LBP on a fortnightly basis or less frequently Discussion The main findings of this study were that Australian football code participants appear to experience LBP more frequently and severely than the non-athletic population. The difference was more evident in the elite group compared to the semi-elite group. We postulate that the higher intensive of play may be associated with the increased low back pain status. Of interest was the finding that elite, semi-elite and non-athletic groups all reported the same average age of first onset LBP although reported etiology differed. Strengths of our study included the use of validated questionnaires to quantify the intensity (QVAS) and quality of LBP (MPQ-SF). This has not been performed previously when assessing athletic LBP sufferers. Functional disability associated with LBP was not determined. This important aspect was not investigated as it was felt that validated questionnaires in use to determine these parameters were not created for an elite athletic population and were likely to be irrelevant to their high-level functional demands, making comparisons vexed. Limitations exist in the study conducted. Firstly, the convenience sample taken for the non-athletic population is not a random population sample and may not be representative. However, random sampling not producing a 100% response rate has been discussed as potentially leading to overestimates of LBP in similar research [4]. Our controls were used because of their likely non-elite or semi-elite athletic participation rate and likelihood to be matched for age. Additionally, football club selection was not random and may not be representative, although difficulty arrises in enticing professional clubs into research and it is likely that a random selection would have produced low response rates. As several of the elite competitions have grown in the number of participating clubs, we recommend that future studies consider broader club representation with a higher and more equal player representations from each sport. Secondly, similar to Bahr et al. [2] our study was based on data from self-reported questionnaires and not more objective findings obtained from clinical interview, physical examination or advanced imaging. However, the QVAS and MPQ-SF have demonstrated validity and reliability. Nevertheless, it has been discussed that there may be potential difficulty in comparing the results between elite athletes and non-athletes. A well motivated athlete may under-report pain in order to improve performance, their chances of team selection and for a positive mind frame [32]. Alternatively, pain may be over-reported as it may be provoked easily by intense training and competition requirements and hinder athletic performance [32]. The athlete may therefore place a greater impact on pain. This situation is more of a concern as exaggeration of self-reported LBP and disability may be a predictor for LBP chronicity [33]. However, previous research on amateur athletes found psychosocial issues such as level of satisfaction with coaches or team-mates not to be related to the development of LBP [6]. Psychosocial factors may be more important for the professional elite athlete who has financial, contractual and performance concerns. In support of this, we found elite participants to have higher levels for the sensory, affective and total pain scores of the MPQ-SF, although we do not know why. Furthermore, it has been shown that LBP in former elite athletes is predicted by psychosocial issues such as life dissatisfaction, neuroticism, hostility, extroversion and poor sleep quality [34], whilst a recent prospective 5 year study of an employed group of participants has suggested that the vast majority of incident-adverse LBP events may be predicted not by structural findings or minor trauma but by a small set of demographic and behavioral variables [35]. Future research is required to more broadly investigate confounding variables including psychosocial factors in the elite athlete and their impact and relevance if any to the development of LBP during play and after a career has ended. Thirdly, our study does not quantify what aspect of the Australian football codes is responsible for the increased frequency or severity of LBP. LBP could be related to the type, intensity, duration and/or amount of athletic activity performed. In endurance based sports with low back demands a dose response relationship appears to exist with LBP [4]. Further, whilst there is an increasing trend for reported first onset LBP to be a result of sporting commitments in football code participants, this may not necessarily be due to football code participation. At a young age, people tend to participate in multiple sports and activities. As some questions asked were retrospective in nature, there is likely an element of recall bias, which may have contributed to lower response rates when compared to the rates of questions requiring more recent recall. It may be reasonable to conjecture that regardless of the etiology of the initial LBP, that once a footballer has experienced significant LBP, they remain susceptible to future pain and aggravation or exacerbation. This supports our findings that elite footballers were more likely to report a recent onset of LBP. Although likely to be multi-factorial, one explanation for recurrent LBP in athletes could be that athletes who demonstrate neuromuscular control alterations to sudden trunk loading have an increased risk of sustaining a low back injury [36]. Previously it has been shown that athletes with a recent acute low back injury exhibit altered neuromuscular control strategies for sudden trunk loading [37]. These findings are relevant to the contact Australian football codes but also for the agility, change of direction and sudden stop-start nature of all codes. Lumbar muscle activity during gait functions to control trunk movements [38]. In a non-athletic population, LBP was shown to produce poorly coordinated activity of the lumbar muscles during gait [39], which in a football player may lead to forces being directed at unprotected spinal structures producing subsequent mechanical stress and injury. Greater and more frequent mechanical spinal loading in elite footballers could contribute to both injury and delayed healing response. Similar to the non-athletic population, a situation may exist where LBP fluctuates over time with recurrences or exacerbations and temporary remissions [40,41]. Given footballers are exposed to greater and more frequent mechanical stresses in training and competition, this scenario appears likely. In support of this contention, Green et al. [6] documented that athletes with a history of low back injury with current LBP have a 6 times greater risk for future injury. For athletes with a previous history of low back injury approximately a 3 times greater risk of injury exists [6,36]. Low back injury in athletes may be of further significance as Nadler et al. [42] documented that athletes with resolved LBP from a history of low back injury demonstrate significantly diminished athletic performance in a 20 m shuttle run test compared with a healthy group. Further study is warranted in elite football code participants given the apparent scale of LBP and low back injury present. Future research is also required to document effective management strategies as apart from one short-term small study [43], we are not aware of randomized controlled trials for the treatment or rehabilitation of LBP with subjects drawn from an athletic population. The limitation in using current published evidence based guidelines for LBP management [44,45] is that acute pain advice generally advocates an approach to management that includes advice to: remain active, modify activity, remove only those activities that specifically aggravate and potential replace with other non aggravating activity (relative rest) and to stay at work. For chronic conditions various exercise-based protocols are often recommended. It may be for an athlete that the active approach and the tissue loading from many of the 'stabilization exercises' [46,47] they predictably already perform are etiological or aggravating or ineffective factors. In support of this assertion, there is no significant advantage of additional core-strengthening in reducing LBP occurrence in athletes [48]. It would be equitable to advocate that separate guidelines are required for the athletic population. Questions need to be raised regarding whether LBP normalizes following a career of participation. It is known that former elite athletes are more likely to receive hospital care suffering from musculoskeletal complaints in general [49]. However, in the largest study performed using self-reported questionnaires, it appears that LBP is less common in former elite athletes (29.3% of 937) than in non-athletes (44% of 620) [34]. This is despite an increase in degenerative radiological findings in former elite athletes [32,34]. It is unclear whether participation in certain sports will affect post career pain or the intensity of LBP experienced [32]. Conclusion The main findings of this study were that elite football code participants in Australia compared with age-matched semi-elites and non-athletes have significantly higher levels and more frequent LBP and are more likely to attribute sporting activity as the etiological factor. Despite the age of onset of first time LBP being the same between groups the reported etiology differs. Predisposing factors need to be identified along with optimal methods of management for the athletic population of LBP sufferers given a current and a previous history of low back injury results in functional disturbances and may be detrimental to athletic performance and well-being in the short and long-term. Competing interests The authors declare that they have no competing interests. Authors' contributions HP and WH conceived the idea of the study. All authors were involved in recruitment of subjects and data entry. WH and HP contributed to writing an initial draft document. All authors contributed to the re-writing of this paper. All authors made original contributions to the content of the final manuscript. All of the authors participated in the editing and revisions of the multiple drafts that existed between the initial and final draft. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements No source of funding was used in the preparation of this manuscript. The authors would like to acknowledge Emeritus Prof. Don McNeil for providing assistance with the statistical analysis and the following people who contributed to the study: Darren Denneman (Belconnen), Arthur Huggins (Canberra City), Lawrie McKinna (Central Coast), Chris Gardner (De La Salle), Matthew Stewart (East Coast), John Quinn (Essendon), Ben Black (Guildford), Randall Cooper (Hawthorn), Mark Dijikic (Knights), Steve Freeman (Manly RL), Paul Bloomfield (Manly RL), Chris Hickey (Manly RU, Eastwood), Mark Gale (Manly Soccer), Mary Toomey (Melbourne Storm), Greg Castle (Pennant Hills), Steve Milne (Penrith), Michael Wood (Perth Glory), Garry Nucifora (Queensland Reds), Matthew Hornsby (Richmond), Mendo Cklamovski (Rockdale City), Greg Mum (Southern Districts), Andrew McDonald (South Sydney), Brett Hoskins (St George), Stewart Porter (Sutherland Sharks), Andrew Long (Sydney University), Trevor Walsh (Sydney University). ==== Refs Leboeuf-Yde C Lauritsen JM The prevalence of low back pain in the literature. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1943676009-PONE-RA-08346R110.1371/journal.pone.0005528Research ArticleImmunology/Immune ResponseImmunology/Immunity to InfectionsInfectious Diseases/Bacterial InfectionsAnalysis of Mycobacterium tuberculosis-Specific CD8 T-Cells in Patients with Active Tuberculosis and in Individuals with Latent Infection CD8 T-Cell in TuberculosisCaccamo Nadia 1 * Guggino Giuliana 1 Meraviglia Serena 1 Gelsomino Giuseppe 1 Di Carlo Paola 2 Titone Lucina 2 Bocchino Marialuisa 3 Galati Domenico 3 Matarese Alessandro 3 Nouta Jan 4 Klein Michel R. 5 Salerno Alfredo 1 Sanduzzi Alessandro 3 Dieli Francesco 1 Ottenhoff Tom H. M. 4 1 Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Palermo, Italy 2 Dipartimento di Medicina Clinica e delle Patologie Emergenti, Università di Palermo, Palermo, Italy 3 TB Infection Screening Unit, Department of Clinical and Experimental Medicine, University of Naples “Federico II”, Monaldi Hospital, Naples, Italy 4 Department of Immunohematology & Blood Transfusion and Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands 5 National Institute of Public Health and the Environment, Bilthoven, The Netherlands Pai Madhukar EditorMcGill University, Canada* E-mail: [email protected] and designed the experiments: NC AS FD THMO. Performed the experiments: GG SM GG. Analyzed the data: NC SM. Contributed reagents/materials/analysis tools: GG GG PDC LT MLB DG AM JN MK AS. Wrote the paper: NC FD THMO. 2009 13 5 2009 4 5 e552826 1 2009 10 4 2009 Caccamo et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-γ and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA+CCR7−). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2+/IFN-γ+ and IL-2−/IFN-γ+ T-cell populations; interestingly, only the IL-2+/IFN-γ+ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease). ==== Body Introduction Globally, Tuberculosis (TB) accounts for approximately nine million new cases of disease and around two million deaths every year [1]. TB is presenting new challenges as a global health problem, especially with new threats of HIV coinfection and multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb). TB is transmitted directly from human to human and the control of the infection depends on early identification and proper treatment of individuals with active disease. However, the lack of accurate diagnostic techniques has contributed to the emergence of TB as a threat to global health. To date, there is no simple, rapid, sensitive and specific test that can differentiate active TB from latent infection, and slowly progressive TB. T-cells, T-cell derived cytokines and cytotoxic molecules are crucial for protection against TB. Although a role for CD4 T-cells in protection against Mtb is well documented, there is also a large body of evidence derived from human and non human models that suggests an involvement of CD8 T-cells [2]–[5]. CD8 T-cells contribute to control of Mtb infection by mediating specific effector functions, including IFN-γ and TNF-α production upon recognition of mycobacterial antigens [6]–[8], lysis of infected host cells [6]–[9], and direct killing of mycobacteria [5], [10], [11]. A limited number of studies focused on the T-cell repertoire in Mtb infection, demonstrating clonal T-cell expansion in granulomas from subjects with LTBI [12] and changes in the peripheral blood and pleural fluid T-cell repertoire from TB patients [13]. Furthermore, CD8 T-cells specific for numerous mycobacterial antigens can be isolated at high frequency from human and mouse models, consistent with the hypothesis that CD8 T lymphocytes are constantly being stimulated with antigen [9], [10]. However, there are few studies which have compared the frequency, phenotype and function of antigen-specific CD8 T-cells in TB patients and subjects with latent infection (LTBI). Among them, we ourselves previously found that the frequency of Ag85A peptide-specific CD8 T-cells was reduced in tuberculous children before therapy, but increased after therapy to levels similar to those detected in healthy tuberculin skin test positive children. Ag85A epitope-specific CD8 T-cells during active TB were mainly present among central memory cells and produced low levels of IFN-γ and perforin, which recovered after therapy [14]. In a parallel study, Kaufmann and colleagues found clonal expansion of effector-memory CD8 T-cells in older children with TB, with potential impact on course and severity of disease [15]. However, the CD8 repertoire of children could well be different from that in adult individuals given the different clinical manifestation of TB in children and adults; moreover, little is known about the size, quality and specificity of Mtb-specific CD8 T-cell responses in adult patients with active TB disease compared to treated TB and subjects with LTBI. To start addressing these issues, we have in this study determined the ex-vivo frequencies, phenotype and functional properties of HLA-A0201 CD8 T-cells specific for different peptides of Mtb proteins in adult subjects with LTBI and adult TB patients with active disease, both before and following four months of anti-mycobacterial therapy. Results Ex vivo analysis of circulating epitope-specific CD8 T-cells To determine the ex vivo frequency of peptide-specific CD8 T-cells, PBMC from HLA-A0201 patients with active TB before (T0) and after four months of chemotherapy (T4) and individuals with LTBI were stained with HLA-A0201/tetramers and anti-CD8 antibody and analysed by FACS. Four out of the six selected epitopes (Ag85B p5–13, Esat-6 p82–90, Hsp65 p362–370 and 16 kDa p120–128) were previously identified as CD8 T cell epitopes, while Rv1490 p325–333 and Rv1614 p197–205 are newly identified in this study as candidate epitopes, based upon whole Mtb genome screening for 9-mer peptides sequences with high/intermediate HLA-A0201 binding affinity (see also Table 1). Moreover, a minimum of three and a maximum of all six tetramers marked the CD8 T-cell response in each group of individuals (data not shown). In all instancies, specificity of tetramer staining was confirmed by the negative data obtained both using tetramer of an irrelevant specificity (the HLA-A0201/HIV-1 gag peptide p76–84) and staining PBMC from normal, uninfected donors with Mtb tetramers (see Table 2). 10.1371/journal.pone.0005528.t001Table 1 HLA-A0201 binding of predicted HLA-A0201 binders. Peptide AA sequence rel IC50 Rv1490 p325–333 FLLGLLFFV 2,50 Rv1614 p197–205 FLYELIWNV 1,79 Ag85B p5–13 GLPVEYLQV 7 Esat-6 p82–90 AMASTEGNV 1,25 Hsp65 p362–370 KLQERLAKL 0,25 16 kDa p120–128 GILTVSVAV 2,25 HBV core p47–56 FLPSDYFPSV 1 Peptides were tested for their ability to compete binding of 1.6 µM biotinylated peptide HBV core p47–56 to HLA-A0201 molecules. The concentration of peptide yielding 50% inhibition (IC50) was deduced from the dose-response curve. One of the six peptides bound to HLA-A0201 with high affinity (IC50<1 µM), while the other peptides bound the HLA-A0201 with intermediate affinity (IC50 1–10 µM). Each peptide was tested in at least two separate experiments. Data are expressed as relative (rel) IC50, compared to the IC50 of the standard peptide HBV core p47–56, which was considered as 1. Values are the mean of rel IC50 of two independent experiments with SE being always <10%. 10.1371/journal.pone.0005528.t002Table 2 Ex vivo analysis of frequency of peptide-specific CD8 T-cells. Tetramer LTBI T0 T4 PPD− Healthy Donor 16 kDa 0.3 0.13a 0.22c <0.01 (0.19–0.55) (0.05–0.34) (0.20–0.72) Rv1490 0.54 0.19a 0.3c <0.01 (0.16–0.80) (0.11–0.65) (0.22–0.36) Rv1614 0.65 0.30b 0.3 <0.01 (0.58–0.85) (0.11–0.59) (0.29–0.45) Ag85B 0.8 0.60 0.64 <0.01 (0.56–0.94) (0.31–0.92) (0.24–1.35) Esat-6 0.56 0.29b 0.58d <0.01 (0.33–0.96) (0.17–0.66) (0.18–0.92) Hsp65 0.57 0.41b 0.34 <0.01 (0.42–0.80) (0.31–0.74) (0.22–0.58) HIV-1 gag <0.01 <0.01 <0.01 <0.01 Cumulative data on the frequencies of the tetramer-specific CD8 T-cells in peripheral blood of subjects with LTBI, patients with active TB before (T0) and four months after therapy (T4). Data are presented as median values while interquartile range is shown in brackets. a p<0.01 and b p<0.05 when compared to values in LTBI subjects. c p<0.05 and d p<0.02 when compared to values in TB patients before therapy (T0). Although there was considerable variability in the proportions of CD8 T-cells that bound to single tetramers, an immunodominance hierarchy in epitope-specific CD8 T-cell response was found both in LTBI subjects and in TB patients at T0 and T4 (Figure 1A and Table 2). In LTBI subjects, the mean ex-vivo frequency of peptide-specific CD8+ tetramer+ T-cells was 0.8% for Ag85B, 0.66% for Hsp65, 0.65% Rv1614, 0.57% for Esat-6, 0.49% for Rv1490 and 0.43% for 16 kDa. The ex-vivo frequency of tetramer-specific CD8 T-cells was higher in LTBI subjects than in TB patients (Table 2) and this difference attained statistical significance with most of the studied epitopes (i.e, Esat-6, Rv1614, Rv1490, Hsp65 and 16 kDa). Ag85B peptide was the most immunodominant also in TB patients at T0 and at T4, as estimated by enumerating the frequencies of tetramer-specific CD8 T-cells; however significant differences in frequencies of epitope-specific CD8 T-cells were observed in TB patients before and after chemotherapy. In three instances (Esat-6, Rv1490 and 16 kDa antigens), mean frequencies of epitope-specific CD8 T-cells significantly increased after therapy: the mean frequency of Esat-6-specific CD8 T-cells was 0.39% in patients at T0 and 0.55% in patients at T4 (p<0.02), the mean frequency of Rv1490-specific CD8 T-cells was 0.35% at T0 and raised to 0.41% at T4 (p<0.05), the mean frequency of 16 kDa-specific CD8 T-cells was 0.24% at T0 and 0.38% at T4 (p<0.05) and the mean frequency Ag85B-specific CD8 T cells was 0.65% at T0 and 0.78% at T4. However, the frequencies of CD8 T-cells specific for the two other studied epitopes remained virtually unchanged before and four months after therapy: in fact, the mean frequency of Rv1614-specific CD8 T-cells was 0.38% at T0 and 0.41% at T4, and finally, the mean frequency of Hsp65-specific CD8 T-cells was 0.52% at T0 and 0.55% at T4. 10.1371/journal.pone.0005528.g001Figure 1 Comparison of the frequencies of tetramer+ CD8 T-cells in peripheral blood from LTBI subjects and TB patients with active disease before therapy (T0) and after four months of therapy (T4). (A) In each group tested, LTBI subjects, TB patients at T0 and TB patients at T4, the median proportion of tetramer+ CD8 T-cells was estimated as 100% and the relative percentages of individual tetramer+ CD8 T-cells calculated accordingly. (B) Dot plot analysis of tetramer+ CD8+ T-cell populations of one representative LTBI subject, one TB patient at T0 and one TB patient at T4. Figure 1B shows FACS analysis of the tetramer+ CD8+ T-cells of one representative LTBI subject, one TB patient at T0 and one TB patient at T4. Altogether, these results indicate that (a) the frequencies of Mtb epitope-specific CD8 T-cells during active pulmonary TB disease in adults are lower than in LTBI individuals, but that they increase after anti-mycobacterial therapy; (b) that between 50 and 100% of epitopes selected are recognized by individuals with latent or active Mtb infection, including two new epitopes (Rv1490 and Rv1614) and (c), that there appears to be an immunodominance hierarchy in the recognition of different epitopes of Mtb in individuals with LTBI, as well as in patients with active TB before and after therapy. Phenotypic analysis of tetramer-specific CD8 T-cells CD8 T-cells can be divided into at least four different populations of naive, central memory, effector memory and terminally-differentiated effector memory T-cells, based on the expression of surface markers associated with their maturation [16]. We have compared the phenotype of circulating tetramer-specific CD8 T-cell subsets in HLA-A0201 TB patients at T0 and T4 and in individuals with LTBI. Representative data are shown in Figure 2A and cumulative data are shown in Figure 2B. 10.1371/journal.pone.0005528.g002Figure 2 Phenotypic analysis of tetramer+ Mtb-specific CD8 T-cells. Peripheral blood mononuclear cells (PBMC) were stained with individual tetramers, and anti-CD8, -CD45RA and -CCR7 mAbs to separate functionally distinct subpopulations. After gating on tetramers+ CD8+ cells, the percentage of cells expressing CD45RA and CCR7 was determined. (A) Representative phenotyping data for one subject with LTBI (LTBI), one TB patient before (T0) and one TB patient 4 months after therapy (T4). Numbers in the corners indicate the percentage of positive cells in each quadrant. (B) Summary cumulative data of the phenotype of tetramer+ Mtb-specific CD8 T-cells. Data are presented with box plot reporting the median values and the interquartile range. Black columns = LTBI; grey columns = T0; white columns = T4. The mean frequencies of tetramer-specific CD8 T-cells with a CCR7+ CD45RA+ naive phenotype were found to be comparable in TB patients and in subjects with LTBI. However, in the latter we found that approximately 60% of CD8 T-cells expressed CD45RA but not CCR7, indicating a terminally-differentiated phenotype; this was irrespective on their antigen specificity. In LTBI subjects, about 5% of the specific CD8 T-cells had an effector memory-like phenotype (CD45RA− CCR7−), while less than 5% had a central memory-like phenotype (CD45RA− CCR7+). In TB patients, although cells expressing a terminally-differentiated CD45RA+ CCR7− phenotype still comprised the predominant subset among specific CD8 T-cells, their mean percentage was lower than in subjects with LTBI (35% versus 60%) and remained virtually unchanged before and four months after therapy (35% versus 42%). However, although lower percentages of CD45RA+ CCR7− cells was detectable within all studied tetramer+ CD8 T-cells, none of the differences between LTBI subjects and TB patients before or after therapy attained statistical significance. Conversely, tetramer-specific CD8 T-cells from TB patients contained higher proportions of cells with an effector memory-like (15% in TB patients versus 5% in LTBI subjects) and a central memory-like (10% in TB patients versus 3% in LTBI subjects) phenotype; however, also the frequencies of these two memory subsets did not change significantly before and after therapy. Thus, the data here reported also point to qualitative differences between TB patients and LTBI subjects in their antigen-specific CD8 T-cell compartment and suggest that the pool of terminally-differentiated CD45RA+ CCR7− epitope-specific CD8 T cells is reduced in TB patients. Analysis of cytokine production by peptide-specific CD8 T-cells at the single cell level IFN-γ and IL-2 have been shown to be the most relevant cytokines to define functional populations of antigen-specific CD4 and CD8 T-cells [16]–[18]. With regard to CD8 T-cells, two cell populations can be defined on the basis of the ability to secrete IL-2 and IFN-γ: CD8 T-cells secreting simultaneously IL-2 and IFN-γ (dual IL-2+/IFN-γ+), and CD8 T-cells secreting only IFN-γ (single IFN-γ). To assess these two profiles, we stimulated PBMC of HLA-A0201 LTBI subjects and TB patients with the same individual peptides as those present in tetramers used in this study and determined the proportion of tetramer specific CD8 T-cells that produced IFN-γ and/or IL-2 by intracellular FACS analysis, after short-term stimulation with peptides. Representative data are shown in Figure 3A and cumulative data are shown in Figure 3B. 10.1371/journal.pone.0005528.g003Figure 3 Polyfunctional cytokine production analysis of tetramer+ Mtb-specific CD8 T-cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the same individual peptides as those present in tetramers and were stained with mAbs to CD8, IFN-γ and IL-2, or with isotype-control mAbs. After gating on CD8+ cells, the percentage of cells expressing IFN-γ and IL-2 was determined. (A) Representative intracellular cytokine staining data in one subject with LTBI, one TB patient before therapy and one PPD− healthy donor. Numbers in the corners indicate the percentage of CD8+ cytokine-positive cells in each quadrant. (B) Summary cumulative data of the IFN-γ and IL-2 secretion capability of tetramer+ Mtb-specific CD8 T-cells in LTBI subjects (white bars) and TB patients with active disease before therapy (black bars). The data are expressed as the percentage of CD8+ T-cells that are IFN-γ+/IL-2− or IFN-γ+/IL-2+. The values reported are the mean percentage of the different subset analysed for each group tested ± standard deviations (SD). p<0.001 and p<0.01 when compared to values in LTBI subjects. Irrespective of the peptide, in subjects with LTBI 35%–45% of CD8 T-cells secreted both IL-2 and IFN-γ. The remainder of CD8 T-cells (55% to 65%) secreted only IFN-γ. In contrast, TB patients had a lower frequency of single IFN-γ-secreting cells against all tested peptides, but attained statistical significance only for Hsp65 peptide-specific CD8 T-cells. However, the most impressive finding was the consistent reduction of dual IL-2+/IFN-γ+ cytokine-secreting CD8 T-cells in TB patients, highly suggestive of a more restricted functional profile of Mtb-specific CD8 T-cells during active disease. Because of limited blood sample volume, it was possible to do intracellular cytokine staining after therapy only in five out of the patients at the first study time point. The results obtained showed, that four months after therapy all these five patients had still a dominance of IFN-γ-only secreting cells for all peptides tested except for the 16 kDa peptide. However, the limited number of patients after therapy did not allow statistical analysis of data. These results indicate for the first time that the percentage of double IFNγ+/IL-2+ producing CD8 T-cells is significantly higher in LTBI subjects than in TB patients before therapy, suggesting a protective role of the two cytokines jointly in association with antigen specific CD8+ T-cell responses towards Mtb in latently infected healthy subjects. Figure 4 shows peptide-specific CD8 T-cell producing single IFN-γ+ or double IFN-γ+/IL2+: each portions of a pie chart indicates the mean percentage of peptides-specific T cells that responded with one or two functions. 10.1371/journal.pone.0005528.g004Figure 4 Peptides-specific CD8 T cell responses in LTBI subjects, in TB patients at T0 and T4. Peptides-specific CD8 T cell responses are shown as a pie chart. Each portion of a pie chart indicates the percentage of peptides-specific T cells that responded with one or two functions, i.e. producing IFN-γ alone or the combination of IFN-γ and IL-2 (see legend). Discussion CD8 T-cells play a critical role in chronic viral infection, but during recent years their role has gained increasing attention also in Mtb infection. In the present study, we investigated the ex-vivo frequencies, multifunctional cytokine production and memory phenotype of circulating CD8 T-cells specific for different peptide-nonamers of Mtb proteins in adult HLA-A0201 subjects with LTBI and in TB patients before (T0) and after four months of anti-mycobacterial therapy (T4). The ex-vivo frequencies of circulating tetramer specific CD8 T-cells in TB patients before therapy was lower than in LTBI subjects, but increased at four months after therapy to comparable percentages detected in subjects with LTBI; this pattern was consistently found for all tested tetramers. Thus, the frequency of circulating Mtb-specific CD8 T-cells is halved during active TB, compared to LTBI individuals. The reason of the reduced antigen-specific CD8 T-cell frequencies in TB patients at the beginning of therapy and their recovery after four months is not known, but the simplest explanation is that in TB patients large numbers of CD8 T-cells are sequestered at the site of disease and repopulate the peripheral blood compartment after successful anti-mycobacterial therapy. The phenomenon of sequestration of antigen-specific cells has been widely observed in TB for both CD4 and CD8 T-cells [14], [19], [20]. The data here reported also point to qualitative differences between TB patients and LTBI subjects in their antigen-specific CD8 T-cell compartment: while approximately 60% of antigen-specific CD8 T-cells in LTBI expressed a terminally-differentiated phenotype (CD45RA+CCR7−), in TB patients this was only 35% of antigen-specific CD8 T-cells. Conversely, TB patients had higher proportions of cells with an effector memory-like and a central memory-like phenotype. Interestingly, the phenotype of the CD8 T-cell population did not change significantly four months after therapy. These findings are somewhat surprising on the basis of our previous study of CD8 T-cell phenotype in children with TB, where the antigen-specific CD8 T-cell distribution pattern consistently changed four months after therapy, with a significant recovery of terminally differentiated effector memory T-cells and decreased frequencies of central memory T-cells [14]. Another study reported expansion of effector-memory CD8 T-cells in children with TB [15], which express a CD28 and CD27 double negative phenotype. However, care should be taken in correlating the phenotype with the functional properties. For instance, CD45RAhi CD8 T-cells may accumulate during chronic viral infections in elderly individuals representing a pool of apoptosis-resistant memory cells that retain replicative potential [21]. The reason for the reduction of antigen-specific CD8 T-cells during active TB is unknown. As discussed above, one explanation we favour is that these cells are sequestered at sites of infection. Accordingly, a significantly high percentage of Mtb Ag85A epitope-specific CD8 T-cells was previously reported in the cerebrospinal fluid (CSF) of a child with TB meningitis [14]. Alternatively, it is possible that the low frequency of specific CD8 T-cells in active TB could be the consequence of sustained in vivo mycobacterial stimulation, which causes their apoptosis. For example, high levels of bacteria (such as occurs in TB patients due to the inability to contain and prevent their spread) could result in chronic stimulation of CD8 T-cells and induce their apoptosis. A final aim of our study was to assess the capability of Mtb-specific CD8 T-cells to coproduce IFN-γ and IL-2, which is thought to be an indication of their multi-functionality and which has been associated with protective immunity [22]. Our results show that while in subjects with LTBI there was a high percentage of IL-2+/IFN-γ+ and IL-2−/IFN-γ+ peptide-specific CD8 T-cells, the IL-2+/IFN-γ+ population was consistently reduced in TB patients, highly suggestive of a restricted functional profile of Mtb-specific CD8 T-cells during active disease. While to our knowledge there has been no study comparing the cytokine response of CD8 T-cells at a single cell level in LTBI subjects and patients with active TB, a recent study on the CD4 T-cell response to Esat-6 and CFP-10 reported that there was a shift in the IFN-γ and IL-2 cytokine profile, notably from a dominance of IFN-γ-only T-cells in active tuberculosis to a dominance of IFN-γ/IL-2-double secreting T-cells [18]. These results, together with those reported in this paper, suggest distinct T-cell functional signatures between subjects which control Mtb infection (LTBI individuals) and those who do not (active TB disease). Accordingly, studies on CD8 T-cell responses in chronic infections such as HIV, CMV, EBV and HCV [23], [24] have highlighted signatures of protective antiviral immunity: poly-functional (i.e. IL-2 and IFN-γ secretion) and not mono-functional (i.e. IFN-γ only secreting) CD4 and CD8 T-cell responses represent correlates of protective antiviral immunity in chronic viral infections. Furthermore, the levels of antigen load modulate the phenotypic and functional patterns of the T-cell response within the same virus infection. Accordingly, Lalvani and colleagues demonstrated that functional CD4 T-cell heterogeneity is also associated with changes in Mtb antigen load: in active disease, in which antigen load if high, IFN-γ is secreted from two functional subsets, namely IFN-γ-only and IFN-γ/IL-2 dual secreting T-cells, whereas after therapy when antigen load is low, IFN-γ is predominantly secreted from IFN-γ/IL-2 dual secreting CD4 T-cells [18]. Although more extensive phenotyping of Mtb-specific IFN-γ- and IL-2-secreting T-cells is beyond the scope of this study, previous studies have identified a relationship between the function and phenotype of memory CD4 T-cells and proposed that the IL-2-only secreting cells are typical of central memory T-cells that persist after antigen clearance while the IFN-γ/IL-2- and IFN-γ-only secreting T-cells are typical of effector memory T-cells [20], [22]. Accordingly, a recent paper reported that in children vaccinated with BCG, specific CD8 T-cells identified by intracellular IFN-γ secretion, displayed a predominant CD45RA−CCR7− effector memory phenotype, while a central memory population (CD45RA−CCR7+) was the second most common [25]. Phenotypic and functional signatures of CD8 T-cells could be used as an immunological marker of mycobacterial load, to monitor the response to treatment, to evaluate new therapies for active tuberculosis and the efficacy of new vaccines in clinical trials where new biomarkers are needed. Moreover, phenotypic and functional signatures of CD8 T-cells could also be used to monitor individuals latently infected with Mtb at a high risk of progression to active tuberculosis, such as those with HIV coinfection or on anti-TNF therapy. Materials and Methods Human subject Peripheral blood was obtained from 13 HLA-A0201 positive adults with TB disease (7 men, 6 women, age range 50–58 years) from the Dipartimento di Medicina Clinica e delle Patologie Emergenti, University Hospital, Palermo, and Monaldi Hospital, Naples, Italy, and 9 HLA-A0201 LTBI subjects (5 men, 4 women, age range 30–45 years) and 5 tuberculin (PPD)− negative healthy subjects (3 men, 2 women, age range 35–55 years). TB patients had clinical and radiological findings consistent with active pulmonary TB [26]. Diagnosis was confirmed by bacteriological isolation of Mtb in 12 patients and 1 further patient was classified as having highly probable pulmonary TB on the basis of clinical and radiological features highly suggestive of TB that were unlikely to be caused by another disease and a decision was made by the attending physician to initiate anti-tuberculosis chemotherapy, which resulted in an appropriate response to therapy. All patients were treated in accordance with italian guidelines and received therapy for 6 months. Treatment was successful in all participants as evidenced by no clinical or radiographic evidence of current disease, the completion of anti-tuberculosis chemotherapy and sterile mycobacterial cultures. Peripheral blood was collected before (T0) and 4 months after chemotherapy (T4). The follow-up time point of four months after starting therapy following was chosen on the basis of previous studies by our and other groups [19], [27]–[29] which have demonstrated change in many different immune responses in TB patients at this time point after therapy, including the CD8 T cell phenotype in childhood TB [14]. None of the TB patients had been vaccinated during infancy with BCG, or had evidence of human immunodeficiency virus (HIV) infection, or was being treated with steroid or other immunosuppressive or anti-tubercular drugs at the time of their first sampling. Tuberculin (purified protein derivative, PPD) skin tests were considered positive when the induration diameter was larger than 5 mm at 72 hrs since injection of 1 U of PPD (Statens Seruminstitut, Copenaghen, Denmark). The study was approved by the Ethical Committee of the Dipartimento di Medicina Clinica e delle Patologie Emergenti, University Hospital, Palermo, and Monaldi Hospital, Naples, Italy where the patients were recruited. Informed consent was written by all participants. For the identification of LTBI subjects, in the absence of a gold standard, the most widely used diagnostic test remains the tuberculin skin test, based on the delayed-type hypersensitivity reaction that develops in Mtb infected individuals upon intradermal injection of PPD. However, this test suffers from many limitations, including false-negative results, especially in some high-risk groups, and false-positive results in BCG-vaccinated individuals or in subjects exposed to non-tuberculous mycobacteria [30]. Moreover, in vitro release of IFN-γ by T lymphocytes upon stimulation with the Mtb-specific antigens Esat-6 and CFP-10 (the T-SPOT.TB test-Oxford Immunotec, Oxford, United Kingdom and the QuantiFERON-TB Gold test-QFT-G, Cellestis, Victoria, Australia), was performed for all patients, for all LTBI subjects that resulted positive, and for PPD− healthy donors included in the present study [31], [32]. Individuals with LTBI were defined as healthy people with a positive tuberculin skin test and no symptoms and signs of active TB. All of the LTBI subjects were health care workers, and thus very likely to be close contacts of TB index cases. Moreover, all the LTBI subjects included in this groups have not been vaccinated with BCG. All the subjects were HLA typed serologically. The HLA subtype, A0201, was confirmed by PCR amplification technique using sequence-specific oligonucleotide primers. HLA-A0201 and β2-microglobulin Recombinant HLA-A0201 was over expressed in E. coli, purified as described [33] and dissolved in 8M urea. The integrity of the protein was confirmed by TOF-MALDI mass spectrometry using insulin as an internal reference. Human β2-micro-globulin was purchased from Sigma (St. Louis, MO) and dissolved in H2O. Heavy chain (50 µM) stock solutions were stored at −20°C until use. Synthetic peptides A total of 6 nonamer peptides derived from the sequence of the proteins of Mtb and containing HLA-A0201-binding motifs [34] were prepared using solid-phase/Fmoc chemistry, as described in detail elsewhere [19], [35], [36]. The peptides were of 90% purity, and their homogeneity was confirmed by analytical reverse-phase high-performance liquid chromatography, mass spectroscopy, and amino acid composition analysis. The sequences of the peptides were: Ag85B p5–13 (GLPVEYLQV), Esat-6 p82–90 (AMASTEGNV), Hsp65 p362–370 (KLQERLAKL), 16 kDa p120–128 (GILTVSVAV), Rv1490 p325–333 (FLLGLLFFV) and Rv1614 p197–205 (FLYELIWNV). Whereas the former 4 peptides have been identified previously, the latter two are newly identified in this study as candidate epitopes, based upon whole Mtb genome screening for 9-mer peptides sequences with the high and intermediate predicted HLA-A0201 binding affinity as indicated in Table 1. The two peptides derived from Rv1490 and Rv1614 were identified after a full genome-wide screening with the HLA_BIND algorithm. The two selected peptides had the highest predicted score for binding to HLA-A0201, and there are reported strong associations between peptide binding HLA-A0201 affinity and epitope recognition for CD8 T-cells in tuberculosis [37]. The in silico approach matched the strategy previously used to identify CD8 T-cell epitopes across the entire Mtb genome [17], [38]. Candidate HLA-A0201 binding peptides in Hsp65 were selected using the MOTIFS software described previously [39]. Positive scores were given for each potential anchor residue found in the peptide, and negative scores were given to inhibitory residues. The overall peptide score was the sum of the scores for individual anchor and inhibitor residues. HLA-A0201-peptide binding assay HLA-A0201 was titered in the presence of 100 fmol standard peptide to determine the HLA concentration necessary to bind 20–50% of the total fluorescent signal [35]. All subsequent inhibition assays were then performed at this concentration. HLA-A0201 was incubated in 96-well plates (polypropylene, serocluster, Costar) at RT (pH 7) for 24 h with 0.5 µl β2M (15 pmol) and 1 µl (100 fmol) fluorescent labelled peptide in 92.5 µl assay buffer (100 mM Na-phosphate, 75 mM NaCl, 1 mM CHAPS), 2 µl protease inhibitor mixture (1 µM chymostatin, 5 µM leupeptin, 10 µM pepstatin A, 1 mM EDTA, 200 µM pefabloc) and 2 µl of the peptides of which HLA-binding capacity was to be determined. As a standard peptide we used FLPSDC(Fl)FPSV. The HLA-peptide complexes were separated from free peptide by gel filtration on a Synchropak GPC 100 column (250 mm×4.6 mm; Synchrom, Inc., Lafayette, Indiana). Fluorescent emission was measured at 528 nm on a Jasco FP-920 fluorescence detector (B&L Systems, Maarssen, The Netherlands). As HPLC running buffer, assay buffer containing 5% CH3CN was used. The percentage of labelled peptide bound was calculated as the amount of fluorescence bound to MHC divided by total fluorescence. The concentration of peptide inhibitor yielding 50% inhibition (IC50) was deduced from the dose-response curve. Each peptide was tested in at least two separate experiments. Data are expressed as relative (rel) IC50, compared to the IC50 of the standard peptide HBV core p47–56, which was considered as 1. Binding affinities of peptides to the HLA-A0201 molecule were defined as high (IC50<1 µM), intermediate (IC50 1–10 µM) and low (IC50>10 µM). Note that, although the binding affinity of the Ag85 peptide is intermediate, it has been shown that is able to induce potent CD8 CTL activity [35]. Generation of HLA-A0201-Peptide Tetramers Tetrameric HLA-A2-peptide complexes were prepared as follows: recombinant HLA-A0201 and human β2-microglobulin, produced in Escherichia coli, were solubilized in urea and injected together with each synthetic peptide into a refolding buffer consisting of 100 mM Tris (pH 8.0), 400 mM arginine, 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione. Refolded complexes were purified by anion exchange chromatography using DE52 resin (Whatman) followed by gel filtration on a Superdex 75 column (Amersham Pharmacia Biotech). The refolded HLA-A0201-peptide complexes were biotinylated by incubation for 16 hrs at 30°C with BirA enzyme (Avidity, Denver). Tetrameric HLA-peptide complexes were produced by the stepwise addition of extravidin-conjugated phycoerythrin (PE) (Sigma) to achieve a 1∶4 molar ratio (extravidin-PE/biotinylated HLA class I). The PE-labelled HLA-A0201 tetramer complexed with the HIV-1 gag peptide p76–84 (SLYNTVATL was obtained from Proimmune Ltd. (Oxford, UK) and used as a negative control of tetramer staining. Tetramer staining and immunophenotyping PE-labelled HLA-A0201 tetramer complexes loaded with the Mtb peptides Ag85B p5–13 (GLPVEYLQV), Esat-6 p82–90 (AMASTEGNV), Hsp65 p362–370 (KLQERLAKL), 16 kDa p120–128 (GILTVSVAV), Rv1490 p325–333 (FLLGLLFFV) and Rv1614 p197–205 (FLYELIWNV) were used throughout. Tetramer staining was carried out as described in detail previously: peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples by Ficoll-Hypaque (Sigma) density centrifugation. PBMC were incubated in U-bottom 96-well plates, washed twice in phosphate buffered saline (PBS, Euroclone, Milan, Italy) containing 1% fetal calf serum (FCS, Sigma) and stained for 30 min at 4°C with PE-labelled tetramers (3 µl), washed and subsequently stained with FITC-labelled anti-CD8 mAb (clone HIT8a, BD Biosciences, San Josè, CA) and analyzed by flow cytometry on a FACS Calibur analyzer with the use of the CellQuest software (BD Biosciences). Viable lymphocytes were gated by forward and side scatter and the analysis was performed on 100.000 acquired events for each sample. To assess the phenotype of tetramer+ T-cells, cells were stained with FITC-labelled anti-CD8 mAb, APC-labelled anti-CCR7 (clone 3D12) mAb and PE-Cy5-labelled anti-CD45RA mAb (clone HI100) all from BD Biosciences in incubation buffer (PBS-1% FCS-0,1% Na azide) for 30 min at 4°C. Cells were then washed twice in PBS 1% FCS and analyzed by flow cytometry as previously described. Analysis was performed on 100.000 acquired events for each sample. Intracellular cytokine staining PBMC (106/ml) were stimulated with peptides (1 µg/ml, final concentration), in the presence of monensin for 6 hrs at 37°C in 5% CO2. The cells were harvested, washed and stained with APC-conjugated anti-CD8 mAb (BD) in incubation buffer (PBS-1% FCS-0.1% Na azide) for 30 min at 4°C. The cells were washed twice in PBS-1% FCS and fixed with PBS-4% paraformaldehyde overnight at 4°C. Fixation was followed by permeabilization with PBS–1% FCS–0.3% saponin–0.1% Na azide for 15 min at 4°C. Staining of intracellular cytokines was performed by incubation of fixed permeabilized cells with PE-labelled anti-IFN-γ (clone B27) and FITC-labelled IL-2 antibody (clone MQ1-17H12) or an isotype-matched control mAb. All mAbs were from BD Biosciences. Cells were acquired and analysed by FACS as described above. Statistical considerations Negative control (background) values for cytokine staining were not subtracted from peptide-induced responses. For phenotype distribution analysis we used a cut off of a minimum number of 50 events, with a mean of 250 events for all tetramers tested and for each group of individuals tested. Nonparametric Mann-Whitney U test was used to determine statistical differences in the distribution of the results. Values of p<0.05 were considered significant. Data were analyzed using statistical software SYSTAT 11 (Systat Software). We would like to thank Sarah Klein who actually did the MTB genome scan for potential HLA-A0201 peptide-binding sequences. We thank Annemieke Geluk for making available results from peptide/HLA-A2 binding experiments. Competing Interests: The authors have declared that no competing interests exist. Funding: This work has been supported by grants from the Ministry for Instruction, University and Research (MIUR-PRIN to FD), the University of Palermo (60% to FD and NC) and the European Commission European Commission within the 6th Framework Programme, contract no. LSHP-CT-2003-503367 to FD and THMO, (the text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information) and the Bill and Melinda Gates Foundation, Grand Challenges in Global Health (GC6#74, GC12#82). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 WHO 2008 Global Tuberculosis Control: Surveillance, Planning, Financing Geneva World Health Organization Available at http://www.who.int/entity/tb/publications/global_report/2008/pdf/fullreport.pdf 2 Flynn JL Goldstein MM Triebold KJ Koller B Bloom BR 1992 Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA 89 12013 12017 1465432 3 Liebana E Girvin RM Welsh M Neill SD Pollock JM 1999 Generation of CD8+ T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis. Infect Immun 67 1034 1044 10024540 4 Koga T Wand-Wurttenberger A DeBruyn J Munk ME Schoel B 1989 T cells against a bacterial heat shock protein recognize stressed macrophages. 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Mol Immunol 32 975 981 7477003 37 Teixeira A Benckhuijsen WE de Koning PE Valentijn AR Drijfhout JW 2002 The use of DODT as a non-malodorous scavenger in Fmoc-based peptide synthesis. Protein Pept Lett 9 379 385 12370025 38 Klein MR Hammond AS Smith SM Jaye A Lukey PT 2002 HLA-B35-restricted CD8(+)-T-cell epitope in Mycobacterium tuberculosis Rv2903c. Infect Immun 70 981 4 11796635 39 D'Amaro J Houbiers JG Drijfhout JW Brandt RM Schipper R 1995 A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide binding motifs. Hum Immunol 43 13 8 7558924	19436760	PMC2678250	CC BY	2021-01-05 16:18:29	yes	PLoS One. 2009 May 13; 4(5):e5528
==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central 1477-7525-7-61918348210.1186/1477-7525-7-6ResearchPsychometric characteristics of the ankylosing spondylitis quality of life questionnaire, short form 36 health survey, and functional assessment of chronic illness therapy-fatigue subscale Revicki Dennis A [email protected] Anne M [email protected] Michelle P [email protected] Robert L [email protected] Lynda C [email protected] Stephen P [email protected] Center for Health Outcomes Research, United Biosource Corporation, Bethesda, MD, USA2 Formerly Abbott Laboratories, Global Health Economics & Outcomes Research, Abbott Park, IL, USA3 Abbott Laboratories, Abbott Immunology, Parsippany, NJ, USA4 Galen Research, Manchester, Lancashire, UK2009 30 1 2009 7 6 6 15 9 2008 30 1 2009 Copyright © 2009 Revicki et al; licensee BioMed Central Ltd.2009Revicki et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ==== Body The authors have withdrawn this article from the public domain because the conclusions drawn may not be supported by the data. In the light of this situation, BioMed Central regrets that this article is no longer available. The authors apologise to all parties for the inconvenience.	19183482	PMC2679727	CC BY	2021-01-04 17:34:40	yes	Health Qual Life Outcomes. 2009 Jan 30; 7:6
==== Front PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1954338008-PLPA-RA-1419R310.1371/journal.ppat.1000480Research ArticleImmunologyImmunology/Immune ResponseImmunology/Immunity to InfectionsImmunology/Innate ImmunityInfectious DiseasesInfectious Diseases/HIV Infection and AIDSInfectious Diseases/Viral InfectionsVirology/Effects of Virus Infection on Host Gene ExpressionVirology/Host Antiviral ResponsesVirology/Immune EvasionVirology/VaccinesInnate Immune Sensing of Modified Vaccinia Virus Ankara (MVA) Is Mediated by TLR2-TLR6, MDA-5 and the NALP3 Inflammasome Innate Immune Sensing of MVADelaloye Julie 1 Roger Thierry 1 Steiner-Tardivel Quynh-Giao 1 Le Roy Didier 1 Knaup Reymond Marlies 1 Akira Shizuo 2 Petrilli Virginie 3 Gomez Carmen E. 4 Perdiguero Beatriz 4 Tschopp Jürg 3 Pantaleo Giuseppe 5 Esteban Mariano 4 Calandra Thierry 1 * 1 Infectious Diseases Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland 2 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 3 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland 4 Centro Nacional de Biotecnología, CSIC, Ciudad Universitaria Cantoblanco, Madrid, Spain 5 Laboratory of AIDS Immunopathogenesis, Immunology and Allergology Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland McFadden Grant EditorUniversity of Florida, United States of America* E-mail: [email protected] and designed the experiments: JD TR QGST TC. Performed the experiments: JD DLR MKR. Analyzed the data: JD TR QGST DLR TC. Contributed reagents/materials/analysis tools: JD TR QGST SA VP CEG BP JT GP ME TC. Wrote the paper: JD TR ME TC. 6 2009 19 6 2009 5 6 e100048012 11 2008 21 5 2009 Delaloye et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNβ-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNβ and IFNβ-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1β. Transcription of the Il1b gene was markedly impaired in TLR2−/− and MyD88−/− BMDM, whereas mature and secreted IL-1β was massively reduced in NALP3−/− BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNβ and IL-1β by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity. Author Summary Modified vaccinia virus Ankara (MVA) is a highly attenuated, replication-deficient, poxvirus currently developed as a vaccine vector against a broad spectrum of infectious diseases including HIV, tuberculosis and malaria. It is well known that robust activation of innate immunity is essential to achieve an efficient vaccine response, and that poxviruses have developed numerous strategies to block the innate immune response. Yet, the precise mechanisms underlying innate immune sensing of MVA are poorly characterized. Toll-like receptors (TLR), RIG-I-like receptors (RLR) and NOD-like receptors (NLR) are families of membrane-bound and cytosolic sensors that detect the presence of microbial products and initiate host innate and adaptive immune responses. Here, we report the first comprehensive study of MVA sensing by innate immune cells, demonstrating that TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways play specific and coordinated roles in regulating cytokine, chemokine and interferon response to MVA poxvirus infection. Delineation of the pathways involved in the sensing of MVA by the host could help designing modified vectors with increased immunogenicity, which would be of particular importance since MVA is considered as a leading vaccine for HIV/AIDS vaccine following the recent failure of an adenovirus-mediated HIV vaccine trial. ==== Body Introduction Attenuated poxviruses are currently being developed as vaccines vectors against various infectious diseases including HIV, malaria and tuberculosis [1]. Modified vaccinia virus Ankara (MVA) and NYVAC are highly attenuated poxvirus strains due to multiple deletions of viral genes and are replication-deficient in human cells. MVA and NYVAC are immunogenic and safe and have been shown to be excellent vaccine vectors for the expression of foreign antigens. MVA is a leading vaccine candidate for delivery of HIV genes with efficient induction of T-cell mediated immune responses [1]–[3]. Profiling of the immune responses triggered by poxvirus vaccine vectors is critical not only for optimal design of vaccine vectors but also for anticipating potential harmful interactions between naturally acquired or vaccine-induced immune responses against the vaccine target. This is indeed an important lesson learned from the adenovirus type 5 (Ad5) HIV vaccine (MRKAd5) STEP trial. Pre-existing neutralizing antibodies against the Ad5 vaccine vector were found to increase the relative risk of HIV infection [4],[5]. Hence the need for extensive assessments of vaccine-induced innate and adaptive immune responses to prevent unexpected adverse events. Sensing of invasive pathogens by sentinel innate immune cells is a fundamental feature of the host antimicrobial defense response. Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I) like receptors (RLRs) and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) have recently emerged as central innate sensors of viruses [6]. Virus sensing by TLR occurs at the cell surface and in the endosomal compartment. At the cell surface, TLR2 or TLR4 recognize either DNA (herpes viruses) or RNA viruses (respiratory syncitial, hepatitis C and measles viruses). In the endosomal compartment, TLR7, TLR3 or TLR9 sense single stranded (vesicular stomatitis virus, Sendai, West Nile and influenza viruses) and double stranded (reovirus) RNA viruses, and DNA viruses (herpes simplex viruses, cytomegalovirus), respectively [7]–[13]. Two members of the cytosolic pattern recognition RLR receptors, RIG-I (also known as DDX58) and melanoma differentiation-associated gene 5 protein (MDA5) (also known as helicard), have been shown to function as sensors of RNA viruses [14]–[19]. RIG-I detects 5′-triphosphate of ssRNAs and short dsRNAs, while MDA5 preferentially recognizes long dsRNAs. NALP3 (NLRP3 also known as cryopyrin) is a member of the NLR family which have been involved in the sensing of both DNA (adenovirus) and RNA (rotavirus, Sendai and influenza viruses) viruses [20],[21]. NALP3, ASC and pro-caspase 1 form a multimeric cytosolic molecular complex known as the NALP3 inflammasome that controls the processing of the IL-1β cytokine precursor pro-IL-1β into IL-1β [22]. Sensing of viruses by TLRs, RLRs and NLRs activates intracellular signalling pathways resulting in the expression of pro-inflammatory cytokines and type I interferons that then act on innate immune cells to limit viral replication and promote the adaptive immune response. Here we report that the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways are the main innate sensors of MVA in the macrophage and that they induce a cytokine response profile characterized by a vigorous chemokine, IFNβ and IL-1β production. Beyond the dissection of the molecular bases of MVA recognition by the innate immune system the present data are likely to help design MVA vaccine vectors with improved immunogenicity. Results Innate immune responses elicited by MVA The profile of innate immune responses elicited by MVA was first examined by RT-PCR and ELISA in a mouse model of poxvirus infection [23]. MVA infection induced a robust innate immune response in peritoneal cells, peritoneal lavage fluid, splenocytes and splenocyte homogenates characterized by the production of pro-inflammatory cytokines (TNF, IL-1β, IL-6, IL-12p40), chemokines (IP-10/CXCL10, RANTES/CCL5, MCP-5/CCL12, MIP-2/CXCL2) and type I interferon (IFNβ) mRNA and protein (Figure 1A and B and data not shown). Infection of human whole blood with MVA also induced a vigorous innate immune response characterized by an abundant production of chemokines (IL-8/CXCL8, MIP-1α/CCL3 and IP-10) and less abundant production of pro-inflammatory cytokines (TNF, IL-1β, IL-6) (Figure 2). Interestingly, MVA was previously shown to down-regulate IL-8 and IL-1β mRNA expression in human monocyte-derived dendritic cells [24],[25], suggesting that MVA infection may induce the production of various patterns of cytokine depending upon the cell-type studied. 10.1371/journal.ppat.1000480.g001Figure 1 MVA stimulates cytokine, chemokine and IFNβ production in vivo. BALB/c mice were injected i.p. with MVA (107 PFU). Peritoneal cells (A) and peritoneal lavage fluid (B) were collected 12 h after infection as described in Materials and Methods . TNF, IL-1β, IL-12p40, IP-10, RANTES and IFNβ mRNA contents of peritoneal cells were analyzed by RT-PCR (A). Results are expressed as the ratio of cytokines, chemokines or IFNβ mRNA levels to that of HPRT. AU: arbitrary units. Cytokine concentrations in peritoneal lavage fluid were measured by ELISA (B). Data are means±SD of triplicate samples from one experiment comprising three mice per experimental condition and are representative of two independent experiments. p<0.05 for all conditions when comparing PBS versus MVA. 10.1371/journal.ppat.1000480.g002Figure 2 TNF, IL-1β, IL-6, IL-8, MIP-1α and IP-10 release by human whole blood exposed to MVA. Whole blood from 3 healthy volunteers (#1, 2 and 3) was incubated for 24 h with (+) or without (−) MVA (MOI 1) in triplicates. Cell-free supernatants were collected to quantify the concentrations of TNF, IL-1β, IL-6, IL-8, MIP-1α and IP-10. Data are means±SD of triplicate samples from one experiment. MVA significantly increased cytokine production (p<0.05 for all conditions). Dissection of the molecular mechanisms of MVA-induced innate immune responses was preformed in PMA-differentiated human THP-1 macrophages and primary human macrophages. Flow cytometry analyses performed with GFP-expressing MVA (MOI 5) indicated that MVA rapidly infected THP-1 cells (Figure 3A and B). More than 60% of cells became GFP positive within 2 h followed by a progressive decline of GFP fluorescence thereafter, which could result either from MVA-induced apoptosis as observed in human HeLa and monocyte-derived dendritic cells [24],[25] or from the shutting down of protein synthesis through activation of the PKR pathway by MVA [26]. Indeed, the number of apoptotic cells increased from 5% at 6 h to 35% at 24 h post-infection as assessed by annexin V and propidium iodine staining (data not shown). 10.1371/journal.ppat.1000480.g003Figure 3 MVA induces the production of cytokines, chemokines and IFNβ by human macrophages. Human THP-1 cells (A–E) and primary human macrophages (F) were infected with GFP-positive (A, B) or wild-type (C–F) MVA (MOI 5). Expression of viral-derived GFP protein by THP-1 cells analyzed by flow cytometry (A, B). Cytokines, chemokines and IFNβ production by THP-1 cells stimulated for 24 h with MVA as assessed by the Luminex technology (C) or by ELISA (D). IL-8 (CXCL8), MIP-1α (CCL3), RANTES (CCL5), IP-10 (CXCL10) and IFNβ mRNA levels were analyzed by RT-PCR and results expressed as the ratio of chemokines or IFNβ to HPRT mRNA levels. AU: arbitrary units (E, F). Data are means±SD of duplicate (C) or triplicate (D to F) samples from one experiment and are representative of one (C) to three (D to F) independent experiments. p<0.05 for all conditions (D to F). The profile of cytokines and chemokines released by MVA-infected THP-1 cells was analyzed with the Luminex technology. Twenty four h after infection, 12 of the 30 mediators analyzed (see Materials and Methods ) were detectable in cell-culture supernatants. Similarly to the results obtained with human whole blood (Figure 2) and in agreement with a recent report by Lehmann et al. [27], MVA induced the production of large quantities of chemokines (IL-8, MIP-1α, MIP-1β/CCL4, MCP-1/CCL2, RANTES and IP-10). MVA also induced large amounts of IFNβ and of IL-1ra, but small amounts of pro-inflammatory cytokines (TNF, IL-1α, IL-1β, IL-6 and IL-12p40) (Figure 3C and D). Kinetics and patterns of chemokines and type I interferon mRNA expression were similar in MVA-stimulated THP-1 cells and primary human macrophages (Figure 3E and F). We then also examined the production of cytokines and chemokines induced by two other vaccinia virus (i.e. the attenuated NYVAC strain and the virulent Western Reserve strain). When compared to MVA, NYVAC induced low levels of IL-8, IL-1β and IFNβ and no TNF, IL-6, MIP-1α, RANTES or IP-10 (Figure S1). The virulent Western Reserve strain of vaccinia virus was observed to also induce low levels of IL-8 and IFNβ in THP-1 cells, but no IL-1β, MIP-1α or IP-10 (Figure S2 and data not shown). Altogether, these results indicated that the innate immune response induced by MVA in human macrophages was characterized by a powerful chemokine production and a less abundant production of pro-inflammatory cytokines probably related to the attenuation of MVA [28]. In contrast, the NYVAC and Western reserve strains stimulated less powerful chemokine and cytokine responses, that most likely reflect differences in the expression of immunomodulatory genes in the genome of MVA, NYVAC and Western Reserve [24],[25]. TLR2, TLR6 and MyD88 are critical for IFNβ-independent chemokine production after MVA infection TLRs have been shown to play an important role in the sensing of viruses and in the initiation of the anti-viral host defense response [29],[30]. Analyses of the TLR repertoire used by the host for sensing of MVA were conducted in bone marrow-derived macrophages (BMDMs) isolated from TLR1−/−, TLR2−/−, TLR4−/−, TLR6−/−, MyD88−/− and TRIF−/− mice and the read-out was the expression of IFN-independent chemokine MIP-2 and of IFNβ. MVA-induced MIP-2 production by BMDMs was completely abrogated in TLR2−/−, TLR6−/− and MyD88−/− cells but not in TLR1−/−, TLR4−/− and TRIF−/− cells, which produced amounts of MIP-2 similar to that of wild-type cells (Figure 4A). In contrast, the amount of IFNβ produced by TLR2−/−, TLR6−/− and MyD88−/− BMDMs was similar to that of wild-type cells (Figure 4B), a finding consistent with the notion that activation of the TLR2 pathway is not implicated in the production of type I IFNs. Similar results were obtained with THP-1 cells stably transduced with a lentiviral delivery system expressing a short hairpin RNA (shRNA) targeting the expression of the TLR2 gene (Figure S3). All together, these results indicated that the activation of the TLR2-TLR6-MyD88 pathway was required for the induction of IFNβ-independent chemokines in MVA-stimulated macrophages. Experiments conducted with NYVAC and the Western Reserve strain of vaccinia virus confirmed that TLR2 was required for IL-8 production by THP-1 cells (Figure S1 and S2). 10.1371/journal.ppat.1000480.g004Figure 4 TLR2, TLR6 and MyD88 are critical for IFNβ-independent chemokine production after MVA infection. MIP-2 (A) and IFNβ (B) produced by wild-type, TLR1−/−, TLR2−/−, TLR4−/−, TLR6−/−, MyD88−/− and TRIF−/− bone marrow-derived macrophages infected with MVA (MOI 5 and 20) or stimulated with lipopolysaccharide (LPS, 100 ng/ml), Pam2CSK4 (P2CSK4, 10 µg/ml), Pam3CSK4 (P3CSK4, 10 µg/ml) for 24 h. Data are means±SD of triplicate samples from one experiment and are representative of 2 to 4 experiments. Endocytosis is required for IL-1β and IFNβ production Vaccinia virus penetrates into target cells either by endocytosis or by membrane fusion in a low pH-independent manner [31]. To determine the contribution of endocytosis to MVA-induced intracellular signalling and cytokine production, THP-1 cells were treated with cytochalasine D, an actin-depolymerizing drug that blocks the endocytotic trafficking, or with chloroquine, a lysosomotropic weak base to neutralize the acidic environment of endocytic vesicles. IL-1β and to a lesser extend IFNβ production were inhibited by cytochalasine D and chloroquine treatment. The inhibition was not related to drug toxicity because chloroquine did not affect IL-8 production and cell viability (Figure 5 and data not shown). The reason why the inhibition of cytokine production (particularly IFNβ) was only partial after treatment with the inhibitors remains uncertain. The data suggest that additional non-endocytic pathways may play a role in the production of IFNβ. In agreement with a key role for membrane-bound TLR2 for IL-8 induction, the production of IL-8 was not reduced after cytochalasine D or chloroquine treatment (Figure 5). UV treatment of MVA causing a nearly complete (i.e. 90%) inhibition of the expression of the early C6L gene (data not shown) did not affect IL-1β, IL-8 and IFNβ production (Figure 5). Although one cannot completely rule out a contribution of residual viral protein synthesis, these observations support the view that induction of cytokines by MVA is most likely independent of viral gene synthesis [32]–[34]. Overall, endocytosis of MVA was required for IL-1β and IFNβ release suggesting a role for intracellular pattern recognition receptors in the production of these cytokines. 10.1371/journal.ppat.1000480.g005Figure 5 Endocytosis is required for IL-1β and IFNβ production after MVA infection. THP-1 cells were preincubated for 1 h with or without cytochalasin (2 µM) or chloroquine (100 µM) prior to exposure to MVA or UV-treated MVA (MOI 20). Cell-culture supernatants were harvested after 6 h (IL-1β) or 24 h (IFNβ and IL-8) and cytokine concentrations were measured by ELISA. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments. MVA is sensed by MDA-5 and not by RIG-I The RLR family of cytosolic pattern recognition receptors has been implicated in the sensing of RNA viruses [35], but very little is known about their role in host response to DNA viruses. Extending the observations by Guerra et al. who noted an increased expression of RIG-I and MDA-5 mRNA in human dendritic cells infected with MVA [24], we observed that MVA caused a time-dependent increase in RIG-I, MDA-5 and IPS-1 mRNA and protein expression in THP-1 cells (Figure 6A and B). RIG-I and MDA-5 mRNAs rose within 3 h of infection and remained elevated for up to 24 h (Figure 6A). In vivo, MVA up-regulated RIG-I and MDA-5 mRNA levels in peritoneal cells and splenocytes (Figure S4). When compared to MVA, NYVAC induced lower levels of MDA-5 and, to a lesser extent, RIG-I and IPS-1 mRNA and protein expressions (Figure S1 and data not shown). Using shRIG-I, shMDA-5 and shIPS-1 THP-1 cells (Figure S5), we then examined whether RIG-I and MDA-5 were involved in MVA-induced IFNβ production. IFNβ and IP-10 mRNA and protein levels were markedly reduced in shMDA-5 and shIPS-1 cells, but not in shRIG-I cells. By contrast, the time-course and magnitude of the IL-8 and IL-1β production was similar in shMDA-5, shIPS-1, shRIG-I and control THP-1 cells (Figure 7A and B). Sensing of MVA by the MDA-5/IPS-1 pathway is therefore critical for the production of IFNβ and IFNβ-dependent chemokines in macrophages. In line with these data, the production of IFNβ, but not of IL-8, was also dependent on the MDA-5/IPS-1 pathway in cells infected with NYVAC and the Western Reserve strain of vaccinia virus (Figure S1 and S2). 10.1371/journal.ppat.1000480.g006Figure 6 MVA up-regulates the expression of RIG-I, MDA-5 and IPS-1 mRNAs and proteins. RIG-I, MDA-5 and IPS-1 mRNA and protein expression by RT–PCR (A) and Western blot (B). THP-1 cells were infected with MVA (MOI 5) for the indicated time. Results are expressed as the ratio of RIG-I, MDA-5 or IPS-1 mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of three independent experiments. AU: arbitrary units. *p<0.05. 10.1371/journal.ppat.1000480.g007Figure 7 MVA is sensed by MDA-5 and not by RIG-I. THP-1 cells stably transduced with control, MDA-5, RIG-I or IPS-1 shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time. IFNβ, IP-10, IL-8 and IL-1β mRNA and protein expression by RT-PCR and ELISA (A–B). Results are expressed as the ratio of IFNβ, IP-10, IL-8 or IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of four independent experiments. AU: arbitrary units. Concentrations of IFNβ and IL-8 in cell-culture supernatants were measured 24 h after stimulation. shMDA-5 and shIPS-1 THP-1 cells produced significantly less IFNβ and IP-10 mRNA and protein than control cells as measured 24 h post-infection (A and B) (p<0.05). Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1β expression and processing IL-1β is a key cytokine of antimicrobial host defenses, whose expression is regulated at a transcriptional and post-transcriptional level [36]. IL-1β is likely to play an important role during poxvirus infection, as suggested by the fact that poxviruses encode for IL-1β decoy receptor and disrupt intracellular IL-1 receptor signalling [37],[38]. We therefore examined whether activation of the TLR2-MyD88 pathway was implicated in the activation of the IL1b gene. As shown in Figure 8A, up-regulation of IL-1β mRNA was markedly impaired in TLR2−/− and MyD88−/− BMDMs infected with MVA, indicating that activation of the TLR2-MyD88 signalling pathway is critical for transcription of the IL1b gene during MVA infection. Secretion of mature IL-1β p17 in response to endogenous and exogenous danger signals requires the cleavage of the inactive pro-IL-1β precursor by the cysteine protease caspase-1. Conversion of pro-caspase-1 into caspase-1 is tightly regulated by the NALP3 inflammasome composed of NALP3, ASC and pro-caspase-1 [22]. To examine the contribution of the NALP3 inflammasome in the production of IL-1β triggered by MVA, we analyzed the expression of pro-IL-1β and IL-1β p17 in THP-1 cells deficient in NALP3, ASC or caspase-1 [39]. Knocking down of either one of the three components of the NALP3 inflammasome (i.e. NALP3, ASC or caspase-1) was associated with a massive reduction of mature and secreted IL-1β (Figure 8B and C). Similar results were obtained in THP-1 cells infected with NYVAC (Figure S1) and in NALP3−/− BMDMs infected with MVA (Figure 8D and E). Of note, in THP-1 cells and in BMDMs the expression of pro-IL-1β was unaffected by the absence of either NALP3, ASC or caspase-1 clearly indicating that NALP3 inflammasome does not itself regulate the transcriptional and translation control of the IL-1β precursor. The NALP3 inflammasome was also dispensable for activation of the IRF3 transcription factor and IFNβ secretion (Figure S6). Altogether, these data demonstrate that IL-1β production after MVA infection requires a crosstalk between TLR2-MyD88 (initiation of the transcription and translational of IL-1β) and the NALP3 inflammasome (processing of pro-IL-1β into mature IL-1β). 10.1371/journal.ppat.1000480.g008Figure 8 Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1β expression and processing. (A) Wild-type, TLR2−/− and MyD88−/− BMDMs were primed overnight with ultra-pure LPS (100 ng/ml) and infected with MVA (MOI 5). IL-1β mRNA expression was quantified by RT-PCR (p<0.05 for TLR2−/− or MyD88−/− vs. wild-type BMDMs). THP-1 cells stably transduced with control, NALP3, ASC and caspase 1 (casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time (B–C). (B) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (C) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection (p<0.05 for cells transduced with NALP3, ASC and casp1 shRNAs vs. control shRNA). LPS-primed wild-type and NALP3−/− BMDMs were infected with MVA (MOI 5 in D) for 6 h (D–E). (D) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (E) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection. Results are expressed as the ratio of IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments (p<0.05 for NALP3−/− vs. wild-type BMDMs). MVA activates the NF-κB, ERK1/2, JNK, IRF3, IRF7 and STAT-1 signalling pathways Poxviruses have been reported to activate the NF-κB, ERK1/2 and JNK pathways in epithelial and fibroblastic cell lines [40]–[43] and IRF3 and IRF7 in dendritic cells [24],[25]. Having identified the pathogen recognition receptors implicated in macrophage response to MVA (TLR2-TLR6, MDA-5 and NALP3), we next examined which downstream signalling pathways are activated for the expression of cytokines, chemokines and type I IFNs. Kinetics studies of NF-κB, ERK1/2 and JNK MAP kinases and IRFs activation were performed in THP-1 cells (Figure 9A). Electrophoretic mobility shift assay revealed that NF-κB nuclear content peaked 3 h after MVA infection. Phosphorylation of the ERK1/2 and JNK MAP kinases was between 1 and 6 h after infection. IRF3, which is essential for transcription of the IFNB gene, was detected 3 h after infection, peaked at 6 h and rapidly decreased thereafter. IRF7 was detected 3 h after infection and levels remained unchanged for 24 h. Phosphorylation of signal transducer and activator of transcription 1 (STAT-1), a critical target of IFNβ signalling required for the transcriptional activation of IFNβ-dependent genes, was first detected 3 h post-infection and gradually increased until 24 h (Figure 9A). The functional significance of the increased binding activity of NF-κB and phosphorylation of the IRF3 was confirmed by showing that MVA increased the transcriptional activities of multimeric-κB and IRF3-dependent-IFNβ promoter luciferase reporter vectors in transiently transfected THP-1 cells (Figure 9B and C). Confirming the importance of NF-κB and ERK1/2 in mediating innate immune response to MVA infection, pre-incubation of THP-1 cells with drugs (i.e. NEMO and U0126, see Materials and Methods ) selectively inhibiting the NF-κB and ERK-1/2 signalling pathways impaired, albeit to a different extent, IL-1β (70% and 65% inhibition), IL-8 (75% and 72% inhibition) and IFNβ (28% and 42% inhibition) mRNA expression (p<0.05 for all conditions). Therefore, consistent with the fact that several pattern recognition receptors are engaged in the sensing of MVA by the innate immune system, multiple intracellular signalling pathways, including NF-κB, MAP kinases and IRFs were found to be activated upon infection of THP-1 macrophages with MVA. Of note, NYVAC induced very weak induction of intracellular signalling (i.e. NF-κB, ERK-1/2, IRF3 and STAT-1) and low levels of cytokines and IFNβ when compared with MVA (Figure S1) which is likely due to the expression of different patterns of immunomodulatory genes by these two poxviruses [24],[25]. 10.1371/journal.ppat.1000480.g009Figure 9 MVA activates the NF-κB, ERK1/2, JNK, IRF3, IRF7 and STAT-1 signalling pathways. Electrophoretic mobility shift assay of NF-κB DNA binding activity and Western blots of phosphorylated ERK1/2 (P-ERK1/2), JNK (P-JNK), IRF3 (P-IRF3) and STAT-1 (P-STAT-1) and total ERK1/2, JNK and IRF7 (A). Nuclear (NF-κB) and cytosolic (ERK1/2, JNK, IRF3, IRF7, STAT-1 and tubulin) extracts were prepared from THP-1 cells infected with MVA (MOI 5) for the indicated time. Results are representative of three independent experiments. The retarded complex detected by EMSA was dose-dependently inhibited by cold wild-type but not mutant NF-κB oligonucleotide, and supershifted using anti-p65 antibody (data not shown). NF-κB- (B) and IRF3- (C) mediated transcriptional activities measured in THP-1 cells transiently transfected with trimeric κB sites or IRF3-dependent IFNβ promoter luciferase reporter vectors and infected with MVA (MOI 5 and 20) for 18 h. Results are expressed as the ratio of luciferase activity to Renilla luciferase activity. Data are means±SD of triplicate samples from one experiment and are representative of four independent experiments. p = 0.05, 0.02, 0.04 and 0.02 for MVA-infected (MOI 5 and 20) vs. control cells. Discussion Analyses of pattern recognition receptors engagement by poxviruses are essential for improving our understanding of the pathogenesis of this important class of DNA viruses and for designing new viral vaccine vectors with improved immunogenicity. Dissection of the molecular bases of innate immune responses elicited by the attenuated poxvirus MVA strain in human macrophages revealed a critical role for TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways in the production of chemokines, IFNβ and IL-1β. These observations provide novel information on MVA recognition by sentinel innate immune cells and highlight the existence of potential differences between attenuated and non-attenuated poxviruses in the engagement of or recognition by innate sensors. Up to now the retinoic acid-inducible gene-I-like receptors (RLR) RIG-I and MDA-5 had been viewed as master cytosolic sensors of RNA viruses [29]. However, recent observations suggested a role for the RLR pathway in the recognition of DNA viruses. Mouse embryo fibroblasts deficient in IPS-1 displayed reduced induction of IFNβ in response to MVA lacking the E3 protein [44]. Adenovirus and HSV1 have also been shown to replicate at much higher titers in RIG-I mutant than in RIG-I wild-type human hepatoma cell lines [45]. Moreover, microarray analyses revealed that RIG-I and MDA-5 expression was upregulated in human monocyte-derived dendritic cells infected with MVA [24]. Here we also showed that MVA caused a strong up-regulation of RIG-I, MDA-5 and IPS-1, yet only MDA-5 and IPS-1 were found to mediate MVA-induced IFNβ and IFNβ-dependent chemokine production by macrophages (Figure 10). As anticipated, transcriptional activation of IFNb and IFNb-dependent chemokine genes was associated with the activation of IRF3 and IRF7 and STAT-1. To the best of our knowledge this is the first demonstration of a direct role for MDA-5 in innate sensing of a DNA virus. Moreover, the MDA-5/IPS-1 pathway was also implicated in the production of IFNβ by macrophages infected with the NYVAC and the Western Reserve strains of vaccinia virus (Figure S1 and S2). 10.1371/journal.ppat.1000480.g010Figure 10 Pathways activated by MVA in the macrophage. Infection of macrophages with MVA stimulates the TLR2-TLR6-MyD88, MDA-5/IPS-1 and NALP3 inflammasome pathways leading to the activation of NF-κB, ERK-1/2, JNK, IRF3, IRF7 and STAT-1 that are involved in the transcriptional activation of genes encoding for cytokines, chemokines and type I IFN. At the cell surface, MVA is sensed by the TLR2-TLR6 heterodimer that induces the production of IFNβ-independent chemokines (IL-8, MIP-1 and MIP-2) (1) and pro-IL-1β (2). Upon virus entry into the cell, cytosolic MVA or MVA-derived viral components (possibly envelope or core proteins, early mRNA or DNA) activate the MDA-5-IPS-1 pathway to release IFNβ (3) and subsequent induction of IFNβ-dependent chemokines (such as RANTES, IP-10) following activation of the type I IFN receptor (4). Finally, MVA infection leads to the activation of the NALP3 inflammasome (composed of NALP3, ASC and pro-caspase 1) enabling caspase-1 processing, pro-IL-1β maturation and IL-1β secretion (5). For simplicity, the same diagram for MVA is shown outside and inside the cell. RIG-I has been shown to be involved in the induction of TNF and type I IFN by myxoma poxvirus in human macrophages [46]. Yet, silencing of MDA-5 was associated with a small (about 25%) but clear reduction of macrophages response to myxoma virus suggesting that both RIG-I and MDA-5 were implicated, albeit to various degree, in innate immune response to myxoma virus. The nature of the component(s) of DNA viruses activating the RLR pathway remains to be identified. Obvious candidate molecules include, envelope or core proteins, early mRNA and DNA itself. Unless RLR engagement is used primarily to the virus own benefit, it is likely that poxviruses have developed antiviral escape strategies interfering with the host RLR antiviral defense pathway. In line with this assumption, the dsRNA binding protein E3 of vaccinia virus has been reported to inhibit IPS-1 signaling, IRF3 phosphorylation, cytokine and IFNβ production [47]–[49]. Should inhibitors of the RLR pathway be identified in the MVA genome, gene deletion might provide an opportunity to generate new MVA vaccine vectors with increased immunogenicity. In addition to RLR, profiling of the cytokine response induced by MVA in the macrophage revealed a key role for the heterodimeric TLR2-TLR6 complex and the adapter protein MyD88 in the production of IFNβ-independent chemokines (such as IL-8, MIP-1α, MIP-1β and MIP-2) (Figure 10). Innate immune recognition of the vaccinia virus has also been shown to depend on TLR2 and MyD88 [32]. The present observation is one of the few examples of viral recognition mediated by TLR2 heterodimers. Recognition of human cytomegalovirus has been shown to be mediated by a TLR2-TLR1 heterocomplex and that of hepatitis C virus by either TLR2-TLR1 or TLR2-TLR6 [50],[51]. The facts that TLR2 is expressed at the cell surface and that the inhibition of endocytosis or UV-irradiation of MVA did not affect IL-8 production by macrophage suggest that a component of the MVA envelope or a core protein is responsible for the activation of the TLR2-TLR6-MyD88 pathway. However, the nature of the viral component likely to serve as ligands for these TLR2-TLR1/TLR6 heterodimers has so far remained elusive. Other TLRs have also been implicated as innate sensors of poxviruses. Ectromelia virus, the causative agent of mousepox, was shown to be recognized by mouse dendritic cells in TLR9 dependent manner [33]. In contrast, responses of dendritic cells to MVA was both TLR9-dependent (up-regulation of CD40) and TLR9-independent (up-regulation of CD69 and production of IFNα and IL-6) [33],[52]. Although we did not perform experiments with TLR9-deficient macrophages in the present study, the data obtained with MyD88 deficient cells clearly rule out the implication of TLR9 in MVA-induced IFNβ and IFNβ-dependent chemokines. However, we cannot exclude the involvement of TLR9 in the production of IFNβ-independent chemokines. Finally, in a mouse model activation of TLR3 contributed to the pathogenesis of Western Reserve vaccinia virus [53]. In contrast, experiments conducted with TRIF-deficient macrophages clearly showed that the production of chemokines and IFNβ induced by MVA was TLR3-independent in the present study. Taken together these observations demonstrate that TLRs may exert a two-sided role in poxvirus infections acting on the one hand as key initiators of the host anti-poxvirus defense response and on the other hand as important mediators of viral pathogenicity and tissue damage. The other important intracellular innate immune sensor of microbial products and endogenous molecules is the NALP3 inflammasome that controls the processing and maturation of the cytokines IL-1β and IL-18 [22]. Here we show that MVA is a potent activator of the NALP3 inflammasome and of IL-1β release by macrophages. IL-1β and IL-18 are key mediators of the host antimicrobial defense response and several lines of evidence suggest that these cytokines are likely to play an important role in host defenses against poxvirus infections. For example, the B15R gene of the vaccinia virus encodes an IL-1β decoy receptor blocking the activity of IL-1β and IL-18 and inactivation of B15R gene reduces the virulence of the vaccinia virus [38],[54]. Furthermore, poxviruses release IL-18 binding proteins inhibiting IL-18 activity and vaccinia viruses A46R, A52R, N1L and, K1L gene products have been shown to disrupt the IL-1 receptor intracellular signaling pathway at multiple levels [37],[55]. Interestingly, we observed that MVA stimulated the release of large amounts of the IL-1 receptor antagonist by macrophages (Figure 3C) adding further support to the view that IL-1 is an important target of the poxvirus antiviral escape strategy. Finally, consistent with the notion that the NALP3 inflammasome plays an important role in host defenses against poxviruses, several inhibitors of caspase-1 and ASC, like CrmA (cowpox virus), M13L-PYD (myxoma virus) and PYD-only (shope fibroma-virus) have been identified in the genomes of several poxviruses [56]–[58]. Crosstalks between TLRs and NLRs have been demonstrated to occur in the course of bacterial infections, such as between TLR5 and the IPAF inflammasome after exposure to flagellated bacteria or the flagellin protein itself [59]–[61]. To the best of our knowledge, however, the present data provide the first demonstration of a crosstalk between the TLR and NLR pathways in the context of a viral infection (Figure 10). While TLR2 and MyD88 were necessary to induce IL-1β mRNA expression (Figure 8A), the NALP3 inflammasome was absolutely required for the processing of pro-IL-1β and IL-1β secretion (Figure 8B and C). Dual activation pathways coupling MVA recognition to IL-1β may provide the host with an increased capacity of fine tuning of its cytokine response. In summary, the present data show that the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways exert both specific and coordinated functions in the sensing of MVA infection and in the regulation of cytokine, chemokine and IFNβ responses (Figure 10). After the unfortunate failure of the adenovirus type 5 HIV vaccine STEP trial due to issues related to natural immunity against this virus, the attenuated MVA and NYVAC strains of poxvirus have become attractive vaccine vectors against HIV/AIDS. Arguments supporting the use of MVA and NYVAC as vaccine vectors include excellent immunogenicity and safety profiles and limited pre-existing immunity to poxvirus in the population at risk of HIV infection due to the abandon of vaccine campaigns after the eradication of smallpox in the 1970s. The present findings are therefore likely to provide important information relevant to the study of the pathogenesis of poxvirus infections, the understanding of antiviral escape mechanisms of poxvirus and may help to design new vaccine vectors with increased immunogenicity. Materials and Methods Ethics statement All animal procedures were approved by the Office Vétérinaire du Canton de Vaud (authorizations n° 876.5, 876.6, 877.5 and 877.6) and performed according to our institution guidelines for animal experiments. Mice Eight to ten-week-old female BALB/c and C57BL/6 mice were purchased from Charles River Laboratories (L'Arbresle, France) and were acclimatized for at least one week before experimentation. MyD88−/−, TRIF−/−, TLR1−/−, TLR2−/−, TLR4−/−, TLR6−/− and NALP3−/− C57BL/6 mice have been described previously [62]–[68]. Mice were bred and housed in specific pathogen free conditions. Cells and reagents The human monocytic THP-1 cell line (American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium containing 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 IU/ml of penicillin, 100 µg/ml of streptomycin (all from Invitrogen, San Diego, CA) and 10% heat-inactivated FCS (Sigma-Aldrich, St. Louis, MO). THP-1 cells differentiated into macrophages by treatment with 0.5 mM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 24 h were used in all experiments except those for reporter gene analyses. THP-1 cells stably expressing control, NALP3, caspase-1 and ASC shRNA have been described previously [69],[70]. THP-1 cells expressing TLR2, IPS-1, MDA-5 and RIG-I shRNA were generated using lentiviruses expressing hairpins directed against TLR2, IPS-1 and MDA-5 (5 for TLR2, 5 for IPS-1, 2 for MDA-5 and 5 for RIG-I) produced with the second-generation pMD2-VSVG and pCMV-R8.91 packaging plasmids as described previously and cultured in the presence of 5 µg/ml puromycin [71]. The sequence of the hairpins selected that gave the best targeting of TLR2, IPS-1, MDA-5 and RIG-I were AAACCCAGGGCTGCCTTGGAAAAG, CAAGTTGCCAACTAGCTCAAA, CCAACAAAGAAGCAGTGTATA and AAACCCAGGGCTGCCTTGGAAAAG, respectively. Levels of expression of targeted genes were analyzed by real-time PCR using specific oligonucleotides (Table S1) and the most efficiently silenced THP-1 subsets were selected for further studies (i.e. cell lines #1 in Figure S2). Peripheral blood mononuclear cells from healthy donors (recruited by the Blood Center, Lausanne, Switzerland) were purified by Ficoll-Hypaque density gradient (GE Healthcare, Uppsala, Sweden). Macrophages were obtained by culturing adherent PBMCs cells for 6 days in RPMI 1640 with Glutamax. Bone marrow-derived macrophages (BMDMs) isolated from wild-type, TLR1−/−, TLR2−/−, TLR4−/−, TLR6−/−, MyD88−/− and TRIF−/− mice were cultured for 7 days in IMDM (Invitrogen) containing 50 µM 2-mercaptoethanol and monocyte-colony stimulating factor to obtain BMDMs. All media were supplemented with 10% FCS, 100 IU/ml of penicillin and 100 µg/ml of streptomycin. In selected experiments, cells were stimulated with 100 ng/ml Salmonella minnesota ultra pure LPS (List Biologicals Laboratories, Campbell, CA), 10 µg/ml polyinosine-polycytidylic acid (poly(I∶C), Invivogen, San Diego, CA), 1–10 µg/ml S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine×3 CF3COOH (Pam2CSK4) or N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine×3 HCl (Pam3CSK4) lipopeptides (EMC microcollections, Tuebingen, Germany), or treated with 50 µg/ml of anti-IFNβ antibodies (BioLegend, San Diego, CA), 2 µM cytochalasine D, 100 µM chloroquine (Sigma-Aldrich), 10 µM SB203580 (p38 inhibitor), 10 µM U0126 (MEK1/2 inhibitor) or 50 µg/ml NEMO-binding domain binding peptide (IkB kinase inhibitor) (Calbiochem-Novabiochem, Nottingham, UK). MVA, NYVAC and WR production, in vitro and in vivo models of infection MVA and NYVAC were cultured in chicken embryo fibroblasts and WR in HeLa cells. Viruses were purified by two sucrose cushions and titrated on BHK-21 and BSC-40 cells as previously described [24],[72]. Cells were infected with MVA, NYVAC or WR at various multiplicities of infection (MOI 1, 5 or 20 pfu/cell). After 1 h of contact with cells, the virus inoculum was removed and fresh medium added to the cultures. Cell-culture supernatants and cells were collected at different time points after infection and processed for flow cytometry, Luminex technology, ELISA, RNA extraction, and Western blot analyses. In selected experiments, MVA suspension (0.2 ml in 24-well plates laid on ice) was irradiated by a 15-min exposure to a 365-nm UV bulb at a distance of 4 cm. UV-irradiation caused a 90% inhibition of the expression of C6L early gene as determined by RT-PCR using oligonucleotides (5′-3′ sense and antisense at position −19541/−19503 and −19071/−19090 in MVA019L) AACTGCAGAAATGAATGCGTATAATAAAGCCGATTCGTTTTCTTTAGAG and CGGGATCCTTACTTGTCATCGTCGTCGTTCTTGTAGTCCSTGTTTAGGAAAAAAfAAATATC. MVA did not propagate in THP-1 cells as demonstrated by the absence of infective viral particles in cell-culture supernatants collected 24 h after infection (data not shown). For whole blood assay, 100 µl of heparinized whole blood collected from 3 healthy volunteers were diluted 5-fold in RPMI 1640 medium containing MVA (MOI 1) and incubated for 24 h at 37°C in the presence of 5% CO2. Samples were centrifuged, and cell-free supernatants were stored at −80°C until cytokine measurement. For in vivo studies, 2×107 PFU of MVA in 1 ml phosphate-buffered saline (PBS) were injected intraperitoneally into BALB/c mice. After 12 h, a peritoneal lavage was performed. The supernatant obtained after centrifugation of the lavage fluid was collected for cytokine measurement by ELISA whereas the cell pellet was processed for gene expression analysis by RT-PCR. Spleens were collected from the same animals to quantify cytokine protein and mRNA expression levels. Flow cytometry To follow cell infection, THP-1 cells were infected (MOI 5) with a GFP-expressing mutant MVA, whereas all other experiments used wild-type MVA. The percentage of GFP-positive THP-1 cells was measured 0, 2, 4, 6, 12 and 24 h after infection. MVA-induced cell apoptosis was determined 6 h and 24 h post-infection using the Annexin-V FITC apoptosis detection kit according to manufacturer's recommendations (BD Biosciences, Erembodegem, Belgium). Acquisition and analysis were performed using a FACS Calibur (BD Biosciences) and FlowJo 8.5.3 software (FlowJow, Ashland, OR). Measurement of cytokine production A screening of mediators produced by MVA-infected THP-1 cells was performed with the human cytokine Bioplex assay (Bio-Rad, Hercules, CA) using the Luminex technology (Luminex Corporation, Austin, TX) available at the Cardiomet Mouse Metabolic Evaluation Facility, Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland. Thirty mediators were tested: TNFα, IL-1α, IL-1ra, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IFNγ, RANTES, IP-10, MIP-1α, MIP-1β, MCP-1, eotaxin, fractalkine, TGFα, EGF, VEGF, GM-CSF, G-CSF and sCD40L. The concentrations of human IL-1β (Bender MedSystems, Vienna, Austria), IL-8, (BD Biosciences), IP-10, MIP-1α (R&D) and IFNβ (PBL Biomedical Laboratories, Picataway, NJ) in whole blood assay and cell-culture supernatants were measured by ELISA. TNF and IL-6 concentrations were measured by bioassay as described elsewhere [73]. Mouse IL-1β, MIP-2 (R&D) and IFNβ were quantified by ELISA (Biomedical Laboratories, Picataway, NJ). RNA analysis by quantitative real-time polymerase chain reaction Total RNA was isolated from THP-1 cell lines, human monocytes/macrophages, peritoneal cells and splenocytes using the RNeasy kit (Qiagen, Hombrechtikon, Switzerland). Reverse transcription of 1 µg of RNA was performed using the ImProm II RT System kit (Promega, Dübendorf, Switzerland). Quantitative PCR was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems, Rotkreuz, Switzerland) using the Power SYBR Green PCR Master Mix (Applied Biosystems) and primer pairs listed in Table S1. All samples were tested in triplicates. Amplifications consisted of a denaturation step at 95°C for 15 sec and an annealing/extension step at 60°C for 60 sec, with the 9600 Emulation mode. For each measurement, a standard made of successive dilutions of a reference cDNA was processed in parallel. Gene specific expression was expressed relative to the expression of HPRT in arbitrary units (A.U.). Gene specific over HPRT ratios were validated using the house-keeping gene ACTB (human studies) or Gapdh and Actg1 (mouse studies). Transfection and reporter assay THP-1 cells were seeded at 5×104 cells per well in 24-well plates. The following day, cells were transiently transfected with 700 ng of multimeric κB site [73] and IFNβ promoter [74] luciferase reporter vectors together with 70 ng of a Renilla luciferase control vector (Promega) using jetPEI™ transfection reagent (Polyplus-transfection SA, Illkirch, France). Twenty-four h after transfection, cells were infected with MVA. Luciferase and Renilla luciferase activities were measured 24 h latter using the Dual-LuciferaseTM Reporter Assay System (Promega). Results were expressed as relative luciferase activity (the ratio of luciferase to Renilla luciferase activity). Western blot analysis THP-1 cells were washed with ice cold PBS and lysed for 5 min at 4°C with the M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology Inc, Rockford, IL). Reaction mixtures were centrifuged 5 min at 14'000 rpm. Protein concentration of supernatants was determined using the bicinchoninic acid protein assay (Pierce Biotechnology). Cell-lysates were electrophoresed through 12% (w/v) polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were incubated with antibodies directed against RIG-1, MDA-5, IPS-1 (Apotech Corporation, Epalinges, Switzerland), cleaved IL-1β, total- and phospho-p44/42 (ERK1/2), and -JNK MAP Kinases, phospho-IRF3 (Cell Signalling Technology, Danvers, TX), caspase 1 (Santa Cruz, Santa Cruz, CA), phospho-STAT-1 (BD Biosciences), IRF7 (Zymed, San Franciso, CA) and tubulin (Sigma). After washing, membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibody (Pierce). Signals were revealed using the ECL Western blotting Analysis System (GE Healthcare). Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared and analyzed by EMSA [73]. Briefly, protein concentration of cell extracts was measured using the Bradford-dye assay (Bio-Rad). Two µg of nuclear extracts were incubated for 15 min at room temperature with a radio-labeled consensus NF-κB probe (Santa Cruz). Reaction mixtures were electrophoresed through 6% non-denaturing polyacrylamide gels. Gels were dried and exposed to X-ray films. Supershift experiments using anti-p65 antibody (sc-109, Santa Cruz) were performed as previously described [75] (data not shown). Statistical analyses Comparisons among treatment groups were performed by two-tailed paired Student's t-test. p values less than 0.05 were considered to indicate statistical significance. Supporting Information Figure S1 Comparison of macrophage responses to MVA and NYVAC. THP-1 cells (A, B, D, F) and THP-1 cells stably expressing control, TLR2, MDA-5, RIG-I, IPS-1, NALP3 and caspase-1 shRNAs (C, E) were infected with MVA and NYVAC (MOI 5). Cytokines, chemokines and IFNβ production were assessed by ELISA (A, E). IL-8, IFNβ, IP-10, MIP-1α and RANTES mRNA levels were analyzed by RT-PCR and expressed as reported in Figure 1 (B, C). Electrophoretic mobility shift assay of NF-κB DNA binding activity and Western blot analyses of RIG-I, MDA-5, IPS-1, phosphorylated ERK1/2 (P-ERK1/2), IRF3 (P-IRF3) and STAT-1 (P-STAT-1) and tubulin (D, F). Data are means±SD of triplicate (A, B, C, E) samples from one experiment and are representative of two to three independent experiments. ND: not detected (A). (0.12 MB PDF) Click here for additional data file. Figure S2 TLR2 and MDA-5 contribute to the production of IL-8 and IFNβ by human macrophages infected with the Western Reserve strain of vaccinia (WR). THP-1 cells (A–B) and THP-1 cells stably expressing control, TLR2, MDA-5, RIG-I and IPS-1 shRNAs (C) were infected with WR (MOI 5). IL-8 and IFNβ mRNA levels were analyzed by RT-PCR and expressed as reported in Figure 1 (A and C). IL-8 and IFNβ production by THP-1 cells stimulated for 24 h with WR were assessed by ELISA (B). Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments. shTLR2 THP-1 cells produced significantly less IL-8, whereas shMDA-5 and shIPS-1 THP-1 cells produced significantly less IFNβ than control cells (C) (p<0.05). (0.02 MB PDF) Click here for additional data file. Figure S3 TLR2 contributes to the production of IFNβ-independent chemokines by THP-1 macrophages infected with MVA. THP-1 cells stably expressing control and candidate shRNA (#1 and #2) directed against TLR2 were obtained as described in Materials and Methods . (A) TLR2 mRNA content was analyzed by RT-PCR. Results are expressed as the ratio of TLR2 mRNA levels to that of HPRT. (B) shControl and shTLR2 THP-1 cells were infected with MVA (MOI 5 unless specified) or stimulated with Pam3CSK4 (1 µg/ml) for 24 h unless otherwise specified. IL-8, MIP-1α, IFNβ and IP-10 mRNA contents were analyzed by RT-PCR. Results are expressed as the ratio of IL-8, MIP-1α, IFNβ and IP-10 mRNA levels to that of HPRT. (C) IL-8 concentrations were measured by ELISA. Data are means±SD of triplicate samples from one experiment and are representative of three (A, B) or two (C) independent experiments. AU: arbitrary units. shTLR2 THP-1 cells produced significantly less IL-8 and MIP-1α mRNA (B) and IL-8 protein than control cells (C) (p<0.05). (0.02 MB PDF) Click here for additional data file. Figure S4 MVA infection increases RIG-I and MDA-5 mRNA expression in vivo. BALB/c mice were injected i.p. with MVA (107 PFU). Peritoneal cells (A) and splenocytes (B ) were isolated 12 h after infection as described in Materials and Methods . RIG-I and MDA-5 mRNA contents were analyzed by RT-PCR. Results are expressed as the ratio of RIG-I and MDA-5 mRNA levels to that of HPRT. AU: arbitrary units. Data are means±SD of triplicate samples from one experiment comprising three mice per experimental condition and are representative of two independent experiments (p<0.05 for all conditions). (0.01 MB PDF) Click here for additional data file. Figure S5 Generation of THP-1 cells expressing reduced levels of RIG-I, MDA-5 and IPS-1. THP-1 cells stably expressing control and candidate shRNA (#1 and #2) directed against RIG-I, MDA-5 and IPS-1 were obtained as described in Materials and Methods . RIGI, MDA-5 and IPS-1 mRNA contents were analyzed by RT-PCR and expressed as reported in Figure 1. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments. (0.01 MB PDF) Click here for additional data file. Figure S6 The NALP3 inflammasome is dispensable for activation of the IRF3 transcription factor and IFNβ secretion. THP-1 cells stably expressing control, NALP3, ASC and caspase 1 (casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) (A, B) or cultured with (+) or without (−) monosodium urate monohydrate (MSU) cristals crystals for the indicated time (A) or 6 h (B, C). Western blots of intracellular phosphorylated IRF3 and tubulin (A) and IFNβ (B) and IL-1β (C) concentrations measured by ELISA in cell-culture supernatants. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments. p<0.05 for cells transduced with NALP3, ASC and casp1 shRNAs vs. control shRNA (C). (0.12 MB PDF) Click here for additional data file. Table S1 Oligonucleotides used in RT-PCR analyses. (0.02 MB PDF) Click here for additional data file. The authors have declared that no competing interests exist. Research was conducted as part of the Poxvirus T Cell Vaccine Discovery Consortium (PTVDC) under the Collaboration for AIDS Vaccine Discovery with support from the Bill & Melinda Gates Foundation. J.D. is supported by a grant from the Swiss National Science Foundation (number 313600-115680). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Gomez CE Najera JL Krupa M Esteban M 2008 The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseases and cancer. Curr Gene Ther 8 97 120 18393831 2 Artenstein AW 2008 New generation smallpox vaccines: a review of preclinical and clinical data. Rev Med Virol 18 217 231 18283712 3 Johnston MI Fauci AS 2007 An HIV vaccine–evolving concepts. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1955717709-PONE-RA-08292R110.1371/journal.pone.0006049Research ArticleDevelopmental Biology/EmbryologyDevelopmental Biology/Molecular DevelopmentDevelopmental Biology/Morphogenesis and Cell BiologyTbx1 Regulates the BMP-Smad1 Pathway in a Transcription Independent Manner Tbx1 Regulates Smad1 SignalingFulcoli F. Gabriella 1 5 Huynh Tuong 2 Scambler Peter J. 3 Baldini Antonio 1 2 4 5 * 1 Telethon Institute of Genetics and Medicine, Naples, Italy 2 Institute of Biosciences and Technology, Texas A&M University Health Sciences Center, Houston, Texas, United States of America 3 Institute of Child Health, London, United Kingdom 4 University Federico II, Naples, Italy 5 Institute of Genetics and Biophysics, CNR, Naples, Italy Sham Mai Har EditorThe University of Hong Kong, China* E-mail: [email protected] and designed the experiments: AB. Performed the experiments: FGF TH. Analyzed the data: FGF TH AB. Contributed reagents/materials/analysis tools: PS. Wrote the paper: AB. 2009 25 6 2009 4 6 e604922 1 2009 27 5 2009 Fulcoli et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Tbx1 is a T-box transcription factor implicated in DiGeorge syndrome. The molecular function of Tbx1 is unclear although it can transactivate reporters with T-box binding elements. We discovered that Tbx1 binds Smad1 and suppresses the Bmp4/Smad1 signaling. Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity. In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction. Expression of Tbx1 in transgenic mice generates phenotypes similar to those associated with loss of a Bmp receptor. One phenotype could be rescued by transgenic Smad1 expression. Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation. ==== Body Introduction Tbx1 is a T-box transcription factor required for pharyngeal and cardiovascular development of humans and mice [1]–[3]. Mutations in TBX1 cause DiGeorge syndrome [4]–[6] and its molecular functions are unknown, but it can transactivate reporters with T-box binding elements [6]–[8]. T-box proteins, including those of the Tbx1 subfamily may interact with histone modifying enzymes H3K27-demethylase and H3K4-methyltransferase and thus modulate gene expression [9]. We and others have identified a number of genes potentially targeted by Tbx1 [7], [10]–[13], but the mechanism(s) by which it can regulate the transcription of these genes and how it controls developmental pathways is unclear. A major obstacle to understanding these mechanisms is our poor knowledge of the molecular interactors of Tbx1. One of the best studied developmental functions of Tbx1 is in heart development, where it is required to sustain proliferation of mesodermally-derived cardiac progenitors of the second heart field (SHF), a cardiac progenitor cell population that contributes to the development of most of the heart, including the outflow tract and right ventricle [7], [14]. Recent data have shown that loss of function of Tbx1 is associated with increased expression of differentiation markers of the myocardium, suggesting that Tbx1 may also regulate negatively cardiomyocyte differentiation [13]. The mechanisms and gene networks that regulate the homeostasis of the SHF cell population are not completely understood. However, it is clear that major signaling systems, such as the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP), as well as transcription factors such Nkx2.5, Isl1, Tbx1 and Foxh1 contribute to specification, proliferation and/or maintenance of this population [15]. In particular, it has been shown that Nkx2.5 regulates negatively the expression of Bmp2, establishing an Nkx2.5/Bmp2/Smad1 negative feedback loop that regulates proliferation of cardiac progenitors of the SHF [16]. Here we show that Tbx1 contributes to this network in an unexpected manner, i.e. by binding to Smad1, interfering with Smad1-Smad4 dimerization and suppressing its transactivation ability. Results Tbx1 binds Smad1 To identify Tbx1 interacting proteins in mammalian cells, we performed affinity purification of Tbx1-interactors complexes followed by identification of co-purified proteins. To this end, we assembled a mammalian expression vector (referred to as P19-Tbx1-PA) coding for a fusion protein consisting of Tbx1 fused to protein A (PA) via a tobacco etch virus (TEV) protease cutting site (Figure 1a). We then generated stably transfected P19Cl6 cell lines expressing the Tbx1-PA fusion protein as well as a control protein without Tbx1 (P19-PA, Figure 1b). We selected P19CL6 cells because they can differentiate into cardiomyocytes upon treatment with DMSO [17] and, during this process, they express endogenous Tbx1, most clearly after 4 days of treatment (Figure S1). Thus, we treated P19-Tbx1-PA and P19-PA cell lines with DMSO for 4 days, and then we obtained nuclear extracts, which we subjected to affinity purification using IgG resin, and proteolytic elution, using TEV protease, to recover Tbx1-interacting protein complexes. Eluates were separated by SDS-PAGE (Figure 1c) and processed for Western blotting analyses. We tested antibodies against several candidate interactors, among which anti phospho-Smad1/5/8, because of its relevance to cardiovascular development and because of previous evidence of interaction with other T-box proteins [18], [19]. This antibody was positive when tested against the affinity-purified material (Figure 2a). Reciprocal coimmunoprecipitation of the two proteins transiently expressed in NIH 3T3 cells confirmed the interaction in mammalian cells (Figure 2b). We then extracted proteins from E9.5 wild type (WT) embryos and carried out co-immunoprecipitation with an antibody against Tbx1. Western blot analysis after immunoprecipitation revealed Smad1-specific immunoreactivity in co-immunoprecipitated material (Figure 2c). The interaction could not be demonstrated in cytosol extracts, indicating that it occurs in the nucleus (Figure 2d). We next tested whether mutant isoforms previously associated with a DiGeorge syndrome phenotype are capable of binding Smad1. We found that the mutant TBX1F148Y (missense mutation in the T-box region [4]) can still bind Smad1, while the mutant TBX1G310S (missense mutation in a conserved region downstream to the T-box region [4]) cannot (Figure 2e). In addition, we tested a T-box mutant (TBX1G145R), which has been shown to prevent DNA binding [20]. Also this isoform interacted with Smad1 (Figure 2e). Overall, these data suggest that the critical region for this interaction resides downstream to and outside the T-box region. We next tested whether Tbx1 could co-immunoprecipitated with other Smad proteins. We found that P-Smad2, Smad4, Smad5, and Smad6 do not interact with Tbx1 (Figure S2). 10.1371/journal.pone.0006049.g001Figure 1 Identification of Tbx1 interactors using affinity purification. Mouse P19-Cl6 embryonic carcinoma cells were stably transfected with plasmid expressing TEV-protein-A alone or C-terminally fused to Tbx1 and induced to differentiate with 1% DMSO. a) Constructs used to generate stably transfected P19CL6 cell clones. b) Western blot analysis of P19-Tbx1-PA and P19-PA (control) cell extracts. Proteins were separated by gel electrophoresis on 10% SDS-PAGE gel and immunoblotted with human IgG F(c), which recognize protein A. c) Colloidal coomassie-stained 10% SDS-PAGE gel containing nuclear extracts from P19-Tbx1-PA (lane 1) and P19-PA (lane 3) cells affinity purified by binding to IgG-Sepharose and then enzymatically eluted by cleavage with TEV protease. Compared with non-purified nuclear extract from Tbx1-TEV-PA cells (lane 2). 10.1371/journal.pone.0006049.g002Figure 2 Tbx1 interacts with Smad1. a) Western blot analysis of affinity-purified nuclear extracts from P19-Tbx1-PA (lane 1) and P19-PA (lane 3) cells, compared non-purified nuclear extracts of P19-Tbx1-PA cells (lane 2), using an antibody anti-Phospho-SMAD1/5/8. b) Western blot analyses of reciprocal coimmunoprecipitation (IP) experiments using the antibodies indicated. NIH3T3 cells were co-transfected transiently with Tbx1-cmyc and Smad1-Flag expression vectors. c) Western blot analyses of nuclear extracts from wild type E9.5 mouse embryos immunoprecipitated with an anti-Tbx1 antibody (lanes 1 and 2) or with anti-rabbit IgG (lane 3) and revealed with an anti-SMAD1 antibody (lane 1 and 3) and an anti-Phospho-SMAD1/5/8 antibody (lane 2). d) Immunoblotting with anti-Smad1 antibody of nuclear extracts of wild type mouse embryos (E9.5) coimmunoprecipitated with anti-Tbx1 (NE-IP), total nuclear extracts (NE), total cytoplasmic extracts (CE), and cytoplasmic extracts coimminoprecipitated with anti-Tbx1 (CE-IP). e) Coimmunoprecipitation of WT TBX1, TBX1G145R, TBX1F148Y, TBX1G310S and Flag-SMAD1 transiently transfected in NIH 3T3 cells. The TBX1G145R and TBX1F148Y mutants physically interact with Smad1, while TBX1G310S is unable to bind Smad1 indicating that Glycine 310 is important for this interaction. f) Luciferase assay using Cos7 cells transfected with the SMAD-responsive reporter NTK-tetramer-luc. Transfected SMAD1 activates the reporter while increasing amounts (5 to 100 ng) of co-transfected TBX1 suppresses it. Tbx1 modulates negatively the Bmp4- Smad1 signal transduction To determine whether the interaction between Tbx1 and Smad1 has functional consequences, we overexpressed Tbx1 and assessed the transactivation ability of Smad1. We carried out a luciferase assay in Cos-7 and C2C12 cells with the Smad-responsive reporter NTK-tetramer-luc, which contains four copies of a Smad consensus-binding element [21]. The reporter was activated by transfection of a SMAD1 expression vector in Cos7 cells or by adding BMP4 to the culture media of C2C12 cells (Figure 2f and Figure S3, respectively). In both cases we observed that increasing amounts of transiently transfected TBX1 is capable of suppressing Smad1- or BMP4-induced activation of the reporter (Figure 2f and Figure S3a). TBX1 expression did not affect the level of P-Smad1/5/8, Smad1 or the inhibitory Smad6 (Figure S4). To assess the role of transcriptional activity for the Bmp-Smad suppression activity, we expressed a mutant isoform of TBX1 (G145R) that carries a T-box mutation, which prevents DNA binding [20]. As shown in Fig. 3a, TBX1G145R was unable to transactivate a T-box reporter, but it was still able to bind Smad1 (Figure 2e) and to suppress the Smad1 signaling in the luciferase assay, indicating that the anti-Smad activity of TBX1 is independent from transcriptional activity. We obtained the same results by activating the Smad reporter with BMP4 (Figure S3b). 10.1371/journal.pone.0006049.g003Figure 3 Transactivation ability of Tbx1 is not required for Smad pathway suppression; Tbx1 interferes with Smad1/Smad4 binding. a) A luciferase assay showing the inability of the TBX1G145R mutant to transactivate a T-box reporter construct in Jeg3 cells. Error bars indicate the standard error mean. b) A luciferase assay with a SMAD reporter showing that the mutant is capable of suppressing SMAD transactivation. c) Western blot analyses of nuclear extracts from C2C12 transfected with Tbx1 and SMAD1-flag expression vectors (as indicated). The top two rows are samples immunoprecipitated with an anti-flag antibody. The bottom two rows are non-immunoprecipitated nuclear extracts from the same samples. Note the strong reduction of Smad4 co-immunoprecipitated with Smad1 in the presence of transfected TBX1. Tbx1 interferes with Smad1-Smad4 binding The Bmp-Smad1 signal transduction pathway requires binding of phosphorylated Smad1 to Smad4 to transactivate target genes [22]. To address the mechanism by which TBX1 suppresses Bmp-Smad signal transduction, we tested whether TBX1 interferes with SMAD1 to SMAD4 binding. To this end, we transfected SMAD1 (tagged with flag) with or without increasing amounts of TBX1 into BMP4-treated C2C12 cells and carried out co-immunoprecipitation of nuclear extracts using an anti-flag antibody. Western blot analysis of immunoprecipitated material using an anti-Smad4 antibody revealed, in the absence of transfected TBX1, the presence of Smad4, which was strongly reduced in samples co-transfected with TBX1. This effect was dosage-dependent (Figure 3c). In the same samples, the presence of TBX1 did not affect the level of Smad1, and did not affect the level of Smad4 in protein extracts before immunoprecipitation (Figure 3c). These data suggest that TBX1 inhibits the Bmp-Smad signaling pathway by interfering with Smad1-Smad4 interaction. Transgenic expression of Tbx1 in mice mimics the Bmpr1a loss of function phenotype If TBX1 has an inhibitory effect on the Bmp-Smad1 pathway, then ectopic expression of Tbx1 during mouse development may mimic loss of function phenotypes associated with loss of Bmp-Smad1 in the same tissues. To test the ability of Tbx1 to suppress Smad1 signaling during mouse development, we have used a mouse transgenic line, named COET (for Conditional OverExpression of Tbx1), expressing Tbx1 upon Cre-mediated recombination [23]. We selected to cross this transgenic line with the Ap2aIREScre/+ driver [24] that expresses Cre in the ectoderm and neural crest tissues, both of which require Bmp-Smad1 signaling. Indeed, ectodermic deletion of the Bmp receptor gene Bmpr1a caused cleft lip [25], and conditional ablation of the same gene in neural crest cells caused cardiac outflow tract defects [26]. Ap2aIREScre/+; COET animals did not survive after birth (data not shown) and examination of E18.5 embryos revealed bilateral cleft lip (Figure 4a–a') and cardiac outflow tract (OFT) defects (Figure 4b–b') in all animals examined (n>20), and control animals exhibited a normal phenotype (Figure 4a and b, and data not shown). Moreover, consistent with the hypothesis of a suppression of the Bmp-Smad1 pathway, we observed reduced expression of the Bmp-Smad1 target gene Msx1 [27] in E9.5 and E10.5 Ap2aIREScre/+;COET embryos (Figure 4c-f'). 10.1371/journal.pone.0006049.g004Figure 4 Ectopic expression of Tbx1 in mouse embryos suppresses the Smad1 pathway in vivo. a–a') A Ap2aIRESCre/+;Coet embryo (a') at E18.5 shows bilateral cleft lip, compared with a control littermate (a). b–b') Three-dimensional reconstruction from digital images of histological sections of E16.5 hearts from control (b) and Ap2aIRESCre/+;Coet embryos. The cavities of the right (RV) and left (LV) ventricles, as well as the great arteries are shown in red. Note the large ventricular septal defect (VSD), and the common origine of the aorta and pulmonary arteries from the right ventricle, a condition known as double outlet right ventricle (DORV). (c–f') Whole-mount in situ hybridization analysis of Msx1 expression in WT (c, d, e, f) and Ap2aIRESCre/+;Coet embryos (c', d', e', f') at E9.5 (c–d') and E10.5 (e–f'). Mutant embryos show reduced expression in the maxillary region (white arrows) and in the second pharyngeal arch (black arrows). (g) Cleft lip present in Ap2aIRESCre/+;Coet embryos (compare with panel a) is rescued by the FSMAD1 transgene. Transgenic expression of Smad1 rescues partially the Tbx1 over expression phenotype If the Ap2aIREScre/+;COET associated phenotype is due to functional depletion of Smad1, then providing an additional source of Smad1 to the affected tissues may ameliorate the phenotype. To test this idea, we generated a mouse transgenic line that expresses SMAD1 upon Cre-induced recombination (Figure S5). This transgenic line, named Fsmad1, was made with the same procedure and construct used to generate the COET line, except that we used a SMAD1 cDNA instead of the Tbx1 cDNA. Ap2aIREScre/+;Fsmad1 E18.5 embryos were grossly normal (data not shown), suggesting that additional Smad1 expression in the ectoderm and neural crest, where endogenous Smad1 is normally expressed, did not cause any obvious developmental anomaly. Next, we crossed COET;Fsmad1 mice with Ap2aIREScre/+ mice and examined the progeny at E18.5. No obvious abnormalities were detected in Ap2aIREScre/+;Fsmad1, but, as expected, all the Ap2aIREScre/+;COET examined (n = 7) exhibited cleft lip and cardiac outflow tract defects (similar to those shown in Figures 4a' and b', respectively). However, with one exception, none of the Ap2aIREScre/+;COET;Fsmad1 embryos examined exhibited cleft lip (n = 6, Figure 4g), however, they did exhibit outflow tract defects. Thus, excess Smad1 expression was sufficient to rescue the cleft lip phenotype of Ap2aIREScre/+;COET embryos but not the heart phenotype. Discussion This study identified Smad1 as a novel interactor for the Tbx1 protein, and the only one that has been validated with endogenous proteins from embryos to date. Nowotschin et al. have shown that Tbx1 and Nkx2.5 interact in cell cultures, although they have not shown that the two endogenous proteins interact in embryo tissues [28]. While we did not map the regions of interaction between Tbx1 and Smad1, we demonstrate that a disease-associated missense mutation of Tbx1 in the C-terminal region of the protein is sufficient to prevent binding. These data suggest that perturbance of the Tbx1-Smad1 interaction may be part of the pathogenetic mechanism of DiGeorge syndrome. Xbra, a Xenopus T-box protein, also binds to Smad1, and this interactions appears necessary to prevent Xbra from activating Goosecoid, but how exactly this is effected is unclear [18]. We show that Tbx1 modulates negatively Smad1-dependent transactivation by interfering with Smad1-Smad4 interaction. Using mouse transgenic models we could reproduce in vivo the Tbx1-mediated Smad1 repression. This could be revealed by the occurrence of a phenotype similar to that caused by loss of Bmpr1a, and by reduced expression of a BMP target, Msx1, in Ap2aIREScre/+;COET embryos. In these embryos, Msx1 expression appeared downregulated only in some tissues. This may be due to tissue-specific Cre expression or to different, tissue specific mechanisms of Msx1 regulation. In any case, the phenotype of ectopic expression of Tbx1 could be partially rescued by transgenic expression of Smad1 in the same tissues. The heart phenotype caused by ectopic expression of Tbx1 in Ap2aIREScre/+;COET embryos could not be rescued by transgenic expression of Smad1. This could be because Tbx1 ectopic expression in the neural crest may cause more perturbances than simply BMP suppression, perhaps in early development of neural crest cells destined to populate the heart. Among the various developmental roles, Tbx1 is thought to maintain proliferation of mesodermally-derived cardiac progenitors of the second heart field (SHF) [14], a migratory cell population that enters the heart in a relatively late stage of its development [15]. Prall and coworkers [16] showed that Smad1 ablation in the SHF enhances cell proliferation, and thus it has been proposed as a negative modulator of cardiac progenitors proliferation. Thus, it is conceivable, that Tbx1, by repressing Smad1, contributes to maintenance of cell proliferation. Recently it has been shown that Chordin (Chrd), a BMP antagonist, is a mild modifier of the Tbx1 mutant phenotype. Indeed, Choi and Klingensmith [29] showed that loss of Chrd enhances the craniofacial phenotype of Tbx1 mutants. This effect can be interpreted on the basis of our findings, i.e. at least part of the Tbx1 mutant phenotype is due to excessive BMP signaling, thus removing an antagonist of BMP in a Tbx1 mutant background further enhances the excess of BMP. The heart phenotype was not affected by Chrd mutation presumably because this gene is not expressed in heart tissues. Tbx1 has been shown to regulate, directly or indirectly, several of the major signaling systems, i.e. the fibroblast growth factor (FGF) signaling [7], [10], [30], [31], the Retinoic acid signaling [12], [31], the Delta-Notch1 signaling [32] and the BMP/Smad1 signaling (this work). Thus, we propose that Tbx1, by modulating positively the FGF and negatively the BMP-Smad1 signaling systems plays a central role in the homeostasis of cardiac progenitor cells of the SHF. Finally, and perhaps most surprisingly, we show that the Smad1 modulatory effect of Tbx1 is not dependent upon DNA binding, therefore, this represents the first example of a transcription-independent function of a T-box transcription factor. Materials and Methods Constructs and cell lines To generate the FSMAD1 transgene construct, the Flag-SMAD1 cDNA was excised from pCMV5-FlagSMAD1 [33] and cloned 3′ to a loxP-flanked neomycin resistance cassette with 3 polyadenylation-sites (the backbone plasmid is a kind gift from Drs. A. Simeone and F. Tuorto, Institute of Genetics and Biophysics, Naples). C2C12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. The human choriocarcinoma-derived placental JEG3 cell line was maintained in Minimum Essential Medium (SIGMA) supplemented with 10% fetal bovine serum. P19CL6 cells were grown in Dulbecco-Modified Minimal Essential Medium supplemented with 10% fetal bovine serum. For differentiation of P19CL6 cells, the culture medium was supplemented with 1% DMSO (Sigma-Aldrich). To generate stably transfected cell lines, 107 P19CL6 cells were electroporated with 10 µg of DNA of an expression vector containing the cmv promoter driving a mouse Tbx1 cDNA fused with a TEV target site and a Protein A coding cDNA (the plasmid backbone has been described in ref. [34]. Using the same procedure but with an expression vector encoding the TEV site, Protein A but not Tbx1, we obtained a control cell line, P19-PA. Selection was performed using G418 (200 µg/ml). Resistant clones were picked, expanded and tested for expression of the transgenic proteins by western blotting. Nuclear/Cytoplasmic Extract Isolation Cells were cultured in 10 cm dishes until 60–70% confluence and transfected with Fugene6 (Roche) following the manufacturer protocol. After 24–48 h after transfection, cells were washed, scraped in cold PBS, and collected by centrifugation using a refrigerated centrifuge at 1000 rpm for 10 min. The pellet was resuspended in 5 pellet volumes of cold CE buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% v/v NP40, 1 mM DTT and 1X protease inhibitors, adjusted to pH 7.6), and centrifuged at 1400 rpm for 4 min. The nuclei were washed with 5 pellet volumes of cold CE buffer without detergent and spinned as above at 1400 rpm for 4 min. 2x pellet volume of NE buffer were added to the nuclear pellet incubating on ice for 30 min. Nuclear and cytosolic extracts were recovered spinning at maximum speed for 30 min to pellet any nuclei. Affinity purification Native affinity purification was performed with strains P19-Tbx1-PA and P19-PA as previously described [35]. Nuclear extracts were transferred to Poly-Propylene Chromatography Columns (10 ml volume; Pierce), which contained 2 ml of pre-washed (with IPP150: 10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.1% NP-40) IgG sepharose beads (Amersham). Columns were sealed and slowly rotated for 2 h at 4°C. After three washes with 10 ml of IPP150 buffer and one wash with 10 ml of TEV cleavage buffer (910 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.1% NP-40; 0.5 mM EDTA; 1 mM DTT), we added 1 ml TEV cleavage buffer and 10 µl of TEV enzyme (Amersham). Protein eluates were concentrated and loaded on a 10% SDS-acrylamide gel. Proteins were stained with colloidal coomassie or transferred into PVDF membrane (Amersham) for Western blotting analyses. Co-Immunoprecipitation For co-immunoprecipitation experiments, cells were lysed at 4°C in Nonidet P-40 lysis buffer. Extracts were quantified using a modified Bradford procedure (Bio-Rad Laboratories, Hercules, CA). Co-immunoprecipitation of TBX1 and Flag-SMAD1 was accomplished using an antibody-coupling gel to precipitate the bait protein and co-immunoprecipitate the interacting prey protein. Anti-Tbx1 (Zymed Laboratories) or anti-Flag M2 antibody (SIGMA) was coupled to an amine-reactive gel (ProFound co-immunoprecipitation kit, Pierce) using slow agitation at 4°C O.N. The precipitated protein complexes were run on a 10% SDS-acrylamide gel, and analysed by Western blotting. Luciferase assay Cos-7 cells were grown in 24-well plates and transfected with 100 ng of NTK-tetramer-luc vector [21], 100 ng of pCMV5-FlagSMAD1 and 5 ng to 100 ng of h-TBX1 (a TBX1 expression vector obtained by cloning a human cDNA into the CMV expression plasmid pCDNA3). C2C12 cells were grown in 24-well plates, transfected with 100 ng of NTK-tetramer-luc vector, 5 ng to 100 ng of h-TBX1 and stimulated with 50 ng/ml of Human recombinant BMP4. JEG3 cells were grown in 24-well plates and transfected using Fugene-6 (Roche) with 100 ng of 2xTtkGL2 vector [6], 5 ng of β-Gal-expression vector, and 100 ng of h-TBX1. In all the experiments, the total amount of transfected DNA was adjusted with empty vectors so that all samples received the same amount. Light emission of extracts was measured using a luminometer. Mouse Mutants and Breedings All the experiments involving mice were done according to a protocol reviewed and approved by the Institutional Animal Care and Use Committee of Institute of Biosciences and Technology, in compliance with the USA Public Health Service Policy on Humane Care and Use of Laboratory Animals. The mutant AP2αIREScre used in this study has been previously reported [36]. The COET transgenic mouse line has been described previously [23]. The FSMAD1 transgenic line was generated as follows. The FSMAD1 construct (described above) was linearized and electroporated into feeder-free E14Tg2A.4 embryonic stem cells (strain 129/Ola, BayGenomics). Cells were selected with G418, and 96 resistant clones were screened by southern blot using a probe specific for the Flag-SMAD1 sequence. 2 positive clones were tested for SMAD1 expression with Western blotting analysis after Cre recombination. One of the clones was injected into C57Bl6 blastocysts to obtain chimeric mice. Founders were backcrossed into the C57/Bl6 strain and maintained in a mixed genetic background C57/Bl6-129/Ola. FSMAD1 transgenic mice were crossed with COET mice (same genetic background) to generate COET; FSMAD1 mice for use in timed matings. COET; FSMAD1 mice were crossed with AP2αIRESCre/+ animals to generate embryos of the appropriate stage and genotype. The FSMAD1 transgene was genotyped with the following primers: 1) 5′- caaagacgacgatgacaagg -3′ and 2) 5′- agctcaaggccttttccagt -3′. Phenotypic analyses of mutant embryos were carried out by gross morphological analyses, embryo dissection and hystological analyses. In some cases, we used digital images of 10 mm-thick histological sections to carry out three-dimensional reconstructions using the software AMIRA 4.1.2 (Mercury Computer Systems). In-situ hybridization Whole mount in situ hybridization was performed by standard methods. The mouse Msx1 probe was kindly provided by James F. Martin (Institute of Biosciences and Technology, Houston). Sources of antibodies and proteins The anti-Tbx1 antibody was obtained from Zymed Laboratories; the anti-P-Smad1 (Ser463/Ser465) from Chemicon; The anti Smad1, P-Smad2 (Ser465/Ser467), Smad4, Smad5, and Smad6 where all purchased from Cell Signaling. Recombinant proteins BMP4 and TGFβ1 were obtained from R&D Systems. Supporting Information Figure S1 Endogenous Tbx1 expression during cardiomyocite differentiation of P19Cl6 cells. RT-PCR analyses for the indicated mRNAs were performed on differentiating P19Cl6 cells. The Tbx1 amplification signal is more clearly evident starting from day 4 of differentiation. (1.71 MB TIF) Click here for additional data file. Figure S2 P-Smad2, Smad4, 5, and 6 do not interact with Tbx1. Nuclear extracts of mouse P19-Cl6 cells induced to differentiate with 1% DMSO were purified by binding to IgG-Sepharose, digested with TEV protease and immunoblotted with (a) anti-Smad5, (b) anti-Smad4 and (c) anti-Smad6 antibodies. Lane 1: Purified nuclear extracts of cells expressing Tbx1-TEV-PA. Lane 2: Total nuclear extracts of cells expressing Tbx1-TEV-PA. Lane 3: Purified nuclear extracts of cells expressing TEV-PA alone. (d) NIH-3T3 cells were transiently transfected with TBX1, stimulated with 5 ng/ml of TGFβ1 for 1 hour and protein extracts were coimmunoprecipitated with anti-Tbx1 and immunoblotted with anti-Phospho-Smad2 antibody. (8.51 MB TIF) Click here for additional data file. Figure S3 TBX1 or TBX1G415R are both capable of suppressing a Smad reporter after BMP4 activation. a) Luciferase assay using a Smad reporter (NTK-tetramer-luc) with C2C12 cells exposed to BMP4. Increased amounts of TBX1 expression vector DNA (from 5 ng to 100 ng) is associated with reduced luciferase activity. b) In a similar experiment, a vector encoding the mutant TBX1G145R isoform, which cannot transactivate a T-box reporter, has a similar capacity to suppress the Smad-reporter activity as the wild type isoform. The mean data are representative of three replicates for each condition and the error bars show the standard error. (8.81 MB TIF) Click here for additional data file. Figure S4 TBX1 does not affect the level of P-Smad1/5/8, Smad1 and inhibitory Smad6. Western blot analyses of protein extracts from BMP4-induced and non induced C2C12 cells, with or without TBX1 transfection. None of the immunoreactivities tested in this figure is affected by TBX1 expression. (7.23 MB TIF) Click here for additional data file. Figure S5 Generation of the FSMAD1 transgenic line. a) Schematic representation of the transgenic construct, which includes a chicken b-actin promoter, a loxP-flanked promoterless neo resistance cassette with 3 copies of a polyadenilation signal, and a cDNA encoding human SMAD1 tagged at the N-terminal with flag. b) Southern-blot of BamHI-digested DNA from ES-cell clones (parental and transgenic) probed with a Flag-SMAD1-specific probe. The position of the BamHI restriction sites is indicated on panel a. c) Western-blot analysis of protein extracts from parental and transgenic ES clones after transient Cre recombinase expression, using an anti-Flag antibody. A transgenic protein of the appropriate size is clearly evident in the transgenic cell line. d) PCR analysis of DNA from transgenic embryos using FSMAD1-specific the oligonucleotides 1) 5′- caaagacgacgatgacaagg -3′ and 2) 5′- agctcaaggccttttccagt -3′(the position of primers is schematically indicated on panel a. (4.93 MB TIF) Click here for additional data file. We thank Dr. H. Bellen for critical reading of the manuscript, Dr. Vitelli for help with phenotypic analyses, Drs. Rauch and Maxson for providing reporter plasmids, and Hedda Leeming, Guilan Ji and Wei Yu for invaluable technical support. We also thank the Darwin Transgenic core of Baylor College of Medicine for ES cell injection. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was funded by the NIH-NHLBI grant HL064832, grants from the Italian Telethon, and the EU CardioGeNet program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Naiche LA Harrelson Z Kelly RG Papaioannou VE 2005 T-box genes in vertebrate development. Annu Rev Genet 39 219 239 16285859 2 Stennard FA Harvey RP 2005 T-box transcription factors and their roles in regulatory hierarchies in the developing heart. Development 132 4897 4910 16258075 3 Baldini A 2006 The 22q11.2 deletion syndrome: a gene dosage perspective. ScientificWorldJournal 6 1881 1887 17205194 4 Yagi H Furutani Y Hamada H Sasaki T Asakawa S 2003 Role of TBX1 in human del22q11.2 syndrome. Lancet 362 1366 1373 14585638 5 Paylor R Glaser B Mupo A Ataliotis P Spencer C 2006 Tbx1 haploinsufficiency is linked to behavioral disorders in mice and humans: implications for 22q11 deletion syndrome. Proc Natl Acad Sci U S A 103 7729 7734 16684884 6 Zweier C Sticht H Aydin-Yaylagul I Campbell CE Rauch A 2007 Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions. Am J Hum Genet 80 510 517 17273972 7 Xu H Morishima M Wylie JN Schwartz RJ Bruneau BG 2004 Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract. Development 131 3217 3227 15175244 8 Ataliotis P Ivins S Mohun TJ Scambler PJ 2005 XTbx1 is a transcriptional activator involved in head and pharyngeal arch development in Xenopus laevis. Dev Dyn 232 979 991 15736267 9 Miller SA Huang AC Miazgowicz MM Brassil MM Weinmann AS 2008 Coordinated but physically separable interaction with H3K27-demethylase and H3K4-methyltransferase activities are required for T-box protein-mediated activation of developmental gene expression. 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==== Front J Minim Access SurgJMASJournal of Minimal Access Surgery0972-99411998-3921Medknow Publications India 19547693JMAS-04-76Original ArticleLaparoscopic versus open pyeloplasty: Comparison of two surgical approaches- a single centre experience of three years Bansal Punit Gupta Aman Mongha Ritesh Narayan Srinivas Kundu A K Chakraborty S C Das R K Bera M K Department of Urology, Institute of Post Graduate Medical Education and Research, Kolkata, IndiaAddress for correspondence: Dr. Punit Bansal, Department of Urology, Institute of Post Graduate Medical Education and Research, 242 AJC Bose Road, Kolkata- 700 020, India. E-mail: [email protected] 2008 4 3 76 79 21 2 2008 12 8 2008 © Journal of Minimal Access Surgery2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.BACKGROUND: Ureteropelvic junction obstruction (UPJO) causes hydronephrosis and progressive renal impairment may ensue if left uncorrected. Open pyeloplasty remains the standard against which new technique must be compared. We compared laparoscopic (LP) and open pyeloplasty (OP) in a randomized prospective trial. MATERIALS AND METHODS: A prospective randomized study was done from January 2004 to January 2007 in which a total of 28 laparoscopic and 34 open pyeloplasty were done. All laparoscopic pyeloplasties were performed transperitoneally. Standard open Anderson Hynes pyeloplasty, spiral flap or VY plasty was done depending on anatomic consideration. Patients were followed with DTPA scan at three months and IVP at six months. Perioperative parameters including operative time, analgesic use, hospital stay, and complication and success rates were compared. RESULTS: Mean total operative time with stent placement in LP group was 244.2 min (188-300 min) compared to 122 min (100-140 min) in OP group. Compared to OP group, the post operative diclofenac requirement was significantly less in LP group (mean 107.14 mg) and OP group required mean of (682.35 mg). The duration of analgesic requirement was also significantly less in LP group. The postoperative hospital stay in LP was mean 3.14 Days (2-7 days) significantly less than the open group mean of 8.29 days (7-11 days). CONCLUSION: LP has a minimal level of morbidity and short hospital stay compared to open approach. Although, laparoscopic pyeloplasty has the disadvantages of longer operative time and requires significant skill of intracorporeal knotting but it is here to stay and represents an emerging standard of care. LaparoscopypyeloplastyUPJO ==== Body INTRODUCTION Open pyeloplasty has been the gold standard for surgical treatment of ureteropelvic junction (UPJ) obstruction, enjoying a long-term success rate exceeding 90%.[1] This procedure requires a muscle incision that entails some degree of morbidity. UPJO causes hydronephrosis and progressive renal impairment may ensue if left uncorrected.[2] The optimum surgical correction of UPJO has been a urological challenge for over a century.[3] Open pyeloplasty originally described by Andersen and Hynes[4] remains the gold standard against which new technique must be compared. The morbidity associated with flank incision, however, has led to development of minimally invasive approaches to UPJ repair. Over the last two decades the treatment approach to UPJ obstruction has evolved from open pyeloplasty to various minimally invasive procedures like endopyelotomy, acucise catheter incision, balloon dilatation and laparoscopic pyeloplasty. These minimally invasive options are reported to been less successful than open pyeloplasty.[5] Laparoscopic pyeloplasty was described first in 1993 by Schuessler et al. Laparoscopic pyeloplasty has developed worldwide as the first minimally invasive option to match success rate of open pyeloplasty. Only one randomised study to compare Laparoscopic and open pyeloplasty has been done by Turk et al in 2002.[6] We analysed the comparison of Laparoscopic and open pyeloplasty in a randomised prospective trial. MATERIALS AND METHODS A prospective randomised study was done from January 2004 to January 2007 in which a total of 28 Laparoscopic and 34 open pyeloplasty were done. All procedures were performed at our institute. The patients had radiographic evidence of UPJO on diuretic renography or hydronephrosis with delayed function on IVP in conjunction with signs and symptoms or deterioration of renal function. Out of the 28 patients for laparoscopy 25 presented with pain and three presented with recurrent urinary tract infection. Thirty patients had pain in open pyeloplasty group while three presented with lump and one patient presented with haematuria after minor trauma. All patients underwent cystoscopy and RGP to confirm the diagnosis before the procedure. Ureteric catheter was left in situ. All laparoscopic pyeloplasties were performed transperitoneally. Patients were placed in lateral kidney position. Four to five trocars were placed to enable dissection, retraction and identification of PUJO. Depending on the anatomical findings at time of dissection dismembered or non dismembered procedures were performed. In case of redundant pelvis reduction, pyeloplasty was performed. Anastomoses were done with 4-0 polyglactin. After completion of posterior layer DJ stent was placed and then anastomosis was completed Drain was inserted adjacent to repair and Foleys catheter was left in the bladder for two days. Drain was removed the next day if the drain output did not increase. Internal stent was removed after the fourth week. Standard open Anderson Hynes pyeloplasty, spiral flap or VY plasty was done depending on anatomic consideration. The patients were randomly admitted for pyeloplasty under four different surgeons. All laparoscopic cases were performed by a single surgeon dedicated to laparoscopy while open cases were performed by different surgeons' expert in open surgery. Ethics committee approval was obtained prior to the study. Patients were followed with DTPA scan at three months and IVP at six months. Thereafter, patients were followed at six months and then annually. The patients were radiologically investigated with DTPA scan depending on symptoms and signs. Peri-operative parameters including operative time, analgesic use, hospital stay, and complication and success rates were compared. Postop patients received transdermal patch 100 mg or 200 mg (Diclofenac) according to severity of pain. Patients were assessed in postop period regarding pain according to the requirement of transdermal diclofenac patch (duration and quantity). The success was defined radiologically as a patent, unobstructed UPJ or improved or maintained renal functional status and symptomatic improvement. Formal chart review was completed with all peri-operative data completed and statistical analysis was done using Fisher exact test, unpaired t test and Marn-Whitney U test. RESULTS The demographics of two groups were similar with regard to sex, age, laterality. None had any significant co-morbid condition. The mean follow-up in open cases was 33.5 months and in Laparoscopic cases was 34.5 months. A total of 28 Laparoscopic pyeloplasties and 34 open pyeloplasties were performed [Table 1]. Two patients in laparoscopic group had VY plasty due to high insertion of ureter and less dilated renal pelvis. Table 1 Details of cases of pyeloplasty Total cases 62 Laparoscopic pyeloplasty 28 Dismembered 26 Vy plasty 2 Associated stone 2 Crossing vessel 4 Open pyeloplasty 34 Dismembered 31 Vy plasty 2 Spiral flap 1 Associatd stone 3 Crossing vessel 4 Mean total operative time with stent placement in LP group was 244.2 min (188-300 min) compared to 122 min (100-140 min) in open group. Total operative time did improve with experience for LP patients as average time reduced to 202 min for last five patients. There was no blood transfusion in any patient. There was no mortality in either group. Compared to open pyeloplasty, the postoperative diclofenac requirement was significantly less in LP group (mean107.14 mg) compared to open group mean (682.35 mg). The duration of analgesic requirement was also significantly less in LP group. The postoperative hospital stay in LP was mean 3.14 Days (2-7 days) significantly less than open group mean 8.29 days (7-11 days) [Table 2]. Table 2 Comparison of laparoscopic and open pyeloplasty Open LP P value Age 29.58 31.64 N.S Sex m/f 20/12 17/11 n.S Side r/l 18/14 16/12 n.S Operating time (min) 122.411 ± 10.60 244.21 ± 41.73 <0.01 Analgesic(mg) 682.35 ± 123.66 107.14 ± 73.00 <0.01 Duration analgesic (days) 3.41 ± 0.61 1.00 ± 0.67 <0.01 Hospital stay (days) 8.29 ± 1.35 3.14 ± 1.29 <0.01 Success 34/34 26/28 N.S We had two cases of UPJO secondary to failed endopyelotomy which required operative duration of 300 minutes. Both patients had a successful outcome. There was only one conversion in laparoscopic group to open surgery as we were unable to remove associated calculus by laparoscopy. There was only one major complication in laparoscopic group. That patient had prolonged drainage of urine (six days) through the drain which subsided with prolonged catheterisation. He had recurrence of symptoms at three months and an obstructive DTPA curve. This was probably secondary to fibrosis caused by leakage of urine that occurred earlier. The patient was managed with endopyelotomy after six months. No patient in open group had recurrence. DISCUSSION The first successful reconstruction of an obstructed UPJO was accomplished in 1892.[7] Since then open pyeloplasty has been the gold standard for UPJO repair and achieves success rates exceeding 90% in contemporary series.[8–10] In 1983 Wicham and Kellet described percutaneous pyelolysis (endopyelotomy) which subsequently gained some popularity.[11] Subsequent evolution in endoscopic physiology and application together with advances in endoscopic technology fostered advances in the field. Current approaches include antegrade percutaneous, retrograde ureteroscopic guided laser and retrograde acusize ® balloon dilatation. The success rate of these minimally invasive options have consistently been less than with open pyeloplasty by 10-30%.[12–14] The varied surgical anatomy of PUJ (huge dilatation, crossing vessels, high insertion of ureter) compromise all of these endourological procedures. These procedures are also associated with a risk of peri-operative haemorrhage and 3-11% patients' required blood transfusion.[1516] Laparoscopic pyeloplasty provides a minimally invasive alternative to repair UPJO. Laparoscopic pyeloplasty was introduced in 1993 by Schussller et al and has developed worldwide as the first minimal option to match success rate of open pyeloplasty.[3] Reconstruction of UPJO can be tailored to anatomical findings at the time of surgery.[17] The feasibility of Laparoscopic pyeloplasty including Anderson Hynes, Fengers, Foleys VY plasty performed through transperitoneal and retroperitoneal approach has been evaluated.[18] Its potential advantages including less postoperative pain, shorter hospital stay and improved cosmesis have been proved in previous comparative series.[19–21] The only disadvantage seems to be longer operative time in published series.[1920] However, Zhang et al,[18] reported less operative time in Laparoscopic group (retroperitoneal) than open group. As laparoscopic surgery becomes more entrenched in resident training, the more complex skills such as intracorporeal suturing become less daunting. Moreover, long operative time may be reduced by skill of intracorporeal knotting and development of new robotic equipment.[21] The performance enhancing feature of Da Vinci robot seems to decrease the difficulty of intra corporeal suturing. In general the reported overall complications rate of laparoscopic pyeloplasty ranges from 4% - 12.7%.[18] In the present study there was only one major complication and only one conversion to open surgery. This is possibly the result of the experience of the surgeon who did laparoscopic cases. Siguriea et al[22] reported success rate in eight of nine patients with secondary PUJO while Sundaram et al[23] reported 89% success rate in secondary procedure and a longer mean operating time of 6.3 h(2.7-10) In our series, we had two secondary cases with operative duration of 300 min. Both patients had a successful outcome. Ram Kumar et al, reported a series of 20 LPs with stone extraction through Laparoscope port. We had five patients with associated stone disease. Three were managed by open approach. One patient being managed laparoscopically had to be converted to open as stone could not be retrieved by laparoscopy. Zhang et al,[18] reported that analgesic requirement was significantly less in LP than open pyeloplasty. The duration and amount of analgesic requirement is significantly less than that in open pyeloplasty in our series. The success rate of laparoscopic pyeloplasty has been reported to be consistently high, at 87-98%.[3] In the present series, we had a success rate of 92.3%. We considered conversion to open as a failure. CONCLUSION LP is a technically sound operation which uses well established principles familiar to urologist. The only disadvantage of Laparoscopic pyeloplasty is longer operative time and requires significant skill of intracorporeal knotting This procedure has a minimal level of morbidity, short hospital stay, better cosmesis compared to open approach. Laparoscopic pyeloplasty has emerged as the standard of care and is here to stay. Source of Support: Nil Conflict of Interest: None declared. ==== Refs REFERENCES 1 Troxel S Das S Helfer E Nugyen M Laparoscopy versus dorsal lumbotomy for ureteropelvic junction obstruction repair J Urol 2006 176 1073 6 16890693 2 Persky L Krause JR Boltuch RL Initial complications and late results in dismembered pyeloplasty J Urol 1977 118 162 4 875213 3 Adeyoju AB Hrouda D Gill IS Laparoscopic pyeloplasty: The first decade BJU Int 2004 94 264 7 15291849 4 Anderson JC HynesW Retro-caval ureter: A case diagnosed preoperatively and treated successfully by a plastic operation Br J Urol 1949 21 209 14 18148283 5 Schuessler WW Grune MT Tecuanhuey LV Preminger GM Laparoscopic dismembered pyeloplasty J Urol 1993 150 1795 9 8230507 6 Turk IA Davis JW Winkelmann B Deger S Richter F Fabrizio MD Laparoscopic dismembered pyeloplasty - the method of choice in the presence of an enlarged renal pelvis and crossing vessel Eur Urol 2002 42 268 75 12234512 7 Kletscher BA Segura JW Le Roy AJ and Patterson DE Percutaneous antegrade endopyelotomy: Review of 50 consecutive 50 cases J Urol 1995 153 701 3 7861513 8 Psooy K Pike JG Leonard MP Long-term follow-up of pediatric dismembered pyeloplasty: How long is long enough? J Urol 2003 169 1809 12 12686849 9 Notley RG Beaugie JM The long term follow-up of Anderson hynes pyeloplasty for hydronephrosis Br J Urol 1973 45 464 6 4748391 10 Nguyen DH Aliabadi H Ercole CJ Gonzalez R Nonintubated Anderson hynes repair of ureteropelvic junction obstruction in 60 patients J Urol 1989 142 704 7 2671411 11 Wickham JE Kellet MJ Percutaneous pyelolysis Eur Urol 1983 9 122 4 6852083 12 Giddens JL Grasso M Retrograde ureteroscopic endopyelotomy using Holmium -YAG laser J Urol 2000 164 1509 12 11025693 13 Baldwin DD Dunbar JA Wells N Mcdougall EM Single centre comparison of laparoscopic pyeloplasty, Acusize endopyelotomy and open pyeloplasty J Endourol 2003 17 155 7 12803987 14 Faerber GJ Richardson TD Farah N Ohl DA Retrograde treatment of ureteropelvic junction obstruction using ureteral cutting ballon catheter J Urol 1997 157 454 8 8996330 15 Badlani G Eshghi M Smith AD Percutaneous surgery for ureteropelvic junction obstruction (endopyelotomy) technique and early results J Urol 1986 135 26 8 3941462 16 Brooks JD Kavoussi LR Preminger GM Schuessler WW Moore RG Comparison of open and endourological approaches to obstructed ureteropelvic junction Urology 1995 46 791 5 7502417 17 Jarrett TW Chan DY Charambura TC Fugita O Kavoussi LR Laparoscopic pyeloplasty: The first 100 cases J Urol 2002 167 1253 6 11832708 18 Zhang X Li HZ Ma X Zheng T Lang B Zhang J Retrospective comparison of Retroperitoneal laparoscopic versus open dismembered pyeloplasty for ureteropelvic junction obstruction J Urol 2006 176 1077 80 16890694 19 Bonnard A Fouquet V Carrricaburu E Aigrain Y El-Ghoneimi A Retroperitoneal laparoscopic versus open pyeloplasty in children J Urol 2005 173 1710 3 15821565 20 Klingler HC Rezmi M Janetschek C Kratzik C Marberger MJ Comparison of open versus laparoscopic pyeloplasty techniques in treatment of uretero-pelvic junction obstruction Eur Urol 2003 44 340 5 12932933 21 Soulie M Thoulouzan M Seguin P Mouly P Vazzoler N Pontonnier F Retroperitoneal laparoscopic versus open pyeloplasty with a minimal incision: Comparison of two surgical approaches Urology 2001 57 443 7 11248616 22 Sundaram CP Grubb RL 3rd Rehman J Yan Y Chen C Landman J Laparoscopic pyeloplasty for secondary ureteropelvic junction obstruction J Urol 2003 169 2037 40 12771713 23 Siqueira TM Jr Nadu A Kuo RL Paterson RF Lingeman JE Shalhav AL Laparoscopic treatment for ureteropelvic junction obstruction Urology 2002 60 973 8 12475652	19547693	PMC2699080	CC BY	2021-01-04 19:39:58	yes	J Minim Access Surg. 2008 Jul-Sep; 4(3):76-79
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1962325608-PONE-RA-07738R110.1371/journal.pone.0006333Research ArticleEcology/Conservation and Restoration EcologyEcology/Environmental MicrobiologyImmunology/Immune ResponseImmunology/Immunity to InfectionsInfectious Diseases/Bacterial InfectionsInfectious Diseases/Fungal InfectionsInfectious Diseases/Protozoal InfectionsSusceptibility to Infection and Immune Response in Insular and Continental Populations of Egyptian Vulture: Implications for Conservation Pathogens in Insular BirdsGangoso Laura 1 2 * Grande Juan M. 3 Lemus Jesús A. 4 Blanco Guillermo 4 Grande Javier 4 Donázar José A. 1 1 Department of Conservation Biology, Estación Biológica de Doñana (CSIC), Sevilla, Spain 2 Department of Ecology and Evolution, University of Lausanne, Biophore, Lausanne, Switzerland 3 Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 4 Department of Evolutionary Ecology, Museo de Ciencias Naturales (CSIC), Madrid, Spain Getz Wayne M. EditorUniversity of California, Berkeley, United States of America* E-mail: [email protected] and designed the experiments: LG JMG JAL GB JAD. Analyzed the data: LG GB JG. Contributed reagents/materials/analysis tools: GB JAD. Wrote the paper: LG JMG JAL GB JAD. Conducted fieldwork: LG JMG JAL GB JAD. Performed pathogen determinations: JG. 2009 22 7 2009 4 7 e633313 12 2008 23 6 2009 Gangoso et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background A generalized decline in populations of Old World avian scavengers is occurring on a global scale. The main cause of the observed crisis in continental populations of these birds should be looked for in the interaction between two factors - changes in livestock management, including the increased use of pharmaceutical products, and disease. Insular vertebrates seem to be especially susceptible to diseases induced by the arrival of exotic pathogens, a process often favored by human activities, and sedentary and highly dense insular scavengers populations may be thus especially exposed to infection by such pathogens. Here, we compare pathogen prevalence and immune response in insular and continental populations of the globally endangered Egyptian vulture under similar livestock management scenarios, but with different ecological and evolutionary perspectives. Methods/Principal Findings Adult, immature, and fledgling vultures from the Canary Islands and the Iberian Peninsula were sampled to determine a) the prevalence of seven pathogen taxa and b) their immunocompetence, as measured by monitoring techniques (white blood cells counts and immunoglobulins). In the Canarian population, pathogen prevalence was higher and, in addition, an association among pathogens was apparent, contrary to the situation detected in continental populations. Despite that, insular fledglings showed lower leukocyte profiles than continental birds and Canarian fledglings infected by Chlamydophila psittaci showed poorer cellular immune response. Conclusions/Significance A combination of environmental and ecological factors may contribute to explain the high susceptibility to infection found in insular vultures. The scenario described here may be similar in other insular systems where populations of carrion-eaters are in strong decline and are seriously threatened. Higher susceptibility to infection may be a further factor contributing decisively to the extinction of island scavengers in the present context of global change and increasing numbers of emerging infectious diseases. ==== Body Introduction Recent research has revealed the worrying conservation status of Palearctic avian scavenger populations [1]–[3]. Besides well-documented threats such as habitat degradation, the decline of wild prey populations and human persecution, the relevance of the combination of two additional factors - changes in livestock managing including the regular use of veterinary drugs, and disease - has recently become apparent [1], [4]–[6]. The increased stabling of livestock together with the ban on abandoning carcasses in the field has severely reduced food availability in the countryside [7], and at the same time, the food now available for vultures increasingly consists of intensively raised livestock that is regularly treated with veterinary drugs (mainly antibiotics)[5]. The direct or indirect ingestion of harmful chemical residues from these drugs may pass on to and directly kill scavengers (e.g. the anti-inflammatory drug diclofenac causes renal failure in Gyps bengalensis and is thought to be responsible of the crash of several vultures populations all across the Indian subcontinent [1], [4]). In other cases, these drugs may induce alterations in their normal intestinal flora, mainly through the acquisition of antibiotic-resistant and/or pathogenic bacteria [5], [6]. Even more critical is the situation of a number of insular populations of scavengers in the Macaronesian and Mediterranean archipelagos. Besides their high dependence on domestic livestock (wild prey populations have almost disappeared [8]), hunting, illegal poisoning, and the effects of pollutants have had a strong negative impact on individual survival [9]–[12]. As a result, several populations, some of them endemic, are at present severely endangered [9], [13], [14]. Within this framework, the characteristics inherent to insular populations (see below) could make insular scavengers especially vulnerable to the arrival of new pathogens mainly associated with the increasing mobility of livestock [15]. Pathogens are powerful selection agents, reducing individual fitness and thus able to drive rapid changes in population size, demographic structure, and the probability of persistence of their host populations [16]. The ecological and evolutionary differences between insular and continental scenarios may cause strong asymmetries in the exposition and susceptibility of vertebrates to pathogens. It has been suggested that insular populations have naturally impoverished pathogen communities [17], [18] and diminished immunocompetence, probably as a result of low exposition and reduced selection for parasite resistance during their evolutionary history [19]–[21]. Moreover, population constraints such as isolation, sedentary habits, high density and a reduction in genetic diversity make insular organisms especially susceptible to infection [22]–[25]. Consequently, pathogen exposure has been involved in the decline and even total extinction of vertebrates inhabiting insular systems [26]–[29]. This process may have become accelerated since growing human intrusion into wildlife habitat may have introduced new pathogens into such areas [5], [17], [22], [30]. The Egyptian vulture (Neophron percnopterus) is a medium-sized Old World scavenger considered to be ‘globally threatened’ whose world population has been recently estimated at 30,000–40,000 mature individuals. Migratory populations in continental Europe have suffered a decline of more than 50% over the last three generations and ongoing declines are occurring in other regions throughout the rest of its range [31]. Several sedentary insular populations still exist in the Macaronesian, Mediterranean, and Ethiopic archipelagos, making it an ideal model for testing whether insular vultures are more susceptible to the effects of the combination of factors outlined above. Taking advantage of parallel long-term studies in two areas of continental Spain and the Canary archipelago [32], [33], we compare here the vulture's vulnerability to pathogen infection and the impact pathogens have on individual health in two populations under two scenarios (insular vs. mainland) with socio-economic similarities (increasing intensification of livestock managing), but differing in ecological and biogeographical constraints (isolation, higher density, sedentarism and lower genetic variability of the insular population). Results (a) Pathogen survey There was a general trend for pathogen prevalence (four of six taxa) to be higher in the insular population for both, fledglings and immature-adult birds. Prevalence of Salmonella spp. and Candida albicans was significantly higher in Canarian than Iberian fledglings (Table 1). The serological typing of Canarian birds showed a high prevalence of antibodies against Salmonella enteritidis (fledglings: 26.47%; immature-adults: 20%) and Salmonella typhimurium (fledglings: 26.47%; immature-adults: 25%) (Table 1). The bacteria Mycobacterium avium was only found in fledglings (6%) and immature-adults (10%) from the Canary Islands; the difference between regions was not significant, however, probably as a consequence of the small sample size. The prevalence of Escherichia coli O-86 was only examined in the insular population, being similar in fledglings (23.53%) and immature-adults (15%) (Fisher exact test, df = 1, p = 0.43). 10.1371/journal.pone.0006333.t001Table 1 Prevalence and comparison of pathogen species in fledglings and immature-adults from the Iberian Peninsula and the Canary Islands. Fledglings Immature/Adults Pathogen species Iberia Canary Islands p Iberia Canary Islands p Candida albicans 0.38 (13/34) 0.68 (23/34) 0.007 0.36 (4/11) 0.55 (11/20) ns Salmonella (all serotypes) 0.06 (2/34) 0.32 (11/34) 0.003 0.09 (1/11) 0.30 (6/20) ns Chlamydophila psittaci 0.59 (20/34) 0.59 (20/34) ns 0.50 (5/10) 0.45 (9/20) ns Trichomonas gallinae 0.56 (19/34) 0.71 (24/34) ns 0.60 (6/10) 0.65 (13/20) ns Mycobacterium avium (culture and PCR) 0.00 (0/35) 0.06 (2/34) ns 0.00 (0/10) 0.10 (2/20) ns Mycoplasma spp. 0.56 (19/34) 0.56 (19/34) ns 0.40 (4/10) 0.40 (8/20) ns Significant differences are shown in bold; ns = not significant (p>0.008 after Bonferroni adjustment). A clear association was found amongst pathogens in the Canarian population. The presence of Chlamydophila was associated with a higher prevalence of Mycoplasma in fledglings (χ 2 = 15.896, df = 1, p<0.001, contingency coefficient = 0.57, p<0.001) (Fig. 1) and with a higher prevalence of Salmonella (χ 2 = 5.09, df = 1, p = 0.024, contingency coefficient = 0.45, p<0.024) and Mycoplasma (χ 2 = 4.85, df = 1, p = 0.028, contingency coefficient = 0.44, p<0.028) in immature-adult birds. We did not find any type of associative patterns in the continental populations (p>0.05 in all cases). 10.1371/journal.pone.0006333.g001Figure 1 Association between the pathogens Chlamydophila psittaci and Mycoplasma spp. in Canarian Egyptian vulture fledglings. (b) Individual immunocompetence Multivariate tests for fledglings showed an overall significant effect of the Population (Wilks' λ = 0.36, F11,29 = 4.70, p<0.001, Partial Eta Squared = 0.64), the pathogens Chlamydophila (Wilks' λ = 0.34, F11,29 = 5.17, p<0.001, Partial Eta Squared = 0.66) and Mycoplasma (Wilks' λ = 0.41, F11,29 = 3.73, p = 0.002 Partial Eta Squared = 0.59) and the interaction between PopulationChlamydophila (Wilks' λ = 0.51, F11,29 = 2.53, p = 0.02, Partial Eta Squared = 0.49) and marginally between PopulationMycoplasma (Wilks' λ = 0.57, F11,29 = 1.97, p = 0.07, Partial Eta Squared = 0.43). Then, pair wise comparisons between estimated marginal means were done using Bonferroni adjustment for multiple comparisons. Canarian fledglings showed significantly lower levels of total white blood cells (F1,39 = 12.28, p = 0.001), heterophils (F1,39 = 10.29, p = 0.003), lymphocytes (F1,39 = 4.87, p = 0.03), monocytes (F1,39 = 24.19, p<0.001) and large lymphocytes (F1,39 = 5.84, p = 0.02). Moreover, Canarian fledglings infected by the pathogen Chlamydophila showed lower levels of lymphocytes (F = 4.80, df = 1, p = 0.034) and small lymphocytes (F = 4.51, df = 1, p = 0.040) and higher levels of basophiles (F = 4.40, df = 1, p = 0.042) than uninfected Canarian individuals. Multivariate tests for immature-adults birds did not show significant effects. Discussion Insular Egyptian vultures had both a higher prevalence and frequency of association of avian pathogens and a poorer immune system. In addition to a reduced capacity for fighting pathogens, other factors such as the use of livestock and host density may explain the differences in pathogen incidence. Most of the reported pathogens are associated with intensively raised livestock [34], [35], [36] and so a higher prevalence of pathogens may be related to a greater reliance on these type of carcasses. However, carcass consumption is lower in insular Egyptian vultures (8% of prey items correspond to intensively raised livestock vs. 25.8% in the Ebro Valley [5]). Alternatively, high host density may increase pathogen spread and transmission efficiency [17]. On Fuerteventura Egyptian vultures come into contact with each other continuously throughout the year, not only at the “vulture restaurant”, but also at other feeding points (corrals) and communal roosts [9]. Mainland populations, on the contrary, are more segregated on their breeding grounds from both an intra- and inter-populational standpoint [37]. Unfortunately, because this species is becoming so rare, it is extremely difficult to test this possibility directly by comparing insular and continental Egyptian vulture populations of differing densities. Within the Canary Island population we found a clear association between different pathogens in both fledglings and immature-adult birds. These associations usually appear when an opportunistic pathogen meets a host already weakened by a previous pathogenic infection [38], [39]. Interestingly, the Egyptian vultures in the Iberian Peninsula are exposed to the same pathogens (in fact, probably more exposed as they rely more on intensively farmed livestock), but no pair of pathogens was found to occur in one individual more or less often than chance would indicate. These results suggest that Canarian vultures are more susceptible to infection by the same pathogens, which would imply that their immune response to them is weaker. Further evidence for this lies in the fact that, besides having higher rates of infection, Canarian fledglings showed lower leukocyte profiles for cells such as heterophils, lymphocytes, and monocytes that are crucial for an adequate innate and/or acquired immune response (see methods for the specific function of each cell type); when faced with infection these cells should circulate in proliferation [40], [41]. Moreover, Canarian fledglings infected by Chlamydophila psittaci showed lower rather than higher levels of lymphocytes and small lymphocytes (although they did have higher levels of basophils), which suggests that the immune response of these birds is inefficient and is not able to properly respond to infections [40], [41]. Finally, the idea that the Canarian population of Egyptian vultures is more susceptible to disease is reinforced by the presence in some birds of Mycobacterium avium, a ubiquitous pathogen generally affecting inmunocompromised animals [42], [43]. The lower immunocompetence is probably more noticeable in nestlings as their immune system is still developing [44]. Hence, the results of the necropsies and egg-content analyses performed (see methods) suggest that pathogens (mainly Salmonella spp., E. coli O-86 and Chlamydophila psittaci) play an important role in breeding failure in the Canarian Egyptian vulture population, which has the lowest known breeding success for this species within its distribution (0.5 fledglings/pair/year, [9]). Thus, disease-related mortality of fully grown fledglings in the nests is relatively high in Canary Islands (6.25% in 2002 N = 16), unlike in continental populations where almost no similar cases have been found in the 70 territories monitored annually (on average) in the Mid-Ebro Valley (1986–2005) [33] and the around 30 territories monitored annually in Cádiz (2000–2007) (authors unpublished). Although no effects of pathogens were detected on the immune system of immature-adult vultures, the association found between Chlamydophila psittaci, Mycoplasma spp., and Salmonella spp. in Canarian birds suggests that the patterns found in fledglings may also exist in full-grown birds. Immature-adults may thus be ‘chronically-infected individuals’, that is, survivors that have developed acquired immunity after early infection by those pathogens. In fact, we found antibodies against Salmonella serotypes in immature-adults, indicating that these individuals had been exposed to this pathogen in the past. Alternatively, these individuals may be merely ‘tolerating’ the infection without eliciting an immune response. This may occur particularly under certain conditions such as stress, behavioural constraints (e.g. breeding), or the bioaccumulative effect of pollutants (heavy metals), which would prevent birds from assuming the high costs of an activated immune system [45]–[47]. In this sense, it should be noted that the sedentary habits of these vultures make them especially sensitive to lead intoxication originating from hunting activities [9], [12]. Lead interferes with the normal regulation of immune functions, leading to increased susceptibility to infection [48]–[50]. This poor immunocompetence may be mediated by the lower genetic variability found in Canarian Egyptian vultures [9], [51]; consequently, negative effects operating on the host's immune system may be expected to occur [25], [52], [53]. Given that we did not conduct any experiments on animals, our results are correlative but still compatible with a scenario of immune naïveté in insular vertebrates; their immune systems have evolved in an environment with a naturally impoverished pathogen community and are incapable of fighting efficiently against newly arrived pathogens [54]–[58]. Pathogen pressure is expected to influence the immune function since immune investment is a balance between costs and benefits, and investment is wasted in an environment without pathogens that need to be fought [59]. The arrival of new pathogens has been favoured by the increasing globalization and intensive management of livestock, which involves the frequent importation of animals to islands from the mainland [60]–[62]. In fact, the importation of sheep, pigs, and goats into the Canary Islands has dramatically increased in recent years (http://www.gobiernodecanarias.org/agricultura/otros/estadistica/default.htm). Consequently, island vultures are now probably more exposed than ever to potentially fatal multiple infections by pathogens typically acquired from livestock [34], [35], and to which they are supposedly naïve and thus far more susceptible [63]. Implications for conservation The scenario described in this study may occur in other insular Mediterranean and Macaronesian systems where populations of scavenger birds of prey are in serious decline and are gravely threatened [14], [32]. Livestock practices are powerful mechanisms of landscape engineering [8], [64], [65], but may also have major implications for the health of wild insular species [66]. The intensification of farming is occurring on a global scale and the disposal of carcasses of intensively raised livestock entails risks for scavengers [5], [6], [67], [68], which may be more serious for insular populations that have a weaker response to the arrival of novel pathogens with which they have had neither opportunity nor time to co-evolve [69], [70]. Not only scavengers but entire island bird communities are interlinked with traditional human activities sharing habitats, vectors, and pathogens with domestic species [66], [71]. Extreme caution should be thus taken when importing foreign livestock into insular systems in order to reduce the irruption of new pathogens into these especially naïve and fragile environments. Materials and Methods Vulture monitoring was carried out on the island of Fuerteventura (Canary archipelago, 1662 km2) where there are 30 breeding pairs plus around 100 non-breeding birds [9] and in the Iberian Peninsula, where there is a widespread population of around 1,500 breeding pairs [72]. We chose two main continental study sites: the mid-Ebro Valley (northern Spain; 100 pairs plus around 200–300 non-breeding birds, 19,000 km2) and Cadiz (southern Spain; 30 pairs, 9.500 km2). The study areas have been well described elsewhere [9], [32], [33]. Island populations show higher densities than that found in continental regions. In Fuerteventura there are 9 birds/100 km2 whereas in the Iberian populations densities are below 2 birds/100 km2 (author's own data). In all these regions vultures regularly feed on carcasses of domestic livestock, both located randomly in the field and left in the so-called ‘vulture restaurants’, that is, artificial feeding stations where supplementary food for scavenger birds is provided [73] (authors' own data). The reliance on artificially supplied livestock carcasses is high for both populations. However, the rate of intensively raised livestock/wild preys in the diet of Egyptian vultures is greater in the Iberian Peninsula [5]. The capture and ringing of the birds was done under permits of the Spanish ‘Ministerio de Medio Ambiente’. Blood sampling and research protocols were authorized by the Regional Governments ‘Consejería de Medio Ambiente’ of Andalucía, Aragón, Navarra and Canarias. Sampling procedures During 2004–2005 over 70 fledglings (36 continental and 34 insular) and 31 immature adults (11 continental and 20 insular) were captured at nest-sites or with cannon-nets. Blood samples (5 ml) were collected from the brachial vein. All the birds were handled following identical protocols and the time spent in collecting samples was almost identical for all individuals to avoid the possible among-individual variation in stress-induced alteration of the immune measurements. Pathogen survey We conducted a comprehensive study of mycoplasmal, chlamydial, bacterial, fungal, and protozoan infections for a total of seven species: Mycoplasma spp., Chlamydophila psittaci, Salmonella spp., Escherichia. coli O-86 (enteropathogenic strain), Mycobacterium avium, Candida albicans, and Trichomonas gallinae. These pathogens are considered to be responsible for emerging infectious diseases [15], [49], [50] and were selected due to their known severe pathogenicity in birds. The effects of infection are variable, ranging from asymptomatic to severe disease with high mortality, as well as embryonic and neonatal mortality. Generally, transmission occurs by direct contact with infected individuals or through the consumption of contaminated food remains [43]. Some of them are primary pathogens, such as the enteric bacteria Salmonella spp., Escherichia. coli O-86 and Chlamydophila psittaci. The first two bacteria have caused disease in scavengers, such as colibacilosis in Red Kites (Milvus milvus), Cinereus vultures (Aegypius monachus) and Egyptian and Bearded vultures (Gypaetus barbatus) [5,6,74; author's unpublished). The list of avian species in which Chlamydophila psittaci infections occur is rapidly increasing. Wild avian species sharing aquatic or moist soil habitats with domestic poultry and granivorous birds may become infected via contaminated water and dust inhalation. The consumption of infected carcasses may transmit C. psittaci to host species that are predators or scavengers of other birds [75]. Host age can affect disease course after C. psittaci infection: adult birds may have asymptomatic infections, while young birds have acute disease [76], [77]. The others are mainly opportunistic pathogens, which generally infect immunodepressed individuals or those individuals first affected by a primary pathogen. At present, this is the main way of pathogen acquisition in scavenging birds in Spain [68]. For example, the widespread pathogen Candida albicans is found in very high prevalence in Black and Griffon vultures (Gyps fulvus) causing severe disease, especially in immunocompromised individuals [6]. 0Most of these pathogens are present in intensively raised livestock [68], [78]. Moreover, Salmonella spp., Escherichia coli O-86 and Candida albicans are saprophytic bacteria normally associated to the accumulation and decomposition of livestock carcasses at vulture's restaurants. Mycobacterium avium is generally associated to extensive livestock from where it can be acquired by scavenger birds [79]. A recent Mycobacterium bovis and serotype VII M. avium outbreak is causing disease and mortality in nestlings and juveniles of Griffon vulture populations in central Spain (authors, unpublished data). As a previous work, after the verification of breeding failure, we developed the analyses of egg content (three unfertile eggs and four embryos) and necropsy of two dead fledglings from Canary Islands. It revealed the presence of several pathogens compatible with fatal septicaemia in eight birds: the commonest pathogens were Salmonella spp., E. coli O-86, and Chlamydophila psittaci, while Erysipelothrix rhusiopathiae was isolated from the corpse of one of the fledglings (authors unpublished). Microbiological isolation Bacterial microflora was sampled from the cloaca, choana, and nares of individuals with sterile microbiological swabs and Amies transport medium. Samples were transported in a cool container to the laboratory within 12 hours of collection and were processed within one to two hours of arrival. For Salmonella, microbiological methods were used for cultivation and isolation, and for serotype identification, as described elsewhere [74]. Serology for Salmonella spp. was performed when no cloacal samples were taken. In this case only Salmonella typhimurium and Salmonella enteritidis antisera were used, because they are the most common serotypes isolated from raptors [80]. Serological tests are satisfactory for establishing the presence and estimating the prevalence of infections [35], [80]–[82]. This rapid whole blood-plate agglutination test used the antigen Difco (TM) Salmonella O Group B Antigen (1–4–5–12) (Becton Dickinson and Company, Maryland, USA). The test was conducted by using the manufacturer's standard instructions [83]. For the determination of Mycobacterium avium, cloacal and tracheal samples taken with sterile swabs were plated on Lowenstein-Jenssen media and incubated for three months. Samples with Mycobacterium growth were stained (Ziehl-Nielsen and auramine rhodamine acid-fast stains) and PCR techniques were used to identify the agent. These techniques have been proved to be adequate for the isolation of this pathogen in wild fauna [84], [85]. The presence of the Mycobacterium was considered to be proven when both cultures and molecular techniques were consistent [86]. Clinical Candida albicans was determined by the examination and sampling of oral cavities. Samples were cultured in standard fungical media (Agar Sabouraud) at 37°C for 48 hours. The presence of Escherichia coli O86 (enteropathogenic strain) was determined by phenotypic and genotypic characterization [87] and by PCR [88]. Mycoplasma spp. and Chlamydophila psittaci were determined by PCR. For the determination of strains we followed the protocols published by [89]. Trichomonas gallinae was determined by direct visualization in warm physiologic solution, culture [90] and PCR [91]. Immune assays We evaluated individual immunocompetence by measuring several cellular and humoral immune system parameters. After blood samples collection, approximately 4 ml was transferred to a lithium-heparinized tube and immediately refrigerated at 4–6°C. In addition, two blood smears were obtained immediately for each individual and fixed for three minutes with methanol and stained with May-Grünwald Giemsa stains for haematological parameter determination. Details of the haematological techniques and parameters were standard and can be found elsewhere [40], [92], [93]. Leukocyte concentrations provide information on circulating immune cells which can be used as an indicator of health [40]. The immunological function of each of the white blood cells (WBC) types has been reviewed extensively elsewhere [41], [94], [95]. Briefly, heterophils are the primary phagocytic leukocyte and mediate innate immunity against novel pathogens. Lymphocytes are involved in several immunological functions, such as immunoglobulin production and modulation of immune defence [40]. Eosinophils and basophils play a role in the inflammation process [40], [94] and the first are associated with defence against parasites [96]. Finally, monocytes are long lived phagocytic cells associated with defence against infections and bacteria [40]. The total WBC count was determined by counting all leucocytes in a Neubauer chamber and multiplying the raw data by 200 to obtain the final values [40]. The proportion of different types of leucocytes was assessed on the basis of an examination of a total of 100 leucocytes under oil immersion. Plasma was separated by centrifugation at 3,000 r.p.m. for 10 minutes within eight hours of extraction and then stored at −20°C. Plasma samples were used for protein plasma electrophoresis and serology tests (immunoglobulins: α, β and γ –globulins) [97]. Statistical analyses (i) Prevalence The prevalence of six pathogen species (number of infected individuals/number of sampled individuals) in insular and continental populations was compared using contingency tables (χ2 test), which accounts for the nature of the prevalence data (frequencies) and the unbalanced sample sizes [98]. The bacterium E. coli O-86 (enteropathogenic strain) was examined only in the insular population. As a previous step, we compared the prevalence between the two continental populations. We found no differences (p>0.05 in all cases) and so the data from the two continental populations were pooled (hereafter Iberia). We kept the data pool separated in age groups because it is well known that the immune system of adult birds differs from that of fledglings since the later need some time to mature and be efficient [99]. Since multiple tests were carried out we adjusted the α-level using the Bonferroni correction for each data set (fledglings and immature-adults). The association between pathogen species was analyzed by means of contingency tables (χ2 test) controlling for the variable “population” and measured by means of contingency coefficients. (ii) Effect of pathogens To determine whether the effect of pathogens on the immune response differed between populations we used multivariate analyses of variance MANOVA, which allow an overall test of the effects of the explanatory variables evaluating cellular and humoral immune response (differential counts of WBC: total WBC, heterophils, lymphocytes, large lymphocytes, small lymphocytes, monocytes, eosinophils and immunoglobulins: α, β and γ –globulins). In order to avoid inherent variance to different immune response between fledglings and immature-adult individuals [97], we carried out separate factorial-MANOVA analyses for each age class (sum of squares type III). When normality was not attained (Shapiro-Wilk tests), variables were transformed accordingly. The Levene contrast was applied in order to test the equality of the error variances for each response variable (p>0.05 in all cases). Population (1 = continental or 2 = insular) and six pathogen species were included as factors in each analysis. Whenever the sample size was appropriate, we considered the interactions between the population and each pathogen species. Moreover, we controlled for the possible effect of the remaining variables evaluating immune system and body condition (mean corpuscular volume MCV, mean corpuscular haemoglobin MCH, and mean corpuscular haemoglobin concentration MCHC) and included them as covariates in the model. We would like to thank M. de la Riva, A. Trujillano, D. Lagares, O. Ceballos, J.L. Tella, D. Serrano, I. Luque, A. Cortés, E. Ursúa, D. Gómez, J.M. Aguilera, J.A. Pinzolas, A. Bueno, J.C. Albero, J.M. Canudo, J.L. Rivas, E. Alcaine, J.L Lagares, P. Martínez, A. Legaz, P. Oliva, A. Pastor, and J.A. Pérez-Nievas for their help during the fieldwork. The staff of the Bardenas Reales Natural Park and the Consejería de Medio Ambiente del Cabildo Insular de Fuerteventura provided logistical support. We would like to thank very much J.L. Tella for his valuable comments on early drafts of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was funded by Research Projects REN 2000-1556/GLO, CGL2004-00270/BOS, CGL2007-61395/BOS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Green RE Newton I Shultz S Cunningham AA Gilbert M 2004 Diclofenac poisoning as a cause of vulture population declines across the Indian subcontinent. J Appl Ecol 41 793 800 2 Rondeau G Thiollay JM 2004 West African vulture decline. Vulture News 51 13 33 3 Koenig R 2006 Vulture research soars as the scavengers' numbers decline. 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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central 1471-2458-9-2411960438310.1186/1471-2458-9-241Research ArticlePremarital Sexual Behavior among male college students of Kathmandu, Nepal Adhikari Ramesh [email protected] Jyotsna [email protected] Geography and Population Department, Mahendra Ratna Campus, Tribhuvan University, Kathmandu, Nepal2 Center for Research on Environment Health and Population Activities (CREHPA), Kathmandu, Nepal2009 15 7 2009 9 241 241 18 9 2008 15 7 2009 Copyright © 2009 Adhikari and Tamang; licensee BioMed Central Ltd.2009Adhikari and Tamang; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In Nepal, as in other Asian countries, the issue of sexuality still remains a taboo. Despite this fact, an increasing number of sexual activities is being reported by Nepalese students. This trend warrants serious and timely attention. Due to the sensitivity of the topic of premarital sexuality, youth receive inadequate education, guidance and services on reproductive health. The main objectives of this paper are to explore the sexual behavior especially focusing on prevalence of premarital sex among college men and to investigate the factors surrounding premarital sexual behavior. Methods A cross-sectional survey of college students was conducted in April-May 2006. A self-administered questionnaire was completed by 573 male students. Association between premarital sex and the explanatory variables was assessed in bivariate analysis using Chi-square tests. The associations were further explored using multivariate logistic analysis. Results Despite the religious and cultural restrictions, about two-fifths of survey respondents (39%) reported that they have had premarital sex. The study has also shown that substantial proportions of students indulge in sexual activities as well as risky sexual behavior. Sex with commercial sex workers, multiple sex partners, and inconsistence use of condom with non-regular partner was common among the students. Less than two in five male students (57%) had used condom at the first sexual intercourse. The prevalence of premarital sex varied on different settings. Older students aged 20 and above were more likely to have premarital sex compared with younger students aged 15–19. Men who had liberal attitude towards male virginity at marriage were almost two times more likely to have engaged in premarital sex compared to their counterparts who have conservative attitude towards male virginity at marriage. Moreover, those students who believe in Hindu religion were more than two times (OR = 2.5) more likely to have premarital sex compared with those who follow other religions. Furthermore, those men who have close unmarried friends who have experienced premarital sexual intercourse were eight times (OR = 8.4) more likely to be sexually active compared to those who did not have such sexually active friends. Conclusion Prevalence of premarital sexual intercourse and risky sexual behavior are not uncommon in Nepal. Young people are exposed to health hazards due to their sexual behavior; hence sex education should be provided. School or college based sexuality education could benefit even out-of-school youths, because their partners often are students. ==== Body Background Nepal presents an important setting for addressing the sexual and reproductive health needs of young people as one-third of the country's population is aged 10–24 [1]. Data from the Central Bureau of Statistics also indicates that the percentage of never married people aged 10 and above is increasing over time (male 28% in 1961 to 39% in 2001; female 15% in 1961 to 30% in 2001) in Nepal [2]. In Nepal, as in other Asian countries, strong norms persist that prohibit premarital sexual contact between young men and women and the topic of sexuality largely remains a taboo. Due to decreased age at menarche and increasing tradition of later age at marriage, the customary attitude has been changing. Declining influence of family, increasing urbanization, migration and the exposure to mass media have collectively contributed to major changes in social and sexual behavior among adolescents [3]. Due to social restrictions, disclosure of premarital sexual activities is rare; however, few studies that have been conducted in Nepal indicate a growing trend towards premarital sexual activities among adolescents [4-6]. A study conducted by Tamang et al. in 1999 among Nepali men in the border towns of Nepal showed that a significant proportion of sexually experienced young unmarried (18–24 years) male who are residents of the border towns (54%) and non-residents (40%) had engaged in sex with a non-regular partner in the last 12 months preceding the survey. Higher proportion of the married non-resident young men (46%) compared with unmarried non-resident men (18%) were involved in casual sex and a large majority of the non-resident young men (67%) cited a commercial sex worker (CSW) as their last casual sex partner. Although regular use of condoms during sex with non-regular partners was generally low, only a small proportion of them considered themselves to be at risk of contracting sexually transmitted infections (STIs) and HIV/AIDS [5]. Another study of the young factory workers in Kathmandu revealed that 20% of unmarried boys and 12% of unmarried girls aged 14–19 years were sexually experienced (penetrative sex). Interestingly, the mean age for first sexual debut was the same for both the boys and the girls (15 years) [4]. Similarly a survey conducted among teenagers in seven districts of Nepal showed risky sexual behaviour especially among young boys. About 22% of the boys interviewed had premarital sexual experience and only two thirds of them used condom. The number of boys who had sex with multiple partners was also high [6]. Although nation wide and extensive research on young people's needs and behaviors in Nepal is rare, existing data indicate that young people do not have adequate access to appropriate information and services about sexual and reproductive health issues [4-6]. Even though young people are taught subjects on health and population at school levels which includes basic information on fertility, mortality, human organ, menstruation, sexually transmitted infections including HIV/AIDS, they are poorly informed about sexual and reproductive health mainly due to lack of comprehensive education about sexual and reproductive health [7]. The design and delivery of appropriate national level services for adolescents has been constrained by long-held traditional beliefs and ideologies. As a result of inadequate or ineffective services and information, young people often experience negative reproductive health consequences, including unplanned pregnancies and HIV/AIDS [3]. Many literatures suggest that the individual, family and peer variables have considerable influence on the sexual behavior of the youth. However, it is one of the least researched topics in Nepal. To fill the gap, it is thus imperative to study the factors surrounding premarital sex in the context of Nepal in order to inform policy makers and planners and to develop appropriate and timely intervention programs to prevent high risk sexual behavior such as premarital sex. This article is based on a study conducted in 2006 in Kathmandu. Although the study was conducted among both male and female college students, the article focuses on data collected from male college students. The study sought to explore the sexual behavior especially focusing on prevalence of premarital sex among college men and to investigate the factors surrounding premarital sexual behavior. This study is the first of its kind conducted among college students. The findings of the study address the gap in knowledge by providing descriptive information on premarital sex that could help program managers of I/NGOs and the Government of Nepal to design appropriate and timely education-based interventions in institutions of secondary and higher education. Methods The data used in this paper comes from a cross-sectional survey on attitude and behavior towards premarital sex among college students of Kathmandu Nepal carried out in 2006. Data for this paper was from 573 male students studying in 12 colleges affiliated to Tribhuvan University (TU) in Kathmandu, the capital of Nepal. The scientific committee which includes ethical review board of University Grant Commission (UGC) Nepal has approved the proposal and provided funding for this study. Two-staged random sampling technique was applied in order to sample the college students. The first stage of sampling included random selection of 12 colleges affiliated to Tribhuvan University (TU) in Kathmandu. In order to select these 12 colleges, a list of all the private and public colleges affiliated to Tribhuvan University and those located in Kathmandu valley (includes three districts, namely Bhaktapur, Lalitpur and Kathmandu) was obtained from the office of the Vice Chancellor in Kathmandu. This list included colleges that provide intermediate (commonly known as Grade 11 & 12), undergraduate and graduate degrees. In the second stage, two classes were selected randomly from each sampled college. These classes were not differentiated by subject. The number of students in each class ranged from 40 to 60 students. Since all the colleges were co-educational, all male and female students present on the day of the interview in the sampled classes were requested to participate in the study. Female and male students were interviewed separately in different classrooms. Due to the sensitive nature of the study, a self-administrated structured questionnaire (Additional file 1) was used to obtain information from the students. The questionnaires were first developed in English and then translated into Nepali language. The questionnaires were pre-tested among college students in a non-selected college and later refined as required. While most of the questions were close-ended, a few open-ended questions were also included. The survey assessed four items that pertained to premarital sex: 1) Experience of sexual intercourse, 2) Age at first sexual debut, 3) First partner and 4) Use of condom during first sexual intercourse. Unmarried respondents were asked 'Have you ever had sexual intercourse?' and married respondents were asked 'Have you ever had sexual intercourse before you got married?'. These two questions were from two separate questionnaire items. In addition to this, the respondents were also asked about their age at first sexual debut, first partner and use of condom during first sexual intercourse. All completed survey questionnaires were entered into a database after manual coding and validation. Data entry and validity checks were performed for all the questionnaires by using computer software dBase IV. The cleaned and validated data was transferred into SPSS for further processing and analysis. Verbal informed consent was obtained from the participants before they were enrolled in the study. Consent form was written in the local language stating the study's objectives, nature of participant's involvement, risk and benefits, and confidentiality of the data. Students were requested to read the consent form carefully. They were given clear options on voluntary participation. It was also made clear that they could refuse to answer any questions and terminate the interview when they desired. None of the approached students refused to participate in the study. Confidentiality of information was ensured by removing personal identifiers from the completed questionnaires. The names of sampled colleges were not made public and thus not possible for anyone outside the research team to trace reported incidents of sexual behavior to respondents. Respondents were protected from any possible adverse repercussions of participating in the study. Both bivariate and multivariate techniques were applied to identify the factors associated with the likelihood of having premarital sexual intercourse. Chi-square test was used to test an association between the variables. The variables were also examined in the multivariate analysis (Binary logistic regression) in order to identify the significant predictors after controlling for other variables. During the process of analysis, multi-collinearity among the variables was assessed and the least important variables were removed from the logistic model. Results A large majority of the respondents (85%) were young (15–24 years). A large majority of the men (88%) were unmarried and 91 percent of sample students were from outside of Kathmandu valley. Students covered in this study were from 67 districts out of 75 districts of the country which means many (59%) lived either with friends or alone in Kathmandu (Table 1). More than half of the men were currently pursuing their undergraduate degree. Awareness of HIV/AIDS was universal among the male college students and knowledge of at least one mode of transmission of HIV/AIDS was also universal. A large majority of the students (91%) had taken up a subject in school or college related to reproductive health. Almost all students in the study reported that sex education is necessary for youth before getting married (table not shown). Table 1 Selected background characteristics of the respondents Characteristics % Age group 15–19 35.8 20–24 49.2 25–29 12.7 30 and above 2.3 Median age 21.0 Marital status Unmarried 88.3 Currently married 11.7 Districts Kathmandu valley (3 districts) 9.2 Outside Kathmandu valley (64 districts) 90.8 Level of education Intermediate 27.7 Undergraduate 53.9 Graduate degree 18.3 Type of accommodation With family 41.0 Alone 19.0 With friends 40.0 Total 100.0 N 573 Sexual Activities The study shows a continuum of sexual behavior ranging from kissing, fondling to sexual intercourse. For example, more than half of the male (57%) had experienced kissing, while three-fifths of them (60%) reported that they placed their hand on a girl's breast. Similarly, more than a third (35%) reported that they placed their hand on a girl's sex organ. Dating in Nepali context seems to be less common compared to other non-penetrative sexual activities. Slightly less than half of the respondents (46%) reported that they experienced dating. Overall, nearly half the men, both married and unmarried men (47%) had experienced sex while more than one third of the study respondents (39%) had premarital sex. Underreporting of such sexual activities is highly possible due to the sensitive nature of the study. Another proxy to measure experience of premarital sex is to ask if the respondent has close unmarried friend who has experienced premarital sex. As expected, more than half the respondents have a close friend who has had premarital sex (Table 2). Table 2 Nature of sexual activities performed % Experience of kissing a girl 57.4 Experience of dating 44.5 Experience of placing hand on a girl's breast 60.2 Experience of placing hand on a girl's sex organ 34.9 Experience of sexual intercourse 46.9 Experience of premarital sex 39.1 Having close unmarried friend with experience of premarital sex 53.0 N 573 Prevalence of premarital sex varied, depending on different settings. Slightly higher proportion of men in the age group of 20 years and above had premarital sex compared to younger men (below 20 yrs). Students who have higher education level reported higher percentage of premarital sexual experience. For example, around one third of men who were studying in intermediate level (35%) and more than two-fifths of students pursuing graduate degree (43%) had premarital sexual experience. Regarding marital status, higher proportion of unmarried men (40%) had premarital sex compared with currently married (33%) respondents. Similarly, level of premarital sexual activities is higher among those students whose permanent residence was outside Kathmandu valley (40%) compared with those who reside in Kathmandu permanently (32%). Regarding living arrangement, the proportion of those students who lived alone had more premarital sex experience compared to others. For example, more than two-fifths of those men who lived alone (43%) had premarital sex while the percentage is less than two-fifths for those who live with family (37%). Similarly, a significantly higher percentage of premarital sex was observed among students who have liberal attitude towards premarital sex compared to those who have conservative attitude towards the same. For example nearly half (46%) of the men who have liberal attitude towards female virginity (disagreed on the following statement: 'women should be virgin at marriage') had engaged in premarital sex. Similar response was found for the attitude towards male virginity (Table 3). Table 3 Premarital sexual experience by selected background characteristics Premarital sex Total Yes No % Number Age group 15–19 34.6 65.4 100.0 205 20 and above 41.6 58.4 100.0 368 Level of education Intermediate 35.2 64.8 100.0 159 Undergraduate 39.8 60.2 100.0 309 Graduate degree 42.9 57.1 100.0 105 Marital status Married 32.8 67.2 100.0 67 Unmarried 39.9 60.1 100.0 506 District Outside Kathmandu valley 39.8 60.2 100.0 520 Kathmandu valley 32.1 67.9 100.0 53 Living arrangement With family 36.6 63.4 100.0 232 Alone 43.4 56.6 100.0 106 With friends 39.6 60.4 100.0 235 Attitude towards female virginity* Conservative 33.4 66.6 100.0 305 Liberal 45.5 54.5 100.0 268 Attitude towards male virginity* Conservative 30.0 70.0 100.0 290 Liberal 48.4 51.6 100.0 283 Family structure Joint family 39.6 60.4 100.0 139 Nuclear family 38.9 61.1 100.0 434 Religion* Non-Hindu 20.0 80.0 100.0 35 Hindu 40.3 59.7 100.0 538 Has a friend who has experienced premarital sex* No 14.9 85.1 100.0 268 Yes 60.3 39.7 100.0 305 Total 39.1 60.9 100.0 573 Note: * = p < .001, ** = p < .01, * = p < .05 Prevalence of premarital sex varied according to respondent's religion. For example, higher proportions of Hindu men (40%) were likely to have premarital sex compared to non-Hindu men (20%). It is also found that behavior of peers has positive effect on the prevalence of premarital sex. For instance, prevalence of premarital sex is far higher among those who have close unmarried friends with sexual experience (60% vs. 15%). Age at First Sexual Intercourse If unprotected, the first sexual event has clear adverse outcomes on health such as unplanned pregnancy for the women and STIs. Thus the first sexual intercourse remains an event of immense social and personal significance. In the current study, the age at first sexual intercourse of male students ranged from 10 to 25 years. About two-thirds of the respondents who had experienced premarital sex had sex before the age of 19. Seven percent reported that they had sexual intercourse before the age of 15 (Table 4). Table 4 Age at first sexual intercourse % Below 15 yrs. 6.7 15–16 25.0 17–18 32.0 19 or more 36.3 Total (age range 10–25 years) 100.0 N 224 First Sex Partner and Condom Use Information regarding first sexual partner was solicited from students who had premarital sex. Over half the male students (55%) had their first sexual intercourse with their girlfriend while a third (32%) reported that their first sexual partner was their friend. It is notable that one out of ten male students (5%) had their first sexual intercourse with a commercial sex worker. Condom use at the time of first sexual intercourse was very low. Less than three out of five students (57%) reported that they used condom during their first sexual intercourse. Sexual Risk Behaviour Number of Sex Partners Over half of the male students (55%) reported that they had more than one sex partner and about one in three sexually active men (31%) had three or more sex partners. The number of sexual partners for these college students ranged from 1 to 15 (Table 5). Table 5 Number of sex partners How many sex partners did you have (total)? % One 45.1 Two 23.7 Three and more 31.3 Average number of sex partners 2.4 SD 2.1 Ranges 1–15 Total 100.0 N 224 Sexual experiences with Commercial Sex Worker and Condom Use Sexually active unmarried respondents were further asked about their sexual experiences with a commercial sex worker (CSW) and their condom use with them. The results show that more than a quarter of them (23%) had sexual intercourse with a CSW while only less than a half of the male students (49%) who had sexual intercourse with CSWs had used condom during every act of sexual intercourse (Table 6). Table 6 Sex with CSW and condom use % Have you ever had sex with CSW? Yes 22.8 No 77.2 Total 100.0 N 224 How often did you use condom with CSW? Every act of sexual intercourse 49.0 Sometimes 45.1 Never 5.9 Total 100.0 N 51 Multivariate analysis Logistic regression analysis was used to measure the strength of the association between various individual, family, peer's characteristics and the probabilities of being sexually active before marriage among these male students. Three models were used in the analysis. In the first model, individual factors were incorporated. In the second model, family characteristics were added and in the third model peer characteristics were included. After assessing multicollinearity in the variables, it was found that 'attitude towards male virginity at marriage' and 'attitude towards female virginity at marriage' were highly correlated (r = 0.8). So the variable 'attitude towards female virginity' was not entered in the logistic model. The analysis found that men who have a liberal attitude (boys shouldn't be a virgin at marriage) towards male virginity at marriage were about two times more likely to have premarital sexual experience compared to those who have conservative attitude towards male virginity at marriage. The variable which was significant in the first model retained significance even after inclusion of family characteristics in the second model. The reduction of odd ratio of the variable after inclusion of family characteristics indicated that the family characteristics were also important predictors for being sexually active among unmarried men. Model 2 further explained that those students who believe in Hindu religion were about three times (OR = 2.6) more likely to have premarital sex compared to those who believe in the Muslim religion (Table 7). Table 7 Estimated odds ratio (OR) for having premarital sex among college-going men by selected predictors Model I Model II Model III Individual characteristics Age group 15–19 1.0 1.0 1.0 20 and above 1.34 1.39 1.69* Level of education Intermediate 1.0 1.0 1.0 Undergraduate 0.93 0.89 0.52* Graduate degree 0.88 0.79 0.48* Marital status Married 1.0 1.0 1.0 Unmarried 1.30 1.29 1.73 District Outside Kathmandu valley 1.0 1.0 1.0 Kathmandu valley 0.82 0.84 0.97 Living arrangement With family 1.0 1.0 1.0 Alone 1.32 1.39 1.28 With friends 1.05 1.07 1.07 Attitude towards male virginity Conservative (ref) 1.0 1.0 1.0 Liberal 2.16* 2.15* 1.91** Family characteristics Family structure Joint family 1.0 1.0 Nuclear family 0.91 0.81 Religion Non-Hindu (ref) 1.0 1.0 Hindu 2.63* 2.99* Peer characteristics Has close unmarried friend who has experienced premarital sex No (ref) 1.0 Yes 9.2* Intercept 0.28 0.124 0.033 -2 log likelihood 741.7 736.1 611.1 Cox & Snell R square 0.043 0.052 0.238 Note: * = p < .001, ** = p < .01, * = p < .05 Model three presents the final results after adding peer characteristics. Even after inclusion of peer characteristics in the third model, the individual variable and one family-level variable were still statistically significant. Furthermore, the variables 'age', 'level of education', 'having close unmarried friends who have had premarital sex' had statistical significant effect on experience of premarital sex after controlling for other variables. Older students aged 20 and above were about two times (OR = 1.7) more likely to have premarital sex compared with younger students aged 15–19. Unexpectedly, those students who have studied undergraduate and graduate degree level of education were less likely to have premarital sex compared with those who have intermediate education only. Those men who had close unmarried friends who had experienced premarital sex were almost eight times more likely to be sexually active compared with those who did not have such friends (Table 7). Discussion and conclusion This study is first of its kind in Nepal and attempted to investigate the influencing factors surrounding premarital sexual behavior among college men. Although premarital sex is socially unacceptable in Nepal, the study has shown that the proportion of students having sexual intercourse before marriage is considerably high. Due to the sensitive nature of the issue, this proportion may still be underreported. However another variable in the study shows that more than half of the college students have friends who have had premarital sexual experience. This variable is an indication of the high prevalence of premarital sex among college male students. The study also showed that risky sexual behavior is common among college men. Condom use at the time of first sexual intercourse was very low among these men. More than a quarter of the male students (23%) had had sexual intercourse with a commercial sex worker. Although awareness about HIV/AIDS and mode of transmission of HIV/AIDS were universal among the male students, it is discouraging to note that only less than half the students (49%) who had sexual intercourse with CSWs had used condom at every act of sexual intercourse, which indicates these sexually active men are more at risk The increase in premarital sex among men who are attending school/college may be due to the fact that they have greater independence (not living with family) from their families and increasing access to young women for sex. The prevalence of premarital sex varied with different settings. The bivariate analysis showed that some of the individual characteristics, family variables and peer characteristics had significant association with experience of premarital sex. Individual characteristics such as attitude towards male and female virginity, family characteristics such as religion and peer characteristic such as peer sexual behavior have significant association with having premarital sex among college-going men. The multivariate analysis corroborated some of the findings of the bivariate analysis. In the multivariate analysis, age, attitude towards male virginity, religion and peer sexual behaviour were found to have statistical significant association with experience of having premarital sex after controlling for other variables. The association between age cohort and premarital sex is substantial. The present study found a positive association between age cohort and premarital sex, but it cannot be concluded that the likelihood of premarital sex is declining among the younger cohort; the association is undoubtedly an artifact of the truncated exposure among the younger cohort. Research has shown that young people who identify with a fundamentalist Protestant group have less permissive attitudes toward premarital sex and these young people are likely to be less sexually active [8]. The present study also supported this finding as students who have liberal attitudes toward premarital sex were more likely to engage in premarital sex. However, some of the students who had conservative attitude had also engaged in premarital sex. One of possible reason for this finding could be peer pressure. Peer role is important in changing personality, attitude and behavior of persons. There is further evidence that in all societies, peer behavior is a model for individual behavior, and this is certainly true in matters of sexuality among adolescents and youths [9]. Even in the present study, the sexual behavior of the peer was positively associated with premarital sex. Sometimes peer pressure upon a person can lead him or her to engage in sex through associated behaviors such as drinking alcohol and seeking CSWs. A study showed that two thirds of the men perceived that after drinking with friends, refusal to visit a brothel upon their friends' request has caused misunderstanding with them [10]. Similarly another study among school aged adolescents in Kenya (1993) showed that males who socialized with sexually experienced peer were nearly seven times more likely to have sex than those whose peers were not sexually experienced [11]. Sensitive issue such as sexuality is difficult to discuss among family members but it is easier to discuss among peers. Therefore, the impact of peer group plays a significant role in influencing views, attitudes and sexual behavior of individuals. Although this paper presents a worthy picture regarding premarital sexual behavior, it cannot be generalized to all college students in Kathmandu valley as the sample was taken only from those colleges that are under the umbrella of Tribhuvan University. Furthermore, since the study was conducted among college students, it also excludes an important group such as out-of-school individuals. In short, we would like to summarize that many findings from our study are in line with findings from previous studies on premarital sexual behavior. This paper not only provides empirical evidence on the importance of individual characteristics, familial role and peer factors on premarital sexual behavior of male college students in Nepal, but also draws attention to the prevalence of premarital sexual behavior among young, college-going youths. This paper seeks to fill the gap in knowledge by providing descriptive findings on premarital sex among college male students and to garner interest from policy-makers to develop appropriate reproductive health programs to combat negative impact of such behavior. Our findings suggest that it is necessary to reinforce reproductive and sexual health education among college students and provide them with convenient and optional services that are easily accessible. There is a need to provide comprehensive education on sexual and reproductive health (SRH) issues such as safer sex and HIV/AIDS in order to make responsible and healthy decisions to protect them from situations and behaviors that would place them at risk of HIV transmission. It is further recommended that a qualitative research should be conducted in order to design appropriate intervention that address the problems and needs of the youths by involving the young people themselves. Competing interests The authors declare that they have no competing interests. Authors' contributions RA, Lecturer of Mahendra Ratna Campus, Tahachal, Kathmandu conceived and designed the study. He carried out the data collection, conducted data analysis and interpretation of the data. JT was involved in the data analysis and interpretation of the datav. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Questionnaire on the study entitled "Survey on Attitude and Behavior towards premarital sex among college students of Kathmandu Valley". The questionnaire includes various aspects of information regarding premarital sex. Click here for file Acknowledgements University Grant Commission, Nepal provided funding for this study. The authors also wish to thank the students for participating in the study. ==== Refs Ministry of Health [Nepal], New Era, and ORC Macro Nepal Demographic and Health Survey 2001 2002 Calverton, Maryland, USA: Family Health Division, Ministry of Health; New Era and ORC Macro CBS Population Monograph of Nepal 2001 Central Bureau of Statistics, Kathmandu Gubhaju BB Adolescent Reproductive Health in the Asian and Pacific Region, Asian Population Studies Series No, 156 2001 Economic and Social Commission for Asia and the Pacific, Thailand Tamang A Nepal B Puri M Shrestha D Sexual Behaviour and Risk Perception among Young Men Engaged Border towns of Nepal Asia Pacific Population Journal 2001 16 195 210 Puri M Sexual Risk Behavior and Risk Perception of Unwanted Pregnancy and Sexually Transmitted Infection among Young Factory Workers in Nepal Kathmandu, Nepal 2002 UNAIDS and UNICEF Survey of Teenagers in Seven Districts of Nepal Kathmandu, Nepal 2001 Tamang A Nepal B Providing Adolescent Health Services: The Nepalese experience Sexual and Reproductive Health: Recent Advances, Future Directions 2000 I 379 398 Thornton A Camburn D The Influence of the Family on Premarital Sexual Attitudes and Behavior Demography 1987 24 323 340 3678537 10.2307/2061301 Cerdana GP Omg-Chang Chang Hui-Sheng Lin Te-Husing Sun Cernada Ching-Ching Chen Implication for Adolescent Sex Education in Taiwan Studies in Family Planning 1986 17 181 187 3750359 10.2307/1966935 Van Landingham M Grandjean N Some Cultural Aspects of Male Sexual Behavior Patterns in Thailand Paper presented at International Conference on Sexual Subcultures, Migration and AIDS Bangkok, February 27 to March 3 1994 Kiragu K Zabin L The Correlates of Premarital Sexual Activity among School-age Adolescents in Kenya International Family Planning Perspectives 1993 19 92 97 10.2307/2133242	19604383	PMC2717085	CC BY	2021-01-04 17:40:33	yes	BMC Public Health. 2009 Jul 15; 9:241
==== Front Microb Cell FactMicrobial Cell Factories1475-2859BioMed Central 1475-2859-8-391961931810.1186/1475-2859-8-39ResearchA novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles Nangia Yogesh [email protected] Nishima [email protected] Nisha [email protected] G [email protected] C Raman [email protected] Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160036, India2 Institute of Nanotechnology, Northwestern University, Evanston, IL, USA2009 20 7 2009 8 39 39 27 3 2009 20 7 2009 Copyright © 2009 Nangia et al; licensee BioMed Central Ltd.2009Nangia et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The synthesis of gold nanoparticles (GNPs) has received considerable attention with their potential applications in various life sciences related applications. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system, which has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. In the present study, we have demonstrated the synthesis of gold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines in India. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization via charge capping was investigated. Results A bacterial isolate capable of gold nanoparticle synthesis was isolated and identified as a novel strain of Stenotrophomonas malophilia (AuRed02) based on its morphology and an analysis of its 16S rDNA gene sequence. After 8 hrs of incubation, monodisperse preparation of gold nanoparticles was obtained. Gold nanoparticles were characterized and found to be of ~40 nm size. Electrophoresis, Zeta potential and FTIR measurements confirmed that the particles are capped with negatively charged phosphate groups from NADP rendering them stable in aqueous medium. Conclusion The process of synthesis of well-dispersed nanoparticles using a novel microorganism isolated from the gold enriched soil sample has been reported in this study, leading to the development of an easy bioprocess for synthesis of GNPs. This is the first study in which an extensive characterization of the indigenous bacterium isolated from the actual gold enriched soil was conducted. Promising mechanism for the biosynthesis of GNPs by the strain and their stabilization via charge capping is suggested, which involves an NADPH-dependent reductase enzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process. ==== Body Background Synthesis of GNPs and subsequent linkage to biomolecules has contributed immensely in various life sciences related applications such as drug-delivery, gene transfer, bioprobes in cell and tissue analysis for visualization of micro- and nano-objects, and for observation of the biological processes at nano-scale etc. [1-5]. These nanoparticles, in general, are synthesized using a number of synthetic procedures in various polar and non-polar media [6-8]. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system. This has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. Microorganisms, such as bacteria, yeast and fungi, are known to produce inorganic materials either intra- or extracellularly [9-12]. These microorganisms play an important role in remediation of metals through reduction of metal ions. Some of these microorganisms can survive and grow even at high metal ion concentrations. They are often exposed to extreme environmental conditions, forcing them to resort to specific defense mechanisms to quell such stresses, including the toxicity of foreign metal ions or metals [13]. The toxicity of metal ions is reduced or eliminated by changing the redox state of the metal ions and/or precipitation of the metals intracellularly, thus, forming the basis of the synthesis of nanoparticles [14]. However, the actual mechanism for the biosynthesis of GNPs by different microorganisms and their stabilization via charge capping is still not well understood. It was shown in one of the earlier studies that the possible mechanism of biosynthesis of silver nanoparticles might involve the reduction of silver ions due to the electron shuttle enzymatic metal reduction process. The enzyme involved in the synthesis of silver nanoparticles may be nitrate reductase present in microorganism, which may be induced by the nitrate ions and reduced silver ions to metallic silver [15]. Duran et al also suggested the possible mechanism of biosynthesis of silver nanoparticles by Fusarium oxysporum strains (fungi). They proposed the involvement of enzymatic electron shuttle relationship for the formation of Ag+ ions and the subsequent formation of silver nanoparticle [16]. Although there are reports on the biochemical steps involved in metallic nanoparticles synthesis by microorganisms [17-20], there are virtually no reports available which may elucidate enzymatic basis of gold nanoparticle synthesis by Stenotrophomonas maltophilia. In this paper, we report the rapid synthesis of GNPs by Stenotrophomonas maltophilia (Acc No GQ220749), a novel bacterial strain isolated from soil samples from the Singhbhum gold mines (located in the Jharkhand state of India). A possible mechanism of biosynthesis of gold nanoparticles from gold chloride (HAuCl4) involves the role of specific NADPH-dependent reductase enzyme present in the organism that converts Au3+ to Au0 through electron shuttle enzymatic metal reduction. Results and discussion Isolation and characterization of strain capable of synthesisizing gold nanoparticles Pure colonies isolated from the gold enriched soil were characterized for their morphological and physiological characteristics by various biochemical tests using the Bergeys Manual of Determinative Bacteriology [21] as summarized in Table 1. The screened strain is aerobic, motile, mucoid and yellow in Nutrient Agar medium (Himedia Labs, India). SEM micrographs revealed that the cells of strain AuRed02 are oval shaped, with discrete lipopolysaccharides layers on its surface causing the bacteria to glue to each other as evident from SEM imaging (Fig. 1). No production of acid was noted from the selected carbohydrates such as adonitol, arabinose, fructose, galactose, inositol, inulin, lactose, mannitol, mannose, rhamnose, sorbitol and xylose. The isolated strain named as AuRed02 produced acid from maltose, dextrose and trehalose, showing typical characteristics of Stenotrophomonas maltophilia. The strain gave a positive lipolytic activity and was able to utilize citrate as a carbon source. Table 1 Biochemical tests of the isolated strain Stenotrophomonas maltophila AuRed02 Gram staining Negative Catalase test Positive Citrate test Positive Oxidase test Negative Glucose o/f test Negative Methyl red test Negative Gelatin test Positive Carbohydrate utilization tests Adonitol Negative Arabinose Negative Cellobiose Positive Dextrose Positive Fructose Negative Galactose Negative Inositol Negative Inulin Negative Lactose Negative Maltose Positive Mannitol Negative Mannose Negative Rhamnose Negative Salicin Positive Sorbitol Negative Sucrose Positive Trehalose Positive Xylose Negative Figure 1 SEM image of Stenotrophomonas maltophilia cells. Scale bar corresponds to 1 μm. The cells were imaged after reaction with gold chloride solution for 8 hrs. The 16S rDNA sequence analysis revealed that strain AuRed02 clustered homologue being Pseudomonas Sp. (Acc. No. FJ211222). At species level, strain AuRed02 shows the highest level of sequence similarity with Stenotrophomonas maltophilia strain: BL-15 (99%) (AB194325) as shown in phylogenetic tree (Fig. 2). Figure 2 Phylogenetic tree made in MEGA 3.1 software using Neighbor joining method. Characterization of gold nanoparticles synthesized by Stenotrophomonas maltophilia A solution of gold chloride in a suspension of cell mass of Stenotrophomonas maltophilia changed progressively from light yellow to cherry red at temperature 25°C showing formation of gold nanoparticles. Control experiments, without the addition of biomass as well as with heat-killed cells, showed no change in color of suspension (Fig. 3a) confirming the formation of GNPs in the presence of biomass only. The kinetics of the reaction was studied using UV-vis spectroscopy by recording spectra from the colloidal gold solutions obtained after disrupting the cells, which were resuspended with HAuCl4 solution for different time intervals. The spectra revealed a strong absorption at nearly 530 nm after 8 hrs of incubation at 25°C, gradually showing a red shift with time at 25°C (Fig 4 curves bottom to top). The intense plasmon resonance band indicated the formation of spherical gold nanoparticles of approximately 40 nm in diameter [18]. Size distribution of GNPs was confirmed by coating a drop of the supernatant solution of disrupted cell suspension by TEM imaging. The images demonstrate gold nanoparticles possessing an average diameter of 40 nm (± 15%) as depicted in (Fig. 3b). Cryo TEM imaging of the thin sections of stained AuRed02 strain after reacting the biomass with gold chloride solution for 8 h showed the presence of gold nanoparticles on the inner cytoplasmic membrane (see Additional file 1, S1). It is likely that some gold ions (Au3+) cross the cell barrier through ion-transport channel and are reduced by the enzymes present on the cytoplasmic membrane and within the cytoplasm. Energy dispersive spectroscopy analysis (EDS) confirmed the presence of gold nanoparticles in the suspension. The spectra (Fig. 3c) present major Au peaks at approximately 2 keV besides several other peaks of C, O, and Si which might be due to the chemical composition of the sample substrate used in the EDS analysis. These results show that the strain Stenotrophomonas maltophilia could effectively synthesize GNPs of different sizes by resuspending the biomass for different time intervals in the presence of HAuCl4 (Fig. 4). The colored solution of GNPs remained stable for more than 2-weeks of storage at 4°C indicating the capping of GNPs with some charged groups. FTIR analysis of these GNPs further confirmed the capping of GNPs by phosphate groups (900 cm-1) (see Additional file 1, S2). Figure 3 (A) GNPs synthesised by Stenotrophomonas maltophilia (tube A). Control experiments: gold chloride incubated without the biomass (tube B) and with heat-killed cell mass (tube C). (B) TEM image recorded from a drop-coated film of disrupted cell suspension. Scale bar corresponds to 200 nm. (C) Energy Dispersive Spectra of synthesized GNPs showing Au peaks as major constituent. Figure 4 UV-vis spectra of GNPs solutions prepared by resuspending the biomass of Stenotrophomonas maltophilia for different time intervals in the presence of 1 mM HAuCl4. Promising mechanism aspects of biosynthesis by Stenotrophomonas maltophilia In microorganisms, the possible mechanisms of resistance against metal ions usually involve biosorption, bioaccumulation, extra-cellular complexation, efflux system, alteration of solubility and toxicity via reduction or oxidation [22]. Our study suggested that the biosynthesis of GNPs and their stabilization via charge capping in Stenotrophomonas maltophilia involved NADPH-dependent reductase enzyme that converts Au3+ to Au0 through electron shuttle enzymatic metal reduction process. For further confirmation, biomass was incubated with varying concentrations of NADPH (from 0.05 mM to 0.8 mM NADPH) and change in color of solution was monitored spectrophotometrically (Fig 5a) and visually (Fig. 5b). Control experiments, without the addition of either cell free extract (C1) or cell free extract without NADPH (C2), showed no change in color of suspension. However, addition of NADPH in the cell free extract at varying concentrations (C3 to C7) showed the synthesis of GNPs with gradual increase in color intensity (Fig. 5b). This confirms the formation of GNPs only in the presence of both biomass and NADPH. Zeta potential measurements of the GNPs showed a peak at -16.7 mV (see Additional file 1, S3), suggesting capping of GNPs by negatively charged phosphate ions from NADP. Based on these experimental findings, a schematic representation of the potential mechanism of gold nanoparticles synthesis by Stenotrophomonas maltophilia through enzymatic reduction is proposed (Fig. 6). The enzyme involved in the synthesis of metal nanoparticles may be a specific reductase present in microorganism, which may be induced by the specific ions and reduced metal ions to metallic nanoparticles. Figure 5 (A) UV-vis spectra of GNPs synthesis by adding different concentrations of NADPH in the solution of suspended biomass along with HAuCl4 (C3 to C7) and (B) shows the GNPs synthesis by adding different concentrations of NADPH in the solution of suspended biomass along with HAuCl4 (tubes C3 to C7). In controls (C1 and C2), either cell mass (C1) or NADPH (C2) was not added. Figure 6 Proposed synthesis mechanism of GNPs by Stenotrophomonas maltophilia through enzymatic reduction. Conclusion The process of synthesis of well-dispersed nanoparticles using a highly efficient microorganism Stenotrophomonas maltophilia has been reported in this study leading to the development of an easy bioprocess for synthesis of GNPs of desired size and shape. The results presented demonstrate that a specific NADPH-dependent enzyme present in the isolated strain reduces Au3+ to Au0 through an electron shuttling mechanism leading to the synthesis of nearly monodispersed GNPs. This green route of biosynthesis of GNPs is a simple, economically viable and an eco-friendly process. Methods Isolation and characterization of bacteria from the gold enriched soil Soil samples from the gold enriched sites near famous Singhbhum gold mines, Jharkhand state, India were used as inoculum, serially diluted and plated onto Nutrient Agar media (Himedia, India). The plates were incubated at 30°C for 24 hrs. The colonies obtained were further subcultured on Nutrient Agar supplemented with 1 mM HAuCl4 (Sigma, India) and incubated at 30°C for 24 hrs. In one of the isolates, the light yellow color of HAuCl4 changed to wine red indicating that the organism was utilizing HAuCl4 for the synthesis of GNPs. The morphological and physiological characterization of the selected isolate was carried out by biochemical tests using the Bergeys Manual of Determinative Bacteriology [21]. The cell morphology was investigated using Zeiuss-EVO 40 scanning electron microscope (SEM). Further characterization of isolate was done by means of 16S rRNA gene analyses [23]. The 16S rDNA sequencing was done by M/s Bangalore Genei, India. To obtain stable gold nanoparticles, the conditions of the characterized isolate were optimized for different concentrations of gold chloride, production media, temperature, pH and time. The characterized isolate was inoculated into 50 ml sterile Nutrient Broth (NB) and subsequently 1 gm of wet biomass was harvested after 16 hrs of incubation. The biomass obtained was washed thrice with deionised water (pH 7.0) and added to a 100 ml Erlenmeyer flask containing 1 mM gold chloride solution prepared in de-ionized water spiked with 400 mg Yeast Extract. The Erlenmeyer flasks were incubated at 25°C for 8 hrs to isolate the nearly monodisperse spherical gold nanoparticles. To isolate the pure GNPs, cells were disrupted using 0.2% (v/v) Triton X-100. The disrupted samples were centrifuged at 3500 rpm for 15 min at 4°C and washed thrice with deionized water to remove cell-debris. The supernatant was used for the characterization of GNPs. Biosynthesis of gold nanoparticles and their characterization To determine the peak time-point of maximum gold nanoparticles synthesis, the absorption spectra of the supernatants were taken at different time intervals and recorded using a Hitachi U2800 spectrophotometer. TEM studies were carried out using Jeol 2100 microscope operating at 120 kV accelerating voltage. Samples were prepared by placing a drop of GNPs solutions on carbon-coated TEM grids. The films on the TEM grids were allowed to dry for 5 min at room temperature before analysis. Energy dispersive spectroscopy analysis (EDS) was carried out to confirm the synthesis of gold nanoparticles using FEI E-SEM Quanta 200. Particle size and charge distribution (zeta potential) was analyzed using dynamic light scattering system (Beckman Coulter, USA) by illuminating the colloidal gold solution with He-Ne Laser (633 nm) in a sample cell. Competing interests The authors declare that they have no competing interests. Authors' contributions YN and NG carried out the isolation and characterization work of bacterial isolate described in the paper. NW took part in the characterization of gold nanoparticles and described the mechanism aspects of biosynthesis. GS did all TEM analysis and CRS coordinated the research as well as the manuscript preparation. All authors read and approved the final manuscript. Supplementary Material Additional file 1 S1: Cryo TEM imaging of the thin sections of stained AuRed02 strain; S2: FTIR analysis of GNPs; S3: Zeta potential measurements of the GNPs. Click here for file Acknowledgements This work was funded by a grant from the joint Indo-Russia Integrated Long Term Programme (ILTP), which we gratefully acknowledge. Authors gratefully acknowledge Mr Kumar Rajesh for necessary technical support. ==== Refs Mirkin CA Letsinger RL Mucic RC Storhoff JJ A DNA-based method for rationally assembling nanoparticles into macroscopic materials Nature (London) 1996 382 607 609 8757129 10.1038/382607a0 Jana NR Gearheart C Murphy CJ Wet Chemical Synthesis of High Aspect Ratio Cylindrical Gold Nanorods J Phys Chem B 2001 105 4065 4067 10.1021/jp0107964 Xiao Y Patolsky F Katz E Hainfeld JF Willner I Plugging into Enzymes": Nanowiring of Redox Enzymes by a Gold Nanoparticle Science 2003 299 1877 1881 12649477 10.1126/science.1080664 Thomas M Klibanov AM Conjugation of gold nanoparticles enhances poly ethylenimine's tranfer of plasmid DNA into mamalian cells Proc Natl Acad Sci USA 2003 100 9138 9143 12886020 10.1073/pnas.1233634100 Salata OV Application of nanoparticles in biology and medicine J Nanobiotechnol 2004 2 3 9 10.1186/1477-3155-2-3 Mandal S Elvakannan PR Sumant P Pasricha R Sastry M Synthesis of a stable gold hydrosol by the reduction of chloroaurate ions by the amino acid, aspartic acid Proc Indian Acad Sci (Chem Sci) 2002 114 513 520 10.1007/BF02704195 Turkevich J Stevenson PC Hillier J A study of the nucleation and growth processes in the synthesis of colloidal gold Disc Farad Trans 1951 11 55 75 Zhou X Khoury JME Qu L Dai L Li Q A facile synthesis of aliphatic thiol surfactant with tunable length as a stabilizer of gold nanoparticles in organic solvents J Colloid Interface Sci 2007 308 381 384 17291518 10.1016/j.jcis.2007.01.040 Ahmad A Senapati S Khan MI Ramani R Sriniwas V Sastry M Intracellular synthesis of gold nanoparticles by a novel alkalotolerant actinomycete Rhodococcus species Nanotechnology 2003 14 824 828 10.1088/0957-4484/14/7/323 Senapati S Ahmad A Khan MI Sastry M Kumar R Extracellular Biosynthesis of Bimetallic Au-Ag Alloy Nanoparticles Small 2005 1 517 520 17193479 10.1002/smll.200400053 Mukherjee P Roy M Mandal BP Dey GK Mukherjee PK Ghatak J Tyagi AK Kale SP Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungus T. asperellum Nanotechnology 2008 19 75103 10.1088/0957-4484/19/7/075103 Pum D Sleytr UB The application of bacterial S-layers in molecular nanotechnology Trends Biotechnology 1999 17 8 12 10.1016/S0167-7799(98)01221-9 Silver S Bacterial resistances to toxic metal ions Gene 1996 179 9 19 8991852 10.1016/S0378-1119(96)00323-X Klaus T Joerger R Olsson E Granqvist CG Silver-based crystalline nanoparticles, microbially fabricated Proc Natl Acad Sci USA 1999 96 13611 13614 10570120 10.1073/pnas.96.24.13611 Kalimuthu K Babu RS Venkataraman D Bilal M Gurunathan S Biosynthesis of silver nanocrystals by Bacillus licheniformis Colloids and surfaces B 2008 65 150 153 10.1016/j.colsurfb.2008.02.018 Duran N Marcato PD Alves OL Souza G Esposito E Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains J Nanotechnology 2005 3 8 Mukherjee P Ahmad A Mandal D Senapati S Sainkar SR Khan MI Ramani R Parischa R Ajayakumar PV Alam M Kumar R Sastry M Fungus-Mediated Synthesis of Silver Nanoparticles and Their Immobilization in the Mycelial Matrix: A Novel Biological Approach to Nanoparticle Synthesis Nano Lett 2001 1 515 519 10.1021/nl0155274 Mukherjee P Senapati S Mandal D Ahmad A Khan MI Kumar R Sastry M Extracellular Synthesis of Gold Nanoparticles by the Fungus Fusarium oxysporum ChemBioChem 2002 3 461 463 12007181 10.1002/1439-7633(20020503)3:5<461::AID-CBIC461>3.0.CO;2-X Shahverdi AR Minaeian S Shahverdi HR Jamlifar H Nohi AA Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: A novel biological approach Process Biochem 2007 42 919 923 10.1016/j.procbio.2007.02.005 He S Guo Z Zhang Y Zhang S Wang J Gu N Biosynthesis of gold nanoparticles using the bacteria Rhodopseudomonas capsulata Materials Letters 2007 61 3984 3987 10.1016/j.matlet.2007.01.018 Holt JG Krieg RN Sneath PHA Staley JT Williams ST Bergey's Manual of Determinative Bacteriology 1994 9 Williams and Wilkins, Baltimore Husseiny MI Abd El-Aziz M Badr Y Mahmoud MA Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa Spectrochimica acta A 2007 67 1003 1006 10.1016/j.saa.2006.09.028 Woese CR Gutell R Gupta R Noller HF Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids Microbiol Rev 1983 47 621 669 6363901	19619318	PMC2719591	CC BY	2021-01-04 17:40:48	yes	Microb Cell Fact. 2009 Jul 20; 8:39
==== Front Indian J UrolIJUIndian Journal of Urology : IJU : Journal of the Urological Society of India0970-15911998-3824Medknow Publications India 19718288IJU-23-35810.4103/0970-1591.36704Original ArticleA comparative study of intratumoral chemotherapy in advanced childhood common solid tumors Rahi Rajeev Vijyendra K. Sharma S. P. Aryya N. C. #Shukla R. C. ^Pradhan S. §Singh T. B. †Gangopadhyay A. N. Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, India Department of Paediatric Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, India# Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, India^ Department of Radiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, India§ Department of Radiotherapy and Medical Oncology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, India† Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, IndiaFor correspondence: AN Gangopadhyaya, Department of Pediatric Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 211 005, India. E-mail: [email protected] 2007 23 4 358 365 © Indian Journal of Urology2007This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Advanced and inoperable solid tumors in children are great killer despite aggressive multimodality treatment. Intravenous chemotherapy, due to high dose of drug given systemically, at times leads to abandonment of therapy due to systemic toxicities. To overcome this problem lots of studies are going on to explore alternative modes of giving anticancer drugs so as to decrease the systemic toxicities of the drugs and increase their therapeutic index at the same time. Aim: The study was conducted to know the results of anterior intratumoral chemotherapy and its comparison to anterior intravenous chemotherapy. Materials and Methods: Forty patients of advanced inoperable solid tumors in children (Wilms' tumor and neuroblastoma) between 2000-2004 were randomly allocated to two groups. Group A (20 patients) was given intratumoral chemotherapy while Group B (20 patients) was given intravenous chemotherapy. Both the groups were compared in terms of reduction in size and volume, resectability of tumor, histopathological changes and side-effects of chemotherapeutic drugs. The Institute's ethics committee approved this study. Results: Males were predominant in both type of cases (Wilms' tumor and neuroblastoma) in both the groups (Group A and Group B). Mean age in the study was 3.27 years. All cases in Group A had Stage III disease except three cases which had Stage IV disease (one case of Wilms' tumor and two cases of neuroblastoma) while in Group B only two cases had Stage IV disease (one case of Wilms' tumor and one case of neuroblastoma). Intratumoral chemotherapy was found to be superior over intravenous chemotherapy in terms of reduction of size and volume (63% in Group A vs. 22% in Group B). The resectability was 70% in the intratumoral group in comparison to 40% in the intravenous group. The overall good histopathological response was 71% in Group A as opposed to 0% in Group B. Moreover, the incidence and severity of side-effects of chemotherapy and morbidity was less in intratumoral chemotherapy. Mortality was also low in Group A (5%) in comparison to Group B (20%). Conclusion: In this study intratumoral chemotherapy was found to be superior over intravenous chemotherapy in terms of better and early tumor regression, minimal side-effects, better tumor resectability and well response on histopathological criteria. This study is still going on at our center where different drug combinations, different drug doses, their toxicities, their mechanisms of action, their serum levels and long-term results of intratumoral mode of chemotherapy are to be evaluated thoroughly in future. Advanced solid tumorsintratumoral chemotherapyintravenous chemotherapyneuroblastomaWilms' tumor ==== Body Advanced and inoperable solid tumors in children are a great killer despite aggressive multimodality treatment. The most common solid tumors in children under 15 years of age are neuroblastoma, Wilms' tumor, lymphoma and rhabdomyosarcoma. These account for 28.1% of all cancer in children and their incidence is 7.3%, 6.1%, 11.3% and 3.4% respectively.[12] Besides lymphoma, Wilms' tumor and neuroblastoma comprise more than 80% of total childhood solid malignancies, so we concentrated our study only on Wilms' tumor and neuroblastoma. In our setup, due to illiteracy and lower socioeconomic status, cases of Wilms' tumor and neuroblastoma present in the advanced stages and are inoperable at the time of initial presentation. Though there have been many advancements with the collaboration of pediatric oncologist, surgeon and radiation therapist the prognosis for advanced solid pediatric malignancies still remains poor.[3–5] There is striking improvement in the survival of malignancies after the incorporation of anterior chemotherapy into the treatment regimens that previously relied only on surgery, radiotherapy to control local as well as systemic disease along with nutritional and hematological support.[6] Though intravenous chemotherapy has remained the conventional mode of anticancer treatment for years, the concentration of the drugs reaching the target tumor cells is less while systemic toxicities are more because the drugs remain in the circulation for a longer period in higher concentration. So to decrease the morbidity and mortality related to neoadjuvant chemotherapy, several authors explored an alternative mode of giving anticancer drugs. Some improvement in survival has been noted by alternating effective groups of chemotherapeutic agents to overcome or prevent resistance and by continuous infusion rather than bolus administration of drugs.[7–8] Similarly, alternative routes of administration of chemotherapeutic agents have been tried in advanced solid tumors but mostly for palliation. In 1976, intraarterial transcatheter occlusion of abdominal tumor was tried by Goldstein et al.[9] Intraarterial chemotherapy has been tried in hepatocellular carcinoma, advanced pancreatic, breast and liver secondaries with colorectal carcinoma.[10–18] Intraperitoneal chemotherapy has also shown promising antitumor effect on ovarian carcinoma, peritoneal carcinomatosis and advanced gastric carcinoma.[19–21] Livraghi T et al.,[22–23] reported fine needle percutaneous intratumoral chemotherapy under USG guidance on 12 selected neoplastic adult patients not responding to conventional treatment. Partial or total pain control, stable disease and response was observed in 60% of patients. They stated that intratumoural chemotherapy could be an alternative treatment for tumor unresponsive to conventional chemotherapy. As most of the cancer patients in our setup are in advanced stage, debilitated and their tolerance to systemic chemotherapy is very poor. Intraarterial and intraperitoneal chemotherapy, however, have better response and less systemic toxicities, but these routes require high skill, elaborate setup and are much more expensive than conventional intravenous route which is associated with a high morbidity rate. So to overcome this problem our study was conducted to evolve a better and safer alternative modality of treatment in advanced inoperable common pediatric solid malignancies which is most promising, technically simple, has much less systemic drug concentration as compared to that of systemic route so as to achieve minimal systemic toxicity and better tumor response to the chemotherapy. MATERIALS AND METHODS The study was conducted in the Department of Pediatric Surgery with cooperation of the department of radiology, radiotherapy and medical oncology and pathology in a University Hospital. The period of study was from July 2000 to June 2004. Forty patients of advanced inoperable solid tumors (nephroblastoma and neuroblastoma) not amenable to surgical excision primarily were randomly allocated to two groups (Group A and B) after confirming the diagnosis by FNAC. Group A consisting of 20 patients (Wilms' tumor—13 and neuroblastoma—seven) were treated by intratumoral chemotherapy comprising vincristine, actinomycin D and adriamycin through 26G spinal needle under aseptic precautions and USG guidance in same doses and schedule as systemic chemotherapy as per standard schedule. Adriamycin and actinomycine D were given as single dose while vincristine was given weekly for six weeks (vincristine-1.5 mg/m2, adriamycin-50 mg/m2 and actinomycin D-45 μg/kg). Injection Hyaluronidase was added to the drugs to enhance its local distribution. Group B consisting of 20 patients (Wilms' tumor—14 and neuroblastoma—six) were treated by intravenous chemotherapy in same doses and schedule. The ethical body of the university permitted the study design. Informed consent was taken from parents after explaining the procedure and relevant investigations were done. All relevant investigations were repeated at every session of chemotherapy. All patients were evaluated by two senior consultants on the basis of clinical examination, sonographic and CT scan findings for the inoperability of the tumor. Volume of tumor under USG guidance was calculated from formula 0.523 × Product of three maximal perpendicular dimensions of the tumor Patients were evaluated before, during and after chemotherapy as per the set proforma. The symptoms and side-effects of drugs in both groups were noted. Supportive therapy was given in the form of whole blood, platelet concentrate and FFP as and when required. After removal of the tumor both the groups received subsequently intravenous chemotherapy as per standard schedule. Ten slides from each specimen were taken for detailed microscopic examination for comparing various histopathological changes occurring following chemotherapy in the two groups. Z-value was calculated, using the following equation, for statistical analysis. Method for calculating ‘z’ value for statistical analysis Z=P1-P2P1q1/n1+P2q2/n2(Where P1=the proportion in the first sample, P2=the proportion in the second sample, n1=Size of the first sample, n2=Size of the second sample, q1=1-P1 and q2=1-P2.) If calculated value of Z >1.96 then it is significant at 5% (P<0.05). RESULTS Patient characteristics There was overall male preponderance. Among the Wilms' tumor cases 62% were males in Group A and 64% in Group B. Among the neuroblastoma cases 71% were males in Group A and 67% in Group B. Among the Wilms' tumor cases 38% were present in the two to four years age group in Group A and 42% in Group B. Among neuroblastoma cases 57% were in the two to four years age in Group A and 50% in Group B. [Table 1] Table 1 Patient characteristics Group A Group B Wilms' tumor (%) Neuroblastoma (%) Wilms' tumor (%) Neuroblastoma (% Sex Male 8 (62) 5 (71) 9 (64) 4 (67) Female 5 (38) 2 (29) 5 (36) 2 (33) Age in years Up to 2 4 (31) 2 (29) 4 (29) 2 (33) 2–4 5 (38) 4 (57) 6 (42) 3 (50) Above 4 4 (31) 1 (14) 4 (29) 1 (17) Stage of the disease III 12 (92) 5 (71) 13 (93) 5 (83) IV 1 (8) 2 (29) 1 (7) 1 (17) Response to treatment [Table 2] Table 2 Treatment response Group A Group B Wilms' tumor (%) Neuroblastoma (%) Wilms' tumor (%) Neuroblastoma (% Reduction in volume of the tumor > 50% 9 (69) 3 (50) 3 (25) 4 (25) 25–50% 3 (23) 2 (33) 4 (33) 1 (25) <25% 1 (8) 1 (17) 5 (42) 2 (50) Resectability Resected 9 (75) 5 (83) 6 (60) 2 (50) Not resected 3 (25) 1 (17) 4 (40) 2 (50) Histological response Well responded 6 (67) 4 (80) 0 (0) 0 (0) Partially responded 2 (22) 1 (20) 4 (67) 1 (50) Not responded 1 (11) 0 (0) 2 (35) 1 (50) Reduction in size and volume Though there was initial apparent increase in tumor volume in the intratumoral group ultimately on comparing intratumoral to intravenous chemotherapy, we found intratumoral chemotherapy to be better in terms of reduction of size by clinical examination [Figure 1] and reduction of volume by USG [Figure 2] and down-staging of tumor to allow resectability. Figure 1 A Wilms' tumor case in Intratumoral Group Figure 2 Volume reduction after chemotherapy in both Wilms' tumor and Neuroblastoma Resectability and histological response The resected specimens of Wilms' tumor were examined histopathologically and divided into three groups on the basis of Zuppan criteria[24] and SIOP studies. Type I: Well responded group: Microscopic examination showed extensive necrosis, focal fibrosis and inflammatory cells which were mainly eosinophillic. There was also presence of hemorrhage, hyaline degeneration and some glands. No malignant cells could be detected in any section. Type II: Partially responded group: Microscopy revealed predominance of atrophic dilated glands with scanty blastemal/tumor cells. Angiomatous malformations were also seen in some sections Type III: Non-responders: Showed focal areas of mianly blastemal/tumor cells with few glands separated by dense fibrocollagenous tissue. There were no inflammatory cells, necrosis or hemorrhage seen. Similarly, based on the observations of Matsuoka et al., SIOP XXXth meeting, resected specimens of neuroblastoma were divided into three groups depending on the ratio of the area of viable immature tumors (rosette-fibrillary and round cell types) to that of total tumor tissue, including histologically modified tissues such as hemorrhage, fibrosis, necrosis and changes in differentiation status, usually from less differentiated to well-differentiated subtypes of tumors. In nine out of 13 patients (69%) of Wilms' tumor in Group A, surgical resection was possible after intratumoral chemotherapy. Six (67%) of them showed well response on histopathological criteria while two (22 %) responded partially and the remaining one (11%) had no response at all in Group A; similarly, in five patients of neuroblastoma out of seven (72%) in Group A surgical resection was possible after completion of intratumoral chemotherapy. Of which four of them (80%) responded well and one patient (20%) had partial response [Figures 3, 4]. Figure 3 Resectability and outcome in two modalities of chemotherapy Figure 4 Histological response in two modalities of chemotherapy The overall well response on histopathological criteria was 71% in Group A and nil in Group B. ‘z’ value 5.90 was statistically significant [Figure 5]. Figure 5 Overall histopathological response in two modalities 3. Side-effects of chemotherapy Patients in both the groups were also evaluated for the side-effects of chemotherapeutic drugs during the course of study. There was local pain and features of colitis in the intratumoral group, which subsided within two to three days and were well controlled with standard analgesics and symptomatic treatment, but the overall incidence and intensity of side-effects related to anticancer drugs, was low in Group A patients. Moreover, the requirement of blood transfusion was also low in Group A in comparison to Group B [Table 3]. The decrease in nausea, alopecia and leucopenia was statistically significant among Wilms' tumor cases [Table 4]. Table 3 Side-effects of chemotherapeutic drugs Group A Group B Wilms' tumor (%) Neuroblastoma (%) Wilms' tumor (%) Neuroblastoma (% Skin and Mucous Membrane 0 9 (70) 5 (71) 8 (57) 0 (0) I 2 (15) 2 (29) 1 (7) 4 (66) II 2 (15) 0 (0) 2 (14) 1 (17) III 0 (0) 0 (0) 3 (21) 1 (17) Nausea and Vomiting 0 7 (55) 4 (57) 0 (0) 1 (17) I 2 (15) 1 (14) 4 (29) 1 (17) II 4 (30) 3 (29) 6 (43) 2 (33) III 0 (0) 0 (0) 4 (29) 2 (33) Leucopenia 0 6 (46) 2 (29) 4 (29) 0 (0) I 6 (46) 3 (42) 4 (29) 4 (67) II 1 (18) 2 (29) 2 (13) 1 (17) III 0 (0) 0 (0) 4 (29) 1 (17) Fever 0 9 (69) 3 (43) 4 (29) 1 (17) I 1 (8) 3 (43) 5 (36) 2 (33) II 3 (23) 1 (14) 3 (21) 2 (33) III 0 (0) 0 (0) 2 (14) 1 (17) Alopecia 0 8 (62) 0 (0) 0 (0) 0 (0) I 3 (23) 3 (43) 5 (36) 2 (33.33) II 2 (15) 2 (29) 2 (14) 2 (33.33) III 0 (0) 2 (29) 7 (50) 2 (33.33) Loss of appetite 0 11 (85) 6 (86) 2 (41) 1 (17) I 2 (15) 1 (14) 10 (71) 3 (66) II 0 (0) 0 (0) 1 (7) 1 (17) III 0 (0) 0 (0) 1 (7) 0 (0) Requirement for blood transfusion (anemia) 0 8 (62) 1 (14) 2 (14) 0 (0) I 3 (23) 3 (43) 6 (43) 1 (17) II 1 (8) 2 (29) 2 (14) 2 (33) III 1 (8) 1 (14) 4 (29) 3 (50) Local pain 0 10 (77) 6 (86) 8 (57) 4 (67) I 1 (8) 1 (14) 4 (29) 1 (17) II 2 (15) 0 (0) 2 (14) 1 (17) Colitis 0 12 (92) 6 (86) 0 (0) 0 (0) I 1 (8) 1 (14) 0 (0) 0 (0) II 0 (0) 0 (0) 0 (0) 0 (0) Sterile pus 0 12 (92) 0 (0) 0 (0) 0 (0) I 1 (8) 0 (0) 0 (0) 0 (0) II 0 (0) 0 (0) 0 (0) 0 (0) 0 to III is the grade of toxicity depending on its severity Table 4 Side-effects of chemotherapeutic drugs Wilms' tumor Z value Neuroblastoma Z value Group A Group B (P value) Group A Group B (P value) No. % No. % No. % No. % Nausea 4 31 10 71 2.307 (<0.05) 3 43 4 67 0.888 Leukopenia (<4000/cmm) 1 8 6 43 2.378 (<0.05) 2 29 2 33 0.183 Alopecia 2 15 9 64 3.037 (<0.01) 4 57 4 67 0.358 Local pain 2 15 - 1.54 1 14 - 1.083 Colitis 1 8 - 0.94 1 14 - 1.083 Sterile pus 1 8 - 1.085 - - - Fever 3 23 5 36 0.728 1 14 3 50 1.457 Loss of appetite - - 2 14 1.521 1 17 1.099 Blood transfusion 2 15 6 43 1.683 3 43 5 83 1.676 Stomatitis/mouth ulcer + skin rashes 2 15 5 36 1.261 - - 2 33 1.734 DISCUSSION Advanced and inoperable solid tumors in children are difficult to manage in spite of advances in cancer research, mainly because of advanced disease process leading to poor general condition and intolerance to multimodal therapy. The international society of pediatric oncology (SIOP) has promoted the use of preoperative chemotherapy with or without radiotherapy to increase the resectability and to minimize the surgical complication rate.[2526] The response to conventional intravenous chemotherapy is not only varied in advanced and inoperable solid tumors but is associated with higher incidence and severity of side-effects. Moreover, the poor tolerance to conventional intravenous chemotherapy in already malnourished patients leads to the postponement of the chemotherapy at times which increases the morbidity and mortality related to the disease process. Intraarterial and intraperitoneal chemotherapy, however, have better response and less systemic toxicities, but these routes require high skill, elaborate setup and are very much expensive. It may seem inappropriate to group two pathologies together in the study but as morbidity and mortality related to neoadjuvant chemotherapy and surgery in these patients with advanced and inoperable intraabdominal solid tumors is very much at our center, we explored an alternative route of giving anticancer drugs which not only reduces the morbidity and mortality related to neoadjuvant chemotherapy and surgery but is also cheap and technically simple. So we selected Wilms' tumor and neuroblastoma case as in our setup cases of both of these tumors present not only in an advanced and inoperable state but have very high morbidity and mortality related to the neoadjuvant chemotherapy and surgery thereafter. In our study, the anticancer drugs were given directly into the tumor under USG guidance to decrease the stage and make them operable. While giving anticancer drug directly into the tumor, we used USG/ Doppler probe so that the drug was given in depot form (making it sure that the drug does not enter the systemic circulation or does not extravasate out of the tumor). After this the drug is absorbed in the circulation slowly and in small amounts which takes care of the systemic metastasis. But the maximum concentration of the drugs remains at the site of the lesion without bulk load in the systemic circulation that not only raises the therapeutic index of the drugs and causes lesser systemic toxicities in advanced disease but also produces better and earlier tumor regression as compared to intravenous chemotherapy. The improvement in the general condition of the patients, the regression in the size (clinically), regression in volume (ultrasonographically) and resectability (on CT scan) of the tumor were assessed after completion of one cycle of chemotherapy (six weeks). In those cases where tumor was not amenable to surgical excision, the chemotherapy cycle was repeated after a gap of two weeks up to maximum of three cycles before surgery. A few cases required three cycles before surgery. The difference in number between the two arms for >50% reduction in volume was statistically significant [Figure 2]. So intratumoral chemotherapy was used not only to increase the resectability of the tumor and to decrease the surgical complication rate thereafter but also to decrease the side-effects of the conventional intravenous neoadjuvant chemotherapy. After surgery both the groups received intravenous chemotherapy to complete the chemotherapy schedule as per the standard protocol. Patients in both the arms are being followed up till date. More then 90% of the patients are still in our follow-up and are doing well (no recurrence of metastasis till date). Duration of follow-up is 36-60 months. Seventy per cent cases in Group A could be successfully excised after one course (six weeks) of intratumoral chemotherapy in comparison to only 40% cases after intravenous chemotherapy (Group B). Unlike cases with intravenous chemotherapy, those receiving intratumoral chemotherapy did not have much adhesion with the surrounding tissue, suggesting that the needle injections (26G spinal needle) of chemotherapeutic drugs under USG guidance do not extravasate out of the tumor and it is a safe method. It was observed during surgery that neovascularization and edema was significantly less in the intratumoral group as compared to the intravenous group, maybe due to the high concentration of chemotherapeutic drugs acting on target tumor cells (adriamycin, actinomycin D and vincristine used in our study do not require hepatic metabolization for its actions). All cases in Group A had Stage III disease except three cases which had Stage IV disease (one case of Wilms' tumor and two cases of neuroblastoma) while in Group B only two cases had Stage IV disease (one case of Wilms' tumor and one case of neuroblastoma). The cases in Group A who presented with metastasis e.g. tumor thrombus in inferior vena cava, secondary deposits in paraaortic nodes or supraclavicular lymph nodes and liver also responded well with disappearance of metastatic deposits (clinically, radiologically and histologically) at the end of six cycles (three cycles preoperatively and three cycles postoperatively) of chemotherapy. Thus intratumor chemotherapy has not only acted locally but systemically also.(The hypothesis behind this is that the drug, via the microcirculation of the tumor, is absorbed in systemic circulation slowly and in small amounts which takes care of the systemic metastasis as well.) Although one case of advanced neuroblastoma in Group A expired during intratumor chemotherapy because of poor general condition due to extensive primary disease, it had started responding satisfactorily in terms of regression of size and volume of the tumor. The mortality was 5% as compared to 20% in Group B which is statistically significant. Out of these two modalities of anterior chemotherapy in advanced inoperable pediatric solid tumors, 71% cases showed well response on histopathology in the intratumoral group (Group A) in comparison to none in the intravenous group (Group B). However, 21% in Group A and 63% in Group B showed partial response while 37% of the intravenous chemotherapy group (Group B) showed no response as compared to 8% of the intratumoral group (Group A). Histopathological response is highly significant statistically in well responders [Figure 3]. The study on intratumoral anterior chemotherapy in advanced pediatric solid tumors was conducted as a pilot study. No reference till now is available in the literature. In this study a fixed regimen of chemotherapy comprising vincristine, adriamycin and actinomycin D was given intratumorally in advanced stage disease in two types of tumors and the results were assessed clinically, sonographically and histopathologically. Although intravenous anterior chemotherapy has been the conventional form of treatment for advanced stage tumors, it is toxic in children due to high dose of drugs given systemically which most of the time leads to abandonment of therapy. In this regards, intratumoral chemotherapy is superior over intravenous chemotherapy in terms of better and early tumor regression, minimal side-effects, better tumor resectability and well response on histopathological criteria. Patients in both the arms are still on follow-up but it will not be the right time to comment on long-term results of the intratumoral mode of chemotherapy. All patients who are candidates for neoadjuvant conventional intravenous chemotherapy can receive intratumoral chemotherapy but the patients who will get the maximum benefit of the therapy are those who are poor, those who are undernourished (e.g. low hemoglobin and low serum protein), those having poor general condition due to systemic spread of the disease, those having very large unresectable intraabdominal tumor (on CT scan) and those who have poorly differentiated tumor on biopsy. In case of local skin disease/disorder and bleeding disorders, intratumoral chemotherapy should be used after correction/treatment of the disorder. Patients having multiple systemic metastases (> 4) should receive intravenous chemotherapy along with the hematological and nutritional support in the beginning to take care of systemic disease and this should be followed by intratumoral chemotherapy to make the large bulky tumor resectable so as to decrease the surgical complication rate. CONCLUSION This study is still going on in our center where different drug combinations, different drug doses, their toxicities, their mechanisms of action, serum levels of different drugs and long-term results of intratumoral mode of chemotherapy are to be evaluated. Till now we have found intratumoral chemotherapy was surperior over intravenous chemotherapy in terms of better and early tumor resectability and well response on histopathological criteria. It was found statistically that morbidity and mortality in the intratumoral group was less as compared to the intravenous group. Source of Support: Nil Conflict of Interest: None declared. ==== Refs REFERENCES 1 Parker SL Tong T Bolden S Cancer statistics, 1996 Cancer J Clin 1996 65 5 27 2 Gurney JG Severson RK Davis S Robison LL Incidence of cancer in children in the Untied States Cancer 1995 75 2186 95 7697611 3 Schwann MR Blattner SR Lynch E Weinstein HJ Hic-Com: A 2 month intensive chemotherapy regimen for children with stage III and IV Burktt's lymphoma and B-cell acute lymphoblastic leukemia J Clin Oncol 1991 9 133 8 1985162 4 Cheung NK Heller G Chemotherapy dose intensity correlates strongly with response, median survival and median progression free survival in metastatic neuroblastoma J Clin Oncol 1991 9 1050 8 2033419 5 Antman K Ayash L Elias A Wheeler C Hunt M Eder JP A phase II study of high dose cyclophosphamide, thiotepa and carboplatin with autologous marrow support in women with measurable advanced breast cancer responding to standard therapy J Clin Oncol 1992 10 102 10 1727912 6 Hammond GD Keynote address. 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==== Front J Exp Clin Cancer ResJournal of Experimental & Clinical Cancer Research : CR0392-90781756-9966BioMed Central 1756-9966-28-1021960768910.1186/1756-9966-28-102ResearchSubstance P and beta-endorphin mediate electro-acupuncture induced analgesia in mouse cancer pain model Lee Hyo-Jeong [email protected] Jae-Ho [email protected] Eun-Ok [email protected] Hyo-Jung [email protected] Kwan-Hyun [email protected] Sun-Hyung [email protected] Keun-Sung [email protected] Hee-Jae [email protected] Sung-Hoon [email protected] College of Oriental Medicine, Kyung-Hee University, Seoul 130-701, South Korea2 Medical Center, Kyung-Hee University, Seoul 130-701, South Korea2009 16 7 2009 28 1 102 102 16 4 2009 16 7 2009 Copyright © 2009 Lee et al; licensee BioMed Central Ltd.2009Lee et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Opioid analgesics are generally used to combat the pain associated with cancerous conditions. These agents not only inhibit respiratory function and cause constipation, but also induce other significant side effects such as addiction and tolerance, all of which further contribute to a reduced quality of life for cancer patients. Thus, in the present study, the effects of electro-acupuncture treatment (EA) on mechanical allodynia were examined in a cancer pain mouse model. Methods In order to produce a neuropathic cancer pain model, S-180 sarcoma cells were inoculated around the sciatic nerve of left legs of Balb/c mice. Magnetic Resonance Imaging (MRI) scanning confirmed the mass of S-180 cancer cells embedded around the sciatic nerve. Mechanical allodynia was most consistently induced in the mouse sarcoma cell line S-180 (2 × 106sarcoma cells)-treated group compared to all the other groups studied. EA stimulation (2 Hz) was administered daily to ST36 (Zusanli) of S-180 bearing mice for 30 min for 9 days after S-180 inoculation. Results EA treatment significantly prolonged paw withdrawal latency from 5 days after inoculation. It also shortened the cumulative lifting duration from 7 days after inoculation, compared to the tumor control. Also, the overexpression of pain peptide substance P in the dorsal horn of the spinal cord was significantly decreased in the EA-treated group compared to the tumor control on Day 9 post inoculation. Furthermore, EA treatment effectively increased the concentration of β-endorphin in blood and brain samples of the mice to a greater extent than that of the tumor control as well as the normal group. The concentration of β-endorphin for EA treatment group increased by 51.457% in the blood and 12.6% in the brain respectively, compared to the tumor control group. Conclusion The findings of this study suggest that a S-180 cancer pain model is useful as a consistent and short time animal model. It also indicated that EA treatment could be used as an alternative therapeutic method for cancer pain due to a consequent decrease in substance P and increase in β-endorphin levels. ==== Body Background Pain is a frequent problem in cancer patients. The analgesic ladder for cancer-related pain provided by the WHO involves progressing from non-opioid (e.g., acetaminophen, ibuprofen), weak opioid (e.g., codeine), and finally to strong opioid (e.g., morphine, fentanyl) intervention for pain relief [1]. Some studies have been reported that opioid switching therapy reduced side effects and produced a reduction in pain level [2-4]. But, unfortunately, opioid analgesics often produce poor pain relief against neuropathic cancer pain and also induce adverse side effects such as hormone (e.g., ACTH, cortisol, LH and testosterone) secretion, neurotransmitter (e.g., nicotine, adenosine, GABA and cholecystokinine) release, feeding, gastrointestinal motility, and respiratory activity [5]. Thus, safe and effective complementary therapies for cancer pain have recently been suggested [5-7]. Generally, of the three types of somatic, visceral and neuropathic cancer pain conditions, neuropathic pain is the most predominant in cancer patients due to compression or infiltration of peripheral nerves by malignant tumors [6,8]. Neuropathic pain resulting from nerve injury is characterized by spontaneous pain, allodynia (the perception of normally innocuous stimuli as painful) and hyperalgesia (an increased sensitivity to painful stimuli). However, an animal model for neuropathic cancer pain still remains unclear regarding cancer cell and animal type. Although acupuncture has a long history, its scientific evaluation has only begun rather recently. Acupuncture treatment or electro-acupuncture has been applied to treat a wide range of symptoms, with some success. Electro-acupuncture at acupoint [9]ST36 has been reported to relieve pain and reduce inflammation and cerebral ischemia [10,11]. Early scientific work on manual and electrical stimulation on ST36 was carried out by many researchers [12-16]. The aim of the present study was to evaluate the effects of electro-acupuncture treatment on mechanical allodynia in a mouse model of neuropathic cancer pain, using S-180 sarcoma cells. The analgesic mechanism of this procedure was elucidated in the dorsal horn of the spinal cord of mice using immunohistochemistry for substance P and enzyme immunoassay (EIA) for β-endorphin in blood and brain of mice. Methods Animals Male BALB/c mice weighing 25–30 g were purchased from Daehan Bio Link. The animals were maintained under laboratory conditions of temperature, humidity, and light. Mice were maintained on a 12:12 h dark-light cycle with food and water ad libitum. The animal protocols were approved by an institutional Animal care and use committee at Kyung Hee University. Cell Culture S-180 sarcoma cells (ATCC CCL-8) were grown in Dulbecco's Modified Eagle Medium (DMEM;Gibco BRL, Grand Island, NY) with 100 mL/L heat inactivated (30 min at 56°C) fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C in 50 mL/L CO2. First Experiment Neuropathic Cancer Pain Model To determine the optimal number of S-180 cells that could induce a neuropathic cancer pain model, three different cell numbers (1 × 107(n = 3), 5 × 106(n = 3), and 2 × 106(n = 3)) of S-180 cancer cells were inoculated into the muscular tissue in the immediate vicinity of the nerve near the trochanter, immediately distal to where the posterior biceps semitendinosus branches off the common sciatic nerve. Thereafter, neuropathic cancer pain was comparatively monitored in S-180 treated groups. MRI Scanning MRI scanning was performed to confirm the presence of the tumor mass around the sciatic nerve by anatomical examination. On days 10, 16 and 24 after inoculation, the mice from each group were sacrificed and scanned around the sciatic nerve by MRI. 2nd Experiment Neuropathic Cancer Pain Model Based the number of cells required to create a successful neuropathic cancer pain model, 2 × 106 S-180 cancer cells were inoculated into the muscular tissue in the immediate vicinity of the nerve near the trochanter, immediately distal to where the posterior biceps semitendinosus branches off the common sciatic nerve (Fig. 1A and 1B). Figure 1 A: Experimental scheme for EA treatment in a neuropathic cancer pain model, B: Neruopathic cancer pain model. EA Treatment EA treatment was applied to the EA group only. A stainless steel needle with 0.3 mm diameter was inserted at a depth of 5 mm into the unilateral acupuncture point ST36 (Zusanli) located 0.5 cm below the fibular head of the hinder leg in mice and stimulated with an intensity of 2 Hz (<3 mA) for 30 min daily. The levels of EA treatment were based on values previously reported [10,17]. The proximal end was soldered to a wire that was connected to one of the output channels of an electric stimulator, PG-306 (YoungMok, Japan). As shown Fig. 3, the ST36 (Zusanli) acupoint was located 5 mm below and lateral to the anterior tubercle of the tibia. Electrical stimulation was applied to ST36 point using two outlets via two needles. An electrical pulse with a voltage of 3–5 V, a duration of 0.25 ms and a frequency of 2 Hz was delivered from an EA stimulator. The intensity of stimulation was determined to be minimum voltage to cause moderate muscle contraction. Behavioral Test (Mechanical von Frey test) During a behaviour test, all mice were divided into three groups including a tumor control group (n = 8), EA-treated group (n = 8) and normal group (n = 8). All mice were placed on a wire mesh platform that was fixed in a transparent plexiglass chamber (20 × 10 × 5 cm). This study was performed based on a modified protocol [17]. Behaviour assessment was performed on days 1, 3, 5, 7 and 9 after tumor inoculation. A series of von Frey hairs was applied from below the wire mesh platform to the plantar surface of the left hind paw. The hind paw withdrawal threshold was determined using von Frey hairs weighing from 0.4 g to 4 g. Behavioural tests using von Frey hair on the hind paw of mice were carried out five times in 5 s intervals. A withdrawal response was considered valid only if the hind paw was completely removed from the wire mesh platform. Spontaneous Pain Test The mice from all three groups were observed for signs of mechanical allodynia as spontaneous pain on days 3, 5, 7 and 9 after tumor inoculation. A spontaneous pain test was performed in all the animals placed in a clear plastic chamber with wire grid floors at room temperature. After approximately 1 h acclimatization, the cumulative duration of hind paw-lifting of each mouse was analyzed for 10 min. The test consisted of evoking a hind paw flexion reflex with a hand-held force transducer (electronic anaesthesiometer, IITC Life science, Woodland Hills, CA, USA) adapted with a 0.5 mm2 polypylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the hind paw with a gradual increase in pressure. The end point was characterized by withdrawal of the paw followed by clear lifting and flinching behaviour in the animal. The lifting of the paw as part of grooming behaviour was not taken into account. Immunohistochemistry The specimens of spinal cord dorsal horn of mice were sectioned on a cryostat as 40 μm coronal sections between L3-L5. The sectioned tissues were rinsed in phosphate buffered saline (PBS) with Tween 20 (PBST) about 3 times before use. PBST contains 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4. For immunoassays, the primary antibody was diluted with blocking solution (Vector Laboratories, Burlingame, CA) and tissues were incubated with antibodies against substance P (Abcam Ltd., Cambridge, UK) in a 1:50 ratio, for 48 h at room temperature, with constant agitation. After rinsing in PBS, the sections were incubated for 2 h with the biotinylated rabbit anti-serum (Vector Laboratories, Burlingame, CA) that was diluted to 1:200 in PBST containing 1% normal goat serum. The sections were placed in the Vectastatin™ Elite ABC reagent (Vector Lab., UK) for 1 h. After further rinsing in PBS, the tissues were developed using diaminobenzadine as a chromogen with nickel intensification. These slides were air-dried, cover-slipped and then observed under a light microscope (Carl Zeiss, Germany). Enzyme Immunoassay Blood samples (1 mL) were collected into lavender vacutainer tubes containing EDTA. The tubes were gently rocked several times immediately after collection of blood for anti-coagulation. Blood was transferred from the lavender vacutainer tubes to centrifuge tubes containing aprotinin (0.6 TIU/mL of blood) and gently rocked several times to inhibit proteinase activity. The blood was centrifuged at 1,600 × g for 15 min at 4°C and the plasma was collected. Brain tissues were ground using a Teflon Homogenizer in 2 mL lysis buffer (10 mM Tris-Hcl, pH 7.4) and centrifuged at 12,000 × g for 15 min at 4°C and the supernatant was collected. Plasma and brain samples were stored at -20°C prior to EIAs and then warmed up to 4°C before analysis. The samples were acidified with an equal volume of buffer A (250 μL), centrifuged at 17,000 × g for 20 min at 4°C and equilibrated using SEP-COLUMN (CA, USA) containing 200 mg of C18 (Code RK-SEPCOL-1) by washing once with buffer B (1 mL) followed by three washes with buffer A (3 mL). The acidified plasma solution was added to the pre-treated C-18 SEP-COLUMN. The column was slowly washed with buffer A (3 mL, twice). The peptide was slowly eluted with buffer B (3 mL, once), collected into a polystyrene tube and evaporated to dryness. The levels of β-endorphin were measured using a direct β-endorphin EIA kit from Phoenix Pharmaceuticals (CA, USA). Statistical analysis The data were presented as means ± SD or SE. Student's t test was used for von Frey hair test and a one-way analysis of variance (ANOVA) test was also conducted for immunohistochemistry and β-endorphin assay. Results Morphological changes of S-180 tumor mass around sciatic nerve and induction of neuropathic cancer pain As shown in Fig. 2A, S-180 cells grow rapidly and embedded around the sciatic nerve in a time-dependent manner, which was confirmed by MRI scanning. On day 9 after inoculation, the sciatic nerve was partially embedded by an S-180 tumor mass and on day 24, the sciatic nerve was almost surrounded by the S-180 tumor mass. As shown in Fig. 2B, among the three groups studied (1 × 107, 5 × 106 and 2 × 106 injected groups), neuropathic cancer pain was most steadily induced in 2 × 106 injected group 2 days after inoculation, suggesting that the suitable cell number that induced neuropathic cancer pain was 2 × 106. Figure 2 A: MRI scans of S-180 tumor mass around the sciatic nerve. After inoculation of S-180 tumor cells around the sciatic nerve, MRI scan was performed. (a) On inoculation day (b) 10 days after inoculation (c) 16 days after inoculation (d) 24 days after inoculation. B: S-180 implantation around sciatic nerve-induced neuropathic cancer pain according to cell number in a time course study. Withdrawal latency of left hind paws was measured every 2 days until 17 days after inoculation. Values are expressed means ± SE. Statistically significant differences were recorded after comparison to the control using the student's t test (* p < 0.05, ** p < 0.01). Effect of EA treatment on neuropathic cancer pain As shown in Fig. 3A, EA treatment significantly attenuated paw lifting latency induced 3 days after inoculation by the von Frey test. As shown in Fig. 3B, hind paw-lifting in the tumor control group became apparent when compared to the normal group from day 5 after tumor inoculation and the cumulative paw-lifting duration reached a peak on day 9 where all the mice in the tumor control group showed a slight foot drop in the left hind limb. On the contrary, EA treatment significantly reduced cumulative lifting duration compared to the untreated tumor control group. Figure 3 A: EA treatment increased paw withdrawal latency compared to that of the untreated tumor control. Paw withdrawal latency was measured every 2 days until 9 days after inoculation. Statistically significant differences were obtained, in comparison to the normal control group using the student's t test (* p < 0.05). B: EA treatment reduced cumulative lifting duration of paw compared to untreated tumor control. Cumulative lifting duration of the left hind paws was measured every 2 days until 9 days after inoculation. Statistically significant differences were compared to the normal group using the student's t test (* p < 0.05). Effect of EA treatment on substance P and β-endorphin Nine days after inoculation, immunohistochemistry was performed using antibodies against substance P, in sections of spinal cord dorsal horn of mice. As shown in Fig. 4A, substance P was overexpressed in the tumor control group compared to that of the normal control, suggesting that the tumor mass could activate neuropathic pain-related proteins. On the contrary, EA treatment for 9 days effectively reduced the expression of pain peptide substance P in the dorsal horn section of the spinal cord of mice compared to the untreated tumor control group. To elucidate its analgesic mechanism, the levels of β-endorphin in blood and brain tissues of mice were analyzed after EA treatment. As shown in Fig. 4B, the level of β-endorphin in blood samples of the tumor control group was significantly increased up to 2.8754 ± 0.0278 ng/mL compared to that of the normal group, 1.3236 ± 0.0041. On the contrary, EA treatment significantly increased the β-endorphin levels up to 4.355 ± 0.2972 ng/mL more than the tumor control group, 2.8754 ± 0.0278 ng/mL. Consistently, as shown in Fig. 4C, the level of β-endorphin in the brain tissues of mice within the tumor control group was significantly increased up to 4.0115 ± 0.3848 ng/mL compared to that of the normal group, 2.668 ± 1.069 ng/mL. In contrast, EA treatment significantly increased the level of β-endorphin up to 9.0847 ± 0.5901 ng/mL more than that of the tumor control group, 4.0115 ± 0.3848 ng/mL. Figure 4 A: Representative photographs of a coronal section showing SP expression in the spinal cord. Photographs (200 ×) illustrate SP immunoreactive neurons in the mouse superficial dorsal horn (SDH) of L3–5 levels. (a) Control, (b) Tumor control, (c) EA treated group. Arrows indicate SP positive cells. B&C: EA treatment increased the level of β-endorphin in blood and brain compared to untreated tumor control. B: level of β-endorphin in blood C: level of β-endorphin in brain. Values of β-endorphin are expressed as means ± SE. Different superscripts(a, b, c) indicate p < 0.05 statistical significance between groups using ANOVA test-Turkey's procedure. Discussion Pain is an important symptom in cancer patients. The prevalence of pain depends on tumor type and varies from 5% in patients with leukemia to 52% in patients with lung cancer. The causes of pain are the tumor itself by bone invasion, compression of the spinal cord or neural structures and pressure on hollow organs [6]. Thus, in the current study, we set up a neuropathic cancer mouse model by inoculation of S-180 tumor cells around the sciatic nerve of mice tumor mass. MRI scanning revealed the tumor size and position around sciatic nerve of mice. Ten days after inoculation, the tumor mass was shown to surround half the area around the sciatic nerve while 24 days after inoculation, the S-180 tumor cells embedded most of the gluteal area, inducing neuropathic pain by compression of the sciatic nerve [18]. A behavioural test using von Frey hairs showed that a tumor mass of S-180 cells significantly induced paw hind lifting from 3 days after inoculation and prolonged cumulative lifting duration as a spontaneous pain 5–9 days after inoculation, suggesting that the neuropathic cancer pain mouse model was successfully set up for cancer pain assessment. In contrast, Shimoyama's cancer model that was produced by inoculation of Meth-A sarcoma cells to the vicinity of the sciatic nerve [19] showed that hind paw-lifting, a behavioural sign of spontaneous pain, was at a maximum on day 18 after inoculation of Meth-A sarcoma cells to the vicinity of the sciatic nerve. Therefore, our cancer pain model may induce neuropathic cancer pain more rapidly and consistently within ten days after S-180 cell inoculation compared to Shimoyama's cancer model. These data strongly suggest that our cancer model can be applied for evaluation of in vivo cancer pain control efficacy within a short time. To confirm the roles of pain-related peptides during acupuncture-induced analgesia, immunohistochemical analysis for substance P and enzyme immunoassay for β-endorphin in blood and brain samples of mice were performed in the spinal cord dorsal horn of mice. Substance P is a neuropeptide involved in the transmission of pain impulses from the peripheral receptors to the central nervous system. It belongs to the tachykinin neuropeptide family [20]. EA treatment downregulated the expression of substance P [21], while substance P was overexpressed in the dorsal horn of the tumor control group 9 days after inoculation [22,23]. Endorphins are endogenous opioid polypeptides released in the pituitary gland and the hypothalamus during strenuous exercise and excitement. Although the role of plasma β-endorphin in pain regulation is unclear, these molecules have been reported to correlate inversely with pain levels in cancer pain [24]. In the current study, β-endorphin levels were unexpectedly released twice as much in the blood and brain samples of the tumor control animals than in the normal group. The β-endorphin that is released into the blood cannot enter the brain in large quantities because of the blood-brain barrier [8]. On the contrary, EA treatment significantly increased β-endorphin levels compared to that of the tumor control group. These data support involvement of the endorphin system in the neuropathic cancer pain model presented in this study. In summary, a mass of S-180 cancer cells was embedded around the sciatic nerve as shown by time course MRI scanning. Mechanical allodynia was most consistently induced in the S-180 (2 × 106)-treated group among all the groups studied. In contrast, EA treatment significantly prolonged the paw withdrawal latency and shortened the cumulative lifting duration compared to the S-180 tumor control group. In addition, the overexpression of pain peptide substance P in the dorsal horn of the spinal cord was significantly decreased in the EA-treated group compared to the S-180 tumor control group, 9 days after inoculation. Furthermore, EA treatment effectively increased the concentration of β-endorphin in the blood and brain of mice compared to the S-180 tumor control group. Conclusion The findings in the current study suggest that a S-180 cancer pain model can be a consistent and short time animal model and EA treatment also can be used as an alternative therapeutic method for cancer pain via decrease of substance P and increase of β-endorphin. Competing interests The authors declare that they have no competing interests. Authors' contributions HJL collected the data and drafted the manuscript, SHK designed this study and modified the manuscript, JHL, EOL, HJL, KHK, KSL, and DWN participated in its design and coordination. All authors read and approved the final manuscript. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1973073409-PONE-RA-09308R210.1371/journal.pone.0006898Research ArticlePlant Biology/Agricultural BiotechnologyPlant Biology/Plant Biochemistry and PhysiologyPlant Biology/Plant Growth and DevelopmentPlant Biology/Plant-Environment InteractionsSoybean Trihelix Transcription Factors GmGT-2A and GmGT-2B Improve Plant Tolerance to Abiotic Stresses in Transgenic Arabidopsis GT Factors in Stress ToleranceXie Zong-Ming 1 2 3 Zou Hong-Feng 1 2 Lei Gang 1 2 Wei Wei 1 2 Zhou Qi-Yun 1 2 Niu Can-Fang 1 2 Liao Yong 1 2 Tian Ai-Guo 1 Ma Biao 1 Zhang Wan-Ke 1 Zhang Jin-Song 1 * Chen Shou-Yi 1 * 1 National Key Laboratory of Plant Genomics, Plant Gene Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 2 Graduate School of Chinese Academy of Sciences, Beijing, China 3 Xinjiang Academy of Agricultural and Reclamation Science, Shihezi, China El-Shemy Hany A. EditorCairo University, Egypt* E-mail: [email protected] (JSZ); [email protected] (SYC)Conceived and designed the experiments: ZMX HFZ JSZ SYC. Performed the experiments: ZMX HFZ GL WW QYZ CFN YL AGT. Analyzed the data: ZMX HFZ GL WW QYZ CFN YL AGT BM WKZ JSZ SYC. Contributed reagents/materials/analysis tools: GL WW BM WKZ JSZ SYC. Wrote the paper: ZMX HFZ JSZ SYC. 2009 4 9 2009 4 9 e689819 3 2009 13 8 2009 Xie et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Trihelix transcription factors play important roles in light-regulated responses and other developmental processes. However, their functions in abiotic stress response are largely unclear. In this study, we identified two trihelix transcription factor genes GmGT-2A and GmGT-2B from soybean and further characterized their roles in abiotic stress tolerance. Findings Both genes can be induced by various abiotic stresses, and the encoded proteins were localized in nuclear region. In yeast assay, GmGT-2B but not GmGT-2A exhibits ability of transcriptional activation and dimerization. The N-terminal peptide of 153 residues in GmGT-2B was the minimal activation domain and the middle region between the two trihelices mediated the dimerization of the GmGT-2B. Transactivation activity of the GmGT-2B was also confirmed in plant cells. DNA binding analysis using yeast one-hybrid assay revealed that GmGT-2A could bind to GT-1bx, GT-2bx, mGT-2bx-2 and D1 whereas GmGT-2B could bind to the latter three elements. Overexpression of the GmGT-2A and GmGT-2B improved plant tolerance to salt, freezing and drought stress in transgenic Arabidopsis plants. Moreover, GmGT-2B-transgenic plants had more green seedlings compared to Col-0 under ABA treatment. Many stress-responsive genes were altered in GmGT-2A- and GmGT-2B-transgenic plants. Conclusion These results indicate that GmGT-2A and GmGT-2B confer stress tolerance through regulation of a common set of genes and specific sets of genes. GmGT-2B also affects ABA sensitivity. ==== Body Introduction Transcriptional regulation of gene expression plays a primary role in plant development and in environmental stimuli responses. Expressions of the stress-responsive effector genes are largely controlled by several classes of transcription factors, such as members of the MYB, ERF/AP2, bZIP, WRKY and NAC families, through binding of the corresponding cis-acting elements [1]–[7]. The potential for improving plant tolerance by engineering of stress-regulated transcription factors is highlighted recently [8]. Several classes of transcription factors such as AP2/ERF, DOF, YABBY and Trihelix families are unique to plant so far [9]–[14], suggesting that they may be implicated in plant-specific gene tuning [15]. Members of Trihelix family, also known as GT factors (DNA binding proteins with specificity for GT-elements), are among the first transcription factors identified in plants [16]. GT elements are highly degenerated and the deduced consensus core sequence is 5′-G-Pu-(T/A)-A-A-(T/A)-3′ [14], and are involved in a wide array of plant biological processes. GT elements were first identified in the pea rbcS-3A gene promoter as a light-responsive element named Box II/GT1 box (5′-GTGTGGTTAATATG-3′) [17], [18] and later in many promoters of other genes, some of which were not responsive for light [14]. For instance, a GT element named Site1, found in the ribosomal protein gene rps1 promoter, has been shown to repress transcription in non-photosynthetic tissues or cells [19], [20]. Box II-related/GT-1 like elements found in the promoter region of soybean chs gene and Pr-1A gene from tobacco are likely responsive to elicitor treatments and pathogen infection [21], [22]. The pathogen- and NaCl- induced soybean SCaM-4 gene contains GT-1 like element in the promoter region [23]. Rice GT-2 and tobacco GT1a/B2F were the first two nuclear proteins identified via affinity screening using GT2 sequence and Box II sequence [24]–[26]. Since then, more members of GT factor (trihelix transcriptional factor) family have been isolated from Arabidopsis, pea, soybean and rice [16], [27]–[32]. The trihelix transcriptional factor family has 28 members in Arabidopsis genome [33], 22 members in rice genome [34] and putatively 13 members in soybean genome [11], and is defined based on the highly conserved trihelix domain (helix-loop-helix-loop-helix). Members of trihelix family can be divided into three subgroups that bind to functionally distinct types of GT elements. GT-1-type factors contain only a single trihelix domain that is responsible for specific binding to the Box II core sequence, whereas GT-2-type factors contain twin trihelix domains with the N-terminal one preferentially binding to GT3-bx (5′-GAGGTAAATCCGCGA-3′) and the C-terminal one to GT2-bx(5′-GCGGTAATTAA-3′) [29], [30]. Although AtGT-3a and AtGT-3b are similar to GT-1 in structure that containing one single trihelix motif, both of them bind specifically to GT-3a site (core sequence 5′-GTTAC, i.e. Site 1), not to Box II, GT2-bx or GT3-bx in vitro, suggesting that they belong to a third subgroup of GT factors [27]. It is generally believed that trihelix factors are involved in the regulation of light-responsive genes [14], [35]. The expression of all of the trihelix factors cloned thus far appears to be ubiquitous and independent of light, except that AtGTL1 mRNA is more abundant in siliques, and soybean GmGT-2 and rice rml1 (rice gene regulated by Magnaporthe grisea and light) were down-regulated by light [24]–[26], [29], [30], [32]. Arabidopsis PETAL LOSS (PTL) gene encoding a GT-2-type factor is the first member of trihelix family known to control morphogenesis [28]. A rice Shattering 1(SHA1) gene encoding a GT-1-type factor plays an important role in activation of cell separation, and a mutation in the trihelix domain resulted in the elimination of seed shattering in cultivated rice [31]. More recently, ASIL1, belonging to a new subfamily of the trihelix transcription factors, has been found to function as a negative regulator of a large subset of Arabidopsis embryonic and seed maturation genes in seedlings [36]. Although the roles of the trihelix factors are gradually disclosed, the regulatory function of this kind of transcription factors in abiotic stress response remains largely unknown. In a previous work, we have identified 13 putative trihelix genes in soybean [11]. From these, two abiotic stress-upregulated genes encoding putative GT-2-type proteins (named GmGT-2A and GmGT-2B, respectively) were cloned from soybean in this study. Both GmGT-2A and GmGT-2B overexpression in Arabidopsis plants increased plant tolerance to abiotic stresses. The downstream genes regulated by GmGT-2A and GmGT-2B were also investigated. GmGT-2A and GmGT-2B may represent the first two members of trihelix family that are responsible for the stress tolerance in transgenic Arabidopsis plants through regulation of downstream genes. Results Gene cloning and structural analysis of the trihelix family genes GmGT-2A and GmGT-2B Among thirteen ESTs that belong to trihelix family genes from soybean, two were identified to be responsive to various abiotic stresses. The full-length coding regions of the two corresponding genes were further obtained using RACE method. Because both of them encoded proteins similar to those from the GT-2 group of the trihelix family [36], we named the two genes as GmGT-2A (EF221753) and GmGT-2B (EF221754) respectively and further analyzed. SMART analysis revealed two trihelix domains in both the GmGT-2A and GmGT-2B proteins (Fig. 1A, C). Between these two trihelix domains, a coiled-coil region of 31 residues was noted in GmGT-2A. Four putative nuclear localization signals (NLS) were identified in GmGT-2A (Fig. 1C). However, only three NLSs were found in GmGT-2B and the second NLS appeared not present in this protein (Fig. 1C). It should be noted that an asparagine-rich region was found in the middle of the C-terminal trihelix domains of the GmGT-2A and GmGT-2B but not in other proteins compared (Fig. 1C). This feature possibly suggests a potentially specific function for this region in regulation of the transcription factor activity in soybean. 10.1371/journal.pone.0006898.g001Figure 1 Schematic representation and amino acid sequence alignment of the GmGT-2A and GmGT-2B. (A) Schematic diagram of the GmGT-2A and GmGT-2B. (B) Cluster analysis of the GmGT-2A and GmGT-2B with other GT-2 group factors. The analysis was performed by using the MEGA 4.0 program with neighbor joining method and with 1000 replicates. Numbers on the figure are bootstrap values. The sequences are from soybean and Arabidopsis plants [36]. (C) Multiple alignments of the amino acid sequences from various GT factors. NLS indicates putative nuclear localization signal. Accession numbers are as follows: GmGT-2 (AF372498), GmGT-2A(EF221753), GmGT-2B(EF221754). Arabidopsis gene codes are as in [36]. The amino acid sequences of the GmGT-2A and GmGT-2B were compared with those from other homologous proteins (Fig. 1C). At the whole amino acid level, the GmGT-2A showed 39% identity with the GmGT-2 (O'Grady et al., 2001), but ∼25% identity with GmGT-2B and the other two proteins compared. GmGT-2B showed 22% identity with GmGT-2 and similar identity with other proteins. For the N- and C-terminal trihelix domains, GmGT-2A had 74% and 67% identity with those of GmGT-2, and 40% and 53% identity with those of GmGT-2B. GmGT-2B had 35% and 56% identity with those of GmGT-2. These results indicate that the GmGT-2A is more closely related to GmGT-2 but not the GmGT-2B. Cluster analysis also supports this conclusion (Fig. 1B). In addition, the soybean GmGT-2A, GmGT-2B and GmGT-2 are closely related to the GT-2 group of Arabidopsis but not clustered with other group members from Arabidopsis (Fig. 1B)[36]. GmGT-2A and GmGT-2B gene expressions in different soybean organs and in response to abiotic stresses The expressions of the GmGT-2A and GmGT-2B were examined in different organs of the soybean plants. The results in Figure 2A showed that the GmGT-2A was expressed in a higher level in stems than those in others organs tested, and no expression of this gene was detected in soybean seeds. For the GmGT-2B, its expression was higher in pods but lower in other organs examined. Similarly, the GmGT-2B showed no expression in soybean seeds. 10.1371/journal.pone.0006898.g002Figure 2 Expressions of the GmGT-2A and GmGT-2B. (A) Gene expressions in different organs of soybean plants. RT-PCR method was used and tubulin gene was amplified as a control. (B) Expressions of the GmGT-2A and GmGT-2B in response to ABA and stress treatments revealed by Northern analysis. GmGT-2 expression was also compared. (C) Subcellular localization of the GmGT-2A and GmGT-2B as revealed by GFP fusion proteins. Green fluorescence indicates location of the GFP control or the GFP fusion proteins. Red fluorescence indicates positions of chloroplasts. The soybean seedlings were treated with various stresses and the GmGT-2A and GmGT-2B gene expressions were investigated. Both gene expressions can be enhanced by ABA, cold, drought and salt treatments (Fig. 2B). However, the expression patterns were different. The GmGT-2A transcripts accumulated to a higher level at 12 h after initiation of the treatments with ABA, cold or drought stress, whereas the GmGT-2B expression had peak levels at 1 h and/or 3 h after the three treatments. The upregulation of GmGT-2A expression after 12-h ABA treatment may be an indirect effect. At 12 h after these treatments, the GmGT-2B expression was apparently declined (Fig. 2B). Under salt stress, the GmGT-2A and GmGT-2B inductions were similar in patterns. These results suggest that the GmGT-2A and GmGT-2B may be involved in regulation of plant responses to abiotic stresses. Because the GT-2 gene showed similarity to the GT-2A and GT-2B, we further tested if this gene is responsive to stresses. However, after the four treatments, the GT-2 expression was not significantly altered, indicating that the gene may not be involved in abiotic stress responses (Fig. 2B). Subcellular localization of the GmGT-2A and GmGT-2B Because the GT-2A and GT-2B contained putative NLSs, we examined the subcellular localization of the two proteins. Each of the two genes was fused to the GFP and then transfected into Arabidopsis protoplasts to observe the localization of the GFP fusion proteins. The green fluorescence from GFP control was localized in both nuclear region and cytoplasm whereas the green fluorescence of the GmGT-2A-GFP and GmGT-2B-GFP fusion proteins was abundant in nuclear region of the protoplasts (Fig. 2C). The red fluorescence indicated the position of chloroplasts. These results indicate that the GmGT-2A and GmGT-2B are nuclear proteins. Transcriptional activation, dimerization and DNA binding analysis of the GmGT-2A and GmGT-2B Because the GmGT-2A and GmGT-2B belong to the transcription factors of trihelix family, we studied the transcription activation ability of the two proteins using the yeast assay system (Fig. 3A). Constructs, which harbored various versions of GmGT-2A and GmGT-2B (Fig. 3B), were made in pBD vector and transfected into yeast strain YRG-2. The yeast transformants were examined for their growth on selection medium (SD-His) due to activation of the reporter HIS3 gene, or determined for their β-galactosidase activity due to the activation of the reporter LacZ gene (Fig. 3A). Full-length of the GmGT-2A [GT-2A(FL)] did not had any transcriptional activation ability. Its N-terminal half [GT-2A(NT)] or C-terminal half [GT-2A(CT)] did not have the activity either (Fig. 3C). However, the full-length GmGT-2B had transcriptional activation activity, and its N-terminal half [GT-2B(NT)] also had the activity (Fig. 3C). The C-terminal half [GT-2B(CT)] did not had the transcriptional activation ability. The N-terminal half of the GmGT-2B was further truncated and the N-terminal peptide of 153 residues [GT-2B(N1-153)] were enough to have the activation ability. Further truncations including the GT-2B(N1-89), GT-2B-NGT(90-153) or the middle part of the GT-2B [GT-2B-M(150-424)] did not have any activation ability (Fig. 3C). 10.1371/journal.pone.0006898.g003Figure 3 Transcriptional activation, dimerization and DNA binding analysis. (A) Schematic diagrams for transcriptional activation (left panel) and dimerization (right panel) in yeast assay. (B) Different versions of GmGT-2A and GmGT-2B used for the analysis. FL: full-length protein. NT: N-terminal region. CT: C-terminal region. Numbers indicate amino acid positions. (C) Transcriptional activation activity of different versions of the GmGT-2A and GmGT-2B. Growth of transformants on SD-His and blue color in the presence of X-Gal indicate that the corresponding proteins have transcriptional activation ability. (D) Dimerization analysis of GmGT-2A or GmGT-2B. Growth of the transformants on SD/-Trp-Leu-His plus 10 mM 3-AT (SD-3) and presence of blue color indicate positive interactions. Each version of proteins in pBD vector plus pAD vector, or each version in pAD vector plus pBD vector served as negative controls. (E) Identification of domains in GmGT-2B for dimerization. Others are as in (D). (F) DNA binding analysis. Growth of the transformants on SD/-Trp-Leu-His plus 3-AT indicates that the corresponding proteins can bind to the cis-DNA elements. D1: 5′-catctacagttactagctagt-3′; GT-1bx: 5′-gtgtggttaatatg-3′; GT-2bx: 5′-tggcggtaattaactg-3′; mGT-2bx-1: 5′-tggctttaattaactg-3′; mGT-2bx-2: 5′-tggcgggcattaactg-3′; mGT-2bx-3: 5′-tggcggtacgtaactg-3′; mGT-2bx-4: 5′-tggcggtaattgcctg-3′; CaM-4: 5′-gatccgcgtag-3′; GT-3b: 5′-taagaaaaataa-3′. (G) Transcriptional activation assay in Arabidopsis protoplasts. GALDBD is a negative control and VP16 is a positive control for transactivation ability. The GmGT-2B has transactiation activity whereas the GmGT-2A does not have the activity. The dimerization ability of the two proteins was also analyzed and we find that the full-length protein of the GmGT-2B, the N-terminal half and the C-terminal half can form homodimers respectively (Fig. 3D). Other combinations did not produce any homo- or -herterodimers (Fig. 3D). Each version of proteins in pBD vector plus pAD vector, or each version in pAD vector plus pBD vector did not generate positive response (data not shown). Because the GmGT-2B(NT) can form dimer, we then examined which part is responsible for the dimerization. Figure 3E showed that the middle part GT-2B-M(150–424) can form dimers whereas the N-terminal trihelix NGT or C-terminal trihelix CGT themselves can't form dimers. Other combinations did not generate dimerizations. DNA-binding ability was investigated using yeast one-hybrid system. The effector plasmids harboring the GmGT-2A, GmGT-2B or their truncated forms were made in pAD vector, and the reporter plasmid was made by inserting four tandem repeats of various cis-DNA elements into the pHIS2, which contained a reporter gene HIS3. A minimal promoter was present downstream of the tested cis-DNA elements but upstream from the HIS3 gene. The effector plasmids and the reporter plasmids were transfected into yeast strain Y187 and growth of the transformants on SD/-Trp-Leu-His plus 3-AT indicates binding of the transcription factors to the corresponding cis-DNA elements. Figure 3F showed that the GmGT-2A, GmGT-2B, and their N-terminal or C-terminal truncated versions all can bind to the GT-2bx, the mGT-2bx-2, and the D1 element. However, the C-terminal region of GmGT-2A or GmGT-2B appeared to have low affinity for the D1 element. In addition, the GmGT-2A, its N-terminal and C-terminal region can bind to the GT-1bx (Fig. 3F). The GmGT-2A, GmGT-2B or their truncated versions could not bind to other elements tested. These results indicate that the GmGT-2A and GmGT-2B had common features in DNA binding and the GmGT-2A also had specialty in this ability. The transcriptional activation ability was further examined in Arabidopsis protoplast assay system. Effector plasmids containing the GmGT-2A or GmGT-2B, and a reporter plasmid containing a firefly luciferase (LUC) gene were co-transfected into Arabidopsis protoplasts and the relative LUC activity was determined. Figure 3G showed that the GmGT-2B protein activated higher level of reporter LUC activity than the negative control GALDBD. However, the GmGT-2A did not promote the reporter LUC activity. These results indicate that the GmGT-2B has transcriptional activation ability in the protoplast assay whereas the GmGT-2A does not have this ability. Seedling growth of the transgenic Arabidopsis plants overexpressing the GmGT-2A or GmGT-2B under ABA and osmotic stress To investigate the biological function of the GmGT-2A and GmGT-2B gene in plant, we transformed the two genes driven by the 35S promoter into Arabidopsis plants and homozygous transgenic lines with higher gene expressions were used for further analysis (Fig. 4A). Throughout the plant growth and developmental stages, no significant difference was observed for these transgenic lines in comparison with the wild type plants grown under normal condition. 10.1371/journal.pone.0006898.g004Figure 4 Morphogenesis of GmGT-2A- and GmGT-2B-transgenic seedlings under ABA and mannitol treatment. (A) Transgene expression in various transgenic lines as revealed by Northern analysis. (B) Seedling morphogenesis under ABA and mannitol treatment. (C) Green seedling rate in response to ABA. (D) Green seedling rate under mannitol treatment. For (C) and (D), each data point is average of three experiments (n = 150 for each experiment) and bars indicate SD. Asterisks indicate highly significant difference (P<0.01) from Col-0. Because the GmGT-2A and GmGT-2B expressions were upregulated by ABA and various abiotic stresses (Fig. 2B), we examined the effects of ABA and stress treatment on seed germination and seedling growth of the transgenic plants. Seed germination was not significantly affected by the ABA, mannitol and salt treatments when compared with the wild type plants (data not shown). However, the morphogenesis of seedlings was altered. Under treatments with both 0.5 µM and 0.75 µM ABA, the GmGT-2B-transgenic lines (L11 and L69) exhibited significantly higher green seedling rates (∼30% to ∼40%) when compared with the rates in wild type plants (∼9% to ∼16%) (Fig. 4B, C). Under the same ABA treatments, the GmGT-2A-transgenic lines (L19 and L27) did not show significant change in the green seedling rate in comparison with that in the wild type plants. These results indicate that the GmGT-2B can promote seedling morphogenesis in the presence of ABA, possibly through suppression of ABA function. The transgenic seeds were also germinated on medium with mannitol and the green seedling rates were not significantly changed in the GmGT-2A- and GmGT-2B-transgenic plants (∼70% to ∼77%) in comparison with the rate in wild type plants (71%) (Fig. 4B, D). These results indicate that overexpression of the GmGT-2B conferred reduced sensitivity to ABA in the transgenic plants. Effects of salt stress on the transgenic plants overexpressing the GmGT-2A or GmGT-2B Performance of the GmGT-2A-, GmGT-2B-transgenic plants under NaCl treatment was examined. Under normal condition, all the transgenic lines showed no significant difference when compared with the wild type plants in terms of the phenotype and the survival rate (Fig. 5A, B). Treatments with 75 mM or 125mM NaCl did not affect the survival of all the transgenic lines compared either (Fig. 5A; data not shown). Under 150 mM NaCl treatment, ∼12% of the wild type plants were dead whereas all the GmGT-2A- and GmGT-2B-transgenic plants survived (Fig. 5A, B). The survival rate was further reduced at the 180 mM NaCl treatment, with the wild type plants having a survival rate of around 70%. On the contrary, the GmGT-2A- and GmGT-2B-transgenic plants had a survival rate of more than 90% (Fig. 5A, B). 10.1371/journal.pone.0006898.g005Figure 5 Performance of the GmGT-2A- and GmGT-2B-transgenic plants under salt stress. (A) Growth of the transgenic seedlings in NaCl medium. (B) Survival rate of the salt-treated plants in (A). (C) Comparison of growth of the salt-treated plants in pots. Plants from treatments as in (A) were transferred in pots and the pictures were taken at 8 d and 21 d after transfer. (D) Survival rate of the salt-treated plants in pots from (C). For (B) and (D), each data point is average of three experiments (n = 30 for each experiment) and bars indicate SD. Asterisks indicate highly significant difference (P<0.01) from Col-0. The salt-stressed seedlings on plates (Fig. 5A) were further transferred in pots containing vermiculite and their recovery at 8 d and 21 d was observed. Treatments with 75 mM and 125 mM NaCl did not significantly affect recovery of all the compared plants (Fig. 5C). However, at 150 mM NaCl, the recovery rate was reduced to ∼50% for the wild type plants whereas this rate was more than 80% for all the transgenic lines. At 180 mM NaCl, the recovery for the wild type plants was ∼25%. For both the GmGT-2A- and GmGT-2B-transgenic plants, the recovery rates were more than 90% (Fig. 5C, D). All these results indicate that the GmGT-2A and GmGT-2B conferred plant tolerance to salt stress. The GmGT-2A and GmGT-2B confer drought and freezing tolerance in their transgenic Arabidopsis plants Because expression of the GmGT-2A and GmGT-2B genes can be induced under drought and cold treatments, we investigated if the performance of their transgenic plants was altered under these stresses. Plants (12-day-old) in pots were subjected to drought stress by withholding water for 16 d. After this stress, only ∼40% of the wild type plants survived whereas more than 90% of the GmGT-2A- and GmGT-2B-transgenic plants can survive and grow well (Fig. 6A, B). All the plants under normal condition grew normally (Fig. 6A). Plants that start to have siliques were further treated under drought stress by withholding water from plants for 16 d. The aerial parts of these plants were harvested and the dry weights of the transgenic plants were significantly higher than that of the wild type plants (Fig. 6C). These results indicate that the GmGT-2A and GmGT-2B confer drought tolerance to the transgenic plants. 10.1371/journal.pone.0006898.g006Figure 6 Performance of the GmGT-2A- and GmGT-2B-transgenic plants under drought and freezing stress. (A) Phenotype of the transgenic plants under drought stress. (B) Survival rate of the transgenic plants under drought stress. Each data point is average of three experiments (n = 30 for each experiment) and bars indicate SD. (C) Comparison of plant dry weight after drought stress. Bars indicate SD (n = 30). (D) Water loss in detached leaves from the transgenic plants. Bars indicate SD (n = 3). (E) Water contents in aerial part of the pot-grown plants after withholding water. Bars indicate SD (n = 8). (F) Survival of the transgenic plants under freezing treatment after aclimation or non-acclimation. (G) Survival rate of the transgenic plants after treatment in (F). Each data point is average of three experiments (n = 30 for each experiment) and bars indicate SD. For (B), (C), (E), (G), asterisks indicate a significant difference (P<0.05 and * P<0.01) from Col-0. Water loss represents another parameter for estimation of the plant tolerance to drought stress. Transgenic plants overexpressing the GmGT-2A or GmGT-2B all had reduced water loss in comparison with the wild type plants when detached leaves were used for the desiccation analysis (Fig. 6D). The whole plants (six-week-old) were also withheld from water for various times and the aerial parts were measured for water content. Figure 6E showed that, at day 8 and day 12 after the treatment, the water content was significantly higher in one GmGT-2A-transgenic line and two GmGT-2B-transgenic lines compared to the control plants, suggesting that these transgenic plants are tolerant to drought stress. Nonacclimated or cold-acclimated (5 d at 4°C) 12-day-old seedlings were exposed to freezing temperature for 6 h and then the plants were returned to normal conditions for evaluation of performance after 7-d recovery. All the transgenic lines showed a better growth than the WT control, and the acclimated transgenic plants had higher survival rate after freezing treatment at different temperatures when compared to the nonacclimated transgenic plants (Fig. 6F, G). Under normal condition, all the plants grew very well (Fig. 6F). In addition, the GmGT-2B-overexpressing plants had a higher survival rate than the GmGT-2A-overexpressing plants under both acclimated and nonacclimated condition (Fig. 6G). These results indicate that overexpression of both the GmGT-2A and GmGT-2B improved plant tolerance to freezing. GmGT-2A and GmGT-2B regulate expressions of stress-responsive genes The GmGT-2A and GmGT-2B may function in plant stress tolerance through regulation of downstream genes. We selected 33 stress-responsive genes for further quantitative real-time PCR analysis. Figure 7 showed that 17 genes including AZF1, MYB74, MYB75, PAD3, LTP3, STZ, MYB73, LTP4, At4g30650, UGT71B6, RHL41, COR13, MYB77, CYP707A3, LTI30, RCI3, and DREB2A were enhanced in all the transgenic lines harboring the GmGT-2A or GmGT-2B. The AZF1, STZ and RHL41/Zat12 encoded plant-specific transcription factors with Cys-2/His-2 zinc finger motifs, and can be induced by various stresses. Overexpression of the STZ, Zat10 and Zat12 increased stress tolerance [37]–[39]. The four MYB genes MYB73, MYB74, MYB75 and MYB77 can be induced by salt stress [40], [41]. The LTP3, LTP4, PAD3 and UGT71B6 were involved in ABA responses [42]–[44]. The At4g30650, COR13 and LTI30 were also involved in abiotic stress response [45], [46]. CYP707A3 encodes an ABA 8′-hydroxylase. The cyp707a3 mutant plants are hypersensitive to ABA and exhibited enhanced drought tolerance [47]. RCI3 encodes a peroxidase and overexpression of the gene conferred dehydration and salt tolerance [48]. DREB2A expression does not activate downstream genes under normal growth condition. However, overexpression of its constitutive active form leads to drought stress tolerance and slight freezing tolerance [49]. 10.1371/journal.pone.0006898.g007Figure 7 Altered gene expressions in GmGT-2A- and GmGT-2B-transgenic plants in comparison with the WT (Col-0). For each gene, two transgenic lines were used. The quantitative RT-PCR was used for the analysis. Totally 33 genes were examined for their expressions. Bars indicate SD (n = 4). Two genes MYB90 and At4g02200 were only highly expressed in the GmGT-2A-transgenic plants compared to the control and the GmGT-2B-transgenic plants. Eleven genes including MYB47, MYB15, PAD3, LTP3, LTP4, CBF2, PMZ, COR13, HVA22E, CBF1, and NCED3 were highly expressed in GmGT-2B-transgenic plants compared to the control plants and GmGT-2A-transgenic plants. These genes have been found to be responsive to various abiotic stresses and/or ABA response [41], [42], [50], [51]. The NCED genes have been found to be responsible for the biosynthesis of ABA precursor and are also involved in regulation of plant responses to abiotic stresses [52], [53]. Six genes including LHY1, MYB2, At5g02840, VAMP711, At3g09600, and CCA1 were downregulated in all the transgenic lines. The LHY1, CCA1, At5g02840 and MYB2 have been found to be responsive to stress and/or ABA [41], [54]. Transgenic plants overexpressing MYB2 had higher sensitivity to ABA and showed stress tolerance [55]. Suppression of the VAMP711, a gene encoding a protein for vesicle trafficking, in antisense transgenic plants improved salt tolerance [56].The gene PK1/PK6, which was induced by different stresses [57], was inhibited in the GmGT-2A-transgenic lines but promoted in the GmGT-2B-transgenic lines. These results indicate that GmGT-2A and GmGT-2B regulated a common set of genes as well as specific sets of genes for stress tolerance. Discussion Although roles of trihelix family transcription factors have been discovered in light-relevant and other developmental processes, their functions in abiotic stress response are largely unknown. In the present study, two trihelix family transcription factor genes GmGT-2A and GmGT-2B from soybean were identified to be stress-responsive and conferred stress-tolerance in transgenic Arabidopsis plants through regulation of downstream genes. This study adds the trihelix family members to those transcription factors that can improve plant stress-tolerance. The GmGT-2B exhibited transcriptional activation activity in both the yeast assay and the protoplast assay, whereas the GmGT-2A did not have this activity. This difference in transcriptional activation ability may result in the differential gene expressions in the transgenic plants, with more genes being highly or specifically expressed in the GmGT-2B-transgenic plants (Fig. 7). The activation domain of the GmGT-2B was also analyzed in detail and it appears that the N-terminal peptide of 153 residues is the minimal domain for transcriptional activation. Neither the N-terminal trihelix domain (90–153) nor its N-terminal sequence (1–89) themselves has the ability to activate transcription. The role of the minimal activation domain needs to be further tested in plant system. A rice GT-2 protein has also been found to function as a transcriptional activator. However, the activation domain was not identified [58]. Arabidopsis GT-1 also has transactivation function in both yeast and plant cells [59]. However, ASIL1 functions as a repressor for embryonic and seed maturation genes in Arabidopis seedlings [36]. The present GmGT-2A does not have transcriptional activation ability (Fig. 3C, G). Whether it has repression activity needs further study. The GmGT-2B, unlike the GmGT-2A, has the ability to dimerize, and the dimerization seems to happen through interaction of the middle part of the protein. No heterodimers could be formed between GmGT-2A and GmGT-2B. The biological significance of such interaction is not known. It is possible that the interaction would modify the transcriptional activation ability and then affect the downstream gene expressions. Arabidopsis GT-3a and GT-3b could form homo or heterodimers, and the dimerization domain seemed to be located at the C-terminus. However, no interaction was observed between each of the two proteins with the GT-1 [27]. The trihelix domain is generally believed to be the DNA-binding domain [29], [30]. In the yeast one-hybrid assay, both the N-terminal and C-terminal trihelix-domain-containing region of the GmGT-2A and GmGT-2B can bind to the three elements (Fig. 3F). In addition, the GmGT-2A can bind to the GT-1 bx whereas the GmGT-2B can't. These different features imply that the two genes may play some different roles in plant. However, since the two genes also have common features in gene expression and DNA-binding, they should also have similar functions in addition to their specific functions. Seed germination and seedling morphogenesis can be inhibited by ABA (Fig. 4). The germination rates of the GmGT-2A- and GmGT-2B-transgenic seeds were similar to that of the wild type seeds with ABA treatment (data not shown), indicating that the two genes did not affect ABA-regulated germination process. It is interesting to find that the morphogenesis of the GmGT-2B-transgenic seedlings but not the GmGT-2A-transgenic seedlings was less affected by ABA treatment. This fact suggests that the GmGT-2B-transgenic plants have reduced sensitivity to ABA, and GmGT-2B may function as a negative regulator to suppress the ABA effects on morphogenesis. Downstream gene analysis revealed that a number of genes had much higher expression in the GmGT-2B-transgenic plants than that in the GmGT-2A-transgenic plants (Fig. 7), and these genes may contribute to the reduced ABA sensitivity in the GmGT-2B-transgenic plants. Alternatively, these GmGT-2B-upregulated genes may reflect a positive feedback of the ABA response due to the reduction of ABA sensitivity. In fact, several ABA-regulated or related genes including NCED3, LTP3, LTP4 and PAD3 etc. were enhanced in the GmGT-2B-transgenic plants. These studies on ABA effects may also suggest that GmGT-2B plays larger roles than GmGT-2A in regulation of seedling morphogenesis. The GmGT-2A and GmGT-2B showed differential expression in response to ABA and other stresses. However, expression of their homologue GmGT-2 was not induced by these treatments, suggesting that the GmGT-2A and GmGT-2B have specific roles in abiotic stress responses. Overexpression of both the GmGT-2A and GmGT-2B greatly improved plant tolerance to salt, freezing and drought stresses as can be seen from the survival rates of the transgenic plants, the dry weight and the water loss (Fig. 5, 6). The regulation of stress tolerance may be achieved through control at levels of transcriptional activation, DNA binding, and dimerization and/or by other unknown mechanisms. These controls at different levels will finally affect gene expressions, through which the stress tolerance can be achieved in plants. Actually, many genes have been found to be upregulated in the transgenic plants. Among these, three C2/H2 zinc finger-type transcription factors were increased and may play significant roles since two of the genes STZ and RHL41/Zat12 have been found to promote stress tolerance [37], [38], [51]. DREB2A gene and a peroxidase gene RCI3 were also highly expressed in the GmGT-2A- and GmGT-2B-transgenic plants. These two genes have been found to improve stress tolerance in transgenic plants [48], [49]. Therefore, the GmGT-2A and GmGT-2B may at least activate these gene expressions either through direct binding to promoter of each gene or in a manner of indirect regulation. Expression of a few genes were also suppressed by the two GmGT genes. The VAMP711, a gene encoding a protein related to vesicle trafficking, was downregulated. Suppression of the VAMP711 inhibited the fusion of the H2O2-containing vesicle to the tonoplast, leading to improved vacuolar functions for plant salt tolerance [56]. It is therefore possible that the present two GmGT genes conferred stress tolerance at least through activation of STZ/ZAT and DREB2A transcription factors as well as the antioxidative system. Moreover, the GmGT-2B-transgenic plants appeared to be slightly more tolerant to stresses than the GmGT-2A-transgenic plants did (Fig. 5B, 6G). This difference is most likely due to the higher expressions of the specific genes in GmGT-2B-transgenic plants (Fig. 7). It should be mentioned that overexpression of the transcription factors in Arabidopsis plants might induce tolerance observations not related to activation of specific pathways but rather indirect or pleiotropic effects. Further transgenic analysis in soybean plants may disclose such a possibility. Overexpression of the GmGT-2B gene resulted in reduced ABA sensitivity (Fig. 4), but still improved plant tolerance to salt, drought and freezing stress (Fig. 5, 6). This phenomenon appears to be inconsistent with the notion that ABA insensitivity would generally lead to reduced stress tolerance. However, our results were in line with several reports showing that genes conferring reduced ABA sensitivity can increase stress tolerance. Yang et al. [60] found that Lily hydrophilin gene LLA23-transgenic seeds showed reduced sensitivity to ABA, however, its transgenic plants exhibited tolerance to salt, osmotic and drought stresses. An ABF2-interacting protein gene ARIA-overexpressing plants are hypersensitive to ABA but also sensitive to high osmolarity during germination [61]. Transgenic plants overexpressing GmbZIP44, GmbZIP62 and GmbZIP78 from soybean show reduced sensitivity to ABA but enhanced tolerance to salt and freezing stress [5]. It should be mentioned that the GmGT-2A does not have transcriptional activation activity and could not form homo or heterodimers. However, it still can bind to cis-DNA elements and improve stress tolerance through alteration of gene expressions. The GmGT-2A may need post-translational modification to achieve its transcriptional activation. Other mechanisms may also be involved. Overall, we have identified two GT factors GmGT-2A and GmGT-2B from soybean, whose overexpression differentially regulated seedling morphogenesis and improved plant tolerance to abiotic stresses. The stress tolerance conferred by these two factors is achieved by upregulation of a number of downstream genes. Further study should disclose more about the mechanism through which the two GT factors regulate plant stress responses. Materials and Methods Plant growth Seeds of soybean (Glycin max, Nannong 1138-2) were grown in pots at 25°C under continuous light. Seedlings of 15-day-old were carefully pulled out from the vermiculite, rinsed and subjected to various treatments. For salt treatment, seedlings were immersed with the roots in 150 mM NaCl. For drought stress, seedlings were placed on filter papers at room temperature for air drying. For cold treatment, seedlings were placed in a beaker containing 4°C water. For ABA treatment, seedlings were immersed with the roots in 100 µM ABA. Seedlings were also placed in water at room temperature as a control treatment. After treated for the indicated times, the materials were harvested for RNA isolation. Roots, stems, leaves and cotyledons from 15-day-old seedlings, and flowers, young pods, and seeds from mature plants were also collected for examination of gene expression. Gene cloning Two ESTs representing the middle parts of two trihelix family genes were found to be inducible under various abiotic stresses. 5′- and 3′-RACE were performed to obtain the full-length of the two genes. Finally the two genes were cloned into the pMD18-T vector to generate the original plasmids pMD18-T-GmGT-2A and pMD18-T-GmGT-2B for further use. The coding sequences of the two genes have been deposited into the GenBank under the accession numbers of EF221753 for GmGT-2A and EF221754 for GmGT-2B. Northern hybridization and RT-PCR analysis Total RNA isolation and Northern hybridization followed previous descriptions by Zhang et al.[62]. Gene expressions were also examined by RT-PCR. For GmGT-2A, primers are 5′-AGGAAACCCCGCTAGAGAAC-3′ and 5′-GTTGTTGTCGGTTGTTGTCG-3′. For GmGT-2B, primers are 5′- GTTTTTGCGAGAGCATTGTG-3′ and 5′-AACTAGGGTTCTGGGGAGGA-3′. For GmGT-2, primers are 5′-GATTCCAAGACTTGTCCCTA-3′ and 5′-CCTATCACATTTCACTCCC-3′. Primers used for gene expressions in the transgenic Arabidopsis plants are listed in File S1. Transcriptional activation and dimerization analysis Transcriptional activation analysis was based on previous method [12]. The full-length of the coding region of the GmGT-2A or GmGT-2B gene was cloned into the pBD vector to generate the pBD-GmGT-2A(FL) or pBD-GmGT-2B(FL). The N-terminal region containing N-terminal trihelix domain plus the sequence between the two trihelix domains was also cloned into the same vector to generate pBD-GmGT-2A(NT) or pBD-GmGT-2B(NT). Similarly, the C-terminal region containing the sequence between the two trihelix domains plus the C-terminal trihelix domain was cloned to generate pBD-GmGT-2A(CT) or pBD-GmGT-2B(CT). The N-terminal region of the GmGT-2B was further truncated and pBD-GmGT-2B(N1-89), pBD-GmGT-2B(N1-153) and pBD-GmGT-2B-NGT(90-153) were made. Further more, the C-terminal trihelix domain and the sequence between the two trihelix domains were used to construct pBD-GmGT-2B-CGT(425–492) and pBD-GmGT-2B-M(150–424) respectively. All the primers used for the transcriptional activation analysis are listed in File S1. BD vector and pBD-GAL4 were used as negative and positive controls respectively. Each plasmid was transfected into the yeast strain YRG-2 containing the HIS3 and LacZ reporter genes. The transfected cells were examined for their growth on SD/-His or for the activity of β-galactosidase. For dimerization analysis, the above full-length genes or truncated versions were also inserted into pAD vector to generate pAD-GmGT-2A/2B(FL), pAD-GmGT-2A/2B(NT), or pAD-GmGT-2A/2B(CT). For GmGT-2B, pAD-GmGT-2B-NGT(90–153), pAD-GmGT-2B-CGT(425–492) and pAD-GmGT-2B-M(150–424) were also constructed. The pBD-GmGT-2A/2B and pAD-GmGT-2A/2B were co-transfected into YGR-2 cells, and the transfected cells were observed for growth on SD/-Trp-Leu-His plus 10 mM 3-AT as previously described [63]. The activity of β-galactosidase was also examined. Transcriptional activation assay in Arabidopsis protoplasts Full length sequences of GmGT-2A and GmGT-2B were obtained by PCR with the same primers as used in followed localization experiments. The GAL4 DNA-binding domain (BD)-coding sequence was fused to the above two genes and inserted into the pRT107 to generate effector plasmids pRT-BD-GmGTs. The fusion genes were under the control of 35S promoter. The BD sequence was also fused to VP16 gene to generate positive control effector plasmid. The pRT107 containing the BD sequence was used as negative control. The reporter plasmid containing 5X UAS and 35S promoter upstream of a reporter gene encoding a firefly luciferase (LUC) was used. The effector and reporter plasmids were co-transfected into Arabidopsis protoplasts and the relative LUC activity was determined based on previous descriptions [5]. The experiments have been repeated independently for three times and the results were consistent. Results from one experiment were presented. DNA binding analysis using yeast one-hybrid assay The yeast one-hybrid assay followed previous description [12]. Four copies of each of the cis-DNA element, with SacI and MluI adaptors, were synthesized, annealed and cloned into the reporter plasmid pHIS2, which contains the reporter gene HIS3. Each of the pAD-GmGT-2A/2B(FL), pAD-GmGT-2A/2B(NT), or pAD-GmGT-2A/2B(CT) was co-transfected with each pHIS2 plasmid harboring different cis-DNA elements into the yeast cells (Y187). The transfected cells were examined for their growth on SD/-Trp-Leu-His plus 30 mM 3-AT. Localization of the GmGT in Arabidopsis protoplasts and confocal microscopic analysis The full length sequence of GmGT-2A and GmGT-2B were cloned into the GFP221 plasmid to construct fusion plasmids using specific primers containing BamHI and SalI sites. Primers 5′-CGCGGATCCATGCTGGAAATCTCAACT-3′ and 5′-ACGCGTCGACACTCATAATTGCAATGGA-3′ for Gm-GT-2A, 5′-CGCGGATCCATGTTCGATGGAGTACCA-3′ and 5′-ACGCGTCGACAAACTGATCAAAATCCAA-3′ for Gm-GT-2B were used. GFP221 plasmid containing a 35S-driven GFP gene was used as a control. The fusion construct or control plasmid was then introduced into Arabidopsis protoplasts (http://genetics.mgh.harvard.edu/sheenweb/protocols/) for confocal analysis using a Leica TCS SP5 microscope. Generation of transgenic Arabidopsis plants The coding region of the GmGT-2A and GmGT-2B was amplified from their original plasmids with primers containing BamHI/SacI sites, and cloned into the pBI121 vector. The two genes were driven by the 35S promoter. For GmGT-2A, primers were 5′-gtcggatcc atgctggaaatctcaacttc-3′ and 5′-cgagagctcttaactcataattgcaatgg-3′. For GmGT-2B, primers were 5′-aacggatccatgttcgatggagtaccagacc-3′ and 5′-atcgagctcttaaaactgatcaaaatccaaag-3. The expression plasmids pBI-GmGT-2A/2B were transfected into agrobacterium GV3101 and then transformed into Arabidopsis plants using floral dip method. T3 homozygous plants with higher transgene expression were used for further analysis. Evaluation of stress tolerance for the transgenic Arabidopsis plants Seeds from Arabidopsis thaliana Columbia (Col-0) ecotype or various transgenic lines were sown on Murashige and Skoog medium, stratified at 4°C for 3 d and incubated at 22°C under continuous light. Seedlings were transferred to plates containing ABA or mannitol to observe their effects on seedling morphogenesis after growth for 16 d. For NaCl treatment, 7-day-old seedlings were transferred onto medium containing different concentrations of NaCl and maintained for 16 d. These plants were further transferred into pots containing vermiculite and grown under normal condition for 8 d and 21 d. The pictures were taken and the survival rates of these plants were evaluated at different periods. Freezing treatments were carried out according to Cuevas's method [64]. The tests were carried out in a temperature programmable freezer. Nonacclimated or cold-acclimated (5 d, 4°C) 12-day-old seedlings were exposed to 4°C for 30 min in darkness and subsequently the temperature was lowered at a rate of 2°C per hour. The final desired freezing temperature was maintained for 6 h, and then the temperature was increased again to 4°C at the same rate. After thawing at 4°C for 4 h in the dark, plants were returned to normal conditions. Tolerance to freezing was determined as the capacity of plants to resume growth after 7 d of recovery under control conditions. For drought treatment, 12-day-old seedlings in pots were withheld from water for 16 d at 28°C with relative humidity of 20%. Plants at silique stage were also withheld from water for 16 d and the dry weight was measured and compared. Equal amount of vermiculite was added to each pot for comparison of plant growth and stress response. For water loss measurements, leaves were detached from plants at the rosette stage and weighed immediately on a weighing paper. The weight was measured at designated time intervals. There were three replicates for each transgenic line. The percentage loss of fresh weight was calculated based on the initial weight of the plants [5]. Water content was measured according to previous descriptions with modifications [65]. Six-week-old plants in pots were withheld from water for 3 d, and then measurements were made every 4 d and lasted for 12 d. Aerial parts of eight plants were excised and fresh weight was measured. The materials were dried in an oven at 37°C for 4 d until constant weight. The relative water content was calculated. qRT-PCR analysis Total RNA from aerial parts of four-week-old plate-grown plants was used for reverse-transcription (RT) with MMLV reverse transcriptase according to the manufacture's protocol (Promega). Genes selected and corresponding primers were shown in File S1. Real-time PCR were performed on MJ PTC-200 Peltier Thermal Cycler based on previous descriptions [5]. The real-time PCR results were analyzed using Opticon Monitor™ analysis software 3.1 (Bio-Rad). Statistical analysis The data were subjected to statistic analysis, and analysis of variance was performed using the SPSS 12.0 program. Supporting Information File S1 Primers used for transcriptional activation analysis and qRT-PCR analysis (0.10 MB DOC) Click here for additional data file. We thank Prof Gai Jun-Yi (Nanjing Agricultural University, China) for providing the original seeds of soybean cultivar. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by the National Basic Research Project (2006CB100102, 2009CB118402), National High Tech program (2006AA10Z113, 2007AA021402), and the project from Chinese Academy of Sciences (KSCXZ-YW-N-010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Hu HH Dai MQ Yao JL Xiao BZ Li XH 2006 Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci USA 35 12987 12992 2 Jin H Martin C 1999 Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol 41 577 585 10645718 3 Liu JX Srivastava R Che P Howell SH 2007 Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1975615009-PONE-RA-08817R210.1371/journal.pone.0007048Research ArticleCell BiologyOncologyImmunology/ImmunomodulationA Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells Reprogramed TAM Changes TALChatterjee Shilpak 1 Mookerjee Ananda 3 Mookerjee Basu Jayati 3 Chakraborty Paramita 1 Ganguly Avishek 1 Adhikary Arghya 4 Mukhopadhyay Debanjan 5 Ganguli Sudipta 5 Banerjee Rajdeep 6 Ashraf Mohammad 2 Biswas Jaydip 2 Das Pradeep K. 6 Sa Gourisankar 4 Chatterjee Mitali 5 Das Tanya 4 Choudhuri Soumitra Kumar 1 * 1 Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, India 2 Department of Surgical Oncology, Hospital Unit, Chittaranjan National Cancer Institute, Kolkata, India 3 INSERM, U-563, CHU Purpan, Toulouse, France 4 Department of Molecular Medicine, Bose Institute, Kolkata, India 5 Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India 6 Rajendra Memorial Research Institute of Medical Sciences, Patna, India Rich Benjamin Edward EditorHarvard Institute of Medicine, United States of America* E-mail: [email protected] and designed the experiments: SKC SC AM JMB. Performed the experiments: SKC SC Pc. Analyzed the data: SKC SC AM JMB Pc AG AA. Contributed reagents/materials/analysis tools: AG AA DM SG RB MA JB PD GS MC TD. Wrote the paper: SC AM JMB. 2009 16 9 2009 4 9 e704820 2 2009 18 8 2009 Choudhuri et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis. Methodology/Principal Findings The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-γ but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-β producing CD4+CD25+ T cells toward IFN-γ producing T cells. Conclusion/Significance Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth. ==== Body Introduction Tumor cells escaping from the immune surveillance in immunocompetent individuals reflects inadequate function of the immune system. Induction of tumor specific T cell mediated immunity may block the tumor growth and may give protective anti-tumor immune response [1], [2]. However strong immune suppression in the tumor microenvironment makes the situation more complicated [3], [4]. It has been suggested that the growing tumors produce various chemoattractants that have been implicated in recruitment of monocytes in the tumor site. When monocytes are recruited into the growing tumor site, local cytokine milieu modulates the immunological functions of these newly recruited monocytes and educates them towards tumor-associated macrophages (TAMs) that are immunosuppressive in nature [5], [6], [7]. TAMs promote tumor cell proliferation and metastasis by secreting a wide range of growth and proangiogenic factors as well as various metalloproteinases [8], [9]. TAMs also possess poor antigen presenting ability and effectively suppress the induction of proper anti-tumor T cell response through the production of immunosuppressive cytokines like TGF-β and IL-10 [6], [10], as well as promote induction and infiltration of CD4+CD25+FoxP3+ T cells (Treg) at the tumor site [11]. However, evidence suggested that phenotype of TAMs can be reprogrammed and the presence of IL-12 in its local milieu plays key role in reprogramming of their functional cytokine profile towards proimmunogenic (IL-12 secreting) nature [12]. IL-12 also dictates the orchestration of T cell response towards generation of protective anti tumor response by stimulating T cells and NK cells to produce IFN- γ (13, 14, 15). Induction of IFN- γ production and suppression of IL-4 production by IL-12 has been shown to induce anti-tumor response in murine tumor models [14], [16]. Thus, if TAMs possess functional plasticity, it would be useful target for anti-tumor therapy because skewing them again towards proimmunogenic nature could induce proper anti tumor Th1 response that can effectively reduce tumor growth and metastasis. It has been reported earlier that copper homeostasis plays a vital role in drug resistance in cancer and also found to be essential in mediating several intracellular signals in macrophage [17], [18]. An elevated copper level in macrophage is associated with the production of inflammatory cytokines whereas a copper deficiency attenuates its conventional immunological functions [19]. Previously our laboratory had synthesized a novel copper chelate [Copper N-2(hydroxy acetophenon) glycinate (CuNG)] which was found to be a potent immunomodulator able to elevate the number of CD4+ IFN-γ producing cells in drug resistant tumor [Doxorubicin resistant Ehrlich Ascites Carcinoma (EAC/Dox)] bearing mice [20], [21]. In this study we found that CuNG has direct effect on TAMs and can modulate their functional cytokine pattern, inducing their conversion from immunosuppressive to proimmunogenic nature. Herein we have also found that change in regulatory cytokine profile of TAMs was able to redirect the T helper function, reprogram Treg population and augment the induction of protective immune response in EAC/Dox bearing mice. Similar results were also obtained in case of peripheral blood monocytes from chemo and/or radiotherapy refractory patients. Results CuNG treatment can directly modulate the regulatory cytokine profile of Tumor Associated Macrophages (TAMs) Previous study with the novel copper chelate CuNG revealed its immunomodulatory properties in EAC/Dox bearing mice. CuNG caused augmentation of apoptogenic inflammatory cytokine (mainly IFN-γ) production and resolution of tumors [21]. This finding prompted us to further investigate how this copper chelate (CuNG) induces the production of such inflammatory cytokines. First we have tried to know whether CuNG can directly act on CD4+ T cells [as this population is the predominant source of inflammatory cytokines following CuNG administration (i.m) [21]] to induce IFN-γ production. It was observed that in vitro application of CuNG did not have any significant effect in the IFN-γ production by CD4+ T cells obtained from EAC/Dox bearing mice (Fig. 1A). TAMs play pivotal role in suppression of IFN-γ producing CD4+ T cells (Th1 response) at the tumor site through establishment of immunosuppressive cytokine environment [22]. Therefore, we probed whether CuNG could modulate the functional behavior of TAMs from suppressive to proimmunogenic type so that protective Th1 response can be elicited. To test this possibility, EAC/Dox bearing mice were treated with CuNG (i.m, 5 mg/kg of body weight), TAMs were isolated 15 days following CuNG treatment (i.e., when tumors start to reduce prominently) and intracellular cytokine profile was checked by flow cytometry (Fig. 1B). It was observed that TAMs isolated from CuNG treated group released elevated level of IL-12 compared to the TAMs obtained from untreated EAC/Dox bearing mice (77.36% vs. 22.6%, MFI: 71.89±1.24 vs. 15.35±1.42). On the other hand, production of two major suppressive cytokines, IL-10 (63.21% vs. 94.09%, MFI: 48.68±0.95 vs. 82.44±0.68) and TGF-β (19.21% vs. 70.68%, MFI: 14.77±0.72 vs. 92.35±0.81) was found to be down regulated in the CuNG treated group compared to the untreated control. 10.1371/journal.pone.0007048.g001Figure 1 Both in vitro and in vivo CuNG treatment caused alteration of cytokines profile of TAMs. A) ELISA. B) Flow cytometry. C) Fluorometric analysis. D, E & F) ELISA. In vitro CuNG treatment (2.5 µg/ml) did not change IFN-γ production from CD4+ T cells of TALs of untreated EAC/Dox bearing mice (A). TAMs were purified from peritoneal ascitic fluid of both untreated and 15 days of CuNG treated EAC/Dox bearing mice and labeled with anti F4/80 antibodies and with either intracellular IL-10 or IL-12 or TGF-β or with specific isotype control Abs. Immunofluorescence analysis were performed by flow cytometry. Representative data of 3 independent experiments is presented (B). Purified TAMs were either kept untreated or treated with CuNG in vitro and ROS was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] at different time points. Results are presented as mean±SD of 3 independent experiments (C). Purified TAMs from untreated EAC/Dox bearing mice were plated (2×106 cells/500 µl). Cells were either kept untreated or pretreated with tocopherol (50 µM) for 1 h. Then the cells were further cultured for 12 h, 24 h and 48 h in the presence or absence of CuNG (2.5 µg/ml). The culture supernatants were collected and analyzed for cytokines IL-10 (D), IL-12 (E) and TGF-β (F) by ELISA and results are presented as mean±SE of 3 independent experiments, each experiment having every measurement in triplicate. The result was further confirmed by performing ELISA for IL-12, IL-10 and TGF-β. It was observed that 12 h and 24 h cultures of TAMs in the presence of CuNG did not show any modulation in regulatory cytokine production but 48 h of CuNG treatment caused significant up regulation in IL-12 production (Fig. 1E) (>13.5 fold) whereas the suppressive cytokine TGF-β (Fig. 1F) production was highly (∼17.2 folds) and IL-10 production (Fig. 1D) was moderately down regulated (∼3.71 folds) as compared to the untreated control. These results together indicate that CuNG treatment significantly modulates the production of regulatory cytokines by TAMs. Interestingly, it was observed that CuNG treated TAMs maintain a sustained higher level of reactive oxygen species (ROS; measured in terms of peroxide) till 18 h post treatment compared to untreated TAMs (Fig. 1C). Chelation of ROS with the anti-oxidant tocopherol (50 µM) reversed the nature of CuNG treated TAMs by increasing IL-10 and TGF-β production and decreasing IL-12 generation to levels comparable to untreated TAMs (Fig. 1D, E and F). In vivo administration of CuNG in EAC/Dox bearing mice induce Th1 type response It has well been documented that the cytokine IL-12 plays a pivotal role in Th1 polarization [13], [15]. Since administration (i.m) of CuNG in EAC/Dox bearing mice induce elevated level of IL-12 production by TAMs at the tumor microenvironment, we therefore checked whether in vivo CuNG treatment could modulate the cytokine profile of the tumor associated lymphocytes (TAL) towards Th1 type. Flow cytometric analysis revealed that CD4+ population of TAL from CuNG treated group showed higher percentage of IFN-γ positive population compared to untreated group (21.42% vs. 3.54%). On the contrary, the percentage of IL-4 and TGF-β positive populations in TALs isolated from in vivo CuNG treated animals was found to be greatly reduced compared to that from untreated animals (7.73% vs. 17.59% and 6.18% vs. 19.24%respectively) (Fig. 2). 10.1371/journal.pone.0007048.g002Figure 2 TALs of EAC/Dox bearing mice showed Th1 specific response after in vivo administration of CuNG. Flow cytometry. EAC/Dox bearing mice (n = 12) were treated with CuNG (5 mg/kg of body weight), i.m., 7 days following inoculation and 15 days after CuNG administration TALs were isolated from the ascitic fluid of treated and untreated animals as nonadherent population (method described in material and method section). From isolated TALs, CD4 vs. intracellular IFN-γ, IL-4 and TGF-β production were analyzed by flow cytometry and significantly higher percentage of CD4 population of treated group showed positive for IFN-γ (marker for Th1 response). A representative result is presented here for comparison. Soluble factors from functionally altered TAMs can skew unresponsive CD4+ T cells of untreated EAC/Dox mice towards Th1 type The conventional role of macrophage in tumor rejection through recognition of tumor antigen and participation in induction of anti-tumor T cell response is changed at the tumor site where it seems to produce elevated levels of immunosuppressive cytokines like IL-10 and TGF-β that effectively attenuate the induction of anti tumor response. We have shown here that single administration of CuNG in EAC/Dox bearing mice was able to alter the functional polarization of TAMs from immunosuppressive to proimmunogenic in nature leading to induction of Th1 type of response at the tumor site. These observations prompted us to investigate whether the soluble mediators derived from CuNG treated TAMs are sufficient to redirect the tumor associated unresponsive CD4+ T cells towards Th1 type in the absence of contact dependent signal. To assess this possibility TAMs were isolated from untreated EAC/Dox bearing mice and cultured for 48 h in presence or absence of CuNG. On the other hand TAMs from in vivo CuNG treated mice were cultured in absence of CuNG for 48 h. Following completion of incubation, cell free supernatants were obtained, diluted 2 folds with fresh medium and used to culture CD4+ cell enriched TALs. Following 96 h of incubation, cells were harvested and the levels of different Th1 and Th2 cytokine specific mRNA expressions were studied by semi-quantitative RT-PCR using specific primers. We observed that CD4+ T cell populations cultured with cell free supernatant of either in vitro CuNG treated TAMs or TAMs from in vivo CuNG treated animals manifested significantly elevated levels of expression of Th1 specific cytokine mRNA (IFN-γ) whereas Th2 specific (IL-4) and suppressive (TGF-β) cytokine mRNA expression levels were undetectable or poorly detectable in these two groups. On the contrary, IL-4 and TGF-β mRNA expressions were found to be significantly higher in the untreated control group (Fig. 3A & B). Similar results were obtained when Th1 and Th2 specific cytokine production by CD4+ T cells were analyzed by flow cytometry. CD4+ T cell population cultured with cell free supernatant derived from cultures of either in vitro CuNG treated TAMs or TAMs from in vivo CuNG treated mice, induced augmented IFN-γ production from CD4+ TALs obtained from untreated EAC/Dox bearing mice. Flow cytometric data (Fig. 3C) also revealed that culture supernatant obtained from in vitro or in vivo CuNG treated TAMs highly reduced the percentage of TGF-β and IL-4 producing CD4+ T cells. So both mRNA expression level and intracellular cytokine assay clearly indicate that the pattern of functional cytokine production by a lineage committed CD4+ T cells can be modulated in response to its local cytokine microenvironment created by the antigen presenting cells (like TAMs). 10.1371/journal.pone.0007048.g003Figure 3 Culture supernatant of TAMs, treated with CuNG, caused altered cytokines production by TALs. A) RT-PCR. B) Densitometric analysis. C) Flow cytometry. CD4+ T cells were purified from TALs obtained from untreated EAC/Dox bearing mice and cultured for 96 h with cell free supernatant of TAMs obtained from untreated EAC/Dox bearing mice that were either kept untreated for 48 h or treated with CuNG (48 h treatement) in vitro or with cell free supernatant of TAMs (cultured for 48 h in absence of CuNG) obtained from in vivo CuNG (15 days after treatment, i.e., when tumors start regressing prominently) treated EAC/Dox bearing mice. Cytokine profile was analyzed by semi-quantitative RT-PCR. Purified CD4+ population from TALs (derived from untreated EAC/Dox bearing mice) cultured without any treatment were used as untreated control. After completion of 96 h of incubation equivalent amount of mRNA (2 µg) from TALs of each experimental group was used for RT-PCR analysis and representative data from three independent experiment is presented (A). In all cases GAPDH was used as housekeeping gene control. Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and representative data of three independent experiments is presented here (C). CuNG treated TAMs induce reprogramming of cytokine status of CD4+CD25+ T cells CD4+CD25+FoxP3+ (Treg) are well known culprit for creation of immunosuppressive tumor microenvironment via production of high levels of TGF-β [23], [24]. It has previously been shown by us that CuNG treatment in vivo reduces CD4+CD25+FoxP3+ T cells at the tumor site (21). Since under in vitro condition combination of cytokines produced from CuNG treated TAMs can shift the cytokine profile of CD4+ TALs from TGF-β producing to IFN-γ producing, therefore we tested whether it can reprogram Treg towards Th1 type. For this purpose sorted CD4+CD25+ population from TALs of untreated EAC/Dox bearing mice were labeled with CFSE and co-cultured for 96 h with either untreated TAMs or TAMs treated with CuNG in vitro for 48 h. T cells cultured with untreated TAMs maintained their high TGF-β status while cytokine status of T cells cultured with CuNG treated TAMs was reprogrammed to low TGF-β and high IFN-γ (Fig. 4A). Furthermore, majority of the CD25+ sorted CD4+ cells, when cultured with CuNG treated TAMs, lost their FoxP3 expression vis-à-vis decreasing TGF-β production and increasing IFN-γ generation (Fig. 4B). These cells also lose the characteristic inhibitory property of Treg on proliferation of T cells (Fig. 4C). 10.1371/journal.pone.0007048.g004Figure 4 In vitro CuNG treatment caused reprogramming of Treg. A, B & C) Flow cytometry. CD4+CD25+ Treg populations were purified from TALs of untreated EAC/Dox bearing mice. (A) Treg cells were labeled with CFSE and then cultured for 96 h with cell free supernatant of TAMs (isolated from untreated EAC/Dox bearing mice) either kept untreated or treated in vitro with CuNG for 48 h. Intracellular IFN-γ and TGF-β production was analyzed with respect to specific isotype control by flow cytometry. Representative data of three independent experiments is shown. (B) Treg cells were cultured for 96 h with supernatant of 48 h culture of untreated or in vitro CuNG treated TAMs and fresh medium (1∶1). Intracellular IFN-γ and TGF-β production versus FoxP3 expression was analyzed with respect to specific isotype control by flow cytometry. Representative data of four independent experiments is shown. (C) Tregs (CD4+CD25+ cells) isolated from ascitic fluid of untreated EAC/Dox bearing mice were cultured for 96 h in presence of cell-free supernatants from 48 h cultures of untreated or CuNG treated TAMs (culture supernatant: fresh medium being 1∶1). Now, these cells were washed and cultured with CFSE loaded CD4+ T cells isolated from inguinal and axillary lymph nodes of normal mice (Treg and CD4+ T cells were taken in a proportion of 1∶5) for 96 h. Fluorescence levels of CFSE were measured by flow cytometry. Proliferation of normal CD4+ T cells either in the presence or absence of Treg cells were also analyzed by CFSE fluorescence level. Representative data of 3 independent experiments is presented here. Redirection of the tumor associated CD4+ T cells towards Th1 type can be accounted for by CuNG mediated altered level of IL-10 and IL-12 production from TAMs It is well evident from Fig. 1B that CuNG treatment caused alteration in the levels of IL-10 and IL-12 production by TAMs but did not completely abrogate the IL-10 production. This observation made us curious to further investigate whether the combination of low level of IL-10 and high level of IL-12 that we obtained with TAMs after CuNG treatment would have the same potential as IL-12 alone to make the decision for the generation of the Th1 response. To test this hypothesis, CD4+ TAL from untreated EAC/Dox bearing mice were cultured in the presence of either recombinant IL-10 (rIL-10) or IL-12 (rIL-12) alone or with a combination of rIL-10 and rIL-12 for at least 96 h. The doses of rIL-10 and rIL-12 applied corresponded to those obtained from the ELISA data of IL-10 and IL-12 production by either untreated or in vitro CuNG treated TAMs. Both mRNA expression study (Fig. 5A & B) and intracellular cytokines production assay (Fig. 5C) indicated that CD4+ T cells population cultured in the presence of 0.35 ng/ml of rIL-10 and 2.7 ng/ml of rIL-12 showed a significant up-regulation in production of IFN-γ similar to CD4+ TALs treated with only a single high dose of rIL-12. On the other hand, CD4+ TALs stimulated in the presence of 1.3 ng/ml of rIL-10 and 0.2 ng/ml of rIL-12 (amount similar to the IL-10 and IL-12 obtained from untreated TAMs), did not show any remarkable change in cytokine production pattern from that we observed in case of single high dose of rIL-10 treated or untreated control group. 10.1371/journal.pone.0007048.g005Figure 5 Combination of high IL-12 and low IL-10 can skew induction of Th1 response. A) RT-PCR. B) Densitometric analysis. C, D, E & F) Flow cytometry. CD4+ population from TALs (obtained from untreated EAC/Dox bearing mice) was purified and challenged either with single or combine dose of recombinant IL-12 and IL-10 and cultured for 96 h. Purified CD4+ population from TALs derived from untreated EAC/Dox bearing mice, cultured without any treatment was taken as untreated control. Equivalent amount of mRNA (2 µg) from each experimental group was used for semi-quantitative RT-PCR analysis and in all cases GAPDH was used as housekeeping gene control (A). Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and a representative data is shown (C). CD4+ TALs were co-cultured with untreated TAMs or CuNG treated TAMs either unfixed or fixed with paraformaldehyde or CuNG treated fixed TAMs along with high rIL-12 and low rIL-10. In some cases CD4+ TALs and CuNG treated unfixed TAMs were separated by transwell insert (0.45 µ Meter pore) in culture. After 96 h of culture intracellular IFN-γ and TGF-β production pattern were studied by flow cytometry (D). Mean fluorescence intensity for IFN-γ (Fig. 5E) and TGF-β (Fig. 5F) production by these experimental groups were also analyzed from the flow cytometric statistical data and represented graphically. Representative data from three independent experiments is presented. Interestingly it is also evident from dot plot analysis of flow cytometry data (Fig. 5C) that the percentage of IFN-γ positive cells among CD4+ TALs was higher when cultured in the presence of combination of high rIL-12 and low rIL-10 compared to the high rIL-12 alone (54.42% vs. 36.89%). Interestingly, CuNG treated TAMs, when fixed with paraformaldehyde could not increase IFN-γ production or decrease TGF-β generation in CD4+ TALs. However, when a combination of low rIL-10 and high rIL-12 was introduced in this system, TALs were reprogrammed (Fig. 5D). Moreover, co-culture of CuNG treated TAMs and CD4+ TALs did not significantly increase the level of reprogramming (Fig. 5D). These results indicate that reprogramming of TALs strongly depends on soluble agents (high IL-12 and low IL-10) released from reprogrammed TAMs. However, the cause of lower percentage of IFN-γ producing TALs following rIL-12 treatment alone compared to that following treatment with high rIL-12 and low rIL-10 remained unanswered. To explain this differential response we reasoned that the combination of high rIL-12 and low rIL-10 might block the death of T cells. Presence of small amount of IL-10 in association with IL-12 delayed the death of Th1 population Several reports are corroborating the fact that high level of IFN-γ produced by the Th1 population mediates its own apoptosis by up-regulating both Fas and Fas-L expression [25], [26], [27], [28]. Recently a differential role of IL-10 as an anti-apoptotic mediator protecting the mouse intestinal epithelial cells from IFN-γ or TNF-α mediated apoptosis by diminishing the Fas expression has been shown [29]. These findings prompted us to investigate whether the presence of low level of rIL-10 in combination with high rIL-12 could prolong the Th1 response by interfering with its self-killing mechanism. To address the issue of involvement of IL-10 in prolonging Th1 response, we cultured CD4+ T cells obtained from TALs of untreated EAC/Dox bearing mice either with a single high dose or different combination of rIL-10 and/or rIL-12 for 5 days. Cell death was quantified by means of PI/Annexin V-FITC. It was observed that rIL-12 alone induced high levels of apoptosis while a combination of low rIL-10 and high rIL-12 protected CD4+ T cells from undergoing apoptosis (Fig. 6A). This apoptotic process was found to be associated with caspase 3 activation. An active caspase 3 level in each experimental group was represented by the fluorescence intensity of the cleaved fluoregenic AMC liberated due to cleavage of Ac-DEVD-AMC by active caspase 3. Caspase 3 assay clearly indicates (Fig. 6B) that intensity of active caspase 3 levels in the high rIL-12 treated group increased much faster than high rIL-12 plus low rIL-10 treated group (15.663±0.57 vs. 15.107±1.06 at 72 h, 21.755±0.74 vs. 16.090±0.61 at 96 h and 27.223±0.60 vs. 18.414±0.36 at 120 h respectively). Addition of cell free supernatant derived from 48 h culture of both in vitro CuNG treated TAMs (originally isolated from untreated animals) and in vivo CuNG treated TAMs (obtained from in vivo CuNG treated animals) in the culture of CD4+ TALs from untreated animals resulted in restricted increase of caspase 3 activity (14.16±0.89 at 72 h, 17.59±0.46 at 96 h and 20.36±0.86 at 120 h and 15.65±0.46 at 72 h, 16.64±0.28 at 96 h and 18.93±0.53 at 120 h respectively) while neutralization of IL-10 in corresponding sets resulted in rapid increase of caspase 3 activity (16.33±0.76 at 72 h, 23.22±0.94 at 96 h and 29.65±0.71 at 120 h and 15.23±0.49 at 72 h, 20.99±0.49 at 96 h and 28.02±0.49 at 120 h respectively) (Fig. 6B). 10.1371/journal.pone.0007048.g006Figure 6 Presence of small amount of IL-10 prolonged Th1 response by delaying T cells apoptosis. A) Flow cytometry. B) Fluorometric analysis. C & D) Flow cytometry. Purified CD4+ population from TALs of untreated EAC/Dox bearing mice cultured in the presence of either only rIL-12 or the combination of high rIL-12 (2.7 ng/ml) and low rIL-10 (0.35 ng/ml) or the culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or IL-10 neutralized culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or 48 h of culture supernatant from in vivo CuNG treated TAMs or IL-10 neutralized culture supernatant of in vivo CuNG treated TAMs, for 72 h, 96 h and 120 h. Purified CD4+ population without any treatment was taken as untreated control. Levels of apoptosis were estimated by PI/Annexin V-FITC staining and flow cytometry. Representative data of 3 independent experiments is presented here (A). Purified CD4+ population pre-treated with H2O2 for 30 mins was taken as positive control for active caspase 3 level (Mean fluorescence intensity value of H2O2 control was 39.25±0.67 that was taken as 100% for active caspase 3 level). In each experimental group active caspase 3 levels was represented by % of H2O2 positive control. Results presented are of 4 independent experiments (B). Expression of Fas by CD4+ population of above mentioned experimental groups were also analyzed by flow cytometry. Cells were labeled with Abs specific for CD4 and for surface Fas or with specific isotype Abs and immunofluorescence analysis was performed. Representative result of 4 independent experiments is presented (C). CD4+ TALs were cultured with or without different combinations of rIL-12 and rIL-10 in absence or presence of neutralizing antibody against IFN-γ for 72 h. Fas expression was studied by flow cytometry and representative data of 3 independent experiments is presented here (D). Next, to decipher the mechanism underlying IL-10 mediated inhibition of Th1 polarized CD4+ T cell death we studied the Fas expression in the above population. To assess this possibility levels of Fas expression by CD4+ T cells of untreated EAC/Dox bearing mice cultured either with combination of high rIL-12 and low rIL-10 or high rIL-12 alone, were analyzed at 72 h, 96 h and 120 h. It was evident from the Fig. 6C that initially at 72 h CD4+ T cells cultured in the presence of high dose of rIL-12 alone showed Fas positive population comparable to that of CD4+ T cells cultured with a combination of high rIL-12 and low rIL-10. However, at 96 h and 120 h positive population for Fas expression was significantly higher in CD4+ T cells cultured in the presence of high dose of rIL-12 alone. Addition of cell free supernatant either from 48 h culture of in vitro CuNG treated TAMs or 48 h culture of TAMs from in vivo CuNG treated animals, at a ratio of 1∶1 with fresh medium in culture of TALs obtained from untreated animals resulted in low levels of Fas expression while neutralization of IL-10 in corresponding sets resulted in increased Fas expression (Fig. 6C). These data clearly indicate that presence of low level of rIL-10 in association with high rIL-12 do not interfere with the normal function of IL-12 in inducing Th1 response although delayed apoptosis by diminishing Fas expression thus prolonged the Th1 response. The ratio of rIL-10 and rIL-12 in this combination was almost 1∶8, as obtained by ELISA. So we tested other combinations like 1∶4, 1∶12, 1∶16 and rIL-12 only along with this combination. It was observed that 1∶4 and 1∶8 were the best combinations in terms of low Fas expression (Fig. 6D, upper panel) while with 1∶8, IFN-γ expression was much higher than 1∶4 (data not shown). Neutralization of IFN-γ yielded low levels of Fas expression in all cases (Fig. 6D, lower panel) indicating that IL-12 mediated IFN-γ generation caused Fas expression and T cell death. Thus, IL-12 is the key factor for reprogramming CD4+ cells towards IFN-γ producing Th1 type as well as inducing T cell death through IFN-γ production. CuNG treatment in vitro reduces the immunosuppressive cytokines and induces IL-12 generation in blood monocytes of patients with metastatic cancers Next we have tried to extrapolate our experimental data under clinical condition to see whether CuNG can modulate peripheral blood monocytes from patients to produce similar effect. PBMC isolated from patients with drug-resistant metastatic cancers was treated in vitro with CuNG. After 72 h, CD14+ adherent population was tested for the cytokine status. Untreated CD14+ cells exhibited an alternative activation status marked by high levels of TGF-β and IL10. Interestingly, following CuNG treatment, CD14+ cells exhibited very low levels of TGF-β, lowered IL-10 and high levels of IL-12 (Fig. 7). 10.1371/journal.pone.0007048.g007Figure 7 Treatment of CuNG upregulates IL-12 production by adherent population of PBMC from different cancer patients sample. Flow cytometry. PBMC from different cancer patients were isolated and only adherent population was either treated with CuNG (2.5 µg/ml) or kept untreated for 48 hr. Cells was labeled with Abs specific for surface CD14 and intracellular IL-10 or IL-12 or TGF-β and analyzed by flow cytometry. Discussion Present study establishes a new paradigm whereby the modulation in regulatory cytokine production pattern of tumor associated macrophages (TAMs) by the copper chelate CuNG is an effective strategy to remodel the local cytokines milieu in the tumor microenvironment. This plays a pivotal role in skewing unresponsive and suppressive CD4+ T cell populations towards Th1 type in EAC/Dox bearing mice. Macrophages are the most versatile cells population and are capable of changing their functional polarization in response to the growth factors or cytokines being released in their microenvironment [30], [31]. Cytokine milieu profoundly affects the functional polarization of the macrophages [31], [32]. Several studies indicate that tumor derived factors educate the newly recruited monocytes towards TAMs, which become immunosuppressive in nature [6], [7]. There is a symbiotic relationship between TAMs and cancer cells, where cancer cells attract TAMs and sustain their survival and TAMs in turn produce various growth and proangiogenic factors that promote tumor progression and metastasis [8], [33], [34] as well as effectively thwart the induction of protective anti-tumor response [6]. However, evidences suggest that TAMs retain functional plasticity and could be converted to nonsuppressive and anti-tumorogenic type by creating appropriate cytokine microenvironment [12]. Stout et al. showed that in the presence of IL-12 TAMs rapidly alter their functional phenotype from tumor-supportive and immunosuppressive to inflammatory [12]. In our study we have showed that a novel copper chelate, CuNG, possesses the potential to alter the immunosuppressive phenotype of TAMs by reprogramming its proinflammatory (IL-12) versus immumosuppressive (IL-10 and TGF-β) cytokine production pattern. Current study also demonstrated that both in vitro and in vivo application of CuNG evoked a robust IL-12 and diminished IL-10 and TGF-β production by TAMs and thereby polarize its functional phenotype towards inflammatory. The induction of Th1 response highly depends on the critical level of the two regulatory cytokines, IL-10 and IL-12 [35], [36]. IL-10 inhibits important aspects of cell-mediated immunity whereas IL-12 induces type 1 cytokine production and effective anti-tumor cell mediated response [37], [38]. IL-10 overproduction by TAMs at the tumor site has been implicated in tumor mediated immune suppression [39]. It has also been found that transgenic mice over expressing IL-10 under the control of IL-2 promoter were unable to restrict the progression of immunogenic tumor whereas applying anti IL-10 mAbs in these mice restored the in vivo antitumor response [40]. In addition to IL-10, high TGF-β secretion by TAMs at the tumor microenvironment seems to play a potent immunosuppressive role by inhibiting T cell activation, proliferation and differentiation [11], [33], [39]. In contrast, IL-12, which stimulates Th1-dominant immunity in vivo, was shown to have strong in vivo anti-tumor activity [14], [38]. In our study we have shown that CuNG caused reduced IL-10 and TGF-β and augmented IL-12 production by TAMs at the tumor site. Elevated levels of IL-12 production by TAMs after CuNG treatment altered the cytokine balance at the tumor site and established a beneficial cytokine microenvironment that efficiently countered the immunosuppressive influence as well as skewed the unresponsive CD4+ T cells population towards Th1 type. Moreover, this reprogramming effect was found to be quite independent of cell-cell contact. Persistence of Th1 response is greatly inhibited due to self-killing of T cells due to over production of IFN-γ. This remains a major obstacle for successful tumor immunotherapy. Addition of IL-12 results in hugely elevated levels of IFN-γ [41], which limits T cell survival [42] and shortens Th1 response. Addition of IFN-γ induces a short-lived tumorocidal effect followed by immunosuppression and aggressive tumor growth, limiting its use as an immunotherapeutic agent [42]. High expression of death receptors and caspase 3 in Th1 cells make them prone to apoptosis [25]–[28]. Our study demonstrates that both in vivo and in vitro CuNG treated TAMs maintained a stable balance between IL-10 and IL-12 production where IL-12 levels were ∼8 folds higher than IL-10. This critical balance between these two cytokines was sufficient enough to induce Th1 response, as well as, the presence of small amount of IL-10 limits the self killing mechanism of Th1 cells and thereby prolonged its persistence. Moreover, CuNG treated TAMs could modulate TGFβ producing CD4+CD25+ T cells toward IFNγ producing T cells with concomitant decrease in the level of FoxP3 expression, indicating that Treg can be reprogrammed toward Th1 phenotype. Similar modulation of peripheral blood monocytes from chemo and/or radiotherapy refractory cancer patients from immunosuppressive to pro-inflammatory status could be achieved by in vitro CuNG treatment. This could also modulate Th2 type response to Th1 response (data not shown). The mechanisms underlying modulation of cytokine behavior of TAM by this copper complex is yet to be deciphered and the role of modulation of redox status cannot be ruled out [43]. The current study suggests that CuNG treatment induced a sustained generation of ROS. Inhibition of this ROS with anti-oxidant reversed the cytokine generation status of CuNG-treated TAMs toward untreated TAMs. Ongoing studies in this direction point towards the complex interplay between intracellular signaling events and the increase in reduced glutathione (GSH) level in late hour of CuNG treatment following its initial depletion (2 h) in the context of pattern of modulation of cytokine profile in macrophages (our unpublished observation). In summary, we critically evaluated the anti tumor efficacy of the novel copper chelate (CuNG) for its potential role of modulating TAMs and thereby inducing protective anti tumorogenic Th1 response. Earlier study with CuNG explored its immunomodulatory effects especially against drug resistant tumors [21]. Here we demonstrated that CuNG causes immune modulation in drug resistant cancer bearing individuals by altering functional cytokine pattern of TAMs to establish a proper immune surveillance at the tumor site. These data indicate that CuNG may be used clinically for immunotherapy of different types of drug resistance cancers. Materials and Methods Reagents Penicillin, Streptomycin was purchased from Sigma (USA). Recombinant murine IL-10, IL-12, opt EIA kit for assay of murine cytokines, anti-mouse IFN-γ, IL-10, IL-4, FITC conjugated IL-12 mAb, anti-mouse TGF-β, PE conjugated TNF-α mAb and all human reactive antibodies were purchased from BD Bioscience/BD Pharmingen (USA). Anti CD5 and CD19, F4/80 biotin conjugated mAb (murine) were obtained from eBioscience (USA). The cell culture medium RPMI-1640 and FCS were purchased from Gibco, Invitrogen (USA). Animals and Cell lines Swiss albino mice, obtained from National Institute of Nutrition (Hyderabad, India) and maintained in the institute animal facilities, were used for experimental purpose with prior approval of the Institutional Animal Ethics committee. EAC/Dox, which is resistant against doxorubicin, cisplatin, cyclophosphamide and vinblastine were developed and maintained according to the previously described methods. [44]. Treatment of Animals EAC/Dox bearing mice were kept either untreated or treated with a single dose of CuNG (5 mg/kg of body weight) 7 days following peritoneal inoculation with 1×106 EAC/Dox cells obtained from EAC/Dox bearing mice treated with Dox (48 hrs before acquisition of cells) [21]. Cell isolation and purification Isolation of Tumor associated macrophages (TAM) Total ascitic fluid was drawn and kept at standing position in a 50 ml sterile tube for at least 2 hrs for settling down the tumor cells and then clear fluid from the upper zone was collected. TAMs were isolated from that clear fluid first by negative selection with anti CD5 and anti CD19 and then by positive selection with anti F4/80 using BD IMagnet system (BD Bioscience) according to the manufacturer's protocol and resuspended in RPMI-1640 containing 10% FCS. Flow cytometric data revealed that purity of the separated population was >90%. Isolation of tumor associated lymphocytes (TAL) and purification of CD4+ T cells from TAL For isolation of TALs, total ascitic fluid was drawn, the upper clear zone of the ascitic fluid that remained after the tumor cells settled down was collected and centrifuged and the pellet was resuspended in RPMI-1640 containing 10% FBS and plated over the 90 mm plastic tissue culture plates and kept for at least ∼4 hrs at 37°C under 5% CO2 in air to allow the attachment of adherent cells. Nonadherent cells ( TALs, 95% lymphocytes) were subsequently removed by aspiration, harvested by centrifugation and resuspended in RPMI-1640 containing 10% FCS. In some cases CD4+ populations from TALs were purified by single positive selection with anti CD4-DM particle (BD Biosciences) or Treg were isolated from TAL using Treg isolation kit (BD Biosciences) using BD IMagnet system according to the manufacturer's protocol. Treatment of TAMs and assay of different cytokines production by flow cytometry and ELISA and estimation of ROS generation TAMs from either treated or untreated group were incubated with anti-F4/80-FITC or PE conjugated monoclonal antibody for 45 min at 4°C. After extensive washing, cells were then fixed, and permeabilized and stained with anti IL-12- FITC, anti IL-10- PE and anti TGF-β- FITC mAbs or corresponding isotype controls as described previously [21] and analyzed by flow cytometer (FACS calibur, BD). TAMs from untreated EAC/Dox bearing mice were plated (2×106 cells/ml) in 24 wells plate in the presence or absence of CuNG (2.5 µg/ml). Supernatants were collected after 12 h, 24 h and 48 h and assayed in triplicate for the production of IL-10, IL-12 and TGF-β using opt EIA kit (ELISA kit from BD Bioscience) according to the manufacturer's protocol. Reactive oxygen species (ROS) generation by differentially treated TAMs was measured using dichlorofluorescein diacetate (DCF-DA) using standard protocol described previously [45], [46]. Treatment of CD4+ T cells and Treg of untreated EAC/Dox bearing mice in vitro The purified total CD4+ population from TALs of untreated mice were plated (2×106 cell/ml) and cultured with or with out CuNG (2.5 µg/ml) for 24 h, 48 h and 72 h and the supernatant was taken and assayed in triplicate for the production of IFN-γ using optEIA kit (ELISA kit from BD Bioscience) according to the manufacturer's protocol. In some cases the purified total CD4+ TAL were plated (2×106 cell/500 µl) in presence of 500 µl of culture supernatant obtained after culturing TAMs derived either from in vivo CuNG treated mice or untreated EAC/Dox bearing mice treated in vitro with CuNG (48 h of treatment). In every case TAMs concentration was 2×105 cell/ml. In some cases different doses of recombinant IL-10 and IL-12 either individually of in combination was applied on CD4+ T cells (2×106 cell/ml). In some experiments CD4+CD25+ cells (Treg; 1×106 cell/ml) isolated from tumor site of untreated EAC/Dox mice (≥80% FoxP3+) were first labeled with CFSE (5 µM/ml) and then cultured in AIM V medium either with culture supernatant of in vitro CuNG (48 h of CuNG treatment) treated TAMs or untreated TAMs. For stimulation of CD4+ T cells anti-CD3 antibody (5 µg/ml) and anti CD-28 (1 µg/ml) antibody was also applied in this culture medium and incubated at 37°C with 5%CO2 95%air atmosphere condition. After 96 h of incubation non-adherent populations (90–95% lymphocytes) were collected by centrifugation at 500 g for 10 mins for performing further experiments. For neutralization of IL-10, 100 µg/mL neutralizing anti-IL-10 antibody was used. Assay of different cytokines production from CD4+ T cells by semi-quantitative RT-PCR and flow cytometry For assay of different cytokines by semi quantitative RT-PCR RNA was extracted from purified CD4+ T cells using NucleoSpin RNA II kit (Machery-Nagel) and reverse transcribed using RETROscript (Ambion). Primers specific for murine TGF-β (sense, CTTTAGGAAGGACCTGGGTT; antisense CAGGAGCGCACAAT-CATGTT), TNF-α (sense ATGAGCACAGAAAGCATGATC; antisense TACAGGCTTGTCACTCGA ATT), IFN-γ (sense CTCAAGTGGCATAGATGTGGA; antisense GACCTCAAACTTGGCAATACTC), IL-4 (sense GTCATCCTGCTCTTCTTTCTC; antisense ATGCTCTTTAGGCTTTCCAG), IL-10 (sense ACTACCAAAGCCACAAAGCAG; antisense AAGGAGTCGGTTAGCAGTATG) and GAPDH (sense CCCACAGTAAATTCAACGGCAC; antisense CATTGGGGTTAGGAACACGGA) were used with 2 µg of sample cDNA and amplified with Taq polymerase (Promega) using a Thermal Cycler (Applied Biosystem). For intracellular cytokines staining of CD4+ population the above mentioned staining protocol were used. In brief CD4+ population from different experimental groups were labeled with anti CD4 FITC or PE and intracellular anti IFN-γ FITC or anti IL-4 PE or anti TGF-β PE and immunoflorescence analysis was performed by using FACScaliber (BD Biosciences) with CellQuest software. Caspase assay and Fas expression study of Th1 polarized CD+ T cells Purified CD4+ T cells from TALs of untreated EAC/Dox bearing mice were cultured either with a single high dose of rIL-12 (2.7 ng/ml) or combination of high rIL-12 (2.7 ng/ml) and low rIL-10 (0.35 ng/ml) or culture supernatant of in vitro CuNG (48 h of CuNG treatment) treated TAMs or IL-10 neutralized culture supernatant of in vitro CuNG (48 h of CuNG treatment) treated TAMs or IL-10 neutralized culture supernatant of in vivo CuNG (15 days of CuNG treatment) treated TAMs for 72 h, 96 h and 120 h. Purified CD4+ population without any treatment was taken as untreated control. 30 mins pretreatment with H2O2 (500 µM/ml) was considered as positive control. An active caspase 3 level was assayed by using caspase 3 assay kit (BD Pharmingen) according to the manufacturer's protocol using spectrofluorimeter (Varian). In these groups CD4+ population vs. Fas expression was also studied by flow cytometry. Cytokine assay of adherent population of PBMC isolated from different cancer patients Leftover excess of blood drawn for routine examination of cancer patients refractory to various chemotherapeutics (certified by the Department of Surgical Oncology and Medical Oncology, Hospital Unit, Chittaranjan National Cancer Institute) were collected as sample from the Department of Clinical Biochemistry, Hospital Unit, Chittaranjan National Cancer Institute. Patient profile is given in Table 1. PBMC were isolated by Histopaque™ (Sigma). Adherent population from PBMC was isolated and either kept untreated of treated with CuNG (2.5 µg/ml) for 48 h. Intracellular cytokines were assayed by above mentioned protocol, in brief, adherent cells were scraped off from the plate and labeled with anti human CD14 FITC and intracellular anti human IL-10 PE or anti human IL-12 PE or anti human TGF-β PE and analysis was performed using FACScaliber (BD Biosciences) with CellQuest software. 10.1371/journal.pone.0007048.t001Table 1 Patient profiles. Patient Age (years) Sex Tumor type Remarks 1 66 F CA breast Brain metastasis; unresponsive to radiotherapy, 5-FU and anthracyclin (Epirubicin). 2 50 M CA lung Adrenal metastasis; unresponsive to Pacitaxel, cisplatin and radiotherapy. 3 35 F CA rectum Liver metastasis; unresponsive to 5-FU and radiotherapy. 4 45 M CA cheek Unresponsive to cisplatin, bleomycin and MTX. Statistical analysis Each experiment was done three to five times and results were expressed as mean±SE and Student's t test for significance was done and P<0.01 was considered significant. Flow cytometric data show representative data of at least three independent experiments. Competing Interests: The authors have declared that no competing interests exist. Funding: Indian council of Medical research, New Delhi, India; WWW.icmr.nic.in Grant Number: 5/13/18/2007 NCDIII and 5/13/18/2004 NCDIII. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Van der Bruggen P Traversari C Chomoez P Lurquin C De plean E 1991 A gene encoding an antigen recognized by cytotoxic T lymphocytes on a human melanoma. Science 254 1643 1647 1840703 2 Hung K Hayashi R Lafond-Walker A Lowenstein C Pardoll D 1998 The central role of CD4+ T cells in the antitumor immune response. J Exp Med 188 2357 2368 9858522 3 Zou W 2005 Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer 5 263 274 15776005 4 Whiteside TL 2005 Immunology of head and neck cancer. 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==== Front Indian J Crit Care MedIJCCMIndian Journal of Critical Care Medicine : Peer-reviewed, Official Publication of Indian Society of Critical Care Medicine0972-52291998-359XMedknow Publications India 19742250IJCCM-12-13610.4103/0972-5229.43684Case ReportSuccessful management of massive intraoperative pulmonary fat embolism with percutaneous cardiopulmonary support Sarkar Suman Mandal Krutisundar Bhattacharya Prithwis From: Department of Anesthesiology, IMS Banaras Hindu University, Varanasi-221 105, Uttar Pradesh, IndiaCorrespondence: Dr. Suman Sarkar, Department of Anesthesiology, IMS Banaras Hindu University, Varanasi-221 105, Uttar Pradesh, India. E-mail: [email protected] 2008 12 3 136 139 © Indian Journal of Critical Care Medicine2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.We report a patient who sustained catastrophic pulmonary fat embolism, during open reduction, internal fixation (ORIF) of fracture femur' In our opinion, the use of percutaneous cardiopulmonary support with (PCPS), saved the patient from certain death. Fat embolismorthopedic surgerypercutaneous cardiopulmonary support ==== Body Introduction Pulmonary fat embolism is a life-threatening complication for patients with long-bone fractures undergoing surgery.[1–4] The incidence of the fat embolism syndrome ranges between 0.9 and 2.2%, and the mortality rate has been reported to be 13-87%.[5–8] We report a patient with severe haemodynamic instability following fat embolism, who was successfully treated using percutaneous cardiopulmonary support (PCPS). Case Report A 65 year old woman had a fall in her bathroom, and presented to the hospital with Fracture shaft of femur. She was admitted to hospital for repair of the left femur fracture. An ORIF with an intramedullary nail, was planned to repair the fracture. Intraoperatively basic monitoring with continuous ECG, non-invasive blood pressure, and pulse oximetry was carried out. The procedure was done with epidural anaesthesia, given in the lumbar region -L3-L4 space, using isobaric bupivacaine 0.5% (16ml). The patient was sedated using intermittent Midazolam 2 mg IV, and the patient received oxygen through a venti-mask. After epidural block her blood pressure was 110/64 mm Hg, heart rate was 83 / min, and the SpO2 was 99%. Approximately 15 min after the insertion of the intramedullary nail, the patient suddenly became restless and this excitement progressed to generalized seizures. The oxygen saturation decreased from 99 to 80%. The patient became tachypnoeic, and the ECG showed ventricular bigeminy. Within seconds, she developed profound hypotension and shock The oxygen saturation could not be recorded. The patient was immediately intubated with a 7.5 mm endotracheal tube and positive pressure ventilation commenced. The initial end tidal CO2 was noted to be between 0 and 2.02. An arterial line was inserted, and an arterial blood gas analysis obtained, which showed a pH 7.019 of pCO2 was 20.17 mmHg, and paO2 was 107.57 mm of Hg, with a base deficit of 3.6 meq litre while breathing 100% oxygen. We urged the surgeon to complete suturing the skin as soon as possible. While the surgeon was suturing the skin, we inserted a pulmonary artery (PA) catheter. The systolic pulmonary arterial pressure was 48 mm Hg and the diastolic pressure was 32 mm Hg. A Transthoracic echocardiogram revealed massive dilatation of the right ventricle, the diameter of the inferior vena cava was 26 mm, and diametric change accompanying respiration was not observed. The left ventricular function was normal. We diagnosed the cause for the circulatory collapse as due to acute right heart failure (acute cor pulmonale) The patient then developed atrial fibrillation and subsequently pulseless ventricular tachycardia and required defibrillation, and cardiopulmonary resuscitation. CPR was continued in the operating theatre. Blood gas analysis during CPR showed a pH 7.075 of pO2 - 340.64 mm of Hg, and pCO2 - 41.84 mm of Hg with a base deficit of 16.9 mEq litre while breathing 100% oxygen. The end-tidal carbon dioxide was between 0 and 12 mm of Hg, and oxygen saturation could not be monitored. After successful CPR using epinephrine, atropine, vasopressin, and debfibrillation, continuous infusion of Norepinephrine 2 µg / kg / min and Dobutamine 20 µg / kg / min were started and the rates of infusions were adjusted to maintain the arterial systolic blood pressure >80 mm Hg. Systolic PA pressure was 51 mm Hg and pulmonary capillary wedge pressure (PCWP) was 33 mm Hg. A repeat Transthoracic echocardiogram (TTE) showed that the right ventricle was severely dilated but left ventricular function was maintained. After about an hour of continuous resuscitation, despite optimal maximal doses of pharmacological support, we could not maintain the systolic blood pressure >80 mm Hg; at this stage, we decided to place a portable PCPS. The PCPS is composed of heparin-coated circuits (Carmeda™ Closed Chest Support System), a biopump, a heat exchange unit, and a Maxima™ membrane oxygenator (Medtronic Cardiopulmonary CO, Anaheim, CA, USA). We inserted a 19 Fr drainage cannula into a femoral vein and 17 Fr re-infusion cannula into the femoral artery percutaneously. The initial blood flow was 2.5 litre min and the rotation rate of the biopump was 2500 rpm. The oxygen fraction was 1.0 at 4.0 litre min-1. About one hour after PCPS was started, the patient started showing signs of recovery, Her ECG returned to normal sinus rhythm and her circulatory failure began to improve, her systolic blood pressure was 92 mm Hg and her heart rate was between 90 - 100 beats / min. The PA pressure gradually decreased from 51 to 34 mm Hg, and her general condition became stable. The patient's blood gas analysis at this time showed a pH pH 7.34, pO2 of pO2 -477.34 mm of Hg, and pCO2 - 27.74 mm of Hg with 100% oxygen. The mixed venous oxygen saturation increased from 48 to 75%. The blood flow was maintained at 2.5 litre min and her core temperature had been lowered to less than 34°C for the protection of cerebral function. After the initiation of PCPS and having stabilized her cardiovascular condition, we transferred the patient from the operating theatre to a radioscintigraphic examination room as by this time we had strong suspicion of fat embolism. The radioscintigram showed diffuse multiple defects of blood flow in both lungs. After scintigraphic examination, we brought the patient to the intensive care unit (ICU). We diagnosed the patient as having pulmonary fat embolism from the presence of lipid granules, sampled from the tip of the PA catheter and stained with oil red O.[9] On day one in ICU, the patient's condition was stable on PCPS. On day two, since the patient was cardiovascularly stable, we lowered the PCPS flow to 1.0 litre min and then to 0.5 litre min. Throughout this time, the systolic blood pressure was maintained above 120 mm Hg and her heart rate was between 80 - 90 beats/min. The cardiac output (CO) was more than 3.5 litre min. The PA pressure had been between 25 and 30 mm Hg. We therefore decided to stop and remove the PCPS from the patient. Immediately after disconnecting the drainage cannula from the femoral vein, the patient crashed and had a very low systolic blood pressure with the ECG showing wide QRS complexes. PCPS was resumed immediately. An echocardiogram showed dilatation of the right ventricle, septal akinesis, and hypo kinesis of the left ventricle and the LV ejection fraction was less than 10%. We therefore continued to support the patient on PCPS. We had to give more time for the recovery of her cardiac function. On day three in ICU, the patient;s CO was 4.0 litre min-1 and PCWP 20 mm Hg. On the fourth ICU day, the systolic blood pressure was 140 mm Hg, heart rate between 80 and 90 beats min, systolic PA pressure between 25 and 30 mm Hg, PCWP 20 mm Hg, and cardiac index 3.6 litre m-2 min using a PCPS flow of 1.0 litre min. IV digital subtraction angiography at this stage showed adequate blood supply to both the lungs, except for a small apical part of the left lung. Good wall motion of the heart was observed at the same time. PCPS was then stopped for 30 min and we confirmed that the patient's haemodynamic condition remained stable. We then proceeded to wean the patient off PCPS. When the PCPS drainage cannula was withdrawn from the femoral vein, the PA systolic pressure suddenly increased from 30 to 65 mm Hg. and the ECG showed paroxysmal supra-ventricular tachycardia with a heart rate between 180-190/ min. The arrhythmia was successfully treated with cardioversion. The systolic arterial blood pressure was between 100 and 120 mm Hg. However the PA pressure remained as high as 65 mm Hg and we administered Prostaglandin 0.5 µg / kg. After PGE1 infusion, the PA systolic pressure gradually decreased from 65 to 40 mm Hg. One hour after weaning from PCPS, the patient had a blood pressure of 120/90 mm Hg, 90-100 /min, cardiac index of 3.0 L/min, and mixed venous oxygen saturation of 73%. However, she continued to have high pulmonary pressures So we continued PGE1 at 0.2 µg / kg / min, which gradually relieved pulmonary hypertension and the PA pressure decreased to 25 mm Hg (mean). On the tenth postoperative day, the patient's PA pressure was pressure was 33/15 mm Hg (mean 19 mm Hg) and PCWP was 10 mm Hg, so we discontinued the PGE1 infusion. Sedation was ceased and the patient gradually woke up and was obeying commands. She was extubated, and subsequently transferred to HDU and then shifted to the ward without neurological complications. Discussion In this case report, soon after a clinical diagnosis of cardiovascular collapse due to acute right heart failure secondary to massive fat embolism was suspected, an urgent echocardiogram helped us support our diagnosis. We have then described the successful management of this patient with aggressive resuscitation along with the use of cardiopulmonary bypass. It is known that increase in pulmonary arterial pressure and resistance induce right ventricular failure that may persist for several hours.[1011] Subsequent right ventricular dilatation may result in septal shift resulting in a decrease in left ventricular filling volume, leading to low CO. High right ventricular end-diastolic pressure in addition to low systemic blood pressure results in ischaemia of the right ventricle, potentiating the vicious cycle of right ventricular depression, dysfunction, and death. In our patient we kept cardiac massage going for an hour. Conventional treatment for right ventricular dysfunction includes the use of pulmonary vasodilators, volume loading, use of inotropes / vasopressors, Mechanical circulatory support is indicated for patients with right heart failure who show no improvement in response to conventional therapy.[10–12] PCPS is a powerful resuscitative tool that may be used to support patients with cardiac arrest and improve their survival.[12–14] PCPS is an extracorporeal life support that involves the continuous drainage of venous blood to a pump and membrane oxygenator and re-infusion to a major vein or artery. Venovenous support is the primary mode for respiratory failure, known as extracorporeal membrane oxygenation; this is primarily used in respiratory failure when improved oxygenation is the main goal Venoarterial bypass provides full support for both respiratory and cardiovascular failure and is called as PCPS in Japan. Venoarterial bypass involves accessing the right atrium or inferior vena cava for venous drainage and infusion into femoral artery; this type of support is used for patients who require cardiovascular support. PCPS can be used for patients with circulatory collapse and patients with damaged pulmonary circulation. PCPS drains blood from the right atrium, bypassing the right ventricle and pulmonary circulation, and oxygenated blood is returned to the systemic circulation. Therefore, we think the use of PCPS for patients with severe pulmonary embolism with catastrophic cardiopulmonary failure is worth considering in appropriate patients., The advantage of PCPS is that cannulation of the femoral vessels can be performed relatively quickly percutaneously. PCPS can be started within 15 min. The heparin-coated PCPS circuit can be used for the patients soon after surgery. The main complications of PCPS are hemorrhage, ischemia to lower legs, infection, and hemolysis. Arterial blood flow is mechanically returned into the femoral artery in the antegrade direction to spontaneous heart beat that increases the afterload of the left ventricle and might cause left ventricular dysfunction. Considering the limitation and complications during longer PCPS, we had tried rapid initial weaning from PCPS, but we should have continued the PCPS until we had confirmed the recovery of cardiac function as acknowledged on the fourth postoperative day. Administration of a large dose of PGE1 reduces the afterload of the right ventricle and improves refractory right heart failure. PGE1 is reported to be more pulmonary specific than Nitroglycerin, Sodium Nitroprusside, and Hydralazine, and resulted in the largest decrease in PA pressure.[15] PGE1 induced the largest decrease in PA pressure compared with the other pulmonary vasodilators Isoproterenol, Prostacyclin, and Nifedipine.[16] PCPS maintained our patient's condition well so that we did not use PGE1 while PCPS support was continued. However, retrospectively, we think we should have administered PGE1 from the start of the PCPS, then perhaps the cardiopulmonary status of the patient might have been stabilized better. If conventional pharmacological measures are not capable of stabilizing a patient with severe pulmonary fat embolism, PCPS is worth considering. This case shows that PCPS could provide effective support for the patients with fatal pulmonary fat embolism syndrome. Administration of PGE1 seemed effective for PH after weaning from the PCPS support. Source of Support: Nil Conflict of Interest: None declared. ==== Refs References 1 Marshall PD Douglas DL Henry L Fatal pulmonary fat embolism during total hip replacement due to high-pressure cementing techniques in an osteopolotic femur Br J Clin Pract 1991 45 148 9 1793703 2 Roummen RM Lako SJ Schoots FJ Acute lung damage after bilateral insertion of femoral intramedullary interlocking nails for metastatic bone disease Eur J Surg 1995 161 451 3 7548385 3 Heine TA Halambeck BL Mark JB Fatal pulmonary fat embolism in the early postoperative period Anesthesiology 1998 89 1589 91 9856743 4 Dewey P Femoral nailing and pulmonary embolism J Bone Joint Surg Br 1994 76 677 8 8027168 5 Hulman G The pathogenesis of fat embolism: A review article J Pathol 1995 176 3 9 7616354 6 Dudney TM Elliot CG Pulmonary embolism from amniotic fluid, fat and air Prog Cardiovasc Dis 1994 36 447 74 8184098 7 Mueller C Rahn BA Pfiister U Meinig RP The incidence, pathogenesis, diagnosis and treatment of fat embolism Orthop Rev 1994 23 107 17 8196970 8 Phillips SJ Zeff RH Kongtahworn C Skinner JR Toon RS Grignon A Percutaneous cardiopulmonary bypass: Application and indication for use Ann Thorac Surg 1989 47 121 3 2521442 9 Adolph MD Fabian HF el-Khairi SM Thornton JC Oliver AM The pulmonary artery catheter: A diagnostic adjunct for fat embolism syndrome J Orthop Trauma 1994 8 173 6 8207576 10 Pietak S Holmes J Matthews R Petrasek A Porter B Cardiovascular collapse after femoral prosthesis surgery for acute hip fracture Can J Anaesth 1997 44 198 201 9043733 11 Urban MK Sheppard R Gordon MA Urquhart BL Right ventricular function during revision total hip arthroplasty Anesth Analg 1996 82 1225 9 8638795 12 Johnson MJ Lucas GL Fat embolism syndrome Orthopedics 1996 19 41 8 8771112 13 Yokota K Yasukawa T Kimura M Fujita Y Sari A Right heart failure in the setting of hemorrhagic shock after pneumonectomy: Successful treatment with percutaneous cardiopulmonary bypass Anesth Analg 1997 85 1268 71 9390591 14 Manji M Isaac JL Bion J Survival from massive intraoperative pulmonary embolism during orthotropic liver transplantation Br J Anaesth 1998 80 685 7 9691880 15 Richard PC Myer RH Ronald PG Vasodilator therapy in vasoconstrictor-induced pulmonary hypertension in sheep Anesthesiology 1988 68 552 8 3128144 16 Richard PC Myer RH Ronald PG Hemodynamic profiles of prostaglandine1 isoproterenol, prostacyclin and nifedipine in vasoconstrictor pulmonary hypertension in sheep Anesth Analg 1988 67 722 9 3293483	19742250	PMC2738309	CC BY	2021-01-04 19:31:51	yes	Indian J Crit Care Med. 2008 Jul-Sep; 12(3):136-139
==== Front PLoS GenetPLoS GenetplosplosgenPLoS Genetics1553-73901553-7404Public Library of Science San Francisco, USA 1981656809-PLGE-RA-0618R310.1371/journal.pgen.1000680Research ArticleGenetics and Genomics/Animal GeneticsGenetics and Genomics/BioinformaticsGenetics and Genomics/Cancer Geneticsp63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair p63 and p73 Regulate DNA Repair after DNA DamageLin Yu-Li 1 Sengupta Shomit 2 3 Gurdziel Katherine 3 Bell George W. 3 Jacks Tyler 2 4 Flores Elsa R. 1 * 1 Department of Molecular and Cellular Oncology, Graduate School of Biomedical Sciences, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America2 Department of Biology and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America3 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America4 Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of AmericaFord James M. EditorStanford University School of Medicine, United States of America* E-mail: [email protected] and designed the experiments: YLL SS ERF. Performed the experiments: YLL SS ERF. Analyzed the data: YLL SS KG GWB ERF. Contributed reagents/materials/analysis tools: YLL SS TJ ERF. Wrote the paper: YLL SS ERF. 10 2009 9 10 2009 5 10 e100068014 4 2009 9 9 2009 Lin et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The p53 family activates many of the same genes in response to DNA damage. Because p63 and p73 have structural differences from p53 and play distinct biological functions in development and metastasis, it is likely that they activate a unique transcriptional network. Therefore, we performed a genome-wide analysis using cells lacking the p53 family members after treatment with DNA damage. We identified over 100 genes involved in multiple pathways that were uniquely regulated by p63 or p73, and not p53. Further validation indicated that BRCA2, Rad51, and mre11 are direct transcriptional targets of p63 and p73. Additionally, cells deficient for p63 and p73 are impaired in DNA repair and p63+/−;p73+/− mice develop mammary tumors suggesting a novel mechanism whereby p63 and p73 suppress tumorigenesis. Author Summary p63 and p73 have been identified as important suppressors of tumorigenesis and metastasis. Although they are structurally similar to p53, they have many functions that are unique including roles in development and metastasis. Here we show, using a genome-wide analysis of cells lacking p63 and p73 individually and in combination, that p63 and p73 regulate many unique target genes involved in multiple cellular processes. Interestingly, one of these pathways is DNA repair. Further validation of differentially expressed target genes in this pathway, revealed that p63 and p73 transcriptionally regulate BRCA2, Rad51, and mre11 providing a novel mechanism for the action of p63 and p73 in tumor suppression. These findings have important therapeutic implications for cancer patients with alterations in the p63/p73 pathway. ==== Body Introduction p53 acts as a tumor suppressor gene by transcriptionally regulating a multitude of target genes in response to DNA damage [1]. Induction of these genes results in multiple cellular fates including apoptosis and cell cycle arrest. p63 and p73 share some of the same functions as p53; however, p63 and p73 are structurally more complex containing multiple isoforms [2],[3],[4]. The TA isoforms are structurally more like p53 and contain a transactivation domain while the ΔN isoforms lack this domain and are transcribed from an internal promoter unique to these isoforms [3],[5]. Based on the fact that the TA isoforms are more similar structurally to p53, the TA isoforms were hypothesized and shown to be the major isoforms that induce transcription and are thought to have tumor suppressive functions [3],[5],[6],[7],[8]. In contrast, the ΔN isoforms have been shown to act as dominant negatives against the TA isoforms of p63 and p73 and also against p53. Because of the ability of the ΔN isoforms to act as dominant negatives and their overexpression patterns in human tumors [2],[6],[9],[10],[11], these isoforms have been hypothesized to act as oncogenes [3],[5],[6]. Interestingly, recent data have revealed that the ΔN isoforms of p73 can induce apoptosis, cell cycle arrest and transactivate target genes, such as p21, 14-3-3σ, and GADD45 [12]. Additional studies have demonstrated that expression of ΔNp73β at physiological levels can result in the suppression of cell growth in the presence or absence of p53 indicating that this isoform of p73 may act as a tumor suppressor gene [12]. Similarly, the ΔN isoforms of p63 have also been shown to have the ability to transactivate target genes [13]. In the case of p63, the ΔN isoforms are more highly expressed in epithelial tissues [14], and thus it is not be surprising that the ΔN isoforms transcriptionally regulate genes involved in the morphogenesis and differentiation of the epithelium. Given the structural complexity and expression of p63, p73, and their isoforms, the transcriptional targets of these genes are an area of growing research. We and others have shown previously that p63 and p73 can induce apoptosis in response to DNA damage [2],[8],[15]. Many of the target genes induced by p63 and p73 are shared with p53 [2],[8]. Additionally, we have shown that the p53 family of genes is interdependent on each other in the apoptotic response and in the suppression of tumorigenesis. p53+/−;p63+/− and p53+/−;p73+/− develop some of the same tumor types as p53+/− mice, but the phenotype of the tumors in the compound mutant mice is highly aggressive and metastatic indicative of cooperativity between family members [7],[15]. Mice heterozygous for combinations of the p53 family members develop a novel tumor spectrum compared to p53+/− mice indicative of functions of p63 and p73 independent of p53 [7]. These independent functions suggest that p63 and p73 may have unique transcriptional programs. To understand the transcriptional program of p63 and p73, we made use of MEFs deficient for each of the p53 family members individually and in combination and performed a genome wide analysis using cDNA microarray analysis to determine whether p63 and p73 transcriptionally regulate genes independently of p53 in response to DNA damage. Interestingly, we found that p63 and/or p73 transactivate sets of genes independent of p53. Among these sets of genes are those involved in homologous DNA repair, including Rad51, BRCA2, mre11 and Rad50. p63 and p73 were found to bind to these gene promoters by ChIP assay and to transactivate them as demonstrated by luciferase assay. Surprisingly, the ΔN isoforms of p63 and p73, which have been shown to be weak transactivators, transactivate the Rad51 and BRCA2 genes to high levels. In addition, p63−/−, p73−/− and p63−/−;p73−/− MEFs exhibited an impaired ability to repair their DNA and to survive in a clonogenic survival assay. Additionally, in vivo evidence from p63/p73 mutant mice supports this finding; p63+/−;p73+/− mice develop mammary adenocarcinomas at a high frequency [7]. Here, we show that these mammary tumors lose expression of p63, p73, BRCA2, and Rad51. Our findings indicate that p63 and p73 may suppress tumorigenesis by transcriptionally regulating critical genes in the DNA repair pathway. Results The p53 family of genes cooperates and acts independently in the regulation of transcriptional targets The p53 family members, p63 and p73, have previously been shown to share many of the same target genes as p53 [2],[8]. Additionally, both p63 and p73 have the ability to bind to the p53 consensus binding site. p63 and p73 also have biological activities independent of p53. Consequently, we were interested in determining whether p63 and p73 had unique transcriptional target genes. A cDNA microarray analysis was performed using E1A expressing MEFs deficient for each p53 family member individually (p53−/−, p63−/−, p73−/−) and in combination (p63−/−;p73−/−). These cells were treated with doxorubicin, a DNA damaging agent, to induce apoptosis in wild-type E1A MEFs. p53−/− and p63−/−;p73−/− E1A MEFs have previously been shown to be resistant to this treatment while the p63−/− and p73−/− E1A MEFS are partially resistant to apoptosis [15]. Microarray analysis revealed a large number of genes differentially expressed in the MEFs deficient for each p53 family member. Because we were interested in identifying genes that are transactivated by the p53 family members in response to DNA damage, genes that are down regulated in the absence of the p53 family members were further analyzed. After filtering and statistical analysis using SAM [16], 620 out of 15,488 genes were found to be down regulated in at least one of the single knockout E1A MEF lines compared to wild-type E1A MEFs in response to DNA damage. Eight-six of the 620 genes were down regulated in the p53−/−, p63−/−, and p73−/− E1A MEFs as illustrated by the Venn diagram (Figure 1 and Figure S1). There were also sets of genes that were uniquely regulated by each p53 family member; the p53−/−, p63−/−, and p73−/− MEFs each had 109, 148, and 131 genes down regulated respectively. Lastly, there were sets of genes that were regulated by two family members only; forty-seven were down regulated in the absence of p53 and p63, 41 in the absence of p53 and p73, and 58 in the absence of p63 and p73. The final list of differentially regulated genes was processed through multiple bioinformatic pipelines to identify biological pathways regulated by the p53 family members. Pathway analysis using the web-based KEGG, BioCarta, and GenMAPP databases indicated that the p53 family members regulate numerous pathways including: cell cycle, DNA-damage, p53 signaling, apoptosis, ribosomal proteins, metabolic pathways, and growth factor signaling (Table S1). 10.1371/journal.pgen.1000680.g001Figure 1 Venn diagram illustrating genes down regulated in the absence of the p53 family members. Eighty-six genes (brown) were down regulated in the absence of all three p53 family members. A number of genes were down regulated in the absence of each p53 family member individually; 109 in p53 deficient cells (red), 148 in p63 deficient cells (yellow), and 131 in p73 deficient cells (blue). Several genes were down regulated in the absence of two family members; 47 in the absence of p53 and p63 (orange), 58 in the absence of p63 and p73 (green), and 41 in the absence of p53 and p73 (purple). The putative target genes identified by microarray analysis were analyzed for the presence of a p53 or p63 consensus binding sites using a computer based genome wide search and HMMER1 software [17]. The promoter sequences (defined as 5 kb upstream and downstream of the transcription start site excluding exons) from the 724 down regulated genes were queried, and 700 of these genes were found to have p53 family member motifs. Of these, 669 genes contained p53 family member motif sites with the ideal p53 spacer of 6 nucleotides between the two half sites. Scores were then given to each identified binding site corresponding to how well they matched with previously published p53 or p63 matrices (Table S2). Hierarchical clustering was then performed in the specified knock out MEFs relative to wild-type after DNA damage to highlight patterns between the downregulated genes in the p53−/−, p63−/− and p73−/− E1A MEFs (Figure 2 and Figure S1). Interestingly, many genes were differentially regulated in the various MEF lines (Figure 2) indicating that p63 and p73 have unique target genes. Also, many genes were found to be down-regulated in all mutant cell types supporting the hypothesis that all three transcription factors can transactivate some of the same gene targets (Figure 2). 10.1371/journal.pgen.1000680.g002Figure 2 Heatmap showing unsupervised hierarchical clustering analysis of p53, p63, and p73−/− E1A MEFs after doxorubicin treatment. Each row represents the specified gene. Each column represents the expression level of a specified knock out MEF line relative to the expression level of wild-type MEFs after DNA damage. The red color indicates upregulation, the green color indicates down regulation, while black indicates no significant change of the indicated gene expression. Clustering based on Euclidean distance indicates that p63- and p73-deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. Genes of interest are listed in boxes and are associated with their corresponding location on the heatmap. DNA damage triggers numerous cellular responses including an extensive DNA repair pathway involving numerous genes [18]. Microarray analysis revealed that the p53 family members regulate numerous genes involved in the DNA repair pathway. Many of these genes seemed to be uniquely regulated by p63 and/or p73. After DNA damage, loss of p63 or p73 prevents induction of Brca2 (Figure 2, cluster 4), an essential co-factor in Rad51-dependent DNA repair of double-stranded breaks, and Rad51 itself (Figure 2, cluster 3) [18]. Sequence analysis also indicates that p53/p63 response elements exist in both the promoter and intronic region of Brca2 and Rad51 (Table 1 and Table S2). Clustered with Rad51 are Dbf4, a regulator of Cdc7 and a prognostic determinant for melanoma development, and Gas6, which cooperates with the tyrosine receptor kinase Axl in tumor proliferation and cell survival (Figure 2, cluster 3). We also found additional genes that were uniquely down-regulated in p63−/−, p73−/−, and p63−/−;p73−/− MEFs. These hits indicate that p63 and p73 have roles independent of p53 in the DNA-damage response pathway. For example, expression of Rad50, which forms a complex with mre11 and Nebrin, is found to be down regulated in p63−/−;p73−/− MEFs relative to wild-type MEFs treated with doxorubicin. There are also p53 family response elements upstream of the transcription start site of Rad50 (Table S2). 10.1371/journal.pgen.1000680.t001Table 1 p53 family response elements assayed by ChIP. Element Intron Sequence MM/spacer Binding RAD51-1 1 : −1766 atgCTTGcca acaCTTGatt 4/0 none RAD51-2 1 : −220 ctcCTAGaac tgaagttataa acaCATGaat 8/11 p73 RAD51-3 2 : +867 aaaCAAGcca c aaaCAAGtag 3/1 p73 RAD51-4 2 : +1347 gagCTTGgtg gcaCTTGctt 3/0 none BRCA2 2 : +133 agtCAAGgtg a atgCTTGctt 4/1 p63 & p73 MRE11-1 1 : −744 tggCTTGtgg cctccctggtcgactc tgaCAAGtcc 4/16 none MRE11-2 1 : −712 gtcCATGttg ggtaacttaggctttgctac ggtCTTGtag 6/20 none MRE11-3 1 : −171 gcgCTTGttc aaaaagtctaccctgcaactga gctCATGtta 4/22 p63 Shown are the sequence elements assayed by ChIP analysis. Mismatches are shown in bold-face type. The intron number and location are shown for each element. MM denotes the number of mismatches to the p53/p63 consensus binding site, spacer indicates the number of nucleotides within the spacer region of the putative binding site, and the binding column shows which family member bound to the element by ChIP assay. In addition to genes that are uniquely regulated by p63 and/or p73, genes controlled by all three p53 family members were identified. Mre11, a gene that functions in the repair of DNA double strand breaks, was found to be down-regulated in p53, p63, and p73 deficient E1A expressing MEFs (Figure 2, cluster 1). In addition, sequence analysis revealed multiple p53/p63 response elements (Table S2). Genes with similar expression profiles as mre11 include the growth factor signaling components Ghr and Sos1 as well as the apoptotic components Traf1 and Cathepsin D all of which contain p53 family member binding sites (Figure 2, cluster 1 & 6 & Table S2). Multiple genes involved in other biological processes, including tumor progression, metastasis and development were found to be differentially regulated in the various E1A MEF cells. For example, Mmp2, a gene shown to play a role in embryonic development and tumor metastasis, is also down regulated in the absence of p73 after doxorubicin treatment. Clustered with Mmp2 are many signaling components such as Grb2, Stat1, Map3k14, and Mapk8ip3- all of which have at least one p53 family member binding motif present near its promoter (Figure 2, cluster 5 and Table S2). Interestingly, brachyury, the developmental transcription factor, was identified as a putative p63 target gene (Figure 2, cluster 2). Given the identified roles of brachyury in limb development, cancer, and hematopoetic stem cells and the development phenotype of the p63−/− mouse, this putative target has important biological significance [19],[20],[21],[22]. We found brachyury to contain multiple p53 family response elements both upstream of its transcriptional start site and within the first intron (Table S2). Other p63 dependent genes that cluster with brachyury include Abr, the GAP for the small GTPase Rac, Socs3, involved in cytokine and apoptotic signaling, and the zinc-finger transcription factor Klf9 which is implicated in control of cell proliferation, cell differentiation, and cell fate (Figure 2, cluster 2). Genes involved in DNA repair are not induced in response to DNA damage in the absence of p63 and/or p73 Strikingly, the results from the cDNA microarray indicate that genes in the DNA repair pathway are differentially regulated in MEFs lacking p63 and/or p73 after treatment with DNA damaging agents. To verify these putative transcriptional targets of p63 and p73, quantitative real time PCR was performed. The expression of mre11, BRCA2, Rad51, and Rad50 was examined in wild-type, p53−/−, p63−/−, p73−/− and p63−/−;p73−/− E1A MEFs before and after treatment with doxorubicin for 12 hours and 5 Gy of gamma radiation. Interestingly, mre11, BRCA2, Rad51, and Rad50 are all induced in wild-type E1A MEFs after these treatments (Figure 3). We measured the baseline levels of mRNA of mre11, BRCA2, Rad51, and Rad50 to determine levels of these transcripts prior to DNA damage (Figure S2). After treatment with doxorubicin or gamma radiation, levels of mre11 mRNA are not induced to wild-type levels in p63−/−and p63−/−;p73−/− E1A indicating that p63 may transcriptionally regulate this gene (Figure 3). Similarly, the levels of BRCA2 are significantly lower in p73−/− and p63−/−;p73−/− E1A MEFs than in wild-type or p53−/− E1A MEFs (Figure 3) after treatment with doxorubicin and gamma radiation. Likewise, the Rad51 gene is not induced to wild-type levels in p63−/−, p73−/−, and p63−/−;p73−/− E1A MEFs after treatment with DNA damaging agents (Figure 3), indicating again that p63 and p73 may be critical transcriptional activators of Rad51 after DNA damage. Lastly, Rad50 also showed a pattern indicative of transcriptional regulation by both p63 and p73. The mRNA levels of Rad50 are approximately 4-fold lower in p63−/−;p73−/− E1A MEFs than in wild-type E1A MEFs (Figure 3) after treatment with doxorubicin and gamma radiation. Taken together, these data indicate that mre11, BRCA2, Rad51, and Rad50 may be transcriptional targets of p63 and p73 in response to DNA damage. 10.1371/journal.pgen.1000680.g003Figure 3 Genes involved in DNA repair are differentially expressed in MEFs deficient for p63 and/or p73. Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/−, p63−/−, p73−/− and p63−/−;p73−/−) after treatment with (A) doxorubicin (0.34 µM) for 12 hours or (B) γ radiation (12 hours). The Y-axis shows the fold induction. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represent the mean ± SEM. The asterisk denotes statistical significance compared to wild-type, p<0.001. Loss of p63 and p73 in mice results in mammary adenocarcinomas with low expression of BRCA2 and Rad51 As previously reported, twenty percent of mice heterozygous for p63 and p73 (p63+/−;p73+/−) develop mammary adenocarcinomas [7] (Figure 4), and ninety percent of these tumors lose the wild-type allele of p63 and p73 [7]. Given that BRCA2 plays an important role in the pathogenesis of mammary adenocarcinoma, this made it a relevant biological target for p63 and p73 in mammary tumors. The protein levels of Rad51 was first examined by Western blot analysis using wild-type and p63−/−;p73−/− MEFs. Interestingly, the basal level of Rad51 is lower in p63−/−;p73−/− MEFs compared to wild-type MEFs (Figure 4A). The levels of Rad51 in p63−/−;p73−/− MEFs are not induced in response to gamma irradiation; however, a 2-fold increase in expression of Rad51 was detected in the wild-type MEFs after DNA damage. To determine whether this change in expression pattern of Rad51 was cell-type specific, we performed immunohistochemistry on mammary adenocarcinomas from p63+/−;p73+/− mice where LOH of p63 and p73 had occurred (n = 10) (Figure 4F–4I). Indeed, Rad51 as well as BRCA expression is detected in normal mammary glands (n = 10) of p63+/−;p73+/− mice (Figure 4B and 4D) and is lost in hyperplastic mammary glands (n = 4) and mammary adenocarcinomas (n = 6) in these mice (Figure 4C and 4E). 10.1371/journal.pgen.1000680.g004Figure 4 Low expression of BRCA2 and Rad51 in cells and mammary tumors deficient for p63 and p73. (A) Western blot analysis for Rad51 using whole cell lysates from wild-type and p63−/−;p73−/− MEFs treated with 0 Gy or 10 min (m), 30 m, 1 hour (h), 2 h and 4 h after 5 Gy of gamma irradiation. Actin was used as a control for equal loading. (B–I) Immunohistochemistry (IHC) of normal mammary tissue or mammary adenocarcinomas from p63+/−;p73+/− mice using antibodies as follows: (B) normal mammary tissue from p63+/−;p73+/− mouse using Rad51 antibody, (C) mammary adenocarcinoma from p63+/−;p73+/− mouse using Rad51 antibody, (D) normal mammary tissue from p63+/−;p73+/− mouse using BRCA2 antibody, (E) mammary adenocarcinoma from p63+/−;p73+/− mouse using BRCA2 antibody, (F) normal mammary tissue from p63+/−;p73+/− mouse using p63 antibody, (G) mammary adenocarcinoma from p63+/−;p73+/− mouse using p63 antibody, (H) normal mammary tissue from p63+/−;p73+/− mouse using p73 antibody, (I) mammary adenocarcinoma from p63+/−;p73+/− mouse using p73 antibody. p63 and p73 bind to the promoter regions of Rad51, BRCA2, and mre11 Both the cDNA microarray and real-time RT-PCR data provide evidence that BRCA2, Rad51, and mre11 are transcriptionally regulated by p63 and p73 after DNA damage (Figure 3). Consequently, chromatin immunoprecipitation (ChIP) assay was performed to determine whether p63 and/or p73 could directly bind to the promoter region of these two genes. A subset of putative binding sites identified and summarized in Table 1 were assayed using ChIP. Sites chosen included those with the best scores for p53 and p63. Four putative binding sites were assayed for RAD51 (Table 1). RAD51-1 and 2 are located in intron 1, upstream of the start site, while RAD51-3 and 4 are found in intron 2, downstream of the start site. One putative element was assayed for BRCA2 in intron 2, 133 nucleotides downstream of the start site (Table 1). Lastly, three putative p53 family response elements were queried for mre11: MRE11-1, 2, and 3, located in intron 1, upstream of the start site (Table 1). ChIP analysis was performed using an antibody for p53, p63 or p73 in wild-type, p53−/−, p63−/−, and p73−/− E1A MEFs treated with doxorubicin for 12 hours (Figure 5). Interestingly, p73 was the only p53 family member that binds to the RAD51 promoter after DNA damage treatment. p73 was found to bind to RAD51-2 and 3 in intron 1 and intron 2 respectively. The primers used for this PCR reaction did not distinguish between the two sites; therefore, it is possible that p73 only binds to one of these sites. p63 and p73, but not p53, were found to bind to the response element in BRCA2 after DNA damage (Figure 5). Lastly, p63 was the only family member found bound to the mre-11 promoter at site mre11-3 within intron 1, 171 nucleotides upstream of the start site. The same binding pattern in the ChIP assay was obtained with other DNA damaging agents, such as gamma radiation (data not shown). 10.1371/journal.pgen.1000680.g005Figure 5 p63 and/or p73 bind to intronic regions within genes involved in DNA repair. Chromatin immunoprecipitation (ChIP) analysis using wild-type E1A MEFs (WT) and E1A MEFs deficient for the p53 family members (p53−/−, p63−/− and p73−/−) before (U) and after treatment with doxorubicin (D) for 12 hours. Antibodies used to immunoprecipitate protein-DNA complexes in each cell line are shown in various colors: p53 (red), p63 (blue), and p73 (green). Total input chromatin is shown for each sample (input). Each ChIP was performed using 3 independent MEF lines in triplicate. ΔNp63 and ΔNp73 transactivate Rad51, BRCA2, and mre11 promoters The ChIP results clearly demonstrate that p63 and/or p73 can bind to the promoters of these genes; however to gain a clear indication of which isoforms of p63 and p73 transactivate Rad51, BRCA2, and mre11, luciferase assays were performed with TA and ΔN isoforms of p63 and p73. Regions shown to bind by ChIP assay were used to construct firefly luciferase reporters. pGL3-Rad51-1 was designed by cloning intron 1 containing RAD-51-1 and 2 (Table 1) in to the pGL3 basic vector and pGL3-Rad51-2 containing the elements, RAD51-3 and 4, was cloned in to the pGL3 basic vector. These constructs were transfected in to p63−/−;p73−/− MEFs along with a renilla luciferase gene and one of the following isoforms of p63 or p73: TAp63α, TAp63γ, TAp73α, TAp73β, ΔNp63γ, ΔNp73α, and ΔNp73β. Interestingly, both ΔNp63α and ΔNp73β are the isoforms that transactivate the Rad51 reporter gene to appreciable levels. ΔNp63α transactivates pGL3-Rad51-1 11 fold and ΔNp73β transactivates this reporter 6 fold (Figure 6A). These isoforms more modestly transactivate the pGL3-Rad51-2 reporter indicating that the p63/p73 element resides in intron 1 (Figure 6A and 6B). Surprisingly, the TA isoforms did not transactivate the reporter gene. The p63/p73 family members also transactivate this reporter gene. ΔNp63α and ΔNp73β together can transactivate the Rad51-1 reporter 19 fold (Figure 6A and 6B). Additionally, the other ΔN isoforms that modestly transactivate this reporter alone can transactivate this reporter to higher levels. For example, ΔNp63α along with ΔNp73α can transactivate this reporter gene 9.8 fold, demonstrating additive effects between these family members. 10.1371/journal.pgen.1000680.g006Figure 6 ΔN isoforms of p63 and p73 transactivate Rad51 and BRCA2 luciferase reporter genes. Bar graphs showing fold induction for each luciferase reporter gene in (A–D) p63−/−; p73−/− or (E,F) p53−/−;p73−/− primary MEFs. Reporter genes used are as follows: (A,E) pGL3-Rad51-1 containing the binding elements in intron 1, (B) pGL3-Rad51-2 containing the binding elements in intron 2, (C,F) pGL3-BRCA2 containing the binding element in intron 2, and (D) pGL3-mre-11 containing the binding element in intron 1. Pluses above each bar graph indicate which isoforms of p63 or p73 were transfected in cells with the firefly-luciferase reporter genes. Renilla-luciferase was used as a control for transfection efficiency, and pPERP-luc was used as a positive control. Each experiment was performed 6 times using 3 independent MEF lines. Data are represented as the mean ± SEM. Similar to the experiments for RAD51, the BRCA2 region within intron 1 found to be bound by both p63 and p73 was cloned in to the pGL3 basic vector. Dual-luciferase reporter assay was performed in p63−/−;p73−/− MEFs as described above. Strikingly, the isoform with the highest ability to transactivate this reporter was ΔNp73β with a 4 fold induction. Additionally, ΔNp63α and ΔNp73β can transactivate the reporter 6 fold and other combinations of ΔN isoforms also show increases in transactivation of this reporter (Figure 6C). The ability of p63 and p73 to transactivate the mre11 gene was also tested by luciferase assay. The region shown to bind to p63 by ChIP analysis was cloned in to the pGL3 basic vector to generate pGL3-Mre11. This reporter was induced 3.8 fold by ΔNp63α and ΔNp73β together (Figure 6D). pPERP-luc, which has previously been shown to be responsive to TAp63γ was used as a positive control for these experiments [23],[24]. To determine whether p53 could transactivate these reporters, p53 was transfected with each reporter and luciferase activity was measured. p53 did not induce any of the reporters assayed (Figure 6A–6D). In addition, we performed luciferase assays using the Rad51-1 and BRCA2 reporters in MEFs lacking p53, p53−/−;p73−/− (Figure 6E and 6F) and p53−/−;p63−/− (data not shown). These experiments yielded similar results as those shown in Figure 6A and 6C. Taken together, these data indicate that the trasactivation of Rad51, BRCA2, and mre11 is p53-independent. Loss of p63 and p73 impairs DNA repair Rad51 and BRCA2 are both involved in homologous recombination (HR) DNA repair, one of the major pathways for repair of double strand breaks (DSBs). Cells lacking genes involved in HR, like BRCA2 and Rad51, have been shown to have an impaired ability to repair their DNA [18],[25],[26],[27]. Consequently, we hypothesized that cells lacking p63 and/or p73, which have low levels of these two proteins, may have a defect in repairing DSBs in damaged DNA. To test this hypothesis, wild-type, p53−/−, p63−/−, p73−/−, and p63−/−;p73−/− primary and E1A MEFs were treated with 5 Gy gamma-radiation or doxorubicin to generate DSBs. A comet assay was then performed to determine the DSB repair capacity in these cells. Comet assay, or single cell gel electrophoresis, is a commonly applied approach for detecting DNA damage in a single cell. The unwound, relaxed DNA migrates out of the cell during electrophoresis and forms a “tail” [28]. Therefore, cells that have damaged DNA appear as comets with tails containing fragmented and nicked DNA, while normal cells do not. The degree of DNA damage is represented using the parameter known as tail moment defined as the product of the tail length and the portion of total DNA in the tail. MEFs lacking the p53 family members were treated with DNA damage and incubated for a total of 16 hours allowing the homologous recombination repair to take place. Cells were and harvested at 0 (untreated), 1, and 16 hours for the Comet assay. In all cases, p63−/−, p73−/−, and p63−/−;p73−/− MEFs were found to have the largest tail moment after DNA damage (Figure 7A–7D). The tail moment after DNA damage was significantly higher for p63−/−, p73−/−, and p63−/−;p73−/− primary and E1A MEFs (18.8) compared wild-type samples (p <0.0001). This result indicates that p63 and p73 play a critical role in DNA repair. 10.1371/journal.pgen.1000680.g007Figure 7 Cells deficient for p63, p73, or both p63 and p73 have an impaired ability to repair damaged DNA and exhibit increased sensitivity to ionizing radiation. (A–D) DNA damage (tail moment) detected by the Comet assay at 0 (untreated), 1, and 16 hours in E1A MEFs treated with (A) 5 Gy γ radiation, (B) 0.34 µM doxorubicin or primary MEFs treated with (C) 5 Gy γ radiation, and (D) 0.34 µM doxorubicin. Genotypes are indicated on the x-axis and tail moment is shown on the Y-axis. Three independent MEF lines were assayed for each genotype in triplicate. Asterisks indicate statistical significance compared to wild-type (p <0.0001). (E,F) Clonogenic survival of E1A MEFs of the indicated genotypes following (E) gamma radiation and (F) doxorubicin. (G,H) Clonogenic survival of primary MEFs of the indicated genotypes following (G) gamma radiation and (H) doxorubicin. Percent (%) survival is indicated on the Y-axis for each dose of gamma-irradiation (0, 1, 2, 3 Gy) or doxorubicin (0.34, 0.5, 1 µM) on the x-axis. Three independent MEF lines were assayed for each genotype in triplicate. Data are represented as the mean ± SEM. Loss of p63 and p73 reduces cell survival Because loss of p63 and p73 impair DSB repair by regulating Rad51, BRCA2, and mre11, it is likely that loss of p63 and p73 results in poor cell survival due to the inability to repair damaged chromosomal DNA. To determine whether loss of p63 and p73 results in a decrease in cell survival, a clonogenic survival assay was performed using both primary MEFs and E1A expressing MEFs after treatment with 1, 2 and 3 Gy of gamma radiation and 0.34, 0.5, and 1.0 µM doxorubicin. After 12 hours, cells were replated and assayed for the ability to form colonies. p63−/−;p73−/− E1A MEFs and primary MEFs have an impaired ability to form colonies after gamma radiation indicative of defects in DNA repair (Figure 7E and 7F). A similar result was seen after treatment with doxorubicin in these cells (Figure 7G and 7H). Discussion p53 transactivates a vast network of genes in response to DNA damage [1]. While p63 and p73 can also transactivate known p53 target genes to varying degrees, they play roles in distinct biological functions including development and metastasis and likely have unique transcriptional targets. The advantage of the system employed here is the use of isogenic primary cells with the deletion of a single p53 family member. Here, we used early passage MEFs lacking the p53 family members individually or both p63 and p73 in combination and expressing E1A, which sensitizes them to undergo apoptosis after DNA damage to identify changes in gene expression in this process. We identified sets of genes that are regulated by individual and multiple p53 family members indicating unique and overlapping functions for this family of genes in response to DNA damage. Six hundred twenty out of 15,488 genes queried were regulated by a p53 family member. Genes identified played a role in multiple processes including apoptosis and DNA repair. In addition to engaging pathways predicted to be induced by DNA damage, genes involved in other processes like development and metastasis were also induced. These are biologically significant given the reported developmental, tumor, and metastatic phenotypes of the p63/p73 mutant mice [7],[20],[22],[29]. Lastly, the majority of the targets identified had binding sites that closely fit the p53 and p63 consensus binding site [14],[30],[31] indicating that they may be bona fide direct transcriptional targets of these family members. Indeed, we verified that Rad51, BRCA2, and mre11, genes involved in DNA repair, are direct transcriptional targets of p63 and p73. Given the high prevalence of mammary adenocarcinoma in mice mutant for p63 and p73 (p63+/−;p73+/−), a group of genes of interest are those involved in DNA repair. These genes were induced in wild-type cells and down regulated in the absence of p63 or p73. The mechanism for the tumor suppressive activity of p63 and p73 is not completely understood [6],[7],[32]. Regulation of DNA repair genes by p63 and p73 has not been demonstrated previously and could be a pathway employed by these genes in tumor suppression. Both Rad51 and BRCA2 were found to be direct transcriptional targets of p63 and p73 indicating that these mechanisms may be triggered during tumorigenesis. Interestingly, Rad51 has been shown previously to be repressed by p53 through a site found upstream of the start site [33]. Here, we show that ΔNp63 and ΔNp73 transactivate Rad51 through a distinct element in intron 1 indicating that there is an intricate and complex regulation of this gene by the p53 family and is likely a critical target in tumor suppression by this family. We also showed that transcriptional regulation of Rad51, BRCA2, and Rad51 by p63 and p73 is p53-independent/ It was surprising that the ΔN isoforms of p63 and p73 were more potent transactivators of Rad51, BRCA2, and mre11 than the TA isoforms. The TA isoforms have an acidic N-terminal domain necessary for transactivation [2],[3], and many studies have shown previously that the TA isoforms are more potent transactivators than the ΔN isoforms [2],[8]. Furthermore, the ΔN isoforms are better known for the dominant negative activities that they impose on the TA isoforms of p63 and p73 and p53. Interestingly, a number of recent studies have shown that the ΔN isoforms are capable of transactivating target genes due to a proline-rich transactivation domain that exists in these isoforms [12],[13]. In addition, the ΔN isoforms of p63 are more highly expressed than TAp63 in certain tissues including the skin [14] making the ΔNp63 isoforms likely candidates for gene regulation in these tissues. Taken together, our results indicate that the roles of the ΔN isoforms are more complex than previously appreciated. We have shown previously that E1A expressing MEFs deficient for p63 and p73 are resistant to apoptosis [15]. Paradoxically, we found that p63−/−;p73−/− primary and E1A MEFs are radiosensitive in long-term clonogenic assays. This finding coupled with the inability of p63/p73 deficient cells to repair DNA as shown by Comet assay indicate that p63 and p73 play a critical role in DNA repair. This new finding does not preclude that p63/p73 deficient cells are resistant to apoptosis after acute exposure to DNA damage. These data demonstrate that surviving p63−/−;p73−/− cells are unable to proliferate and establish a colony after DNA damage. This is likely due to defects in the DNA repair mechanisms. Using a genome wide analysis, these studies have revealed novel transcriptional targets of the p53 family members. We have also identified a novel mechanism of the regulation of the DNA repair pathway by p63 and p73. Given the high incidence of mammary adenocarcinoma in p63/p73 mutant mice, these studies have unveiled a potential mechanism for p63 and p73 as tumor suppressor genes. In addition, our studies have revealed further complexity by indicating that the primary transactivators of these DNA repair genes are the ΔN isoforms of p63 and p73. These isoforms have previously been thought to act as oncogenes. More recent data have challenged this notion as these isoforms can also transactivate genes involved in apoptosis and the expression of these isoforms does not provide a growth advantage [12]. These studies provide further evidence that the ΔN isoforms may have some anti-tumor functions such as the ability to engage DNA repair pathways. Future studies using isoform specific knock out mice should yield important insights in to how each of these isoforms contributes to tumor suppression and shed light on the interactions of the complex p53 family. Materials and Methods Preparation of 15 K murine cDNA microarrays The Laboratory of Genetics at The National Institute on Aging (NIA) cloned approximately 15,000 unique cDNAs into the NotI/SalI site of Ampicillin-resistant pSPORT1 vector (Life Technologies). Average insert size of the clones is 1.5 kb (0.5–3 kb). Inserts were amplified for microarray printing following a modified version of the protocol described previously [34]. In 96 well format, bacterial stocks were grown overnight in 2X YT medium (100 µg/ml ampicillin) with agitation. Ten microliters of the overnight bacterial culture was added to 90 µl ddH2O in PCR plates (MJ Research) and denatured at 95° C for 10 minutes. Following denaturation, plates were centrifuged for 10 minutes. To perform PCR, 5 µl of supernatant from each well was used as template in a 100 µl reaction with 3.5 units of AmpliTaq DNA polymerase (Applied Biosystems), forward primer (5′–CCAGTCACGACGTTGTAAAACGAC-3′) reverse primer (5′-GTGTGGAATTGTGAGCGGATAACAA-3′), and deoxynucleotide triphosphates (dNTPs). Amplification was carried out in thermocyclers with a program that contained an initial denaturation step at 95°C for 2 minutes followed by 38 cycles of 30 s at 94°C, 45 s at 65°C, and 3 minutes at 72°C, and a final extension of 5 minutes at 72°C. The amplified inserts were then purified using Montage PCR96 cleanup Filter Plates (Millipore) on a BIO-TEK Precision 2000 Automated Microplate Pipetting System to a purified volume of 100 µl. Thirty-five microliters of each purified PCR product was added to a 384-well plate, and desiccated using a large Savant Speed-vac apparatus, then reconstituted in 7 µl of 3X SSC/1.5 M betaine to a mean concentration of 600 ng/µl. The microarrays were fabricated at the MIT BioMicro Center using Corning GAPS II Gamma Amino Propyl Silane slides. cDNA clones were printed using a BioRobotics Microgrid 600 TAS Arrayer with a 32-pin print head and quill pin microfluidic liquid transfer technology. Cell culture, RNA extraction, and labeling of the cDNA probe All procedures involving mice were approved by the IACUC at U.T. M.D. Anderson Cancer Center and M.I.T. E1A-expressing mouse embryonic fibroblasts (MEFs) (wild-type, p53−/−, p63−/−, p73−/−, and p63−/−;p73−/−) were generated as described previously [15] from passage 1 primary MEFs. 3×106 E1A MEFs were plated on each of 6–15 cm dishes. Twenty-four hours after plating, the cells were treated with 0.34 µM doxorubicin. Twelve hours after treatment, total RNA (150–300 µg) was extracted from treated and untreated E1A MEFs using the RNAeasy Midi Kit (Qiagen). For each microarray hybridization, 100 µg of total RNA prepared from the reference or experimental cells were labeled by incorporating Cy3- or Cy5-labeled dUTP (NEN) using oligo d(T) (MWG) and Superscript II reverse transcriptase (Invitrogen). The resulting probes were purified using the Qiaquick PCR purification Kit (Qiagen) and recovered in a volume of 30 µl ddH20. Microarray hybridization The printed slides were rehydrated, UV cross-linked, and blocked to reduce background using succinic anhydride (Sigma), 1-methyl 2-pyrrolidinone and sodium borate. Each slide was incubated in 60 µl total volume of hybridization solution containing Cy3- and Cy5-labeled target (one probe is the reference invariant target and the other is the experimental target), 1 µg of Mouse Cot-1 DNA (Invitrogen), 0.1 units of poly-A40–60 (Amersham Pharmacia), and 10.1 µg of Salmon Testes DNA (Sigma), 25% Formamide, 5X SSC, 0.1% SDS under a 22×40-mm lifterslip (Erie Scientific Company) at 42°C for 16 hours exactly. The slide was placed in a sealed hybridization chamber (Corning) containing two side wells with a total of 20 µl 3X SSC for humidification in a light-sealed water bath. After exactly 16 hours of hybridization, the slide was washed in 500 ml of 1X SSC, 0.03% SDS for 5 minutes after the lifterslips are gently removed in the wash solution. Then, the slides were washed for 5 minutes in 0.1X SSC, 0.01% SDS followed by 0.1X SSC. Slides were centrifuged in a speed-vac to dry. Each slide was scanned using an arrayWoRx Auto Biochip Reader that employs white light, polychromatic filter-wheel/CCD camera (Applied Precision) at wavelengths corresponding to each analog's emmision wavelength (595 and 685 nm for Cy3 and Cy5, respectively). RNA from each sample was hybridized to four independent cDNA microarrays. For 2 replicates, the invariant target was labeled with Cy3 and the experimental target was labeled with Cy5. For the other 2 replicates for each sample, the invariant target was labeled with Cy5 and the experimental target was labeled with Cy3. The invariant reference target RNA used was extracted from untreated wild type- E1A MEFs. These cells were chosen as a source of reference target RNA because this species of RNA robustly hybridized to a large percentage of genes, and it is relevant to the experimental design. Data processing Hierarchical mapping Microarray images imported from the arrayWoRx scanner were filtered and annotated using the DigitalGenome software (MolecularWare). The resulting spot intensity data was normalized using the rank invariant method [35]. The gene filtering process was performed using SAM [16]. SAM is a statistical technique designed for analysis of microarray data [16] that uses repeated permutations of array data to report the most statistically significant differentially expressed genes between two groups of samples. Using all four microarray replicates, SAM reports an estimate of the median false discovery rate (FDR) for each list of differentially expressed genes. The FDR is the percentage of genes falsely reported as showing statistically significant differential expression. In addition, SAM uses an algorithm based on the Student t-test to determine the q-value of each individual gene, which is the lowest FDR at which the gene is called significant [36]. Using the bona fide biological target of p63, PERP, we used a cut-off FDR of 8.24% to determine our list of significant genes. As a result, 15 genes on our list has a q-value between 5% and 8.24% while the remaining 605 genes have a q-value less than 5%. Heatmaps were generated using functions within the Bioconductor project [37] of the R statistical programming language. Background subtracted and normalized intensity values obtained from the microarray experiment comparing the different cell populations were used. To perform hierarchical clustering of the genes and cell samples, Euclidean distance was used to compute dissimilarity. Identification of p53/p63 binding sites To identify all potential p53-family binding sites, promoter sequences (defined as genomic sequences within 2 kb of the transcription start site which have previously been reported to be enriched for these sites [30]) and intron 1 were extracted for all genes regulated by each p53 family member. These promoter sequences were initially searched for CWWG tetramers separated by a spacer of 5–8 nucleotides. To increase the specificity and score of these predicted sites, both strands were searched with a series of position-specific matrices for p53 [30],[31],[38] and p63 [14],[39] using HMMER1 v1.8 [17] with the “local search” option. To rank sites predicted across multiple matrices, all CWWG tetramer pairs were matched to corresponding HHMER sites and scored using the sum of bit scores. Quantitative Real Time PCR Total RNA was extracted from the E1A MEFs of the genotypes described above using the RNeasy Midi and Rnase-free Dnase kits (Qiagen). RNA was quantified and tested for quality on the Agilent 2100 Bioanalyzer (Agilent Technologies). To generate cDNA, RNA (2 µg) from each E1A MEF line treated with 0.34 µM doxorubicin was used for random hexanucleotide- primed cDNA synthesis. Each 40 µl reaction contained 1X buffer, 10 µM DTT, 1 µg random hexamer, 2 µl of Superscript II (Invitrogen), 0.5 mM each of all four dNTPs, and 80 units of RNase inhibitor (Promega). Using heating blocks, reactions were incubated at 42°C for 1 hour, 70°C for 15 minutes, 37°C for 20 minutes, and 95°C for 2 minutes. RNase H (2 units) (Invitrogen) was added to each reaction following the 70°C incubation. Afterwards, each reaction was diluted with ddH2O to a final working volume of 200 µl. cDNAs (2 µl) were added to 25-µl reaction mixtures containing 12.5 µl of 2X SYBR Green master mix (Applied Biosystems), and 40 nm of gene-specific primers. Primers were designed using Primer Express software (Applied Biosystems). Assays were performed in triplicate with an ABI Prism 7000 Sequence Detector (Applied Biosystems). All data were normalized to an internal standard (18 S ribosomal RNA; TaqMan Ribosomal RNA Control Reagents VIC Probe: Protocol: Rev C, Applied Biosystems) or GAPDH. Chromatin Immunoprecipitation Assay ChIP Assay was performed as described previously, E1A MEFs (wild-type, p53−/−, p63−/−, p73−/−, and p63−/−;p73−/−) were untreated or treated with 0.34 µM doxorubicin for 12 hours, which are the same conditions used for the array and real time PCR. Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products). Immunprecipitated complexes were recovered by Staphylococcus A cells, treated with proteinase K, and DNA was purified. PCR was performed for putative p53 family binding elements. Putative p53 family member binding sites were identified by scanning 1000 bp of the 5′ UTR, exon 1, intron 1, exon 2 and intron 2 for the consensus p53 binding site [31]. These sites are summarized in Table 1. Sequences for primers used are available upon request. Construction of luciferase reporters To generate the pGL3-Rad51 luciferase reporter, DNA was amplified from a BAC clone containing the Rad51 gene (RP23-15121, CHORI BACPAC resources) using primers designed containing the p73 binding site shown by ChIP and 5′ NheI and 3′ XhoI cloning restriction enzyme sites: forward primer (5′- ACTAGCTAGCAGCAGGGCGACCAACCGAC-3′) and reverse primer (5′-CCGCTCGAGTGGCCCTCCCTATCCACAGG-3′). To construct the pGL3-BRCA2 luciferase reporter, the DNA fragment containing the p63/p73 binding site shown by ChIP was amplified from C57/B6 genomic DNA by PCR using the following primers with 5′ XhoI and 3′ BglII cloning restriction enzyme sites: forward primer (5′-CCGCTCGAGAGAGGGATCCGGCGCGTC-3′) and reverse primer (5′-GGAAGATCTGGTCTAAGCTCTGTTGCTCCTG-3′. To generate the pGL3-Mre11 luciferase reporter, DNA was amplified from a BAC clone containing the mre11a gene (RP23-149D5, CHORI BACPAC resources) using primers designed containing the p63 binding site shown by ChIP and 5′ XhoI and 3′ BglII cloning restriction enzyme sites: forward primer (5′- CCGCTCGAGACAGAGAGAACCTCACCGAGAAC -3′) and reverse primer (5′-GGAAGATCTCTGTACCAGGTTCCTCTCCAAG-3′). The resulting amplified DNA fragments were gel-purified (Wizard Prep Kit, Promega) after restriction enzyme digestion and then ligated to pGL3-basic vector (Promega) between the respective cloning sites. Western blot analysis 6×105 wild-type and p63−/−;p73−/− MEFs were plated on 6 cm dishes. Twelve hours after plating, the MEFs were irradiated with 5 Gy of gamma-irradiation and then harvested at 10 minutes, 30 minutes, 1, 2, and 4 hours. The MEFs were lysed on ice in lysis buffer (100 mM Tris, 100 mM NaCl, 1% Nonidet P40, protease inhibitor cocktail (Roche)). Thirty micrograms of each lysate was subjected to electrophoresis on a 10% SDS PAGE for Rad51 and transferred to PVDF membrane. Rad51 was detected using the anti-Rad51 antibody (clone 51RAD01, Neomarkers), and BRCA2 was detected using the anti-BRCA2 antibody (clone H-300, Santa Cruz). Immunohistochemistry Slides were dewaxed in xylene and rehydrated in a graded series of ethanol following standard protocols [7]. Slides were incubated with primary antibodies for p63 (4A4, Santa Cruz), p73 (IMG-259A, Imgenex), Rad51 (clone 51RAD01, Neomarkers), or BRCA2 (clone H-300), Santa Cruz). at a dilution of 1∶100 for 18 hours at 4 deg C. Detection was performed using the Vectastain kit (Vector Labs) followed by the VIP kit or DAB kit (Vector Labs) and counterstained with methyl green (Vector Labs). Ten normal mammary glands and ten mammary adenocarcinomas were stained with each antibody. Dual-luciferase reporter assay p63−/−;p73−/−, p53−/−;p73−/− or p53−/−;p63−/− MEFs were plated on 6-well plates (3.5×105 cells per well). Twelve hours after plating, the MEFs were transiently transfected using Fugene HD (Roche) with 2.5 µg of the following Firefly luciferase reporter plasmids (pGL3-Rad51-1, pGL3-Rad51-2, pGL3-BRCA2) or pPERP-luc [24], 1 µg of Renilla luciferase plasmid (transfection control), and 2.5 µg of empty vector (pcDNA3) or plasmids encoding the p63/p73 isoforms (TAp63α, TAp63γ, ΔNp63γ, TAp73α, TAp73β, ΔNp73α and ΔNp73β) or p53/ In experiments where 2 isoforms of p63 and p73 were assayed simultaneously, 1.25 µg of each isoform was used. After 24 hr, cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega) and a Veritas microplate luminometer (Turner BioSystems). The relative luciferase activity was determined by dividing the Firefly luciferase value with the Renilla luciferase value and the fold increase in relative luciferase activity was determined by dividing the relative luciferase value induced by p63 and p73 isoforms with that induced by the pcDNA3 control vector. Each experiment was performed in triplicate. Clonogenic survival assay E1A MEFs or primary MEFs were plated in 6-well plates (1×106 cells per well) of the following genotypes (wild-type, p53−/−, p63−/−,p73−/−, and p63−/−;p73−/−) [15]. Twelve hours later, MEFs were irradiated with 1, 2, and 3 Gy of gamma radiation or 0.34, 0.5, and 1 µM doxorubicin. After 12 hr, 1200 cells were plated on 10 cm dishes. After 12 days of incubation, the cells were stained with clonogenic reagent (0.25% of 1,9-dimethyl-methylene blue in 50% ethanol). Surviving colonies were counted, and the survival rate was calculated as the ratio of the surviving colonies after DNA damage treatment over the number of colonies for each genotype before treatment. Each experiment was performed in triplicate on three independent MEF lines for each indicated genotype. Comet assay Wild-type, p53−/−, p63−/−, p73−/−, and p63−/−;p73−/− primary and E1A MEFs were plated on 6-well dishes (1.6×105 cells per well). Twelve hours after plating, MEFs were irradiated with 5 Gy of gamma radiation. Cells were harvested 0,1, and 16 hours later for Comet Assay (Trevigen) according to the manufacturer's protocol specific for DSB detection. Briefly, cells were suspended in PBS at a density of 3×105 cell/mL. Twenty microliters of each cell suspension was mixed with 200 µL of melted low melting point agarose (LMA) and 75 µL of this mixture was placed onto the Trevigen CometSlide for electrophoresis. Subsequent to electrophoresis, samples were visualized with SYBR Green I and fluorescence microscopy. Twenty pictures were taken for each sample and at least 135 cells per experiment were examined for comet tails using CometScore software (TriTek Corporation). Three independent MEF lines for each genotype were assayed in triplicate. Student's t test was used for statistical analysis. Statistics All experiments were performed at least in triplicate. Data are represented as the mean ± SEM. Statistics for qRT-PCR, luciferase, clonogenic, and comet assays was performed using Student's t test for comparison between two groups. A p value of 0.05 was considered significant. Supporting Information Figure S1 Heatmap showing unsupervised hierarchical clustering analysis of p53, p63, and p73−/− E1A MEFs after doxorubicin treatment. Each row represents the relative expression level for a gene compared to an untreated isogenic MEF line. The columns represent multiple isogenic MEF lines for each indicated genotype. Only genes that were significantly down-regulated in at least one mutant MEF line and not wild-type MEFs in response to doxorubicin treatment are represented. The red color indicates high expression and the green color indicates low expression, while black indicates no significant change. Clustering based on Euclidean distance indicates that p63 and p73 deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. The GenBank Accession Number is shown in the right hand column. (0.39 MB PNG) Click here for additional data file. Figure S2 Genes involved in DNA repair are differentially expressed in MEFs deficient for p63 and/or p73. Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/−, p63−/−, p73−/−, and p63−/−;p73−/−) before and after (D) treatment with doxorubicin. The Y-axis shows the relative mRNA levels of mre11, BRCA2, Rad51, and Rad50 before and after treatment with doxorubicin (0.34 µM) for 12 hours. GAPDH was used as an internal control. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represented as mean ± SEM. (2.17 MB TIF) Click here for additional data file. Table S1 Pathway analysis of p63 and p73 target genes. (0.11 MB JPG) Click here for additional data file. Table S2 Identified p53/p63 binding sites for genes with differential expression. (1.10 MB XLS) Click here for additional data file. The authors have declared that no competing interests exist. This work was supported by grants to ERF from the Susan G. Komen Foundation (BCTR600208) (http://ww5.komen.org/), American Cancer Society (RSG-07-082-01-MGO) (http://www.cancer.org/), March of Dimes (Basil O'Connor Scholar) (http://www.marchofdimes.com/), Leukemia and Lymphoma Society/Hildegarde D. Becher Foundation (http://www.leukemia.org/hm_lls), and NCI-Cancer Center Core Grant (CA-16672) (http://www.cancer.gov/) (U.T. M.D. Anderson Cancer Center). ERF is a scholar of the Rita Allen Foundation (http://www.ritaallenfoundation.org/) and the V Foundation for Cancer Research (http://www.jimmyv.org/). TJ is an Investigator of HHMI (http://www.hhmi.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Vogelstein B Lane D Levine AJ 2000 Surfing the p53 network. Nature 408 307 310 11099028 2 Yang A Kaghad M Wang Y Gillett E Fleming MD 1998 p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 2 305 316 9774969 3 Yang A McKeon F 2000 P63 and P73: P53 mimics, menaces and more. 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==== Front BMC Womens HealthBMC Women's Health1472-6874BioMed Central 1472-6874-9-271976159810.1186/1472-6874-9-27Research ArticleFactors affecting awareness of emergency contraception among college students in Kathmandu, Nepal Adhikari Ramesh [email protected] Geography and Population Department, Mahendra Ratna Campus, Tribhuvan University, Kathmandu, Nepal2 Institute for Population and Social Research, Mahidol University, Salaya, Thailand2009 17 9 2009 9 27 27 14 6 2009 17 9 2009 Copyright © 2009 Adhikari; licensee BioMed Central Ltd.2009Adhikari; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In Nepal, Emergency Contraception (EC) could play a critical role in reducing unintended pregnancies, but very few people aware about it. This paper aims to investigate the level of awareness and factors influencing awareness of EC among college students. Methods A cross-sectional study was carried out in April-May 2006. Structured self-administered questionnaires were administered to 1,137 college students (573 males and 564 females) in Kathmandu valley. The association between awareness of EC and the explanatory variables were first assessed in bivariate analysis using the Chi-square test. The associations were further explored using a multivariate logistic analysis. Results Only about two-thirds of college students (68%) had ever heard about EC. Bivariate analysis shows that males were more aware (72%) of EC than were females (64%). Similarly, the awareness level was significantly higher among younger, unmarried youth who were from outside Kathmandu Valley, who lived with friends, and who had received reproductive health (RH) education in school/college. The study also found that students' sex, permanent place of residence (district), and RH education are significant predictors of awareness of EC. Males are 1.5 times more likely to be aware of EC compared to females. Furthermore, students who lived in Kathmandu Valley were 41% less likely to be aware of EC than were students from outside Kathmandu Valley. On the other hand, those students who received RH education in school/college were almost nine times more likely to be aware of EC compared to those who did not receive such education. Conclusion Awareness of the EC is low among college students in Nepal. Health education initiatives should target students as they are more likely to be sexually active. There is a need to further educate students about EC which can help to reduce unintended pregnancies, many of which result in unsafe abortion and take a large toll on women's health. ==== Body Background Emergency contraception (EC) is contraception administered after unprotected intercourse. EC is the only method women can use to prevent pregnancy after they have had unprotected sexual intercourse, have experienced a contraceptive failure, have remembered too late that they have forgotten to take their birth control pills, or have been forced to have sex against their will. EC is sometimes referred to as "morning-after" or "post-coital" contraception. EC is intend for occasional or emergency use only and not as a regular means of contraception. Formerly, EC was thought to be effective only within 72 hours, but recent studies have confirmed it is effective for up to 120 hours [1,2]. EC methods include taking special doses of ordinary birth control pills as well as inserting an intrauterine device (IUD). Depending on the method used, EC can reduce women's risk of becoming pregnant from a single act of intercourse by between 75 and 99 percent [3]. Each year, about 210 million women around the world become pregnant [4]. Among them, about 75 million pregnancies (36%) are unplanned and/or unwanted [5]. Unplanned/unwanted pregnancy is one of the leading causes of maternal mortality and morbidity in South Asia. Reasons for such huge numbers of unintended pregnancies in South Asia include a low rate of contraceptive use, method failure, and high unmet need for contraceptives. Each year worldwide, more than 20 million women experience ill health as a result of pregnancy [6]. It is estimated that between 8 and 30 million pregnancies each year result from contraceptive failure either due to inconsistent or incorrect use of contraceptive methods or failure of the method itself [7]. Research studies conducted in the USA have reported that higher rates of unintended pregnancy occur among college-age women, with 60% of pregnancies among 20-24 years old being unintended. The percentage of unintended pregnancy is even higher among 18-19-year-old females (79%) [8]. In Nepal, the data suggest that more than a third (35%) of all pregnancies [9] and 41% of the current pregnancy among currently pregnant women are unintended [10]. The prevalence of premarital sex has been reported as 39% among college males and 12% among college females [11]. A considerable proportion of both males (10%) and females (22%) reported that their first sexual intercourse happened without their consent. These students are at the greatest risk of unintended pregnancy. A study has also found that a large proportion of college students who were studying in Kathmandu (43% of males and 55% of females) did not use a condom during their first sexual intercourse [12]. Research on the awareness of EC among college students may help to inform policy makers and education planners in Nepal. Unfortunately, no research has been conducted in this area among the students in the country. The aim of our study is to investigate awareness levels and factors influencing awareness of EC among college students. We hope this study will provide baseline data to assist policy makers and education planners in developing appropriate evidence-based strategies and curricula in school/college to prevent unintended pregnancy and unsafe abortion. Methods The institutional review board of the University Grant Commission (UGC), Nepal approved this study. The data used in this paper come from a cross-sectional survey on "Attitude and behavior towards pre-marital sex among college students of Kathmandu, Nepal" carried out in 2006. A two-stage systematic random sampling technique was applied, the first stage of which included random selection of 12 colleges in Kathmandu. In order to select these 12 colleges, a list of all the private and public colleges affiliated with Tribhuvan University and located in Kathmandu Valley was obtained from the office of the Vice Chancellor in Kathmandu. This list included colleges which provide intermediate (commonly known as Grade 11 & 12), undergraduate, and graduate degrees. In the second stage, two classes were selected randomly from each sampled college. The number of students in a class ranged from 40 to 60. Since all the colleges were co-educational, all males and females students present on the day of the interview in the sampled classes were requested to participate in the study. Females and males students were interviewed separately in different classrooms. A self-administrated structured questionnaire in the Nepali language was used to obtain information from the students. The questionnaires were pre-tested among college students in a non-selected college and later refined as required. Almost all students in selected classes were present on the day of interview. None of the approached students refused to participate in the study. With regard to awareness of EC, the survey assessed awareness by asking the question, "Have you ever heard about emergency contraception?" [See additional file 1]. A total of 1,137 college students (573 males and 564 females) in Kathmandu Valley were interviewed. Verbal informed consent was obtained from the participants before they were enrolled in the study. The consent form was written in the local language stating the study's objectives, nature of participant's involvement, risk and benefits, and confidentiality of the data. Students were requested to read the consent form carefully. They were given clear options on voluntary participation. It was also made clear that they could refuse to answer any questions and terminate the interview when they desired. Confidentiality of information was ensured by removing personal identifiers from the completed questionnaires. The names of sampled colleges are not made public and thus it is not possible for anyone outside the research team to trace reported incidents of sexual behavior to respondents. Respondents are thus protected against any possible adverse repercussions from participating in the study. All completed questionnaires were entered into a database immediately after being manually coded and validated. Data entry and validity checks were performed for all the questionnaires by using the software program dBase IV. The cleaned and validated data was transferred into the SPSS 11.5 program for further processing and analysis. Both bivariate and multivariate techniques were applied to identify the factors associated with the likelihood of being aware of EC. The Chi-square test was used to test the association. The variables that were significant in the bivariate analysis were reexamined in the multivariate analysis (binary logistic regression) in order to identify the significant predictors after controlling other variables. Results A large majority of the respondents (85% males and 92% females) were in the youth category (15-24 years). An overwhelming majority of the students (88% of males and 83% of females) were unmarried, and 86% (91% of males and 80% of females) were from outside Kathmandu valley. Students covered in this study were from 67 districts out of 75 districts of the country. A majority of the males (59%) and about a quarter of the females (23%) lived either with friends or alone in Kathmandu. More than half of both males and females were currently pursuing their undergraduate degree. A large majority of the students (93% of the females and 91% of the males) had received education related to reproductive health in their respective schools/colleges (table not shown). Overall, only about two-thirds of the college students (68%) had heard about EC. Table 1 shows a clear association between awareness of EC and other background variables such as sex and age of the students, level of education, marital status, permanent place of residence, types of current accommodation, and RH education in school/college. For example, male students were more aware (72%) of EC than were female students (64%). Similarly, a higher proportion of younger students aged 15-19 (73%) than the older students (66%) were aware of EC. Unexpectedly, a higher percentage of students (74%) who had an undergraduate level of education had heard of EC compared to those who had graduate or post graduate education (66%). Regarding marital status, unmarried students were more aware of EC than married students. Similarly, the awareness level was significantly higher among those who were from outside Kathmandu Valley, who lived either alone or with friends, and who had received reproductive health education in school/college. For instance, more than two-thirds of students (70%) who were from outside Kathmandu Valley while less than three-fifths (57%) of those who lived in Katmandu Valley were aware of EC. Likewise, those students who were living with their family members were less aware (66%) of EC than those who were living either alone or with friends (72%). A far higher proportion of the respondents who had received RH education in school/college (72%) had heard about EC than those who had not received RH education (24%) (Table 1). Table 1 Awareness about Emergency Contraception by background characteristics Background characteristics Awareness of EC Number χ2 Yes No Sex of the respondents P < 0.01 Females 64.2 35.8 564 Males 72.3 27.7 573 Age group P < 0.05 15-19 72.5 27.5 418 20 and above 65.8 34.2 719 Marital Status P < 0.05 Married 61.8 38.2 165 Unmarried 69.3 30.7 972 Level of education P < 0.05 Intermediate 74.3 25.7 272 Bachelors degree 66.3 33.7 629 Masters degree 66.5 33.5 236 Permanent place of residence P < 0.01 Outside Kathmandu Valley 70.1 29.9 973 Kathmandu Valley 57.3 42.7 164 Type of current accommodation P < 0.05 With family members 65.9 34.1 665 Without family members 71.6 28.4 472 Received RH education in school/College P < 0.001 No 23.7 76.3 93 Yes 72.2 27.8 1044 Total 68.2 31.8 1137 100.0 Table 2 Odd Ratio (OR) and 95% Confidence Interval (CI) for having awareness about emergency contraception among college students Selected predictors OR 95% CI Sex of the respondents Females (ref.) 1.00 Males 1.50 1.12-2.01 Age group 15-19 (ref.) 1.00 20 and above 0.83 0.58-1.19 Level of education Intermediate (ref.) 1.00 Bachelors degree 0.85 0.57-1.26 Masters degree 0.89 0.53-1.47 Marital Status Married (ref.) 1.00 Unmarried 1.19 0.82-1.72 Permanent place of residence Outside Kathmandu Valley (ref.) 1.00 Kathmandu Valley 0.59 0.41-.86 Type of current accommodation With family members (ref.) 1.00 Without family members 0.96 0.71-1.31 Received RH education in school/College No (ref.) 1.00 Yes 8.92* 5.38-14.78 -2 Log likelihood 1309.4 Cox & Snell R Square 0.094 Note Significant at P < 0.01, ***P < 0.001, ref = reference category These observed associations in bivariate analysis were reassessed by logistic regression to identify adjusted association with the probability of being aware of EC. The results are presented in Table 2. As can be seen from the table, variables such as sex of the students, permanent place of residence (district), and RH education are significant predictors of awareness of EC after controlling for other variables. Males are 1.5 times more likely than females to be aware of EC. Similarly, students who lived in Kathmandu Valley were 41% (OR = 0.59, 95% CI = 0.41-0.86) less likely to be aware of EC than students from outside Kathmandu Valley. Likewise, those students who received RH education in school/college were almost 9 times more likely to be aware of EC compared with those who did not receive such education (Table 2). Discussion The study looked at the awareness level and factors influencing awareness of EC among college students. Research shows that most college students had experienced unprotected sex and unintended pregnancy [8]. In such cases, EC could play a critical role in reducing unintended pregnancies. The awareness of EC among college students in Kathmandu is 66%, which is higher than the level found among university students in Kenya (39%) [13], Ghana (43%) [14] and Cameroon (63%) [15] On the other hand, it was very low compared to the university students, for example, in the USA (94%) [16] and Jamaica (84%) [17]. Although EC is not recommended as a routine family planning method, it is a very useful method after unprotected sexual intercourse to reduce the chance of unplanned or unwanted pregnancies. EC is an effective means of preventing unwanted pregnancies, but unfortunately the large numbers of college-going students are unaware of it. Analysis shows that male students were more likely to be aware of EC than were female students. It could be due to the fact that more males are living with their friends. The other reason could be that males are more open to talking with friends or seniors about issues of sex and sexuality than are females. Similarly, those respondents whose permanent place of residence is outside of Kathmandu were more likely to be aware of EC than were those whose permanent residence is in Kathmandu Valley. It was also found that almost all of the students whose permanent place of residence is Kathmandu were living with their family. This could cause them to be reluctant to talk about family planning methods or sex-related issues with their family members. The other reason might be that the students who live with friends are better informed by their peer groups. In Nepal, students are taught subjects touching on health and population in school, including basic information on fertility, mortality, human reproductive organs, menstruation, and sexually transmitted infections including HIV/AIDS. Although the school or college health curriculum does not include EC, those students who had received RH education in school/college were more likely to be aware of EC than were those who had not. This may be because teachers who teach RH usually teach about risky sexual behavior, including the prevention of unwanted or unplanned pregnancies. Similarly these teachers usually teach the students about the emerging issues in reproductive health. Conclusion Awareness about EC among college level students is low. Health education initiatives should target such students as they are more likely to be sexually active. There is a need to educate students about EC, which can help to reduce unintended pregnancies, many of which result in unsafe abortion and take a large toll on women's health. Education about EC at college levels could benefit even out-of-college youth, because their friends often are students. Competing interests The author declares that they have no competing interests. Authors' contributions RA, Lecturer of Mahendra Ratna Campus, Kathmandu, conceived and designed the study. He carried out the data collection, conducted data analysis and interpretation of the data. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Questionnaire on the study entitled "Survey on Attitude and Behavior towards premarital sex among college students of Kathmandu Valley". The questionnaire includes various aspects of information regarding premarital sex, risky sexual behavior, HIV, emergency contraception etc. Click here for file Acknowledgements The author would like to thank the University Grant Commission, Nepal for providing the funds for this research, to the administrators of all sampled colleges in the Kathmandu Valley for their support and the students for participating in the study. 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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central 1471-2164-10-4551978575110.1186/1471-2164-10-455Research ArticleMicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori) Liu Shiping [email protected] Liang [email protected] Qibin [email protected] Ping [email protected] Jun [email protected] Daojun [email protected] Zhonghuai [email protected] Qingyou [email protected] The Key Sericultural Laboratory of Agricultural Ministry, College of Biotechnology, Southwest University, Chongqing 400715, PR China2 National Engineering Center for Beijing Biochip Technology, Life Science Parkway, Changping District, Beijing 102206, PR China3 Beijing Genomics Institute at Shenzhen, Shenzhen 518083, PR China4 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100000, PR China5 Institute of Agricultural and Life Sciences, Chongqing University, Chongqing, 400030, PR China2009 28 9 2009 10 455 455 18 3 2009 28 9 2009 Copyright © 2009 Liu et al; licensee BioMed Central Ltd.2009Liu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Results Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275). Conclusion We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional analysis of miRNAs in this animal. ==== Body Background MiRNAs are an abundant class of small (~22 nucleotides) noncoding RNAs expressed by a variety of eukaryotic organisms and viruses [1,2], which represent at least 1% of predicted genes within the genomes of individual species [3]. A mammalian genome may contain >500 genes encoding miRNAs [4,5]. Accumulating evidence shows that miRNAs function in a broad range of biological processes, including development, cellular differentiation, proliferation, metabolism and apoptosis [1,6-8]. Organisms devoid of miRNAs undergo arrest during development [9,10]. Recent studies additionally implicate miRNAs in the pathogenesis of human diseases, including cancer and metabolic disorders [11-15]. Moreover, miRNAs are required for normal steroid hormone signaling [16]. Interestingly, Dicer and miRNAs are not prerequisites for the development of zebrafish germline stem cells [17], despite being essential for zebrafish development [9]. However, in Drosophila, miRNA pathways appear essential for stem cell division and for bypassing the G1/S checkpoint of the cell cycle [18]. This dissimilarity may reflect diverse mechanisms in the proliferation and differentiation of pluripotent stem cells [19]. Computational predictions of miRNA targets indicate that these noncoding RNAs regulate hundreds of different mRNAs at the posttranscriptional level, and over 30% of animal genes [6,20]. However, to date, the roles of only a handful of known miRNAs have been experimentally determined. The current repression models of the mechanisms of miRNA-mediated gene silencing are still the subject of considerable debate [21], and many potential targets may actually be pseudotargets, thus preventing miRNAs from binding to their authentic targets via sequestration [22]. B. mori, the characteristic representative of lepidoptera, undergoes four distinctive main developmental stages, defined as embryo, larva, pupa, and adult moth. Silkworms have no characteristic feeding behavior at the adult stage, but display prominent traits, such as silk production, monophagy, and voltinism [23]. Therefore, B. mori is considered an important model organism to investigate various biological phenomena, including development, gene regulation, and morphological innovation [23]. Increasing numbers of miRNAs have been experimentally identified in the silkworm [24-27]. Several of these are temporally regulated or stage-specifically expressed, as observed by microarray analysis at four time-points [24]. However, the lifespan of the silkworm is over 50 days, with each of the four main metamorphosis stages lasting for several to more than 20 days. Therefore, examination of a few time-points is unlikely to reflect the accurate expression patterns of miRNAs over the entire life cycle of this animal [28]. Several heterochronic genes function in a regulatory pathway to influence the timing of cellular development, thereby ensuring a coordinated schedule of developmental events [29,30]. Correlation between miRNA expression and normal behavior is suggested in Drosophila adults [31]. In the present study, particular attention was focused on the detailed temporal profiling of miRNAs responding to developmental stage transitions of silkworms. The microarray data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [32] under accession number GSE18030 . The data revealed wide diversity in miRNA expression, ranging from stage-specific to constitutive patterns. Our study provides a broad overview of miRNA expression in the developing silkworm, and is thus an important initial step in elucidating the functions of miRNAs in this animal. Results General profile of miRNA expression over the whole life cycle of Bombyx mori To determine the expression profiles of miRNAs in the developing silkworm, we performed microarray hybridization with 106 cDNA probes complementary to 92 miRNAs (see Additional file 1). Additional file 1 contains the main information, including mature and putative precursor sequences, foldback arms, 9× genome location data (site distribution, intergenic, intronic, or overlapping CDS; and chromosome), miRbase designation where available, and folding energy. All probes for miRNAs as well as negative and positive controls were printed in triplicate on the microarray slides (see Methods and Additional file 2). Half of the 92 miRNAs passed the filtering expression threshold, 35 were also confirmed by Northern blotting, and 15 (including bantam and miR-1) were robustly expressed from the embryo to adult stages (Additional files 3 and 4). The use of alternative probes for some miRNAs generally resulted in similar expression profiles (for example, in the case of miR-275# and miR-275), strongly confirming probe authenticity. Expression peaks for all established miRNAs were distributed over nine time-points. We observed up-regulation of 7 miRNAs and down-regulation of 10 miRNAs over the whole life cycle of the silkworm (Additional file 4). One new silkworm miRNA, miR-970, presented the highest signal on microarray at the early 1st instar larva stage, which was confirmed by Northern blotting (Additional file 3). Four miRNAs (miR-281-5p, miR-281-3p, miR-79, and miR-317) were not detected until the silkworm hatched (eIL1), and were subsequently expressed at high levels until the birth of the adult moth, in microarray analysis (Additional file 4). miR-124 was typically down-regulated during silkworm development from day 6 embryo to adult moth, and undetectable after the 3rd instar larva stage (Figure 1A, Additional file 4). In contrast, both microarray and Northern blotting analyses confirmed that let-7 and miR-100 were coordinately up-regulated, gradually accumulating from late 1st molt until the 3rd molt stage (Figure 1A, C, Additional file 4). These oscillations were coincident with the traditional watershed in sericulture management between the early larval and late larval stages. miR-29b displayed the highest expression at the embryo stage, and rapidly decreased in level after the 1st molt larval stage, but rose significantly once more in fresh female moths carrying eggs in the abdomen (Figure 1A), raising the possibility of an involvement in oogenesis and embryogenesis. Interestingly, in other organisms, such as the mouse, miR-29b was almost undetectable in the embryo, but extensively expressed in the adult during brain development [33]. The expression patterns of miR-34b and miR-275 fluctuated in a complementary manner (Figure 1B). Specifically, miR-34b expression was restricted to instar larvae and adults, and was absent in the embryo, molt larvae, and pupae (Additional file 4). Ecdysone and Broad-Complex are responsible for the down-regulation of miR-34 in the development of Drosophila [34]. In fact, miR-34 is highly conserved, and its homologs have been widely identified in several other species across phyla. Recent studies show that members of this family, miRNA-34a-c, are direct transcriptional targets of p53, and participate in the suppression of cell proliferation [35,36]. In contrast to miR-34b, a faint expression signal from miR-275 was initially detected at early 3rd molt, followed by slight up-regulation in early 4th instar, early 4th molt, and early 5th instar larvae. This miRNA was highly expressed in early spinning larva and up-regulated to peak levels in the new pupa, but was remarkably absent in new female adults (Figure 1B). The expression profile derived from microarray data was supported by Northern blotting results (Figure 1C). Microarray analyses revealed that miR-92 was exclusively expressed at day 6 embryo, late 2nd molt, and early pupal stages (Figure 1A), but strong expression was confirmed in day 6 embryos only by Northern blotting (Figure 1C). The miRNA profiles obtained with the general whole-life array provide an insight into the stage-specific miRNA transcriptome, and may thus facilitate the identification of sequential classes of miRNAs and their primary targets associated with distinct developmental stages. Figure 1 Temporal expression profile of miRNAs obtained with the whole-life test. (A) Specific miRNAs were up-regulated or down-regulated over the entire life cycle of the silkworm. (B) Comparison of the expression profiles of miR-275 and miR-34b during the life cycle of silkworm. (C) Northern blotting analysis of miRNAs during the silkworm life cycle. 5srRNA and U6 were used as the loading controls. Expression profile of miRNAs in silkworm embryos In the course of embryogenesis, large gene regulatory networks generate refined spatial and temporal patterns of expression [37]. Silkworm embryogenesis is initiated by formation of the zygote nucleus (synkaryon) within about 2 hours after egg deposition, and develops through diverse phases for about 10 days before hatching [38]. Only one time-point (day 6 embryo) was examined using the whole-life array, and this did not provide sufficient information on expression profiles in developing embryos of silkworm. We further assessed 9 and 11 time-points using microarray and Northern blotting, respectively, leading to significant extension of the range of temporal profiles of miRNA expression. In all, 36 unique miRNAs satisfied the expression standard for microarray analysis during embryogenesis (Figure 2A, see Additional file 5). The whole process was divided into two main sections in terms of hierarchical clustering of miRNA expression, with one clear boundary between day 2 and day 3 embryos and the remaining time-points. Figure 2 MiRNA expression patterns during silkworm embryonic development. (A) Hierarchical clustering analysis of the expression of 36 miRNAs obtained from embryos of early 1st instar larvae. The colors represent relative and mean-centered expression for each miRNA: green, low; black, mean; red, high. a and b represent the average signals of each probe printed at three points on each block. (B) Northern blotting analysis of miRNAs in the embryos and early 1st instar larvae. 5srRNA and U6 served as the loading controls. Small and large miRNA transcripts were detected in pre-laid eggs and embryos, and were identified as miR-8, miR-252 and let-7 (indicated with a red arrow in Figure 2B). Accumulating large fragments of these miRNAs suggest that maturation of functional small molecules involves sequential cleavage from larger transcripts [39]. Seven "early" miRNAs were detected in freshly deposited eggs (including bantam, miR-92, and miR-184-3p), which continued to be expressed until the formation of the synkaryon (6 h Em) (Additional file 5). Whole-life analysis revealed that miR-92 expression was restricted to three time-points during the course of development from embryo to moth, and peaked at the day 6 embryo stage (Additional file 4). A more precise examination disclosed a significantly extended existence during embryogenesis and a virtual expression peak in the day 3 embryo (Additional file 5). After fertilization, the synkaryon undergoes synchronous mitosis, resulting in the formation of blastoderm and the subsequent germband. Numerous cleavage nuclei begin to penetrate the periplasm about 10 h after oviposition. However, microarray and Northern blotting confirmed that few miRNAs were expressed at this time (12 h Em) (Figures 2A, B, Additional file 5), possibly because of the impending diapause stage [38]. After a further 12 h (24 h Em), 12 miRNAs were detected soon after egg release from diapause, including the 7 "early" miRNAs and others observed for the first time (miR-252, miR-8, miR-2a, miR-79, and miR-10b-3p). Expression of these miRNAs continued for 24 h (2 d Em), with slight fluctuations during the time of cell differentiation, to successively form the germband, amnion, gastrulation apparatus, and even the appendages of the abdomen and telson. One day later (3 d Em), expression of another group of miRNAs was initiated during the blastokinesis period after formation of the neural groove and abdominal appendages. In view of the finding that morphogenetic processes are severely affected in miRNA-devoid embryos [17], we propose that these miRNAs act concertedly to regulate the morphogenesis oforgans. After blastokinesis, the embryo resumes growth by successively forming dorsal integument, the alimentary canal, setae, the epidermis, and the trachea, possibly involving several genes expressed during formation of larval organs [38]. This occurs around the head pigmentation stage in day 6 embryos. At this time-point, 8 miRNAs were initially expressed (including miR-29b, miR-34b, and miR-124). Once the larval organs form, the serosa is digested and pigmentation of the head and body progresses, but no other important morphological changes occur in the embryo [38]. However, a higher number of miRNAs were initially present in day 9 embryos (9 d Em) at the body pigmentation stage, including miR-281-5p, miR-281-3p, miR-263, miR-970, miR-133, miR-283, and miR-317. During the subsequent 24 h, the young larvae inside the eggshells eat and break the chorion around the micropylar region and emerge [38]. No distinct differences in miRNA expression existed between newly hatched silkworms and day 9 embryos. miRNA expression in silkworm larvae correlates with stage transitions To further investigate the correlation between miRNA expression and stage transition, we sampled throughout the larval stages from newly hatched silkworm to late 3rd molt larva (early larval stages), and early 4th instar larva to late 5th instar larva (late larval stages). Day 2 and day 9 embryos served as controls to ensure comparable results. Twenty unique miRNAs were highly expressed, as shown by microarray experiments, during the early larval stages, including 15 strongly detected miRNAs identified in the whole-life array (Additional file 6). Late 2nd instar larvae expressed the largest number of miRNAs, among which 25 displayed peak levels at this time-point (Additional file 6). The whole-life array revealed exclusive miR-29 expression at the early 1st instar and late 1st molt larval stages. In fact, miR-29 was additionally expressed in late 1st instar, late 2nd instar, and late 3rd molt larvae. miR-92 was absent from the early 1st, 2nd, and 3rd instar larval stages, but highly expressed in late 1st, 2nd, and 3rd instar larvae and late 1st, 2nd, and 3rd molt larvae. The miR-124 level at the late 1st instar larval stage was two-fold higher than that during the early 1st instar larval stage. miR-124 was sharply up-regulated to peak levels from the time of its initial expression in the day 6 embryo, followed by down-regulation, despite waning in early 2nd and early 3rd instar larvae and early 3rd molt larvae, and waxing in late 2nd and late 3rd instar larvae and late 3rd molt larvae. In contrast, miR-100 and let-7 were initially expressed in late 2nd instar larvae, and accumulated to high levels in late 3rd molt larvae, with obvious fluctuations during the early larval stages. miR-34b and miR-275 exhibited complementary changes in patterns in whole-life profiling, but displayed sympathetic vibrations, particularly from the early 2nd instar to late 3rd molt stages, upon more detailed examination (Figure 3B). The presence of miRNAs during the early larval stages was further confirmed by Northern blotting (Figure 4A). Figure 3 Microarray analysis of miRNA expression during the larval stages. (A) Stage-specifically expressed miRNAs during the early larval stages. (B) Comparison of the expression profiles of miR-275 and miR-34b during the early larval stages. (C) Stage-specifically expressed miRNAs during the late larval stages. (D) Comparison of miR-275 and miR-34b expression profiles during the late larval stages. Figure 4 Northern blotting analysis of miRNA expression during the larval stages. (A) Northern blotting analysis of miRNAs during the early larval stages. (B) Northern blotting analysis of miRNAs during the late larval stages (C) A number of miRNAs displayed sex-dependent differences in expression at day 3 and day 7 fifth instar larval stages. 5srRNA and U6 served as the loading controls. In total, 18 of the 20 miRNAs expressed throughout the early larval stages were maintained at high levels during late larval stages (Additional file 7). The two remaining miRNAs, miR-133 and miR-279, showed low levels of expression during the whole 4th molt stage (Additional file 7). miR-100 and let-7 were up-regulated from 1st instar to 3rd molt, maintained over the 4th and 5th larval stages (Additional file 7), and highly expressed from early to late 4th instar larvae and fifth-instar day 2 and day 7 larvae (Figure 3C). miR-274 displayed no expression signal during early larval stages, was initially detected in 5th instar day 2 larvae, and was subsequently up-regulated to peak levels in day 7 larvae. miR-29b was detected at the late embryo and 1st instar larval stages (Figure 3A), but was not expressed from early 4th to 5th instar day 7 larval stages (Additional file 7). miR-92 and miR-124 shared similar patterns with high expression at three time-points, specifically, late 4th larval instar, and day 3 and day 7 5th instar larvae, but was absent from 4th molt larvae. The expression profiles of miR-275 and miR-34b were regulated in a complementary manner throughout the penultimate and final larval stages (Figure 3D). The changing expression profile of miR-275 by microarray was supported by Northern blotting results (Figure 4B). Some miRNAs displayed significant gender-specificity at the day 3 and day 7 fifth larval instar stages (Figure 4C), whereby signal values in females were at least two-fold higher than those in males. The majority of expressed miRNAs oscillated with a clear-cut pattern corresponding to transitions between instar and molt stages, specifically, being higher at the late 1st, 2nd, and 3rd instars and the late 3rd molt, and lower at the early 1st, 2nd, and 3rd instars and the early 3rd molt (Additional file 8A). As with expression patterns at the early larval stages, most miRNAs exhibited a defined expression pattern during the 4th and 5th larval stages, specifically, up-regulation at the 4th instar, down-regulation at the 4th molt, up-regulation at the day 2 to day 3 fifth instar, down-regulation for 2 days, followed by a sharp increase again at day 7 (Additional file 8B). Interestingly, these coordinate expression changes were generally in keeping with ecdysone pulsing [40]. Expression profiles of miRNAs in spinning larvae, pupae and moths To determine the expression profiles of miRNAs at the spinning larva (Sp), pupa (Pu), and adult (Ad) stages, the whole-life test (Additional file 4) was applied from the development of the spinning larva to adult moth at 15 and 14 specific time-points for females and males, respectively. Based on hierarchical clustering, samples from the larval, pupal, and adult stages were clearly separated (Figure 5A). The proximal stages displayed similar miRNA expression profiles. Therefore, miRNA expression patterns may be applied as a developmental marker of silkworm individuals, as suggested for the developing mammalian brain [33]. In total, 15 miRNAs were highly expressed throughout pupal and adult stages by microarray, among which 10 were common to both sexes and 9 were robustly expressed over the whole life cycle (including miR-252, miR-1, and let-7a) (Additional files 9 and 10). A number of miRNAs were also confirmed in females by Northern blotting (let-7a, let-7b, miR-8, and miR-2a) (Figure 5B). Moreover, Northern blotting revealed the presence of miR-263b throughout the metamorphosis process, although high expression was only evident in day 6 and day 7 pupae in microarray experiments. In females, the 55 expressed miRNAs peaked at eight time-points (20 in the day 7 pupa and 10 in the egg-removed moth stage) whereas in males, the 52 expressed miRNAs displayed the highest signals at nine time-points (16 at early prepupal and 11 at day 4 pupal stages). However, specific miRNAs shared the same expression profile in both genders as larva-pupa-adult metamorphosis advanced, and expression of the majority of miRNAs abruptly fell at the day 3 pupa time-point (see Additional file 11). These results raise the issue of whether females and males share the same mechanism in coordinating miRNAs for the larval-pupa-adult transition. Illustratively, five miRNAs were compared between females and males (Figures 5C, D). In the whole-life test, miR-29b was not present in early female pupae, but was strongly expressed in the early female moth (Additional file 4). A more detailed examination revealed its initial expression in both genders at the day 4 pupal stage, and subsequent up-regulation. Whereas expression in both sexes fluctuated in a generally similar manner, miR-29b was far more highly expressed in females. The whole-life array revealed that, in contrast to miR-29b, miR-275 expression peaked in early female pupae, but not in the early female moth (Figures 1B, C). A precise test further revealed strongest expression in the prepupa and day 2 pupa in both females and males (Figures 5C, D). Moreover, both Northern blotting and microarray tests revealed down-regulation of miR-275 in both sexes during this process (Figure 5B, E). Unexpectedly, miR-275 was not expressed in the early female moth with eggs. However, when the eggs were manually removed from the maternal abdomen, miR-275 expression was significantly increased, further confirming its absence in pre-laid eggs and early embryo stages. Although the whole-life array did not confirm the existence of miR-274 in silkworms, the presence of this miRNA was verified in the fifth instar larva from days 2 to 7 (Figure 3C, Additional file 8). Interestingly, the detailed investigation revealed high expression in the spinning larvae of both genders, but no expression in pupae of either sex (Figure 5C, D, Additional files 9, 10), strongly implying an important function for this miRNA in the spinning activity of silkworms. miR-29b and miR-92 appeared to share similar patterns by whole-life profiling, but the detailed assay revealed evident differences between their expression profiles. miR-29b expression peaked in egg-removed moths, whereas miR-92 expression was maximal in the new pupae of both sexes. miR-34b was robustly expressed in females at the early cocoon spinning and new adult moth stages, but was expressed at only low levels in early pupae in the whole-life test (Figure 1, Additional file 4). Its expression in pupae and adults was further established by precise sampling (Figure 5C, D; Additional files 9, 10). miR-34b was not expressed, or was expressed at very low levels, in female pupae during the first 5 days, but was significantly up-regulated in day 6 pupa, and highly expressed in female adult moths with or without eggs. However, this miRNA was also nearly undetectable in the male prepupa and male pupae from 0 h to day 7, but displayed a sharp increase at day 8 and attained its highest expression in the fresh male adult moth, followed by an evident decrease 2 days later. The silkworm moth lives for only several days, and dies soon after mating or laying eggs. In keeping with the development of the silkworm body, the expression levels of all miRNAs in adults of both sexes decreased significantly within 2 days (Additional file 11). Figure 5 Profiling of miRNA expression during pupal metamorphosis. (A) Developmental time-points were grouped using the hierarchical clustering method and gene sets from Additional file 9. Gene names and quantitative miRNA expression levels are presented in Additional file 9. (B) Northern blotting analysis of specific miRNAs in females. (C) Stage-regulated miRNAs in females. (D) Stage-regulated miRNAs in males. (E) Northern blotting analysis of specific miRNAs in males. Northern blots were exposed for different times, and thus the intensities of signals on one Northern blot cannot be directly compared to those from others. A probe against 5srRNA was hybridized to respective blots for comparison. Discussion To the best of our knowledge, this is the first comprehensive investigation of miRNA expression profiles over the whole life-cycle of Bombyx mori. Although previous studies have examined four time-points representing the main developmental stages of the silkworm, to clarify the temporal expression profile of miRNAs [24-26], it is impossible to establish detailed expression patterns at stage transitions over the fifty-day life cycle with limited time measurements. The whole-life test used in the present study provided a general expression profile of miRNAs during silkworm development, and subsequent precise stage-by-stage examinations further confirmed the presence or absence of miRNAs at multiple time-points. The miRNAs exhibiting significant expression changes over the whole life cycle and specific developmental stages are summarized in Additional file 12. For example, the miR-10b-5p/3p pair was down-regulated in whole-life profiling, but stage-by-stage examination revealed up-regulation of these miRNAs during embryogenesis, followed by down-regulation during the early and late larval stages. Several miRNAs, such as miR-34b and miR-305, were up- or down-regulated in whole-life profiling, but in fact, displayed diverse or even opposite regulation patterns at some developmental stages. Despite stable culture conditions of a 12 h light:12 h dark cycle, the temporal niche varies dramatically within the life-cycle of the silkworm, consequently exerting vital pressure on the capacity for temporal adjustment [41]. The Dazao silkworm is a bivoltine strain in which the nature of the diapause is dependent on incubation temperature. Specifically, incubation at 25°C produces only diapause eggs and incubation at 15°C produces exclusively non-diapause eggs [42]. Therefore, the temporal expression profiles of silkworm miRNAs may be under dual control of the developmental program and environmental stimuli, such as the polyprotein precursor mRNA, which is common to diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) [43]. In zebrafish, few miRNAs are expressed within 12 h post-fertilization. An increasing number of miRNAs are detected 1 to 2 days after fertilization and show strong expression when organogenesis is virtually complete [44]. In the early embryo of Drosophila, several miRNAs initiate expression at the onset of zygotic transcription [45]. Their dynamic expression patterns are mediated by tissue-specific enhancers [46]. In the silkworm, a small set of miRNAs weakly accumulated at fertilization (6h Em) or the early embryo stages (see Additional file 5). The maternal Dicer enables zygotic Dicer mutants of zebrafish to live for almost 2 weeks [9]. Emergence of these miRNAs in pre-laid or newly deposited eggs may also be attributed to maternal information, similar to the embryonic diapause induced by the diapause hormone (DH), which is secreted by the maternal subesophageal ganglion during pupal-adult development [43]. MiRNAs are absent in the zygotes of zebrafish, and are primarily detected during the blastula period [47]. The majority of zebrafish miRNAs are expressed in a tissue-specific manner during the late stage of embryonic development [44], and present widely divergent expression profiles throughout the 3 dpf and 5 dpf embryonic brain [48]. In frog (Xenopus laevis), an increasing number of miRNAs are expressed at specific stages as embryonic development proceeds, and are continuously expressed until the tadpole stage [49]. Few miRNAs are detected during the formation of primary germ layers of the chick, but rapid accumulation occurs during organogenesis [50]. Several miRNAs cannot be detected until the late specific stages of mouse (Mus musculus) embryonic development, but their transcription levels are markedly increased thereafter [51]. Overall, these data leave the intriguing question open as to whether and how these miRNAs are implicated in controlling the fate of protein-encoding genes during embryogenesis, consequently contributing to tissue differentiation and organogenesis. During the prepupal and pupal stages of holometabolous insects, imaginal tissues and organs are newly formed from primordial cells and imaginal discs, whereas larval tissues degenerate in the pupa, and pupal tissues are fully lost in the adult [52]. Larval cuticles break down during the last day before pupal molt [53,54]. Oogenesis and ovarian development of B. mori are triggered by 20-hydroxyecdysone (20E) [55], and then pass through various stages for approximately 10 days from the fifth instar larvae to the pharate adult [56]. Hundreds of genes are required for the dramatic morphological changes in wing disc development during metamorphosis, although their intrinsic roles are yet to be established [57]. This in vitro progression is possibly controlled by unidentified non-coding RNAs [58]. Four miRNAs (miR-29b, miR-34b, miR-277, and miR-285) were significantly up-regulated from the spinning larvae to adult stages, and nine (miR-305, miR-275, miR-289, miR-307-3p, miR-274, miR-286, miR-87, miR-315, and miR-92) were significantly down-regulated during this time-course (Additional file 12). These opposite but coordinate expression changes during the non-feeding stages of females and males should pave the way for further analysis of the mechanisms underlying the metamorphosis of insects. The normal formation of a mature insect egg is processed under concerted regulation of the steroid hormone ecdysone and its receptors [59-61]. Simultaneous expression of miR-125 and let-7 during Drosophila development is synchronized with the high- titer ecdysone pulses that initiate metamorphosis [62]. The ecdysone titer in silkworm fluctuates in response to stage transitions during embryogenesis [63], instar larval stages [40], and larval-pupal-adult development [64-66]. The majority of miRNAs are significantly up-regulated as the ecdysone synthesis rate rises at the end of each larval instar. Similarly, miRNAs are clearly down-regulated when ecdysone synthesis falls during the period from molt to instar larval stage. During pupal metamorphosis, several miRNAs were down-regulated in day 3 pupae of both sexes, consistent with a significant decrease in the ecdysone level. Thus, over the whole life cycle of silkworm, expression rhythms of several miRNAs may be coordinated by ecdysone. However, the miRNA expression profiles did not always fluctuate in accordance with the ecdysone titer. For example, miR-275 and miR-274 expression was typically stage-specific and unrelated to ecdysone pulsing. Furthermore, several miRNAs were upregulated at the end of the 3rd and 4th molt stages, presenting abnormal patterns in relation to ecdysone levels. Most miRNAs displayed two expression peaks at the 5th instar larval stage (from day 2 to 3 and from day 5 to 7), whereas the ecdysone peaks on the last day of the 4th instar larva rapidly declined at the early 4th molt, and ecdysone was maintained at a low level until the onset of the wandering stage [65]. It is possible that additional factors, including hormones other than ecdysone, regulate miRNAs, as the expression rhythms were sometimes out of step with the ecdysone pulse. Conclusion Here, we obtained initial temporal measurements of the levels of abundant miRNAs throughout the entire life-cycle of Bombyx mori. The diverse expression patterns of silkworm miRNAs strongly supports the idea that the miRNAs function at different levels to regulate silkworm development. Refined expression patterns corresponding to stage transitions may provide a strong molecular basis for further functional analysis of miRNAs in this animal model. Methods Computational prediction of silkworm miRNAs We initially predicted silkworm miRNAs using miRscan algorithms, as described by Lim and colleagues [67,68], as well as homology searches with PatScan algorithms [69,70] with the first silkworm genome assembly (6× genome data) [71] and the limited number of miRNAs available in the miRBase release 4.0 [72]. The specific sequences that could form a correct hairpin structure (hairpin length larger than 55 bp and at least 6 base pairs in the arm) with low free energy (lower than -25 kcal/mol overall and lower than -0.27 kcal/mol per nucleotide) were submitted to microarray and Northern blotting analyses. Subsequently, we employed all the mature miRNAs from the miRNA repository miRBase 11.0 for a homology search for complementary sites on the silkworm genome with no more than 3 mismatches. Hits were extended on the genome and further filtered by limits based on the folding parameters determined by RNAfold [73,74] and mfold [75]. According to the current nomenclature guidelines [76-78], abbreviated 3 letter prefixes 'bmo' are used to designate the species of B. mori, mature sequences and precursor hairpins are labeled 'miR' and 'mir', respectively, and the different miRNA sequences excised from opposite arms of the same hairpin precursor are currently given names ending with -5p or -3p to distinguish the arms. Silkworm culture and RNA extraction Female moths of the domesticated silkworm (B. mori), Dazao, were allowed to lay eggs for 4 h at 25°C. Developing eggs were incubated at 25°C from oviposition until hatching, the first day being the day of oviposition. When developing eggs were incubated at 25°C, head pigmentation occurred on day 7 (6 × 24 h after oviposition), body pigmentation appeared on day 9 (8 × 24 h after oviposition), and more than 95% of eggs hatched on the tenth day. To prevent entry into diapause, fertilized eggs were treated with a hydrochloric acid solution (4 N) at 46°C for 6 minutes and then rinsed thoroughly with water. After hatching, silkworm larvae were reared on mulberry leaves at 25°C and 85% H.R. under a 12 h light/12 h dark photoperiod, and harvested at the desired developmental stages. To obtain populations of B. mori at various developmental stages, animals were synchronized after oviposition by means of cold storage and acid treatment, keeping the diapause eggs at 4°C for at least three months, followed by 5 min of treatment with hydrochloric acid solution at 46°C. Moreover, developmental landmarks, including hatching, larval molting, mounting, spinning, pupariation and eclosion, were employed for more precise staging. Total RNA was extracted with TRIzol reagent (Invitrogen, Gaithersburg, MD), according to the manufacturer's instructions. Microarray printing and hybridization The miRNA probes (denoted 'SW' followed by a serial number) on the microarray were designed to be complementary to the mature sequences of miRNAs, concatenated up to 40 nt with polyT, and modified with an amino group at the 5'-end. Since probe sets for some miRNAs are present more than once on the array, 106 probes for 92 unique miRNAs were used to establish the existence and profiling of miRNAs in the silkworm (Additional file 2). These comprised 45 probes for the homology-found miRNAs, 2 for special silkworm miRNAs, 4 for the antisense strands of miRNAs, 42 for miRNAs of other organisms, and 13 as replicate probes for several miRNAs. All probes, including the controls, were synthesized at MWG Biotech (Ebersberg, Germany), dissolved in EasyArray spotting solution (CapitalBio, Beijing, China) at a concentration of 40 μmol/L, and printed in triplicate on aldehyde-coated slides (CapitalBio) using a SmartArray-136 spotter (CapitalBio). Low molecular weight RNA (4 μg) isolated using PEG solution precipitation was labeled with fluorescent Cy3 using T4 RNA ligase, according to a previous protocol [79], and hybridized overnight to the microarray in 16 μl hybridization buffer (15% formamide, 0.2% SDS, 3×SSC, 50×Denhardt's) at 42°C. Following hybridization, slides were washed in a SlideWasher-8 instrument (CapitalBio) using washing bufferI (0.2%SDS, 2×SSC) and bufferII (0.2×SSC) and dried. Slides were scanned using a laser confocal scanner, LuxScan 10K-A, and images extracted using LuxScan 3.0 software (CapitalBio). Net signals were calculated by subtracting the local background from total intensities and spots with a negative signal awarded the value 10. To make the inter-slide signals comparable, signals were normalized using a global median method. Flaw spots were excluded for further analysis after visual inspection of the hybridization figures using a self-developed program, "Flaw-Spot-Finder", according to X and Y axes of the spot position on the array. Differentially expressed miRNAs were selected with Significance Analysis of Microarrays (SAM, version 3.0), as described previously [80,81]. The signal values of triplicate spots for each probe on individual slides were averaged, and individual samples hybridized with two replicate slides (indicated as lowercase a and b). The mean signal values were log2 transformed before submission to Gene cluster 2.0 for SOM analysis and Gene Cluster 3.0 for cluster analysis. Microarray data passing the threshold 1,000 were generally confirmed by Northern blotting. A signal value of 1,000 was set as the positive expression threshold. Northern blotting Blots were prepared by electrophoresing 150 μg of total RNA per lane on a denaturing 12% polyacryamide-7 mol/l urea gel at 200 V for 1 h and 300 V for 2 h, followed by electroblotting to Hybond-N nylon membranes (Ambion) using the semi-dry Trans-Blot Electrophoretic Transfer cell (Bio-Rad). After electroblotting, RNAs were fixed to the membrane by UV cross-linking (1000 μJ, HL-2000 HybriLinker; UVP), followed by baking in a vacuum oven at 80°C for 30 min. DNA oligonucleotides complementary to the predicted candidate miRNAs, U6 RNA and 5srRNA were synthesized (Sangon, Shanghai). The 5'-ends of DNA and Decade Markers (Ambion) were labeled with [γ-32P] ATP (Amersham) using T4 polynucleotide kinase (Takara), and subjected to purification using a Purification Cartridge (Ambion). The membrane was pre-hybridized in solution containing 6×SSC, 10×Denhardt's solution, 0.2% SDS and 50 μg salmon sperm DNA (Ambion) at 65°C for about 5 h. Membranes were hybridized in solution containing 6×SSC, 5×Denhardt's solution, 0.2% SDS and 50 μg denatured sheared salmon sperm DNA (Ambion) with 1-5 × 106 cpm eluted radiolabeled oligonucleotide probes at 10-15°C below the calculated dissociation temperature for at least 10 h. Blots were washed three times for 5 min each at 37°C with 6×SSC and 0.2% SDS, and once at 42°C for at least 15 min. After the final wash, blots were wrapped in plastic film and exposed to X-ray film at -70°C for 24 to 72 hours. The former probe was stripped for reprobing by washing at 90°C in 0.1×SSC, 0.5% SDS. Radioactive signals were quantified with the ImageQuant software package (Molecular Dynamics). Abbreviations 0 h Em: 0 hour embryo; 6 h Em: 6-hour embryo; 12 h Em: 12-hour embryo; 24 h Em: 24-hour embryo; 2 dEm: day 2 embryo; 3 dEm: day 3 embryo; 6 d Em: day 6 embryo; 9 dEm: day 9 embryo; eIL1 (0 h IL1): early 1st instar larva; lIL1: late 1st instar larva; lML1: late 1st molt larva; eIL2: early 2nd instar larva; lIL2: late 2nd instar larva; lML2: late 2nd molt larva; eIL3: early 3rd instar larva; lIL3: late 3rd instar larva; eML3: early 3rd molt larva; lML3: late 3rd molt larva; eIL4: early 4th instar larva; lIL4: late 4th instar larva; eML4: early 4th molt larva; lML4: late 4th molt larva; eIL5: early 5th instar larva; 2 dIL5: fifth-instar day 2 larva; 3 dIL5 (f): female fifth-instar day 3 larva; 3 dIL5 (m): male fifth-instar day 3 larva; 5 dIL5 (f): female fifth-instar day 5 larva; 5 dIL5 (m): male fifth-instar day 5 larva; 5 dIL7 (f): female fifth-instar day 7 larva; 5dIL7 (m): male fifth-instar day 7 larva; efSp (0 h fSp): early female cocoon-spinning larvae; 0hPPu: early prepupa; efPu (0 h fPu): early female pupa; efAd (0 h fAd): early female adult; 0 h Ad (EB): fresh adult moth with eggs in its abdomen (0-hour egg-bearing adult moth); 0 hAd (ER): fresh adult moth whose eggs were manually removed (0-hour egg-removed adult moth); 2 dAd (EL): day 2 adult moth after laying eggs (day 2 egg-laid adult moth). Authors' contributions SL conceived and designed the study, performed microarray and Northern blotting hybridization experiments, analyzed data, and wrote the manuscript. QX, PZ and ZX coordinated the study. QL performed miRNA prediction and analysis. LZ, DC and JD performed microarray experiments and generated array data. QX reviewed the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 microRNAs examined in this study. (A) miRNAs subjected to homology searches and two silkworm-specific miRNAs. This set of conserved miRNAs comprised the majority of silkworm miRNAs subjected to homology searches due to the expanding miRBase. Therefore, some miRNAs were not probed in this microarray (such as let-7b). 'SW' followed by a number represents the serial number of one probe printed on the microarray slides. 'Homologs' column includes the corresponding names of miRNAs of other organisms. The 'location' column depicts the genome site of the mature sequence (left) and the precursor (right). The loci of miRNAs were described in the 'locus description' column. The 'arm' column shows that the mature sequence originates from either arm of the precursor. The folding energy of the stem-loop structure is presented on the 'dG' column. Most miRNAs found by homology searches can be localized on the chromosomes ('chr.' Column). The 'str.' column shows the sense or antisense strand encoding miRNAs. (B) Specific antisense sequences were probed to determine their presence or absence. anti-miR-276-5p, anti-miR-124 and anti-miR-263b displayed signals higher than the threshold of 1,000 at the late 3rd molt and 4th molt stages. Moreover, anti-miR-276-5p displayed signals above 1,000 in day 3 fifth instar larva and day 6 female pupa as well as at three time-points of male pupa. These findings suggest that antisense transcriptions of some miRNAs also exist in silkworm. (C) Alternative strands of specific miRNAs. Alternative forms were probed independently as repeats to ascertain whether results were reproducible. Alternative probes of the specific miRNAs clearly yielded identical results (let-7a and let-7a#; miR-275 and miR-275#). (D) miRNAs from other organisms. A large number of miRNAs are highly conserved between species. However, only a few match the primary silkworm genome data. Accordingly, we also probed 42 miRNAs of other organisms. Remarkably, nearly half of these were confirmed using both Northern blotting and microarray. In this table, names, mature sequences, homolog miRNAs, locus description, arm, folding energy (dG, Kcal/mol), chromosome (chr) are shown, and supplementary information is excluded due to limited space. Click here for file Additional file 2 Printing design and oligonucleotides for the miRNA microarray chip. This table presents the probe location on the microarray slide. 'Bock' represents the microarray slide with all printed probes. To obtain convincing results, all probes for miRNAs and controls were printed in triplicate on two parallel blocks. Moreover, a number of time-points were repeatedly sampled and examined with the microarray blocks. Numbers presented in 'Row' and 'Column' represent the loading site on each block. 'ID' is the respective locus for each printing. 'Oligo name' is the probe ID. Click here for file Additional file 3 Whole-life test on miRNA expression in the silkworm by Northern blot hybridization. To obtain general whole-life expression patterns of miRNAs in the silkworm, we performed Northern blot hybridization. 5srRNA and U6 were used as the loading controls. Abbreviations: 6 d Em, day 6 embryo; eIL1, early 1st instar larva; lML1, late 1st molt larva; eIL2, early 2nd instar larva; lML2, late 2nd molt larva; eIL3, early 3rd instar larva; eML3, early 3rd molt larva; eIL4, early 4th instar larva; eML4, early 4th molt larva; eIL5, early 5th instar larva; efSp (0 h fSp), early cocoon-spinning larvae; efPu (0 h fPu), early female pupa; efAd (0 h fAd), early female adult. Click here for file Additional file 4 Identification of miRNAs over the whole life cycle of silkworms. A normalized signal value ≥ 1,000 was considered the positive expression threshold. +, expressed; -, not expressed. #, some miRNAs were hybridized to more than one probe containing several nucleotides more or less at the ends. 15 unique miRNAs were detected at all developmental stages. peak, the highest signal value on microarray. Nor., summarized Northern blot results; reg. regulation types; up, up-regulated expression; down, down-regulated expression NA, not assayed. Click here for file Additional file 5 Identification of miRNAs during embryogenesis by microarray. In total, 36 unique miRNAs were selected as 'expressed' during embryogenesis. +, expressed; -, not expressed; peak, the highest expression level obtained from this test; the 'points' column depicts the total positive time-points; the 'WLE' column shows whole-life expression with the general whole-life test; 'SUM' row represents the total number of expressed unique miRNAs at a specific time-point; E6-, not detected in day 6 embryo with the whole-life test. Abbreviations: 0 h Em, 0 hour embryo; 6 h Em, 6-hour embryo; 12 h Em, 12-hour embryo; 24 h Em, 24-hour embryo; 2 dEm, day 2 embryo; 3 dEm, day 3 embryo; 6 dEm, day 6 embryo; 9 dEm, day 9 embryo; 0 h IL1(eIL1), early 1st instar larva. Click here for file Additional file 6 Identification of miRNAs by microarray during the early larval stages. In total, 62 unique miRNAs were selected as 'expressed' during the early larval stages, of which 23 were not detected in the whole-life test. Twenty unique miRNAs displayed expression signals above the threshold in all the time-points examined from day 9 embryo to late 3rd molt. +, expressed; -, not expressed; peak, highest expression level obtained with this test; the 'points' column presents the total positive time-points; WLE, whole-life expression with the general whole-life test; 'SUM' row represents the total number of expressed unique miRNAs at a specific time-point; E6-, not detected in day 6 embryo with the whole-life test. Abbreviations: 2 d Em, day 2 embryo; 9 d Em, day 9 embryo; eIL1, early 1st instar larva; lIL1, late 1st instar larva; lML1, late 1st molt larva; eIL2, early 2nd instar larva; lIL2, late 2nd instar larva; lML2, late molt larva; eIL3, early 3rd instar larva; lIL3, late 3rd instar larva; eML3, early 3rd molt larva; lML3, late 3rd molt larva. Click here for file Additional file 7 Identification of miRNAs by microarray during the late larval stages. In total, 54 unique miRNAs were selected as 'expressed' during the late larval stages, among which 16 were not detected with the whole-life test. Twenty unique miRNAs displayed expression signals above the threshold at all the time-points examined from early 4th instar larva to fifth-instar day 7 larva. +, expressed; -, not expressed; peak, highest expression level obtained with this test; the 'points' column shows the total positive time-points. 'eIL' column presents miRNAs expressed throughout the early larval stages; 'WLE' column shows whole-life expression with the general whole-life test; 'SUM' row is the total number of expressed unique miRNAs at a specific time-point; E6-, not detected in day 6 embryo with the whole-life test. Abbreviations: eIL4, early 4th instar larva; lIL4, late 4th instar larva; eML4, early 4th molt larva; lML4, late 4th molt larva; eIL5, early 5th instar larva; 2dIL5, fifth-instar day 2 larva; 3dIL5(f), female fifth-instar day 3 larva; 3dIL5(m), male fifth-instar day 3 larva; 5dIL5(f), female fifth-instar day 5 larva; 5dIL5(m), male fifth-instar day 5 larva; 5dIL7(f), female fifth-instar day 7 larva; 5dIL7(m), male fifth-instar day 7 larva. Click here for file Additional file 8 Microarray-based expression levels of miRNAs correlate with stage transitions of larvae. (A) Fluctuating miRNA expression corresponding to stage transitions during the early larval stages. (B) Fluctuating miRNA expression corresponding to stage transitions during the late larval stages. The colors indicate relative and mean-centered expression for each miRNA: green, low; black, mean; red, high. The lowercase letters a and b represent the average signals of each probe printed at three points on individual blocks. Abbreviations of the samples are described in the figure legends to Additional files 6 and 7. Click here for file Additional file 9 Identification of miRNAs in the female silkworms from spinning to adult stages with microarray. In total, 55 unique miRNAs were screened as expressed miRNAs in females across spinning larva, pupa and adult, among which 16 were not detected using the whole-life test. At all examined time-points across these stages, 15 unique miRNAs displayed expression signals above the threshold, and 10 were also expressed throughout in males. Clearly, day 3 pupae expressed the lowest and day 7 pupa expressed the highest number of miRNAs. +, expressed; -, not expressed; peak, Highest expression obtained with this test; the 'points' column depicts the total positive time-points; 'male' column depicts miRNAs expressed in males throughout these stages; 'WLE' column presents whole-life expression patterns obtained using the general whole-life test; 'SUM' row is the total number of expressed unique miRNAs at a specific time-point; E6 -, not detected in day 6 embryo with the whole-life test. Abbreviations: 0 hSp, early spinning larva; 0 hPPu, early prepupa; 0 hPu, early pupa; 12 hPu, 12-hour pupa; 24 hPu, day 1 or 24-hour pupa; 2 dPu, day 2 pupa, 3 dPu, day 3 pupa; 4 dPu, day 4 pupa; 5 dPu, day 5 pupa; 6 dPu, day 6 pupa; 7 dPu, day 7 pupa; 8 dPu, day 8 pupa; 0 hAd(EB), fresh adult moth with eggs in its abdomen; 0 hAd(ER), fresh adult moth whose eggs were manually removed; 2 dAd(EL), day 2 adult moth after laying eggs. Click here for file Additional file 10 Identification of miRNAs expressed by microarray in male silkworms from spinning to adult stages. Overall, 54 unique miRNAs were selected as 'expressed' miRNAs in males across spinning larva, pupa and adult. Among these, 12 were not detected with the whole-life test. In total, 13 unique miRNAs displayed expression signals above the threshold at all the time-points examined across the stages, and 10 were additionally expressed throughout all stages in females. Similar to females, day 3 pupa expressed the lowest, while day 4 pupa expressed the highest number of miRNAs. +, expressed; -, not expressed; peak, highest expression level obtained with this test; the 'points' column presents the total positive time-points; 'female' column shows miRNAs expressed in females throughout these stages; 'WLE' presents the whole-life expression patterns obtained with the general whole-life test; 'SUM row is the total number of expressed unique miRNAs at a specific time-point; E6-, not detected in day 6 embryo with the whole-life test. Abbreviations: 0 hSp, early spinning larva; 0 hPPu, early prepupa; 0 hPu, early pupa; 12 hPu, 12-hour pupa; 24 hPu, day 1 or 24-hour pupa; 2 dPu, day 2 pupa, 3 dPu, day 3 pupa; 4 dPu, day 4 pupa; 5 dPu, day 5 pupa; 6 dPu, day 6 pupa; 7 dPu, day 7 pupa; 8 dPu, day 8 pupa; 0 hAd, fresh adult moth; 2 dAd, day 2 adult moth. Click here for file Additional file 11 Microarray-based expression of miRNAs in spinning larvae, pupae and adults. (A) Fluctuating expression levels in female silkworms during pupal metamorphosis. (B) Fluctuating expression levels in male silkworms during pupal metamorphosis. Both females and males displayed a deep expression trough at the day 3 pupa stage for the majority of miRNAs (indicated with red arrows). Abbreviations are described in the figure legends to Additional files 9 and 10, respectively. Click here for file Additional file 12 miRNAs are regulated in multiple ways during silkworm development. All microarray data were analyzed using SAM 3.0. The miRNAs presented are specifically up- or down-regulated in the whole-life test or stage-by-stage assay. Early larvae comprise the 1st, 2nd and 3rd stages, while late larvae comprise the 4th and 5th stages. Sp-Pu-Ad (f)/(m), from spinning larvae to pupae and adults (females or males); reg, regulated; score (d), the T-statistic value; up, up-regulated; down, down-regulated. Click here for file Acknowledgements We are very grateful to Dr. Keith Mitchelson, and Professor Goldsmith, M.R., for critical reading of the manuscript, and apologize to colleagues whose work we could not cite due to space limitations. 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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central 1471-2202-10-1231978575510.1186/1471-2202-10-123Research ArticleChronic NMDA administration to rats increases brain pro-apoptotic factors while decreasing anti-Apoptotic factors and causes cell death Kim Hyung-Wook [email protected] Yunyoung C [email protected] Mei [email protected] Stanley I [email protected] Jagadeesh S [email protected] Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA2009 28 9 2009 10 123 123 9 6 2009 28 9 2009 Copyright © 2009 Kim et al; licensee BioMed Central Ltd.2009Kim et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Chronic N-Methyl-d-aspartate (NMDA) administration to rats is reported to increase arachidonic acid signaling and upregulate neuroinflammatory markers in rat brain. These changes may damage brain cells. In this study, we determined if chronic NMDA administration (25 mg/kg i.p., 21 days) to rats would alter expression of pro- and anti-apoptotic factors in frontal cortex, compared with vehicle control. Results Using real time RT-PCR and Western blotting, chronic NMDA administration was shown to decrease mRNA and protein levels of anti-apoptotic markers Bcl-2 and BDNF, and of their transcription factor phospho-CREB in the cortex. Expression of pro-apoptotic Bax, Bad, and 14-3-3ζ was increased, as well as Fluoro-Jade B (FJB) staining, a marker of neuronal loss. Conclusion This alteration in the balance between pro- and anti-apoptotic factors by chronic NMDA receptor activation in this animal model may contribute to neuronal loss, and further suggests that the model can be used to examine multiple processes involved in excitotoxicity. ==== Body Background Glutamate is the major excitatory neurotransmitter in vertebrate brain. Glutamate acts on two different classes of receptors, ionotropic glutamatergic receptors and G-protein-coupled metabotropic receptors. The ionotropic receptors are further classified into α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors [1]. Binding of glutamate to NMDA receptors (NMDAR) results in an influx of extracellular Ca2+ into the cell, which leads to the activation of many Ca2+-dependent enzymes such as calpain [2], calcineurin [3], inducible nitric oxide synthase (iNOS) expression [4] and arachidonic acid (AA, 20:4n-6) selective cytosolic phospholipase A2(cPLA2)[5,6]. NMDAR are present throughout the brain and predominantly in frontal cortex and hippocampal CA1 region [7]. Activation of NMDAR also induces signaling cascades involved in learning and memory, synaptic excitability and plasticity, and neuronal degeneration [8]. Overactivation of glutamate receptors can result in the death of neurons through a process termed excitotoxicity. Excitotoxicity has been implicated in several neurodegenerative diseases, including Alzheimer disease [9-11], Huntington disease [12], schizophrenia [13], and bipolar disorder [14-16]. Chronic NMDA administration to rats reduced NMDAR subunits and increased arachidonic acid cascade markers in rat frontal cortex [6]. Similarly, an altered NMDAR subunits [17,18] and increased arachidonic acid cascade markers have been reported in Alzheimers patients[19,20]. Glutamate was reported to trigger DNA degradation, apoptotic cell death, and increase the Bcl-2-associated X protein (Bax) to B-cell lymphoma (Bcl)-2 ratio in cells in vitro [21-24]. In addition, AA was reported to induce apoptosis in vitro by producing mitochondrial damage [25], activating caspases-3 and -9, releasing cytochrome C [26], decreasing expression of brain-derived neurotrophic factor (BDNF) [27], and reducing neuronal viability [28]. Dietary deprivation of n-3 polyunsaturated fatty acids (n-3 PUFAs) in rats increased AA signaling while decreasing BDNF expression in frontal cortex [29,30]. In contrast, chronic administration of mood stabilizers to rats decreased brain expression of cPLA2 as well as AA turnover in brain phospholipids [31]. Mood stabilizers also increased expression of anti-apoptotic Bcl-2 and BDNF in the rat frontal cortex [32-34]. We have established an animal model of excessive NMDA signaling in rats by administering a subconvulsive dose of NMDA for 21 days. This model demonstrates upregulated markers of brain AA metabolism, including increased turnover of AA in brain phospholipids and increased expression of AA-selective cPLA2 and the cPLA2 gene transcription factor, activator protein (AP)-2 [6,35]. It also demonstrates increased brain neuroinflammatory markers, consistent with crosstalk between NMDAR-mediated excitotoxicity and neuroinflammation [4]. In our present study, we wished to see if chronic NMDA administration to rats, as a model of excitotoxicity, also would alter the balance of pro- and anti-apoptotic factors in brain and lead to neuronal death. To the extent that this model represents clinical excitotoxicity, it might be used for drug development and for understanding interactions among different brain processes that lead to cell death. We studied the frontal cortex because we had studied this region previously in this model [4,6]. Methods Animals The study was conducted following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (Publication no. 80-23) and was approved by the Animal Care and Use Committee of the "Eunice Kennedy Shriver" National Institute of Child Health and Human Development. Male CDF-344 rats weighing 200-215 g (Charles River Laboratories; Wilmington, MA, USA) were randomly assigned to a control group (n = 10) that received vehicle (0.9% saline i.p.) once daily for 21 days, or to an NMDA group (n = 10) that received 25 mg/kg i.p. NMDA (Sigma Chemical Co., St Louis, MO, USA) once daily for 21 days. This dose does not produce convulsions but can cause paroxysmal EEG activity [36] and an increase in brain AA metabolism in rats [37]. Three hours after the last saline or NMDA injection, rats were anesthetized with CO2 and then decapitated. The brain was rapidly excised and the frontal cortex dissected, frozen in 2-methylbutane at -50°C, and stored at -80°C until use. Preparation of Cytosolic Fractions Cytosolic fractions were prepared from frontal cortex as previously described [6]. Tissue from control or chronic NMDA rats was homogenized with a Polytron homogenizer in a buffer consisting of 20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 5 mM EDTA, 1.5 mM pepstatin, 2 mM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 U/ml aprotinin, and 2 mM dithiothreitol. The suspension was centrifuged at 100,000 × g for 60 min at 4°C. The resulting supernatant was the cytosolic fraction. Protein concentrations of cytosolic fractions were determined by using a protein reagent (Bio-Rad, Hercules, CA). The frontal cortex nuclear fraction was prepared from the control and NMDA administered rats as previously described [6]. Western Blot Analysis Proteins from cytosolic extracts (65 μg) were separated on 10-20% SDS-polyacrylamide gels (PAGE) (Bio-Rad), and then were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). Cytosolic blots were incubated with primary antibodies for BDNF, Bcl-2, Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter (Bad), and 14-3-3z (1: 1000) (Santa Cruz Biotech, Santa Cruz, CA). The blots then were incubated with appropriate HRP-conjugated secondary antibodies (Bio-Rad) and were visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ) on X-ray film (XAR-5, Kodak, Rochester, NY). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA) and were normalized to β-actin (Sigma) to correct for unequal loading. All experiments were carried out twice with up to 6 independent samples. BDNF and phospho-CREB Protein Levels BDNF and phospho-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) levels were measured in brain cytosolic and nuclear extracts using an ELISA kit according to the manufacturer's instructions (Chemicon International, Temecula, CA). BDNF levels are expressed in pmol/mg protein and phospho-CREB levels were expressed as percent of control. Total RNA Isolation and RT-PCR Total RNA was isolated from frontal cortex of control and chronic NMDA-administered rats using an RNeasy lipid tissue mini kit (Qiagen, Valencia, CA, USA). Expression of BDNF, Bcl-2, Bax, and Bad was determined using specific primers and probes purchased from TaqManR gene expression assays (Applied Biosystems). Data were expressed as the level of the target gene mRNA in brain from NMDA-administered animals normalized to the level of the endogenous control mRNA (β-globulin), and relative to values in brains from control saline-injected rats (calibrator) [38]. All experiments were carried out in duplicate with six independent samples per group. FJB staining Brains from control and NMDA administered rats (frontal cortex) were sectioned coronally (25 μm) on a cryostat (Bright Instrument Company, Ltd., Huntingdon, England) and then mounted on gelatin-coated glass specimen slides. Staining with FJB (Histo-Chem, Jefferson, AR) was performed as described [39]. Briefly, the tissue slides were dehydrated in 70% ethanol and then hydrated with distilled water. After hydration, they were immersed in FJB stain for 20 min at room temperature, washed with distilled water and dried at 50°C for 10 min. The slides were mounted with the cover slip with DPX and examined under a fluorescence microscope. Statistical Analysis Data are expressed as means ± SEM. Statistical significance was calculated using two-tailed, unpaired t-test, with significance set at p < 0.05. Results Decreased levels of anti-apoptotic factors Chronic NMDA administration for 21 days, compared with chronic saline, significantly decreased protein levels of BDNF (75%; p < 0.001) (Figure 1A), Bcl-2 (33%; p < 0.05) (Figure 1B), and phospho-CREB (39%; p < 0.001) (Figure 1C) in rat frontal cortex. The decreases in these protein levels were associated with decreases in their mRNA levels. Thus, chronic NMDA significantly decreased mRNA levels of BDNF (0.6 fold; p < 0.01) (Figure 1D) and of Bcl-2 (0.6 fold; p < 0.01) (Figure 1E). Figure 1 Protein levels of BDNF (A) and Bcl-2 (B) in frontal cortex of control rats (n = 10) and chronic NMDA-treated rats (n = 10), measured using ELISA and immunoblot as described in the method section. Optical densities of immunoblot bands were normalized to b-actin to correct for unequal loading. Values are expressed as percent of control. Phosphorylated CREB (C) was measured in frontal cortex of control rats (n = 8) and of chronic NMDA-treated rats (n = 8) by ELISA, as described in manufacturer's instructions. mRNA levels of BDNF (C) and Bcl-2 (D) in frontal cortex of control rats (n = 6) and of chronic NMDA-treated rats (n = 6), measured using RT-PCR. Data are expressed as mRNA level in frontal cortex of chronic NMDA administered rats, normalized to the endogenous level of β-globulin mRNA, and relative to the control (calibrator), using the ΔΔCT method (means ± SEM, p < 0.05, p < 0.01, *p < 0.001). Increased levels of pro-apoptotic factors In contrast to the reductions in anti-apoptotic factors, chronic NMDA increased protein levels of pro-apoptotic Bad (71%; p < 0.05) (Figure 2A) and Bax (30%; p < 0.01) (Figure 2B). mRNA levels also were increased for both Bad (1.4 fold; p < 0.05) (Figure 2C) and Bax (0.23 fold; p < 0.05) (Figure 2D) by chronic NMDA. Chronic NMDA administration increased the protein level of 14-3-3ζ(50%; p < 0.05) (Figure 3A). Figure 2 Protein levels of Bad (A) and Bax (B) in frontal cortex of control rats (n = 8) and of chronic NMDA-treated rats (n = 8), measured using immunoblot. Optical densities of immunoblot bands were normalized to b-actin to correct for unequal loading. Values are expressed as percent of control. Data are expressed as means ± SEM, p < 0.05, *p < 0.01. mRNA levels of Bad (C) and Bax (D) in frontal cortex of control rats (n = 6) and of chronic NMDA-treated rats (n = 6), measured using RT-PCR. Data are expressed as mRNA level in frontal cortex of chronic NMDA administered rats, normalized to the endogenous level of β-globulin mRNA, and relative to the control (calibrator), using the ΔΔCT method (means ± SEM, p < 0.05, *p < 0.01). Figure 3 A Protein levels of 14-3-3-ζ in frontal cortex of control rats (n = 6) and of chronic NMDA-treated rats (n = 6), measured using immunoblotting. Optical densities of immunoblot bands were normalized to b-actin to correct for unequal loading. Values are expressed as percent of control. Data are expressed as means ± SEM, p < 0.05. B. Representative FJB stained frontal cortex slices from control and chronic NMDA administered rats. Magnification is at 40 × objective. FJB positive neurons were only observed in the brains of chronic NMDA administered rats. Evidence of cell death Chronic NMDA administration increased FJB staining, a marker of neuronal loss, in rat frontal cortex (Figure 3B). Discussion Chronic daily administration of a non-convulsive dose of NMDA to adult male rats significantly decreased frontal cortex protein and mRNA levels of the anti-apoptotic factors BDNF and Bcl-2, and of their transcription factor, phospho-CREB. In contrast, chronic NMDA significantly increased frontal cortex protein and mRNA levels of Bad and Bax and of the protein level of 14-3-3ζ, pro-apoptotic factors, as well as Fluoro Jade-B staining, a marker of neuronal death, in rat frontal cortex. These data can be added to evidence that chronic NMDA under the same administration paradigm increased frontal cortex expression of inflammatory markers (protein and mRNA levels of interleukin-1 beta, tumor necrosis factor alpha, glial fibrillary acidic protein and inducible nitric oxide synthase) [4], decreased frontal cortex NMDAR (NR)-1 and NR-3A subunits, and increased activity, phosphorylation, protein, and mRNA levels of cPLA2 but did not change activity or protein levels of secretory sPLA2 or calcium-independent iPLA2 [6]. Chronic NMDA also increased the DNA-binding activity of AP-2 and its protein levels of AP-2 alpha and beta subunits [6], which are recognized on the promoter region of cPLA2 gene [40] as well as turnover and other kinetic markers of AA metabolism in frontal cortex of rat brain [35]. These changes did not follow administration of a single 25 mg/kg i.p. dose of NMDA and thus were a consequence of long term activation of NMDARs [6]. Together, they provide a profile of an experimental and probably evolving animal model of excitotoxicity, which might be exploited for future drug development and for understanding interactions of processes of excitotoxicity. There is evidence that excitotoxicity plays a role in a number of neuropsychiatric and neurodegenerative disorders, including Alzheimer disease [9-11], Huntington's disease [12], schizophrenia [13], and bipolar disorder [14,16,41]. The effects of chronic NMDA in rats suggest alterations of multiple signaling cascades such as calpain [2], calcineurin [3] and iNOS expression [4] but it may be premature to ascribe a change in one to a change in another. Nevertheless, increased AA metabolism caused by chronic NMDA may be involved in altering the balance between pro- and anti-apoptotic factors, leading in turn to the observed neuronal loss. Increased AA exposure decreased BDNF protein in spinal cord neurons in vitro [27], induced mitochondrial damage [25], activated caspases-3 and -9, released cytochrome C from mitochondria [26] and decreased neuronal viability [28]. Expression of BDNF and Bcl-2 is regulated mainly by CREB [42]. BDNF and Bcl-2 play important roles in cell survival and plasticity, and in growth and differentiation of new neurons and synapses [43]. Increased AA signaling may interfere with transcription of neuronal survival factors [27,44-47]. Downregulation of BDNF and Bcl-2 could occur through a decrease in their transcription factor phospho-CREB [48], as was found in this study. BDNF also may regulate Bcl-2 levels through activation of the MAP kinase cascade and the downstream phosphorylation of CREB protein [49]. Bcl-2 can be repressed by the AP-2 transcription factor [50], resulting in apoptosis. Chronic NMDA in rats increased the DNA-binding activity of AP-2 and protein levels of its alpha and beta subunits [51]. AP-2 also is a transcription factor of the cPLA2 gene, and its overexpression may lead to upregulated cPLA2 activity and of AA signaling upon chronic NMDA administration [51]. Thus, increased AP-2 binding activity or decreased BDNF caused by chronic NMDA may have led to the decreased Bcl-2 expression in the present study. Consistent with the notion that increased AA signaling reduces BDNF expression, rats deprived of dietary essential n-3 PUFAs for 15 weeks demonstrated increased brain AA signaling and reduced mRNA and protein levels of phospho-CREB and BDNF [29,30]. In relation to this, chronic NMDA administration also increased brain cPLA2 activity, phosphorylation, protein, and mRNA levels, as well as AA turnover in brain phospholipids [6,35]. 14-3-3ζ proteins bind the pro-apoptotic protein Bad [52]. Disassociation of 14-3-3ζ from Bad causes dephosphorylation of Bad by protein phosphatase 2A [53], allowing Bad to move from the cytoplasm to mitochondria, where it can displace Bax from Bcl-xL [54] and promote apoptosis. There also may be a more direct mechanism by which AA induces polymerization of 14-3-3ζ and dissociation from Bad [55]. The combination of increased expression of 14-3-3ζ and increased AA signaling [6] caused by chronic NMDA may have contributed to the neuronal loss, which is suggested by the increased FJB staining. Studies also have reported increased protein levels of 14-3-3ζ associated with neurodegenerative disease [56-58]. Increased 14-3-3ζ protein levels caused by chronic NMDA may be a secondary response to the observed increased Bad expression or be due to the increased AA signalling. Further studies are needed to understand the direct role of 14-3-3ζ in NMDA mediated apoptosis. Conclusion Chronic NMDA excitotoxicity may be involved in the apoptosis in neurodegenerative diseases, while targeting the excitotoxicity with drugs may be a useful therapeutic approach in these neurodegenerative diseases by way of reducing apoptosis in brain. Abbreviations AP-2: activator protein-2; BDNF: brain derived neurotrophic factor; Bcl-2: B-cell lymphoma-2; CREB: cAMP response element binding protein; phospho-CREB: phosphorylated CREB; Bax: Bcl-2-associated X protein: Bad: Bcl-2-associated death promoter; Fluoro-Jade B: FJB. Authors' contributions HWK and YCC were carried out the experiments and analysis. SIR and JSR were involved in designing and writing, editing the manuscript. Acknowledgements This work was entirely supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health. We thank Dr Sang-Ho Choi for assistance with florescence microscopy. 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==== Front Indian J Community MedIJCMIndian Journal of Community Medicine : Official Publication of Indian Association of Preventive & Social Medicine0970-02181998-3581Medknow Publications India IJCM-34-5210.4103/0970-0218.44520Original ArticleMusculoskeletal Disorders: Epidemiology and Treatment Seeking Behavior of Secondary School Students in a Nigerian Community O Adegbehingbe Olayinka O Fatusi Adesegun 1A Adegbenro Caleb 1O Late Adeitan Opeyemi 1O Abass Ganiyu 1Akintomiwa Akintunde 1Department of Orthopaedic Surgery and Traumatology, Obafemi Awolowo University, College of Health Sciences, Faculty of Clinical Sciences, Ile Ife, Osun State, Nigeria1 Department of Community Health, Obafemi Awolowo University, College of Health Sciences, Faculty of Clinical Sciences, Ile Ife, Osun State, NigeriaAddress for correspondence: Dr. OO Adegbehingbe, Department of Orthopaedic Surgery and Traumatology, Obafemi Awolowo University, College of Health Sciences, Faculty of Clinical Sciences, Ile-Ife - 220005, Osun State, Nigeria. E-mail: [email protected] 2009 34 1 52 56 © Indian Journal of Community Medicine2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Epidemiological information paucity exists on musculoskeletal disorders (MSD) among secondary school students in Nigeria. We aimed to determine prevalence, pattern, and treatment seeking behaviors (TSB) of MSD in south-west Nigeria. Materials and Methods: A school-based cross sectional study was conducted in four randomly selected secondary schools in Ile-Ife in 2007. All the students were screened for MSD using interviewer-administered questionnaire and physical examination, which involved use of scoliometer and goniometer. Affected children were recommended for treatment and plain radiography taken. Results: A total of 133 students had 204 MSD representing 3.0% prevalence among the 4,441students screened. Eighty-one (60.9%) students had congenital disorders and 52 (39.1%) were acquired. The lower limbs (93.1%) were most commonly affected and 87 (65.4%) students presented with knee deformity. Other abnormalities were limb length discrepancy 6.8%, scoliosis 4.4%, pes planus 3.9%, and poliomyelitis 2.9%. One hundred students (75.2%) had no form of treatment, 18.8% receive treatment in the hospital, 3.7% in traditional healing home and 2.3% in church. Age, family, and school type were significant factors (P < 0.05) in health seeking behavior. The factors affecting treatment outcome were the place of treatment, hospital specific treatment, and reasons for stopping treatment. Conclusion: Treatable cases constitute a large proportion of MSD among secondary school students, but TSB was generally poor. Parental socio-economic and health services factors were related to the health seeking behavior. Strengthening of school health services and improved linkage with orthopedic services, community education on MSD, and education of all cadres of health professionals are recommended. Epidemiologymusculoskeletal disorderschoolscreeningtreatment ==== Body Introduction Musculoskeletal disorders (MSD) constitute an important health problem globally. Injuries and diseases of the musculoskeletal system account for more than 20% of patient visits to primary care and emergency medical practitioners in United State of America.(1) In Africa and developing countries, poverty with its attendant malnutrition, infectious diseases, ignorance and inadequate medical facilities are all associated with the occurrence of MSD.(2–6) Screening programs can identify most cases of previously undiagnosed orthopedic abnormalities, improve our knowledge of the prevalence and pattern of MSD. It can lead to early diagnosis which often is beneficial in altering the natural history of disease.(7–9) The Bone and Joint Decade (2000-2010) has been launched to increase the awareness, encourage research and international cooperation in the prevention and treatment of MSD.(10) This study aimed at determining the prevalence, pattern of MSD, and treatment seeking behavior among secondary school students in Ile-Ife, South-west Nigeria. Materials and Methods The study was conducted in Ife Central Local Government Area (LGA) of Osun State, Nigeria. The LGA has an estimated population of 96,580 from the National Population Commission data. The LGA has its headquarters in Ile-Ife, though a university town is mainly semi-urban in nature. The town has twenty secondary schools made up of twelve private and eight public schools. The total population of students enrolled was 15,180 for the 2006/2007 academic session. Stratified sampling approach was used to select study schools to ensure that both private and public schools were represented as the socio-economic background of the students attending the two groups of schools may be significantly different. A total of four schools were selected: two public schools (Urban Day Grammar School and Oduduwa College) and two private schools (Ibikunle Lawal College and Adventist Grammar school). In each school, all the students were targeted for screening for MSD after the school authority had given approval for the study and informed consent obtained from student above 18 year or parents/guardians of those under 18 years. Participation rate was 100%. Information on the socioeconomic background and medical history of the respondents was obtained through the use of questionnaires administered by trained final year students. Physical examination was carried out by one of the researchers, an orthopedic surgeon (AOO), and involved also the use of stadiometer, weighing balance, scoliometer and goniometer. The footprint ratio or arch index - the ratio of the middle third of the toeless footprint to the total toeless footprint area(11) - was used to quantify pes planus deformity. Individualized plain radiography was taken at the Obafemi Awolowo University Teaching hospitals Complex, Ile-Ife, to confirm deformity and determine Cobb Meyer's angles in scoliosis. Clinical photographs were also used to document some of the significant findings. Data collection took place from July to October 2007. The sample size taken was appropriate to calculate prevalence in this population. The sample size was determined using the Armittage and Perry formula {n=p (1-p) z2 / d2} of single proportion. Where minimum sample size required was (n =384.2) while taken the prevalence (p) of MSD among Nigerian secondary students to be 50% based on non available previous study data, standard normal deviation (z) set at 1.96 corresponding to 95% confidence interval and the degree of accuracy (d) was set at 5%. Data analysis was carried out through the use of SPSS software (version 11.0). Chi-square was used to determine association between discrete variables such as selected factors and health seeking behavior. The alpha error was 0.05 and level of significance p value <0.05. Results A total of 4,441 secondary school students were screened, comprising of 2449 (55.1%) males and 1992 (44.9%) females [Table 1]. The mean age of the students was 13.5 year±4.4 year (range: 9-22year). A total of 133 (3.0%) had musculoskeletal deformities. The total number of MSD cases detected was 204. The age range of students with MSD was 9 to 20 years, with a mean of 14.1 years and standard deviation of 3.6 years. There were 73 (2.8%) males and 60 (3.0%) females who had MSD. The male: female ratio of students with MSD was 6: 5. Table 1 Percentage distribution of MSD by selected socio-demographic factors among students screened in Ile-Ife, Nigeria MSD present (n=133) MSD absent (n=4308) Total (n=4441) Statistical significance Age group 9-15 year 101 3096 3197 P<0.05 16-22 year 32 1212 1244 Sex Male 73 2346 2449 P>0.05 Female 60 1932 1992 School type Private 56 1261 1317 P<0.05 Public 77 3047 3124 MSD = Musculoskeletal disorder [Table 1] shows the distribution of MSD among affected students. A total of 93 students (69.9%) had their deformity in the lower limbs followed by the upper limbs 27 (20.3%). The congenital cases constituted 60.9% of cases and most cases of MSD was bilateral (53.4%). The pattern of MSD is shown in [Tables 2 and 3]. Scoliosis was mainly non-structural among seven students (3.4%) while the remaining two students (1.0%) had structural scoliosis sequel to post poliomyelitis paralysis. Scoliosis was twice common in the females compared to the males. Postural scoliosis was seen in four subjects and three had compensatory scoliosis due to the limb length discrepancy and pelvic inequality. The scoliosis was mild to moderate without any respiratory embarrassment reported by the students affected. Table 2 Distribution of musculoskeletal disorders among affected secondary school students in Ile-Ife, Nigeria MSD distribution Number of student with MSD (n=133) % Side Left 28 21.0 Right 21 15.8 Bilateral 71 53.4 Central (Spine) 13 9.8 Site Back 6 4.5 Pelvis 4 3.0 Hip 3 2.3 Upper limb 27 20.3 Lower limb 93 69.9 Mode Congenital 81 60.9 Acquired 52 39.1 MSD = Musculoskeletal disorder Table 3 Pattern of musculoskeletal disorders among affected secondary school students in Ile-Ife, Nigeria Musculoskeletal deformity No. of MSD % Genu varum 110 53.9 Genu valgum 24 11.8 Limb length discrepancy (Avascular necrosis of the femur) 14 6.9 shortening Scoliosis 9 4.4 Knock knee 16 7.8 Pes planus 16 7.8 Poliomyelitis 6 2.9 Neglected hip dislocation (with pelvic obliquity) 3 1.5 Syndactyl 3 1.5 Congenital talipes equino-varum deformity 1 0.5 Kyphoscoliosis 1 0.5 Erb's palsy 1 0.5 Total 204 100.0 Many students have more than single MSD disorder, MSD = Musculoskeletal disorder Most (80.4%) of the MSD affected the lower limb while 13.2% and 6.4% were located in the upper limbs, the central (spine and pelvis), respectively. Approximately two-thirds (65.4%) of the students with MSD had knee deformities (genu varum-53.9%, genu valgum-11.8% and knock knee-7.8%). The pattern of the MSD varies between students of private and public schools. The public schools accounted for genu varum (53.0%), genu valgum (65.2%), limb length discrepancy (76.9%), scoliosis (83.3%), polydactyl (85.7%), and pelvic obliquity (66.7%). All cases of syndactyl, poliomyelitis, kyphosis, kyphoscoliosis and Erb's palsy were seen only in the public schools. 76.9% of the knocked knee was predominant in private school. Painless pes planus was seen among 8 (6.0%; males=3, females=5; age: 14-16year) students. The males had a higher arch index greater than the females, the difference was not significant (P>0.05). The treatment seeking behavior among students screened in [Table 4] shows that only 33 (24.8%) had previous treatment; 93 (69.9%) had no treatment and 7 (5.3%) did not know their treatment history. Out of those that had previous treatment, twenty-five students (18.8%) received treatment from hospitals while five (3.7%) received treatment from traditional healers and three (2.3 %) from churches. A higher proportion of students from private schools (14 of 58 MSD students; 24.1%) compared to those in public schools (11 of 75 MSD students; 14.7%) sought for hospital treatment. Occupation of the parents did not have significant impact on the treatment seeking behavior (P =0.685). 20.6% of the civil servant children with MSD had no treatment. 35.1% of the students whose mothers were traders had no form of treatment as compared with the 13.2% of those whose fathers were traders had no treatment. 18.4% students with orthopedic disorders that were under 16 years sought for treatment in contrast to the 6.1% in the age group 16-20 years (P < 0.037). All the six (100%) students with deformities whose parents were separated or divorced had no form of treatment, while 21.3% of those with parents (polygamy/monogamy) alive sourced for treatment. Table 4 Factors associated with treatment seeking behavior of secondary school students with musculoskeletal disorders Treatment seeking behavior Number of students with MSD frequency Students with MSD that received hospital care (%) Statistical significance School type P<0.05 Private 58 14.0 (24.1) Public 75 11.0 (14.7) Parents’ occupation P<0.05 Farming 16 3 (18.7) Trading 57 13 (22.8) Civil servant 43 5 (11.6) Artisan 17 4 (23.5) MSD = Musculoskeletal disorder The type of treatment had varied with pattern of MSD. The students with structural scoliosis, post poliomyelitis and congenital non-syndromic talipes equinovarum deformity received different multiple treatment. Among those that sought treatment from health facilities, three (12.0%) had soft tissue operations (elongation of tendo-achilles). 15 (60.0%) had drugs given, 6 (24.0%) had plaster of Paris and 9 (36.0%) received physiotherapy. The treatments were not exclusive of one another. One-third (8 students, 32%) of those that sought hospital treatment did not complete prescribed treatment. Three of the five students treated by the traditional healers said that the treatments offered were difficult and did not complete their treatment. Out of those that received hospital treatment, 16 (64%) were of the opinion that their cases improved while 6 (24%) felt their condition deteriorated and 3 (12.0 %) did not notice any change. The six students who reported poor outcome were cases of severe deformities from paralytic poliomyelitis 5 (19.2%), and Erb's palsy 1 (3.8%). The factors affecting treatment of the six students who did not complete their treatment mainly was as a result of financial problem and the logistics that the lengthy period of treatment entailed. The other reasons were, parents not having time for further treatment and that nobody was available to stay with the student at hospital. A total of 128 of the 133 (96.2%) students with MSD indicated interest in obtaining appropriate treatment if such could be made available to them. When they were followed up immediately, 4.0% of the student who had not received any form of treatment informed their parents and had surgery (Corrective osteotomies-3; triple ankle arthrodesis-1) with uneventful outcome at the teaching hospital. Discussion Determination of the prevalence and pattern of musculoskeletal symptoms is the first step in the effective intervention and prevention of further chronic pain syndromes in young adults.(912) Our study has assessed the prevalence and pattern of MSD in Nigerian population and provided evidence that could inform appropriate interventions. With the paucity of locally available work that focused on community-based secondary schools, our study is a pioneering work in the Nigerian environment and can serve as benchmark values. The characteristics of student population screened revealed a good mix of socio-economic background as students from both private and public schools were included in the study. The preponderance of males than females may be a reflection of the entire population pattern of enrollment in the schools. The recent rapid assessment of the primary school health system in Nigeria(13) showed enrollment data and MSD prevalence to be similar to our study result. The inability to carry out genetic studies due to lack of resources make it impossible to rule out disorders at microscopic level among the majority of our subjects that were not found to have macroscopic MSD. The higher rate of MSD among public schools compared with private schools is likely to be due to the effect of the socioeconomic background, as mostly children of parents in the lower socio-economic class attend public schools in Ile-Ife as in most parts of Nigeria. Congenital MSD is more preponderant than acquired deformity in this study which agreed with the findings of Thanni and Folami in another study carried out in South-west Nigeria,(13) although their study was hospital-based and 60% of their study population was under-5 years. The congenital disorders identified among secondary school may likely be a reflection of factors, particularly ignorance of the conditions, lack of knowledge about treatment possibilities and appropriate treatment sources, inability to afford orthodox care, and poor health seeking behavior. Whereas MSD in our study was found predominantly in the lower limb followed by the upper limbs and the spine, respectively, Whittfield et al. reported that musculoskeletal symptoms were more prevalent in the neck, shoulders, upper back and lower back among secondary schools in New Zealand.(14) The carriage of heavy schoolbags was a suspected contributory factor among the New Zealand secondary school students, which is not the case in the Nigerian environment. In our study, genu varum, genu valgum, knock knee accounted for majority of MSD among the students screened. Earlier work among Nigerian population, consisting mostly of pre-school children,(1315) had reported knee deformity as being the most prevalent MSD. The persistence of knock knee to secondary school level could be due to indifference to cosmetic appearance and the fact that no mortality is associated with knee deformity in an environment where high level of child mortality from various communicable diseases and poverty obtains. Our study showed that the prevalence of pes planus and arch index is similar to the findings among Malawians.(11) The overall prevalence of spine deformities (scoliosis and/or thoracic hyperkyphosis) reported in this study was similar to the 7.8% adolescents with scoliosis reported by Milenkovic et al. 2004.(12) The prevalence of scoliosis similar to our findings was two times higher in girls compared with boys.(121617) The treatment seeking behaviors was generally found to be poor in our study, with the majority of affected students found not to have had any form of treatment. This is in contrast to finding in more developed parts of the world such as United States, where most pupils were reported to have received considerable amount of professional attention.(18–20) The fact that 5.3% of students did not know their treatment history reflects a low level of health communication between these students and their parents. Inclusion of parents in the study would have made it possible for us to have more complete treatment history of the children and better insights into reasons for treatment seeking decisions and behaviors. The significant difference between treatment seeking patterns among school children in private and public schools reflect the possibility that parental socio-economic factors plays a significant role in decisions for treatment seeking. It was interesting to note that none of the affected students in private schools sought treatment from churches and traditional healers. The fact that none of the children from unstable family setting had sought treatment also reflects another dimension of parental background to treatment seeking. The relevance of socio-economic level of parents to treatment was further reflected in the fact that inability to afford medical bill was a major reason for inability to complete prescribed treatment. There is absence of functioning effective, affordable and accessible national health insurance scheme. Several other factors in the health system could also have been contributory to the poor treatment behavior observed. These include: inaccessibility of specialist services with the low number of orthopedic surgeons and traumatologists available in the country; ineffective school health services; and lack of effective social medical services particularly to support those severe deformities secondary to paralytic poliomyelitis and Erb's palsy. Conclusion and Recommendations Treatable cases constitute a large proportion of musculoskeletal disorders (MSD) among secondary students in Nigeria. Parental socio-economic and health services factors were related to the poor health seeking behavior. This could be improved through community education, early detection, and linkage of school health services to facility-based orthopedic services as a major approach. Source of Support: Nil Conflict of Interest: None declared. ==== Refs References 1 Schmale GA More evidence of educational inadequacies in musculoskeletal medicine Clin Orthop Relat Res 2005 437 251 9 16056057 2 Mbamali EI Badoe EA Archampong EQ da Rocha-Afodu JT Principles and Practice of Surgery including pathology in the tropics 2000 3rd ed 1052 3 Huckstep RL The challenge of the third world Curr Orthop 2000 14 26 33 4 Huckstep RL Appliances and operators for poliomyelitis in developing countries: In Instructional course lectures Am Acad Orthop Surg 1999 Vol. 49 5 Huckstep RL Poliomyelitis - A guide for developing countries, including Appliances and Rehabilitation 1983 2nd ed Edinburgh Churchill living stone ELBS and French 6 Craton N Matheson GO Training and Clinical competency in musculoskeletal medicine. 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==== Front Indian J Community MedIJCMIndian Journal of Community Medicine : Official Publication of Indian Association of Preventive & Social Medicine0970-02181998-3581Medknow Publications India IJCM-33-15610.4103/0970-0218.42051Original ArticleProfile of Clients Tested HIV Positive in a Voluntary Counseling and Testing Center of a District Hospital, Udupi, South Kannada A Kumar P Kumar M Gupta A Kamath A Maheshwari S Singh Department of Community Medicine, Kasturba Medical College, Manipal - 576 104, Karnataka, IndiaCorrespondence to:Dr. Megha Gupta, Department of Community Medicine, Kasturba Medical College, Manipal - 576 104, Karnataka, India. E-mail: [email protected] 2008 33 3 156 159 24 10 2007 02 5 2008 © Indian Journal of Community Medicine2008This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: The growing menace created by the HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome) has alarmed not only the public health officials but also the general community. The Voluntary Counseling and Testing Centre (VCTC) services have begun as a cost-effective intervention in reversing this epidemic. Objectives: 1. To study the sociodemographic characteristics of HIV-positive clients and their risk behaviors. 2. To elucidate the reasons for their visit to the VCTC and know the problems anticipated by the clients after revealing their HIV-positive status. Study Design: A cross-sectional record-based study. Materials and Methods: The study was conducted in August 2007 among clients who tested positive for HIV in the VCTC of a district hospital in Karnataka from January to July 2007. Results: Study included 249 individuals, of whom 64.7% were males, 88.7% (age, 15–49 years), married (72.7% males and 84.0% females) and literate (females 71.5% and males 85.7%). A high percentage of nonresponse regarding the pattern of risk behavior was noted among the subjects (males: 42.8% and females: 90.9%). Of the individuals who responded, 91 males (98.9%) and 6 females (75.0%) had multiple heterosexual sex partners, while 1 male had homosexual partner. The figures in females show that two (25%) of them had a history of blood transfusion. The reason for visiting the VCTC were cited as some form of illness (33.3%), confirmation of test results (32.9%), family members diagnosed as HIV positive (12.9%) and 11.6% were referred from Directly Observed Treatment Scheme (DOTS) center. More than three quarter of the sample population anticipated discrimination at the time of medical treatment. Conclusion: People have begun using VCTC services, which reflects a change in their attitude toward HIV. The study provides us a clue to formulate an effective approach to educate people as well as the health personnel who are thought of as one of the important sources of discrimination. DiscriminationHIV positiverisk behaviorVCTC ==== Body Introduction The human immunodeficiency virus (HIV) infection is a global pandemic and has grown into a public health program of unprecedented magnitude. According to the acquired immunodeficiency syndrome (AIDS) epidemic update, December 2007, released by the UNAIDS and World Health Organization (WHO), approximately 33.2 million people are living with HIV/AIDS worldwide.(1) The prevalence rate of HIV in adults varies in different regions from 5% in the Sub-Saharan Africa, 0.3% (Middle East), 0.5% (Latin America), 0.9% (Eastern Europe) to 0.6% in North America (AIDS Epidemic Update 2007).(1) It is estimated that 90% of the HIV-infected persons live in the developing countries with the estimated number of Indians being 2.7 million.(2) Overall, the average prevalence rate of HIV in adults inIndia is approximately 0.36%, and it accounts for 10% of the global HIV burden and 65% of that in the South and South-East Asia.(3) Counseling for HIV and AIDS has become a core element of a holistic model of health care; in this model, psychological issues are recognized as integral to patient management. Both pre- and post-test counseling have become standard components of prevention-oriented HIV antibody testing programs.(4) The Voluntary Counseling and Testing Centre (VCTC) now known as the ICTC (Integrated Counseling and Testing Centre) provides a key entry point for the ‘continuum of care in HIV/AIDS’ for all segments of the population. The sentinel surveillance carried out in October 1999 revealed that the state of Karnataka has a high prevalence of HIV infection.(5) A number of factors contribute to Karnataka's vulnerability to the HIV epidemic. It is bordered by three states that have well-established and further growing HIV epidemics (Maharashatra, Tamil Nadu and Andhra Pradesh). Karnataka shares many demographic and economic ties with these neighboring states. There is an extensive migration to and from these states, and major transportation routes connect Karnataka with them. Certain economic and social factors also contribute to Karnataka's vulnerability to this epidemic. Poverty levels are high, leading to economic pressures that promote commercial sex work. The data collected in the present study from the VCTC of a district hospital in Udupi, Karnataka, may provide important clues regarding the epidemiological profile of HIV-positive individuals. Materials and Methods The present study was conducted in the VCTC of a district hospital in Udupi situated in southern Karnataka. Udupi is flanked by the verdant mountains of the Western Ghats on the east and the vast and the tranquil Arabian Sea on the west. According to the 2001 India census, the district has a population of 963548 with 51% females. The average literacy rate is 83%, higher than the national average of 59.5%. From January to July 2007, of the data of total 2586 attendees at the VCTC who were either volunteers or referred from other institutions, the study included that of 249 HIV–positive patients (9.6%). Information for all the attendees of the VCTC was available from the records maintained at the VCTC regarding variables such as age, gender, marital status, education and occupational status, residence, behavioral patterns, discrimination anticipated, support expected. In the present study, only the data from patients who tested positive for HIV at the VCTC was included. This information was recorded when the client visited the VCTC for the first time and most of them were unaware of their status of HIV infection. HIV was diagnosed by performing enzyme-linked immunosorbent assay (ELISA) by using two different antigens and a rapid test as recommended by the National AIDS Control Organization (NACO). Data was collected and analyzed using the SPSS software version 11.5. However, the current study is subject to certain limitations since it was conducted in a district hospital; therefore, the results are based on the reporting and data collection by the personnel employed in the VCTC. Information regarding certain variables such as socioeconomic status, substance abuse, counseling performed and condom use are not available. All these variables could have unmasked certain behavioral patterns and could have given new dimensions to this study. The study setting being a district hospital decreased its external validity. Results The male population constituted 64.7% (161) of the total study subjects. Table 1 clearly shows the sociodemographic profile of the attendees with positive test result. A majority of the study subjects, i.e., 221 (88.7%) belonged to age group of 15–49 years with 7 (2.8%) subjects being less than 14 years of age. The distribution according to marital status showed that 72.7% of males and 84.0% of females were married of which 11.1% of males and 44.4% of females were divorced, separated or widowed. Table 1 Sociodemographic characteristics of the study subjects Factors Male (%) N = 161 Female (%) N = 88 Age group (years) <15 2 (1.2) 5 (5.7) 15–49 143 (88.9) 78 (88.6) >50 16 (9.9) 5 (5.7) Education Illiterate 23 (14.3) 25 (28.5) Upto 4th standard 20 (12.4) 12 (13.6) Upto 8th standard 66 (41.0) 34 (38.6) Upto 12th standard 45 (28.0) 12 (13.6) College and above 7 (4.3) 5 (5.7) Occupation Unskilled 46 (28.6) 23 (26.1) Semi-skilled 80 (49.7) 17 (19.3) Skilled 19 (11.8) - Professional 6 (3.8) 3 (3.4) Housewife - 30 (34.2) Unemployed 8 (4.9) 10 (11.3) Student 2 (1.2) 5 (5.7) Marital status Married 117 (72.7) 74 (84.1) Unmarried 44 (27.3) 14 (15.9) The literacy rate among the female subjects was found to be 71.5%, while that in males was 85.7%. The most common source of income for males (48.8%) was semi-skilled occupation, such as bidi rolling and fishing. Among females, 30 (34.2%) were housewives and 23 (26.1%) were working as housemaids or laborers. The unemployment rate among the study subjects was 7.3%. All females and 149 males (93.1%) resided with their family members. Approximately half of the study subjects (50.6%) had visited the VCTC voluntarily, while almost a similar percentage (49.4%) of the subjects was referred to the VCTC by another doctor. Among the reasons cited for their visit to the VCTC, illness (medical or surgical: 33.3%) was the leading cause, followed by 32.9% who visited for the confirmation of their test result. More than one–tenth (12.9%) of the study subjects had the family members (spouse/parents) who were positive for HIV and 29 (11.6%) were referred from a Directly Observed Treatment Scheme (DOTS) centre for detection and treatment of tuberculosis. Among the total subjects, 69 males (42.8%) and 80 females (90.9%) did not respond to the question on the pattern of risk behavior followed. Of the subjects who responded, 91 males (98.9%) had multiple sex partners and 1 was involved in homosexual practices. Among the females, six (75.0%) were having multiple sex partners and two (25%) had a history of blood transfusion [Table 2]. Table 2 Risk behavior followed by the study subjects Risk behavior Male no. (%) Female no. (%) Total (%) no. (%) Heterosexual multiple partners 91 (56.4) 6 (6.8) 97 (39.0) Homosexual partner 1 (0.6) 0 1 (0.40) Blood transfusion 0 2 (2.3) 2 (0.80) No response 69 (43.0) 80 (90.9) 149 (59.8) Total 161(100.0) 88 (100.0) 249 (100) The expectation of the subjects regarding the social support after testing positive shows that 31.7% candidates were in favor of individual counseling by counselors, 23.7% preferred family counseling and 18.6% preferred one to one discussion with doctors. The views of the study subjects regarding problems they would face after disclosing their HIV status revealed that a large percentage of subjects (79.1%) believed they would be discriminated at the time of medical treatment. A small number of subjects, i.e., eight (3.2%) anticipated a disturbance in their marital life, and an almost equal number, i.e., five (2.0%) believed that they would be discriminated by their other family members. Discussion The prevalence of HIV seropositivity in VCTC clients in the present study was noted to be 9.6%, which is lower than that reported from a study conducted in a district of West Bengal (17.1%) in 2003.(6) The present study highlights the fact that males contributed to 64.7% of the case load in VCTC with 35.3% being the females. These figures are slightly lower than the national average of 38.4% for females. Such a high proportion of infection rate in females is a cause for concern since this will lead to a proportionate increase in the children being infected due to transmission from mother to child. It is believed that HIV/AIDS affects the bread winners of the society, which is also evident from the results of this study. According to the study, 88.7% of the subjects belonged to the age group of 15–49 years (the most sexually active age group), which is slightly lower than the national figure (90%) and the figure obtained from another study (92.4%) conducted at a VCTC in Darjeeling.(6) The present study clearly indicates that 93% of the infected males and 100% of the infected females are living with their families. The information regarding their disclosure of the test result to their family members is not available and hence it is difficult to say whether such a high level of acceptance by the family, especially toward females will be maintained even after the disclosure or not. The pattern of risk behavior shows that a large percentage of males (98.9%) and females (75%) of those who responded to the study, had multiple sexual partners. However, none of the women was working as a commercial sex worker. Heterosexual contact was the commonest mode of transmission, which is supported by the findings of another study from eastern India.(7) A large part of the married women (44.4%) who were HIV positive were separated, divorced or widowed. This can be explained by the strong family ties and inhibitions that Indian females have as a part of culture. A large part of the study subjects (42.8% males, 90.8% females) did not disclose their risk status in the questionnaire. The figures for the risk status were 29.9% in males and 53.8% females in a study conducted in West Bengal (2003).(6) This can be attributed to the fear of discrimination or punishment, which still prevails in the society toward HIV-infected individuals. The major problem anticipated by the subjects was observed to be from the health personnel at the time of medical and surgical treatment. This could be a reason for only 18% subjects responding in favor of doctors as the best option for their support. More than half of the subjects were in favor of counseling since it is gaining importance in the current era as an important step toward normalizing the attitude to HIV and improving the environment for the prevention of its transmission. Another important finding of the study shows that approximately 3% of the subjects actually believed they would be discriminated by their family members, while the remaining thought that they would be easily accepted. This can be attributed to the increasing awareness among the people by the combined efforts of health care personnel and media. This assumption can also be explained by a large percentage of subjects coming to VCTC on their own without being referred by someone else. This was in contrast to the figures reported from a study in Chennai (2004–05) where only 3 of the total 89 HIV-positive patients had visited voluntarily for testing.(8) The current study highlights the existence of HIV-TB collaboration, which is evident from the study since 29 subjects (11.6%) had been referred from the DOTS centers. Conclusion The high prevalence of seropositivity in the attendees of VCTC in a district hospital in Karnataka highlights the importance of this issue for the policy makers as well as health professionals. The medical fraternity should take a stand and fight against the discrimination of sufferers, rather than ostracizing them to have a positive attitude from HIV sufferers. Increased availability and the use of VCTC services will prove to be a huge potential benefit for the society. Source of Support: Nil Conflict of Interest: None declared. ==== Refs References 1 WHO/UNAIDS AIDS Epidemic Update 2007 12 Available from: http://www.unaids.org/en/HIV-data 2 2.5 million people living in India with HIV, according to new estimates, UNAIDS/NACO/WHO 2007 7 06 Available from: http://www.who.int/mediacentre/news/releases/2007 3 HHS/CDC Global AIDS program (GAP) in India The GAP India Fact sheet Available from: http://www.Cdc.gov/nchstp/od/gap/countries/India.htm 4 Valdiserri RO Moore M Gerber AR Campbell CH Dillon BA Jr West GR A study of clients returning for counseling after HIV testing: implications for improving rates of return Public Health Rep 1993 108 12 8 8434087 5 Sengupta D Rewari BB Shaukat M Mishra SN Study on Clinico-epidemiological profile of HIV Patients in Eastern India J Posgrad Med 2001 15 91 8 6 Jordar GK Sarkar A Chatterjee C Bhattacharya RN Sarkar S Banerjee P Profile of attendees in the VCTC of North Bengal Medical College in Darjeeling district of West Bengal Indian J Community Med 2006 31 237 40 7 Chakravarty J Mehta H Parekh A Attili SV Agrawal NR Singh SP Study on Clinico-epidemiological profile of HIV patients in Eastern India J Assoc Physicians India 2006 54 854 7 17249252 8 Studies on HIV/AIDS Voluntary counseling and testing centre 2004–2005 Chennai National Institute of Epidemiology Available from: http://www.google.com	19876475	PMC2763673	CC BY	2021-01-04 17:46:06	yes	Indian J Community Med. 2008 Jul; 33(3):156-159
==== Front Mol CancerMolecular Cancer1476-4598BioMed Central 1476-4598-8-781978110210.1186/1476-4598-8-78ResearchBlockage of transdifferentiation from fibroblast to myofibroblast in experimental ovarian cancer models Yao Qin [email protected] Xun [email protected] Qifeng [email protected] David A [email protected] Shuzhen [email protected] Beihua [email protected] Ming Q [email protected] Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical Research, School of Medical Science, Griffith University, Gold Coast campus, Southport, Qld 4222, Australia2 Department of Obstetrics and Gynaecology, Qilu Hospital, Shandong University, Ji'nan 250012, Shandong, PR China3 Department of Obstetrics and Gynaecology, Affiliated Hospital of Qingdao University, Qing'dao 266003, Shandong, PR China4 Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Ji'nan 250012, Shandong, PR China2009 27 9 2009 8 78 78 19 5 2009 27 9 2009 Copyright © 2009 Yao et al; licensee BioMed Central Ltd.2009Yao et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Tumour stromal myofibroblasts can promote tumour invasion. As these cells are genetically more stable than cancer cells, there has been enormous interest in developing targeted molecular therapies against them. Chloride intracellular channel 4 (CLIC4) and reactive oxygen species (ROS) have been linked with promoting stromal cell transdifferentiation in various cancers, but little is known of their roles in ovarian cancer. In this study, we examined the functional roles that both CLIC4 and ROS play in the process of ovarian cancer cell-stimulated or TGF-β1 induced fibroblast-to-myofibroblast transdifferentiation. We also examine whether it is possible to reverse such a process, with the aim of developing novel therapies against ovarian cancer by targeting activated transdifferentiated myofibroblasts. Results We demonstrate that TGF-β1 induced or CMSKOV3 activate transdifferentiated myofibroblasts (fibroblasts). These fibroblasts mimic "reactive" stromal myofibroblasts and demonstrate significant up-regulation of CLIC4 expression and increased level of ROS production. Blocking the production of ROS with an antioxidant consequently reduces the expression of CLIC4, and is accompanied by disappearance of α-smooth-muscle actin (α-SMA), a myofibroblast marker, suggesting ROS acts as a signalling molecule that promotes and enhances CLIC4 activities in the myofibroblast transdifferentiaton process. Down-regulation of CLIC4 with a generic agent or specific siRNA both significantly reduces the expression of factors related to the phenotypes and functions of myofibroblasts, such as α-SMA, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), thus reversing the myofibroblast phenotype back to fibroblasts. These results convincingly show that ROS and CLIC4 are responsible for TGF-β1 induced fibroblast-to-myofibroblast transdifferentiaton and down-regulation of both is sufficient to block transdifferentiated myofibroblasts. Conclusion Molecular targeting of ROS and CLIC4 has the potential to develop novel therapies for ovarian cancer. ==== Body Background Ovarian cancer is not only common (1 in 67 women), but also is the most lethal gynaecological disease. It has, for years, been dubbed as a "silent killer" -- causing vague and non-specific symptoms until it is too advanced at the time of diagnosis [1]. Thus, ovarian cancer causes more deaths than any other type of gynaecological malignancies [1-3]. Even with the advent of modern treatments such as optimal cytoreductive surgery and systemic combination of chemotherapies, the 5-year survival rate for patients with advanced ovarian cancer has improved little; it is currently at as low as 10 - 30% [3]. Extensive studies have shed some lights on the complexity of ovarian cancer, and increasing evidence indicates that the disease development and progression are facilitated by interactions between tumour cells and activated stromal cells [4]. The tumour stroma (also referred to as "reactive stroma") is characterized by marked alterations in the phenotype and expression profile of "fibroblast-like cells". These cells commonly express α-smooth-muscle actin (α-SMA) and thus are termed as myofibroblasts [5,6]. Stromal fibroblasts are located at the tumour border near the invasion front. When these cells are activated by the tumour, they have a more profound influence on the development and progression of carcinomas than was previously appreciated [7]. Recent studies on reactive stroma in human breast carcinomas and in prostate cancer subjects have demonstrated that the co-culturing of myofibroblasts with these tumour cells can promote tumour invasion and angiogenesis [8,9]. Using an in vitro tumour-stroma model of skin carcinogenesis, Cat and colleagues (2006) demonstrated that myofibroblasts are recruited into cancer from different sources during cancer development and the invasion progression [10,11]. These cells were differentiated from the fibroblast population within the epithelial stroma after stimulation by transforming growth factor-β1 (TGF-β1) secreted by tumour cells. Tumour-associated myofibroblasts could also be transdifferentiated from non-malignant or epithelial derived carcinoma cells via epithelial-mesenchymal transition [11]. In addition, myofibroblasts could be recruited or derived from distant fibroblasts and bone marrow progenitor cells [12,13]. Although it has been shown that the conversion from fibroblasts to myofibroblasts constitutes the major source of myofibroblasts in tumour stroma, the molecular mechanisms underlying fibroblast-to-myofibroblast transdifferentiation is still not fully understood. Many questions remain, including what molecules are involved in the process and what roles are they playing? A family of intracellular chloride channels comprises seven highly homologous members (CLIC1-7). Recently, it was reported that one of them, the chloride intracellular channel 4 (CLIC4), a chloride channel of intracellular organelles, regulates intracellular pH and cell volume. Besides its presence on the organelle membrane, CLIC4 exists in soluble form in the cytoplasm and nucleus acting as a signalling protein or channel regulator [14]. The transcriptional level of CLIC4 is up-regulated when fibroblasts are induced by TGF-β1 and transdifferentiated into myofibroblasts, and more importantly CLIC4 is highly expressed in myofibroblasts of breast cancers [15]. Results from Littler and colleagues [16] shows that functional activity of CLIC4 depends on its redox state and that oxidative conditions enhance membrane binding and channel activity of CLIC4. More recent findings also reveal that reactive oxygen species (ROS) can alter the level of gene expression associated with cell differentiation, including fibroblast-to-myofibroblast transdifferentiation and epithelial-mesenchymal transition in liver fibrosis and some cancers [10,17,18]. However, the role of ROS in ovarian cancer and its relationship to CLIC4 in fibroblast-to-myofibroblast transdifferentiation process is not well understood. We hypothesized that the factors involved in fibroblast-to-myofibroblast transdifferentiation, such as tumour-cell-derived TGF-β1, promote the generation of intercellular ROS, which in turn acts as a signalling molecule, and initiates up-regulation of CLIC4 expression, thus triggering fibroblast-to-myofibroblast conversion. Agents that block ROS and CLIC4 might inhibit such transdifferentiation, which may delay myofibroblast-dependent tumour progression. Understanding these molecular interactions may assist with the development of novel molecular targeted therapies for ovarian cancer. To test the hypothesis, we designed an experiment modelling ovarian cancer fibroblast-to-myofibroblast transdifferentiation and examined the pathway in which ROS and CLIC4 participates in fibroblast-to-myofibroblast transition induced by TGF-β1 or conditioned media from ovarian cancer SKOV3 cells (CMSKOV3). Furthermore, special agents were employed to down-regulate the production of ROS and CLIC4 in transdifferentiated myofibroblasts and such effects were assessed. Results and Discussion Myofibroblasts in the experimental ovarian cancer stroma have increased CLIC4 expression Recent studies demonstrated that CLIC4 was markedly up-regulated in serum- or TGF-β1-stimulated fibroblasts isolated from breast cancer. These fibroblasts have a gene expression profile similar to myofibroblasts [15,19]. In vivo, the level of CLIC4 expression in the normal ovarian stroma is very low, but is significantly increased in the stroma of breast, kidney, oesophagus and colon cancers [20-22]. We hypothesised that CLIC4 may also play an important functional role in causing stromal fibroblast to myofibroblast transdifferentiation, which is critical in ovarian cancer development and progression. Ovarian surface epithelium is in fact a single layer of mesothelial cells covering the ovarian surface and contiguous with coelomic mesothelium. We recently showed that the expression of CLIC4 was readily detected in epithelia and stroma of these primary epithelial ovarian cancers in 96.7% (29/30) of patients. Moreover, the expression of CLIC4 in cancer stroma was correlated with up-regulation of a well-accepted myofibroblast marker, α-SMA, which was detected in 93.3% (28/30) of the same stroma (Yao et al., manuscript in preparation). In order to define the expression of CLIC4 in transdifferenciated myofibroblasts we performed immunohistochemical staining of CLIC4 and α-SMA1 (Fig. 1A). We used a myofibroblast conversion culture model where ovarian primary fibroblasts and/or human fibroblast MRC-5 cells were co-cultured with CMSKOV3 or TGF-β1. Fig. 1A showed that the myofibroblasts displayed a significant increase in CLIC4 expression both in the cytoplasm and nucleus (p < 0.05) with co-expression of a myofibroblast marker, α-SMA in the cytoplasm in the presence of CMSKOV3 or 10 ng/ml TGF-β1 compared with the serum-free medium control (p < 0.05). Furthermore, the immunoblot and real time qRT-PCR data provides strong evidence that the increased amounts of α-SMA and CLIC4 expression in myofibroblasts were up-regulated both at the transcription and protein levels (P < 0.05) (Fig. 1B). These results confirmed that the expression of CLIC4 was up-regulated during fibroblast to myofibroblast conversion when induced by TGF-β1 or activated by CMSKOV3 suggesting that CLIC4 directly participates in stromal fibroblast activation in ovarian cancer. Figure 1 Immunohistochemical staining of CLIC4 and α-SMA. (A). In cultured fibroblasts (control) and transdifferiated myofibroblasts (activated by CMSKOV3 (CM) and TGF-β1). Scale bar, 50 μm. (B) Western blot of MRC-5 cells or primary ovarian fibroblasts cultured in serum-free medium (control), in CM or TGF-β1 for 48 hours. ROS mediates up-regulation of CLIC4, which leads to myofibroblast transdifferentiation More recent reports have suggested that ROS acts as a signalling molecule of TGF-β1 in the regulation of myofibroblast differentiation in liver fibrosis and an in vitro tumour-stroma model of skin carcinogenesis [10,17]. It can alter gene expression levels by regulating the expression of multiple phenotypic markers, such as α-SMA [10]. When we examined the intracellular ROS levels in MRC-5 fibroblasts treated with 10 ng/ml TGF-β1 or CMSKOV3, we showed a significant increase in the fluorescence intensity using DCF-DA dye staining, indicating high levels of intracellular ROS production (Fig. 2A). Simultaneously, the high level of ROS was accompanied by up-regulation of α-SMA and CLIC4 expressions in the myofibroblasts. However, when the MRC-5 fibroblasts cells were pre-treated with the antioxidant N-acetylcysteine (NAC), which blocked the intracellular ROS, the up-regulation of α-SMA and CLIC4 expressions, stimulated by TGF-β1 or CMSKOV3, were also reduced at both mRNA and protein levels (Fig. 2C). These results showed that not only α-SMA but also CLIC4 transcription and translation levels were significantly up-regulated followed by generation of ROS during TGF-β1 induced fibroblast-to-myofibroblast differentiation. This effect could be blocked by treatment with the NAC. This is the first evidence that showed TGF-β1 or CMSKOV3 stimulated the production of cellular ROS and CLIC4, which correlated with myofibroblast transdifferentiation (Figure 2). Moreover, agents which inhibit ROS alleviated the expression of CLIC4 and α-SMA. Figure 2 ROS-mediated up-regulation of CLIC4 expression during myofibroblast transdifferentiation. (A) CMSKOV3 and TGF-β1 induced oxidative stress in MRC-5 fibroblasts using DCF-DA analysed by fluorescent microscopy. Scale bar, 50 μm. (B, C), Myofibroblast transdifferentiation was dependent on ROS. The expression of α-SMA and CLIC4 was determined by real time RT-PCR and Western blot, using MRC-5 cells treated with or without 5 mM of antioxidant (NAC), before addition of CMSKOV3 and TGF-β1. * P < 0.05. (D), ROS mediated up-regulation of CLIC4 expression in both cytoplasm and nucleus during myofibroblast transdifferentiation. CLIC4 protein expression in cytoplasm and nucleus was determined by Western blot analysis. Fibroblasts cultured in serum-free medium served as control for all the experiments. CLIC4 shares homology with that of the glutathione S-transferase (GST) superfamily, suggesting its function may depend on the cellular redox state. A cysteine at position 35 preceding the N-terminal transmembrane domain is redox sensitive and is modified by glutathione [16]. CLIC4 subcellular locations are critical in its physiological functions [23]. Since CLIC4 is upregulated in myofibroblasts, we further assessed the expression of CLIC4 in the nucleus and cytoplasm during myofibroblast differentiation using Western blot. As shown in Fig. 2D, the expression of CLIC4 increased not only in the cytoplasm but also in the nucleus, particularly when fibroblasts were treated with TGF-β1 or CMSKOV3. Pre-treatment with antioxidant NAC also reduced both cytoplasmic and nuclear CLIC4 expressions. Taken together, these data suggest that TGF-β1 or CMSKOV3 increases cellular ROS, which induces up-regulation and activity of CLIC4, leading to myofibroblast transdifferentiation. Down-regulation of CLIC4 reverses the myofibroblast phenotype A previous study reported that up-regulation and nuclear translocation of CLIC4 was responsible for expression of keratinocytic differentiation markers by altering chloride content and pH of the nucleus. Treatment of keratinocytes with a chloride channel inhibitor blocked the chloride ion flux by nuclear-specific CLIC4 and reduced the expression level of differentiation biomarker K10 [21]. In addition to the channel activity of CLIC4, there was a report that showed CLIC4 could promote cytoskeleton function by interaction with dynamin, tubulin, actin and 14-33 proteins [24]. Recombinant CLIC4 was shown to reduce cell motility in fibroblasts [15]. Thus, the relationship between CLIC4 and α-SMA seems to be important to the physiological function of myofibroblast cells. Since CLIC4 is the only intracellular chloride channel that can be induced by TGF-β1 in fibroblasts [15], we used a chloride channel inhibitor, indanyloxyacetic acid 94 (IAA-94) to inhibit the function of CLIC4 and found reduced expression of α-SMA and myofibroblast-mediated angiogenesis factors. Using a CLIC4 specific siRNA, we further demonstrated the consequential outcome of reduced CLIC4 expression. When fibroblasts were transfected with specific CLIC4 siRNA prior to treatment with TGF-β1 or CMSKOV3 CLIC4, siRNA efficiently down-regulated CLIC4 transcript levels by an average of 90%. The induction of α-SMA transcript induced by TGF-β1 or CMSKOV3 was significantly blocked in CLIC4 siRNA-transfected fibroblasts (Fig. 3A, B). At the protein level, immunoblot and immunofluorescent data showed that silencing of CLIC4 prevented the expression of α-SMA (Fig. 3C, D). These results are in agreement with those of a previous study by Suh et al., who have reported that α-SMA protein level in fibroblasts increases significantly upon CLIC4 over expression produced by infecting with recombinant CLIC4 adenovirus [20]. Additionally, we found that silencing CLIC4 reversed the up-regulation of angiogenic factors, ie.: hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in TGF-β1 or CMSKOV3 treated fibroblasts (Fig. 3E, F, G). Figure 3 Specific inhibition of CLIC4 expression stops myofibroblast transdifferentiation to fibroblast. MRC-5 cells were transfected with specific CLIC4 siRNA and then treated with or without CMSKOV3 and TGF-β1. Mock transfection with transfection reagent alone and scrambled siRNA (neg siRNA) served as negative controls. The relative levels of CLIC4 (A), α-SMA (B) transcripts and α-SMA protein expression were assessed by real time RT-PCR, Western blot (C) and immunofluorescent staining (D). Scale bar, 50 μm. Quantitative RT-PCR was used to analyse transcription levels of pro-angiogenic factors, VEGF (E), HGF (F) and α-SMA (G) in MRC-5 cells treated with chloride channel inhibitor IAA-94, NAC or CLIC4 siRNA before addition of CMSKOV3 and TGF-β1. * P < 0.05. Angiogenesis plays a key role in tumour development by supplying the tumour with oxygen and nutrients. Recent data shows that myofibroblasts are the major cell type expressing pro-angiogenic factors in cancer stroma such as angiogenic factor VEGF [12]. In this study EGF CMSKOV3 or TGF-β1 activated myofibroblasts have higher levels of expressions of HGF and VEGF when compared to inactivated fibroblasts (Fig. 3). Pre-treatment with a chloride channel inhibitor IAA-94 to block function of CLIC4 lowered HGF and VEGF expression levels in myofibroblasts by 24% - 80%. Furthermore, after fibroblasts were relieved from these treatments, exposure to CMSKOV3 or TGF-β1 restored the expression of factors related to the phenotype and functions of myofibroblasts (data not shown), indicating these repressing activities were due to the activation by CMSKOV3 or TGF-β1, and were not mere cytotoxic effects. Conclusion Ovarian cancer, like most solid tumours, not only acquires adaptive mutations during therapy, but interacts with myofibroblasts, thus promoting further growth and metastasis. Because myofibroblasts are genetically more stable than cancer cells, therapeutic targeting of these myofibroblasts in the stroma has distinct advantage and potential. In this study, we showed that treatment of myofibroblasts with agents blocking ROS or CLIC4 can reverse the phenotype of myofibroblasts, and markedly repress the production of angiogenesis factors associated with myofibroblast transdifferentiation, indicating that ROS or CLIC4 are excellent targets for the development of targeted molecular therapies for the treatment of ovarian cancer. Materials and methods Cell culture and in vitro culture models Both human epithelial ovarian cancer cell line SKOV3 and human foetal lung fibroblast cell line MRC-5 were obtained from Basic Medicine Research Institute, Qilu Hospital, Shandong University, P.R. China. Human ovarian primary fibroblasts were isolated as described previously [25]. These cells were cultured in DMEM (Gibco) containing 10% heat-inactivated foetal bovine serum (Gibco) and 100 units/ml penicillin/streptomycin. The cultures were maintained at 37°C in an atmosphere of 5% CO2. The purity of primary stromal cells was more than 98% confirmed by keratin and vimentin staining [26]. Ovarian fibroblasts between passage 3 and 8 were used for all experiments. Treatment of cells with TGF-β1, conditioned medium from SKOV3 (CMSKOV3), N-acetyl-L-cysteine (NAC) or IAA-94 After SKOV3 cells were cultured in serum-free medium for 48 h, the conditioned medium were collected, clarified by centrifugation and stored at -20°C for future use. When MRC-5 cells and human ovarian primary fibroblasts reached subconfluence, the medium was replaced by serum-free medium, serum-free medium with 10 ng/ml human recombinant TGF-β1 (PeproTech EC Ltd., UK) or CMSKOV3, then cultured for another 48 hours. In order to study the inhibitory functions of NAC and IAA-94, subconfluent fibroblasts were incubated with 5 mM NAC for 4 hours or 10 μM IAA-94 for 24 hours before addition of TGF-β1 or CMSKOV3. Immunochemical and immunofluorescent staining For immunohistochemical staining, the sections were deparaffinized, and rehydrated followed by antigen retrieval. The cells were fixed with 3.7% formaldehyde for immunocytochemical analysis. Afterwards, these sections and cells were blocked with 10% normal goat serum, and then incubated with rabbit anti-CLIC4 polyclonal antibody (Lifespan Biosciences, USA) and rabbit anti-α-SMA monoclonal antibody (Abcam, UK) at 4°C overnight, respectively. After washing with PBS, the slides were incubated with anti-rabbit IgG secondary antibody for 45 min and then incubated with biotinylated horseradish peroxidase solution for 45 min. Finally, the slides were stained with diaminobenzidine and observed under light microscope. For immunofluorescent staining, cells were stained with FITC conjugated anti-rabbit IgG secondary antibody, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Images were recorded using confocal microscope. Western blot analysis Cells were lysed in RIPA lysis buffer plus protease inhibitors. Cell nuclear and cytoplasmic proteins were extracted using Nuclear/Cytosol Extraction Kit (Pierce Biotechnology, Inc., USA). The expressions of CLIC4 and α-SMA were characterized by Western blot using polyclonal anti-CLIC4 antibody and monoclonal anti-α-SMA antibody as primary antibodies. Reaction products of β-actin were used to normalize the intensities between lanes. Analysis of intracellular ROS The intracellular ROS generation was assayed using 2'-7'-dichlorofluorescein diacetate (DCF-DA) (Sigma) dye as previously described [10]. Small interfering RNA (siRNA) transfection The siRNAs used in this study were designed, functional tested by and purchased from Qiagen. The sequences of siRNA were as follows: CLIC4 validated siRNA 5'-GCAGUACAAUGAU UAGUAAdTdT-3' (sense) and 5'-UUACUAAUCAUUGUACUGCdTdA-3' (antisense); negative Control siRNA 5'-UUCUCCGAACGUGUCACGUdTdT-3' (sense) and 5'-ACGUGACACGU UCGGAGAAdTdT-3' (antisense); MAPK1 positive control siRNA 5'-UGCUGACUCCAAA GCUCUGdTdT-3' (sense) and 5'-CAGAGCUUUGGAGUCAGCAdTdT-3' (antisense). Lipofectamine 2000 reagent was used for transfection with a final concentration of 30 nM of each siRNA. After 16 hours of incubation of transfection, cells were replaced with serum-free medium with or without 10 ng/ml TGF-β1 or CMSKOV3 and then incubated for another 48 hours. Real-time Quantative Polymerase Chain Reaction Total RNA was extracted from cells using Trizol reagent (Invitrogen). RNA was reverse transcribed with oligo (dT) and M-MLV reverse transcriptase (Promega). The primers were designed as follows: α-SMA (accession no. NM_001613), 5'-AGGTAACGAGTCAGAGCTTTGGC-3' (forward) and 5'-CTCTCTGTCCACCTTCCAGCAG-3' (reverse); CLIC4 (accession no. NM_013943), 5'-CACGTAAATTTCTGGATGGCAATG-3' (forward) and 5'-ATCACTGGGACAGGTATTGGTGAAC-3' (reverse); VEGF (accession no. NM_001025366), 5'-CCTGGTGGACATCTTCCAGGAGTACC-3' (forward) and 5'-GAAGCT CATCTCTCCTATGTGCTGGC-3' (reverse); HGF (accession no. NM_000601), 5'-GTAAATG GGATTCCAACACGAACAA-3' (forward) and 5'-TGTCGTGCAGTAACAACCAACTC-3' (reverse); β-actin (accession no. NM_001101), 5'-AACTCCATCATGAAGTGTGA-3' (forward) and 5'-ACTCCTGCTTGCTGATCCAC-3' (reverse). β-actin was chosen as the house keeping gene. The quantification was performed using SYBR Green (Takara Biotechnology, Inc., Japan). Samples were analysed using LightCycler® 2.0 Instrument (Roche Applied Science, Switzerland). Statistical analysis The data represented means ± S.D. of three independent experiments. Statistical differences were analysed with the Student's t-test. Differences were considered significant at P-values < 0.05. List of abbreviations CLIC4: chloride intracellular channel 4; DCF-DA: 2'-7'-dihydrodichlorofluorescein diacetate; NAC: N-acetyl-L-cysteine; ROS: reactive oxygen species; siRNA: small interfering RNA; TGF-β1: transforming growth factor-β1 Competing interests The authors declare that they have no competing interests. Authors' contributions QY - day to day bench researcher, generated most of the data, and drafted the manuscript; XQ - initial help to QY on cell culture, immunoblots; QfY - initial help to QY on cell culture, immunblots, and Western blots; DAG - revised the paper and created some figure; SD - initial supervision of Q Y, provided help with interpreting data. BK - initial supervision of QY, provided help with interpreting data. MQW - head of the laboratory, initiated the collaboration, supervised QY on day to day bench work, analysed the data and wrote the manuscript. All authors read and approved the final manuscript. Authors' information QY is a visiting fellow (supported by MW) to Professor Ming Wei's laboratory at the Division of Molecular and Gene Therapies, Griffith University at Gold Coast campus. Acknowledgements This work was supported by the Dr. Jian Zhou Smart State Fellowship from the Queensland state government, and grants from the National Health and Medical Research Council and Cancer Council, Queensland to MQW. ==== Refs Ozols RF Bookman MA Connolly DC Daly MB Godwin AK Schilder RJ Xu X Hamilton TC Focus on epithelial ovarian cancer Cancer Cell 2004 5 19 24 14749123 10.1016/S1535-6108(04)00002-9 Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics, 2008 CA Cancer J Clin 2008 58 71 96 18287387 10.3322/CA.2007.0010 Levi F Lucchini F Negri E Boyle P La Vecchia C Cancer mortality in Europe, 1995-1999, and an overview of trends since 1960 Int J Cancer 2004 110 155 69 15069676 10.1002/ijc.20097 Tlsty TD Coussens LM Tumor stroma and regulation of cancer development Annu Rev Pathol 2006 1 119 50 18039110 10.1146/annurev.pathol.1.110304.100224 Mueller MM Fusenig NE Friends or foes - bipolar effects of the tumour stroma in cancer Nat Rev Cancer 2006 4 F839 49 10.1038/nrc1477 Bhowmick NA Neilson EG Moses HL Stromal fibroblasts in cancer initiation and progression Nature 2004 432 332 7 15549095 10.1038/nature03096 Mahadevan D Von Hoff DD Tumor-stroma interactions in pancreatic ductal adenocarcinoma Mol Cancer Ther 2007 6 1186 97 17406031 10.1158/1535-7163.MCT-06-0686 Orimo A Gupta PB Sgroi DC Arenzana-Seisdedos F Delaunay T Naeem R Carey VJ Richardson AL Weinberg RA Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion Cell 2005 121 335 48 15882617 10.1016/j.cell.2005.02.034 Yang F Strand DW Rowley DR Fibroblast growth factor-2 mediates transforming growth factor-beta action in prostate cancer reactive stroma Oncogene 2008 27 450 9 17637743 10.1038/sj.onc.1210663 Cat B Stuhlmann D Steinbrenner H Alili L Holtkotter O Sies H Brenneisen P Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species J Cell Sci 2006 119 2727 38 16757516 10.1242/jcs.03011 Chaffer CL Brennan JP Slavin JL Blick T Thompson EW Williams ED Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2 Cancer Res 2006 66 11271 8 17145872 10.1158/0008-5472.CAN-06-2044 Guo X Oshima H Kitmura T Taketo MM Oshima M Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer J Biol Chem 2008 283 19864 71 18495668 10.1074/jbc.M800798200 Granot D Addadi Y Kalchenko V Harmelin A Kunz-Schughart LA Neeman M In vivo imaging of the systemic recruitment of fibroblasts to the angiogenic rim of ovarian carcinoma tumors Cancer Res 2007 67 9180 9 17909023 10.1158/0008-5472.CAN-07-0684 Suh KS Malik M Shukla A Yuspa SH CLIC4, skin homeostasis and cutaneous cancer: surprising connections Mol Carcinog 2007 46 599 604 17443730 10.1002/mc.20324 Ronnov-Jessen L Villadsen R Edwards JC Petersen OW Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts Am J Pathol 2002 161 471 80 12163372 Littler DR Assaad NN Harrop SJ Brown LJ Pankhurst GJ Luciani P Aguilar MI Mazzanti M Berryman MA Breit SN Curmi PM Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4 FEBS J 2005 272 4996 5007 16176272 10.1111/j.1742-4658.2005.04909.x Yang KL Chang WT Hung KC Li EI Chuang CC Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity Biochem Biophys Res Commun 2008 373 219 23 18558083 10.1016/j.bbrc.2008.05.192 Radisky DC Levy DD Littlepage LE Liu H Nelson CM Fata JE Leake D Godden EL Albertson DG Nieto MA Werb Z Bissell MJ Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability Nature 2005 436 123 7 16001073 10.1038/nature03688 Suh KS Crutchley JM Koochek A Ryscavage A Bhat K Tanaka T Oshima A Fitzgerald P Yuspa SH Reciprocal modifications of CLIC4 in tumor epithelium and stroma mark malignant progression of multiple human cancers Clin Cancer Res 2007 13 121 31 17200346 10.1158/1078-0432.CCR-06-1562 Suh KS Mutoh M Mutoh T Li L Ryscavage A Crutchley JM Dumont RA Cheng C Yuspa SH IC4 mediates and is required for Ca2+-induced keratinocyte differentiation J Cell Sci 2007 120 CL2631 40 10.1242/jcs.002741 Bohman S Matsumoto T Suh K Dimberg A Jakobsson L Yuspa S Claesson-Welsh L Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis J Biol Che 2005 280 42397 404 10.1074/jbc.M506724200 Stuhlmann D Steinbrenner H Wendlandt B Mitic D Sies H Brenneisen P Paracrine effect of TGF-beta1 on downregulation of gap junctional intercellular communication between human dermal fibroblasts Biochem Biophys Res Commun 2004 319 321 6 15178409 10.1016/j.bbrc.2004.05.004 Suh KS Mutoh M Nagashima K Fernandez-Salas E Edwards LE Hayes DD Crutchley JM Marin KG Dumont RA Levy JM Cheng C Garfield S Yuspa SH The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis J Biol Chem 2004 279 4632 41 14610078 10.1074/jbc.M311632200 Samoszuk M Tan J Chorn G Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts Breast Cancer Res 2005 7 R274 83 15987422 10.1186/bcr995 Suginta W Karoulias N Aitken A Ashley RH Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms Biochem J 2001 359 55 64 11563969 10.1042/0264-6021:3590055 Parrott JA Nilsson E Mosher R Magrane G Albertson D Pinkel D Gray JW Skinner MK Stromal-epithelial interactions in the progression of ovarian cancer: influence and source of tumor stromal cells Mol Cell Endocrinol 2001 175 29 39 11325514 10.1016/S0303-7207(01)00436-1	19781102	PMC2765417	CC BY	2021-01-04 17:46:14	yes	Mol Cancer. 2009 Sep 27; 8:78
==== Front Indian J Crit Care MedIJCCMIndian Journal of Critical Care Medicine : Peer-reviewed, Official Publication of Indian Society of Critical Care Medicine0972-52291998-359XMedknow Publications India 19881173IJCCM-13-1210.4103/0972-5229.53109Research ArticleChanges of splanchnic perfusion after applying positive end expiratory pressure in patients with acute respiratory distress syndrome Sarkar Suman Bhattacharya Prithwis Kumar Indrajit Mandal Kruti Sundar From: Department of Anesthesiology, Intensive Care Unit, IMS Banaras Hindu University, Varanasi-221 105, Uttar Pradesh, IndiaCorrespondence: Dr. Suman Sarkar, Department of Anesthesiology and Critical Care, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, Uttar Pradesh, India. E-mail: [email protected] 2009 13 1 12 16 © Indian Journal of Critical Care Medicine2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Positive end-expiratory pressure (PEEP) improves oxygenation and can prevent ventilator- induced lung injury in patients with acute respiratory distress syndrome (ARDS). Nevertheless, PEEP can also induce detrimental effects by its influence on the cardiovascular system. The purpose of this study was to assess the effects of PEEP on gastric mucosal perfusion while applying a protective ventilatory strategy in patients with ARDS. Materials and Methods: Thirty-two patients were included in the study. A pressure–volume curve was traced and ideal PEEP, defined as lower inflection point + 2cmH2O, was determined. Gastric tonometry was measured continuously (Tonocap). After baseline measurements, 10, 15 and 20cmH2O PEEP and ideal PEEP were applied for 30 min each. By the end of each period, hemodynamics, CO2 gap (gastric minus arterial partial pressures), and ventilatory measurements were taken. Results: PEEP had no effect on CO2 gap (median [range], baseline: 18 [2–30] mmHg; PEEP 10: 18 [0–40] mmHg; PEEP 15: 17 [0–39] mmHg; PEEP 20: 16 [4–39] mmHg; ideal PEEP: 19 [9–39] mmHg; P = 0.19). Cardiac index also remained unchanged (baseline: 4.7 [2.6–6.2] l min−1 m−2; PEEP 10: 4.4 [2.5–7] l min−1 m−2; PEEP 15: 4.4 [2.2–6.8] l min−1 m−2; PEEP 20: 4.8 [2.4–6.3] l min−1 m−2; ideal PEEP: 4.9 [2.4–6.3] l min−1 m−2; P = 0.09). Conclusion: PEEP of 10–20 cmH2O does not affect splanchnic perfusion and is hemodynamically well tolerated in most patients with ARDS, including those receiving inotropic supports. Acute respiratory distress syndromegastric mucosal perfusionpositive end-expiratory pressuretonometry ==== Body Introduction Many recent studies have shown that mechanical ventilation (MV) in patients with acute respiratory distress syndrome (ARDS), using low tidal volumes and high levels of positive end-expiratory pressure (PEEP) reduces the mortality rate and this ventilatory strategy is now accepted as standard practice in patients with ARDS.[12] Although PEEP improves arterial oxygenation, it has adverse hemodynamic effects. PEEP reduces the venous return to the heart and the left ventricular end diastolic volume by pushing the inter ventricular septum towards the left, and thus reduces the cardiac output. These effects are proportional to the PEEP level. Regional perfusion can also be affected by PEEP, independently of cardiac output changes. The splanchnic perfusion is particularly sensitive, and any reduction can compromise its barrier function, promote bacterial translocation, and contribute to the development of multiple organ failure.[3] In experimental models, PEEP has markedly decreased mesenteric and portal blood flow, despite only moderate reductions in cardiac output.[4–8] Similar results have been reported in patients without lung injury.[910] These effects are usually dose related, becoming more pronounced with PEEP levels around 20 cmH2O. Kiefer reported that PEEP did not significantly alter splanchnic circulation in six patients with acute lung injury.[11] Nevertheless, caution should be exercised in extending these results to clinical practice, because only hemodynamically stable patients without adrenergic drugs were studied, and PEEP levels never exceeded 14 cmH2O.[12] Our aim was to evaluate the effects of PEEP levels up to 20 cmH2O on gastric mucosal perfusion and systemic hemodynamics in mechanically ventilated patients with ARDS on hemodynamic support. Materials and Methods Patients The study was performed in the Intensive Care Unit (department of Anesthesiology) of the Banaras Hindu University Hospital, Varanasi, India. Adult, mechanically ventilated patients were considered eligible for the study if they met the following criteria for ARDS during the 24 hours that preceded the study: Acute onset of respiratory failure; diffuse bilateral infiltrates in the chest radiograph involving more than three-fourths of both lung fields; a ratio of partial pressure of O2 (PaO2) to fraction of inspired oxygen (FiO2) of less than 200 mmHg; and a pulmonary arterial occlusion pressure less than 18 mmHg and no cardiac failure. Hemodynamic monitoring included an arterial line and a pulmonary artery catheter. Patients could be on vasopressor or inotropic support (Dopamine 5-20 microgram/kg/min with or without Noradrenaline 10-20 micrograms /min dose), but had to be hemodynamically stable (with a mean arterial blood pressure of more than 60mm of Hg, pulse rate should be less than 100/min and more than 50 per minute) for at least 5 hours before starting the protocol. Patients were excluded if they had any of the following conditions: Pregnancy, pre-existing respiratory dysfunction, cardiac index of less than 2.5 l min−1 m−2, or were receiving enteral nutrition. Interventions A nasogastric tonometer (TRIP® Tonometry Catheter 14F, with biofilter connector for TONOCAP™ Monitor) was inserted into the stomach and connected to an air automated tonometer (TONOCAP™ Monitor; Datex-Engstrom, Helsinki, Finland). All patients were sedated with midazolam, and paralyzed with vecuronium. Neuromuscular relaxation was measured by a Train of Four watch® device. A 40mg intravenous dose of pantoprazole was administered before starting the study. Patients were started on volume controlled ventilation. A pressure–volume curve was obtained for each patient by the airway occlusion technique[13]and ideal PEEP was defined as the lower inflection point + 2 cmH2O, or 12 cmH2O if no lower inflection point was found. PEEP levels of 10, 15, 20 cmH2O, and ideal PEEP, with tidal volumes of 6 ml kg−1, were applied in four consecutive 30 min periods, respectively. Respiratory rate was modified to maintain end tidal CO2 within ±10mmHg of basal. All patients were receiving a constant infusion of 6% hetastarch before the beginning of the study (40–60 ml h−1). Cardiac output was optimized before and during the trial by determining the respiratory variation of systolic arterial pressure.[14] Whenever the variation was more than 10% a 100 ml bolus of 6% hetastarch was infused and the volume status was reassessed. No changes in vasopressor or inotropic support were allowed during the study. If hypotension (mean arterial pressure <60 mmHg) persisted for more than 2 min, the protocol was stopped. Measurements Hemodynamic, ventilatory and tonometric measurements were made at baseline, and at the end of each period, and arterial blood samples taken. Hemodynamic records included mean arterial pressure, heart rate, cardiac output, pulmonary artery occlusion pressure, central venous pressure and left ventricular stroke work index. Cardiac output was measured by thermodilution as the average of three values obtained after injections of 10ml of 5% dextrose in water at room temperature. Mean airway pressure, oxygenation index and PEEP levels were registered. Oxygenation index was calculated as mean airway pressure × FiO2 × 100/PaO2. The CO2gap (gastric partial pressure of CO2[pCO2] minus arterial pCO2) was calculated by comparing simultaneous measurements of tonometric gastric mucosal pCO2 and arterial pCO2. Statistical analysis Results are presented as median and range. The software Statview 5.0 was used to perform the statistical analysis. Nonparametric tests were used because of the small sample size. Data was analyzed with a Friedman test followed by a Wilcoxon signed-rank test if necessary. Results were considered statistically significant at P < 0.05. Results Thirty-two patients with ARDS were enrolled. They had a median (range) age of 47 years, (25 were male and seven were female, and two of the female patients were in postpartum state, four patients had previous history of diabetes) and an Acute Physiology and Chronic Health Evaluation II score of 20 at admission to the intensive care unit. On the day of the study they had a median Sepsis-related Organ Failure Assessment (SOFA)[15] score of 10. All patients fulfilled criteria for ARDS, as defined by the inclusion criteria, during the 24 hours before the study and they had been on mechanical ventilation for 32 (12–72) hours. They were being ventilated with a median PEEP level of 9 (4–12) cmH2 O, had a PaO2/FiO2 ratio of 230 (140–386) mmHg and their respiratory system compliance was 45 (27–60)ml per cmH2O. Twenty-eight patients had sepsis (eight pneumonia and 20 extra pulmonary sepsis), and four had severe head and thoracic injury. Of the septic patients, 24 were in septic shock. No changes in cardiac index or in CO2 gap were found at any of the study periods [Table 1]. Oxygenation index, mean arterial pressure, pulmonary mean arterial pressure, pulmonary artery occlusion pressure, central venous pressure and left ventricular stroke work index also remained stable through the study. Only mean airway pressure and PaO2/FiO2 ratio differed between periods, as expected. Twenty patients required a 100ml bolus of hetastarch during the trial; in no patient was it necessary to repeat it. At baseline, 12 patients had already a CO2 gap of more than 20 mmHg. After starting the protocol with 10 cmH2O PEEP, 24 patients decreased their CO2 gap and eight increased it. When PEEP was increased from 15 to 20 cmH2O, 12 patients increased their CO2 gap, 12 decreased it and in four patients it remained unchanged. Table 1 Respiratory, hemodynamic and tonometric measurements Parameter Baseline (n = 32) PEEP 10 (n = 32) PEEP 15 (n = 32) PEEP 20 (n = 32) Ideal PEEP (n = 32) P PEEP (cmH2O) 9 (4–12) 10 15 20 12 (8–15) Mean airway pressure (cmH2O) 12.1 (8–18.7) 14.3 (12–17) 20.2 (17–22.2) 24.7 (22–26.4) 15.9 (11.5–22.2) 0.0001a OI (cmH2O per mmHg) 5.3 (2.9–12.4) 7 (3–14.5) 6.7 (4.1–12.3) 7 (5–12.3) 6.6 (2.9–12.3) 0.31 PaO2/FiO2 (mmHg) 230 (140–386) 208 (115–412) 281 (151–421) 329 (197–438) 240 (169–469) 0.0009b PaCO2 (mmHg) 34 (31–51) 42 (28–61) 43 (31–66) 46 (36–59) 43 (29–52) 0.09 Cardiac index (I min−1 m−2) 4.7 (2.6–6.2) 4.4 (2.5–7) 4.4 (2.2–6.8) 4.8 (2.7–6.1) 4.9(2.4–6.3) 0.09 LVSWI (g mm−2) 45 (22–71) 43 (22–60) 40 (14–60) 36 (15–58) 42 (14–66) 0.13 MAP (mmHg) 76 (74–103) 79 (69–99) 77 (72–97) 76 (67–93) 75 (71–96) 0.24 PAOP (mmHg) 15 (10–18) 16 (8–20) 16 (11–21) 18 (12–24) 15 (13–23) 0.23 CVP (mmHg) 10 (9–17) 12 (10–19) 13 (11–24) 12 (10–19) 13 (8–18) 0.25 CO2 gap (mmHg) 18 (2–30) 18 (0–40) 17 (0–39) 16 (4–39) 19 (9–39) 0.19 Results are presented as median (range). CVP-central venous pressure; CO2 gap-arterial partial pressure of CO2[pCO2] minus gastric pCO2; FiO2-fraction of inspired oxygen; LVSWI-left ventricular stroke work index; MAP-mean arterial pressure; OI-oxygenation index-defined as mean airway pressure × FiO2 × 100/arterial pCO2; PaO2-partial pressure of O2; PaCO2-partial pressure of CO2; PAOP-pulmonary arterial occlusion pressure; PEEP-positive end-expiratory pressure. a P < 0.05 for all comparisons except baseline versus PEEP 10 and PEEP 10 versus ideal PEEP. b P < 0.05 for all comparisons except baseline versus PEEP 10, baseline versus PEEP 15, baseline versus ideal PEEP, and PEEP 15 versus ideal PEEP Twenty-four of the 32 patients studied, survived (75%). The median length of stay in the intensive care unit was 18 days and the median duration of mechanical ventilation was 11 (5–34) days. Discussion Similar studies have been done previously, but the number of cases studied were very few; we have done this study with a relatively large number of patients and our results show that high PEEP levels (up to 20 cmH2O) do not compromise gastric mucosal perfusion, as assessed by tonometry, and do not affect systemic hemodynamics in most patients with ARDS. This is consistent with the findings of two other studies on the effects of PEEP on splanchnic perfusion in patients with ARDS. However, in contrast to our study, neither of those studies included patients in septic shock or on adrenergic support.[1116] Shock and cardiovascular dysfunction are frequently associated with ARDS. This is an important issue, because hemodynamic safety concerns could preclude the use of high or optimal PEEP levels in that setting, even if necessary. A major finding of our study is that PEEP levels up to 20 cmH2O can be well tolerated, even in patients with ARDS and septic shock. Nevertheless, our trial was relatively short and we cannot exclude the possibility that keeping high PEEP levels for a longer period might result in increased fluid requirements, which could be deleterious in the longer term. Experimental and clinical research has demonstrated that in mechanically ventilated subjects without lung injury, PEEP decreases venous return and, secondarily, cardiac output.[17–19] In addition, Trager and colleagues showed that in patients with acute respiratory failure associated with septic shock, high PEEP levels induced a decrease in cardiac output.[20] In contrast, we found no decrease in cardiac output in our patients tested with increasing PEEP levels when fluid administration was optimized according to the respiratory variation in systolic arterial pressure. A similar result was reported by Kiefer et al. and by Akinci et al.[1116] Possible explanations for these contradictory results are a higher rate of fluid administration and the use of lower tidal volumes in the latter studies. Although we did not determine the upper inflection point of the pressure–volume curve, we think that by keeping tidal volume at 6 ml kg−1 any overdistension of the lungs was minimized. Lung volumes are a critical component of the hemodynamic effects of ventilation.[21] Thus, it seems that it is possible to preserve cardiac output in patients with ARDS, despite the use of high PEEP levels, by optimizing fluid administration and limiting tidal volumes. Gastric mucosal perfusion, as assessed by CO2 gap, also remained unchanged during the PEEP trial. This is consistent with the results reported by Kiefer and Akinci in similar studies. In all these studies cardiac output remained unchanged.[1116] In contrast, Trager reported, in a series of septic shock patients with acute respiratory failure, that an increase in PEEP from 5 to 15 cmH2O induced a decrease in cardiac output associated with a decrease in hepatic vein O2 saturation and in hepatic glucose production.[20] It therefore seems that by avoiding a decrease in cardiac output, splanchnic perfusion can be preserved in a majority of patients. One major limitation of our study is the small number of patients studied. Thus, a type II error cannot be excluded. We did not perform any a priori power analysis because we had no estimation of the possible magnitude of the effects that PEEP could have on gastric tonometry. Another limitation is the rather moderate severity of ARDS in our study. Although all patients fulfilled the criteria for ARDS during the 24 hours that preceded the study, at inclusion their PaO2/FiO2 ratio and their respiratory system compliance were only moderately decreased. Two recent papers provide an explanation for this observation.[2223] They show in patients diagnosed with ARDS that after a few hours of treatment with PEEP or a high FiO2, more than half of the patients present a PaO2/FiO2 ratio of more than 200 mmHg. In addition, the respiratory system compliance increased by more than 10 ml per cmH2O after 6 hours of treatment with PEEP.[23] At inclusion our patients had already been ventilated with a median PEEP level of 9 cmH2O for more than 12 hours, which could have explained the rather improved respiratory performance at baseline. In any event, this improvement demonstrated a less severe ARDS. It is possible that more severely compromised patients might present a lower tolerance to high PEEP levels. Another limitation is that tonometry was the sole method used to assess gastric mucosal perfusion. Nevertheless, Elizalde et al. showed that gastric mucosal blood flow, measured by laser Doppler flowmetry and by reflectance spectrophotometry, is well correlated with gastric intramucosal acidosis in mechanically ventilated patients.[24] Conclusions Our study supports the findings of previous studies suggesting that high PEEP levels do not affect splanchnic perfusion and are hemodynamically well tolerated in most patients with ARDS. Furthermore, our study shows that gastric mucosal perfusion can be well preserved while high PEEP levels are applied even in patients presenting cardiovascular dysfunction and receiving vasopressor support, which is a frequent occurrence in critical care. Future studies should assess the effects of PEEP on splanchnic perfusion in a longer term. Source of Support: Nil Conflict of Interest: None declared. ==== Refs References 1 The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome N Engl J Med 2000 342 1301 8 10793162 2 Amato MB Barbas CS Medeiros DM Magaldi RB Schettino GP Lorenzi-Filho G Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome N Engl J Med 1998 338 347 54 9449727 3 Gutierrez G Palizas F Doglio G Wainsztein N Gallesio A Pacin J Gastric intramucosal pH as a therapeutic index of tissue oxygenation in critically ill patients Lancet 1992 339 195 9 1346170 4 Love R Choe E Lippton H Flint L Steinberg S Positive end-expiratory pressure decreases mesenteric blood flow despite normalization of cardiac output J Trauma 1995 39 195 9 7674385 5 Fournell A Scheeren TW Schwarte LA PEEP decreases oxygenation of the intestinal mucosa despite normalization of cardiac output Adv Exp Med Biol 1998 454 435 40 9889921 6 Fujita Y Effects of PEEP on splanchnic hemodynamics and blood volume Acta Anesthesiol Scand 1993 37 427 31 7 Lehtipalo SB Arnelov C Frojse R Johansson G Winso O PEEP can induce splanchnic ischemia during critical reductions in regional perfusion pressure Intensive Care Med 2000 26 S375 8 Arvidsson D Almquist P Haglund U Effects of positive end-expiratory pressure on splanchnic circulation and function in experimental peritonitis Arch Surg 1991 126 631 6 2021348 9 Berendes E Lippert G Loick HM Brussel T Effects of positive end-expiratory pressure ventilation on splanchnic oxygenation in humans J Cardiothorac Vasc Anesth 1996 10 598 602 8841866 10 Aneman A Eisenhofer G Fandriks L Olbe L Dalenback J Nitescu P Splanchnic circulation and regional sympathetic outflow during peroperative PEEP ventilation in humans Br J Anaesth 1999 82 838 42 10562775 11 Kiefer P Nunes S Kosonen P Takala J Effect of positive end-expiratory pressure on splanchnic perfusion in acute lung injury Intensive Care Med 2000 26 376 83 10872128 12 De Backer D The effects of positive end-expiratory pressure on the splanchnic circulation Intensive Care Med 2000 26 361 3 10872126 13 Levy PS Corbeil C Albala M Pariente R Milic-Emili J Jonson B A method for studying the static volume-pressure curves of the respiratory system during mechanical ventilation J Crit Care 1989 4 83 9 14 Gunn SR Pinsky MR Implications of arterial pressure variation in patients in the intensive care unit Curr Opin Crit Care 2001 7 212 7 11436530 15 Vincent JL Moreno R Takala J Willatts S De Mendonca A Bruining H The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine Intensive Care Medicine Intensive Care Med 1996 22 707 10 16 Akinci IO Cakar N Mutlu GM Tugrul S Ozcan PE Gitmez M Gastric intramucosal pH is stable during titration of positive end-expiratory pressure to improve oxygenation in acute respiratory distress syndrome Crit Care 2003 7 R17 23 12793886 17 Dorinsky PM Whitcomb ME The effect of PEEP on cardiac output Chest 1983 84 210 6 6347545 18 Liebman PR Patten MT Manny J Shepro D Hechtman HB The mechanism of depressed cardiac output on positive end-expiratory pressure (PEEP) Surgery 1978 83 594 8 347615 19 Pick RA Handler JB Murata GH Friedman AS The cardiovascular effect of positive end-expiratory pressure Chest 1982 82 345 50 7049595 20 Trager K Radermacher P Georgieff M PEEP and hepatic metabolic performance in septic shock Intensive Care Med 1996 22 1274 5 9120128 21 Pinsky MR Recent advances in the clinical application of heart-lung interactions Curr Opin Crit Care 2002 8 26 31 12205403 22 Ferguson ND Kacmarek RM Chiche JD Singh JM Hallett DC Mehta S Screening of ARDS patients using standardized ventilator settings: Influence on enrollment in a clinical trial Intensive Care Med 2004 30 1111 6 14991096 23 Estenssoro E Dubin A Laffaire E Canales HS Saenz G Moseinco M Impact of positive end-expiratory pressure on the definition of acute respiratory distress syndrome Intensive Care Med 2003 29 1936 42 12955187 24 Elizalde JI Hernandez C Llach J Monton C Bordas JM Pique JM Gastric intramucosal acidosis in mechanically ventilated patients: Role of mucosal blood flow Crit Care Med 1998 26 827 32 9590311	19881173	PMC2772258	CC BY	2021-01-04 19:31:49	yes	Indian J Crit Care Med. 2009 Jan-Mar; 13(1):12-16
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 1995669209-PONE-RA-10602R110.1371/journal.pone.0007971Research ArticleCell Biology/Cell SignalingMolecular Biology/RNA-Protein InteractionsOncology/Neuro-OncologyDifferential SELEX in Human Glioma Cell Lines Glioma Specific AptamersCerchia Laura 1 Esposito Carla Lucia 2 Jacobs Andreas H. 3 4 Tavitian Bertrand 5 de Franciscis Vittorio 1 * 1 Istituto per l'Endocrinologia e l'Oncologia Sperimentale del CNR “G. Salvatore”, Naples, Italy 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli “Federico II”, Naples, Italy 3 European Institute of Molecular Imaging (EIMI), University of Muenster, Muenster, Germany 4 Laboratory for Gene Therapy and Molecular Imaging, Max Planck Institute for Neurological Research, Cologne, Germany 5 CEA/DSV/DRM Service Hospitalier Frederic, Joliot, INSERM ERM 103, Orsay, France Jin Dong-Yan EditorUniversity of Hong Kong, Hong Kong* E-mail: [email protected] and designed the experiments: LC VdF. Performed the experiments: LC CLE. Analyzed the data: LC AHJ BT VdF. Contributed reagents/materials/analysis tools: AHJ BT VdF. Wrote the paper: LC VdF. 2009 24 11 2009 4 11 e797126 5 2009 29 10 2009 Cerchia et al.2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers. ==== Body Introduction A major challenge in oncology aims at the characterization of the heterogeneity of disease by defining more reliable diagnostic/prognostic factors and at developing effective anticancer therapeutics selectively targeting tumor cells. Indeed, cancer is a complex disease characterized by the accumulation of several and often unknown molecular alterations that cause genetic instability, cell proliferation, and acquisition of an increasingly invasive phenotype resistant to therapeutic treatments. In turn the heterogeneity of malignant cells combined to the variability of the patient's genetic background create different cancer phenotypes with distinct clinical outcomes. By reducing the degree of uncertainty on the clinical status of individual patients, the simultaneous analysis of multiple biomarkers improves the possibility to distinguish between two even close tumor types and predict distinct therapeutic responses. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity (within the low nanomolar range) to a target molecule by providing a limited number of specific contact points imbedded in a larger, defined three-dimensional structure [1], [2], [3], [4], [5]. Aptamers are isolated by the Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and since from their first description in 1990 [1], [6], aptamers soon became a valuable research tool and show great application prospected in fundamental research, drug selection and clinical diagnosis and therapy. At present, aptamers against many kinds of proteins have come into clinical test phase [7], [8], [9]. Recently, nucleic acid aptamers have been selected against whole living cells, with the advantage of a direct selection of ligands without prior knowledge of the target molecules, notably by using as target red blood cells [10], leukemia cells [11] small lung cancer cells [12] and rat brain tumor microvessels [13]. Nonetheless, the use of complex cells as targets has been shown to enable the identification of aptamers that bind large cell surface-specific markers, in their native conformation. Indeed, by applying SELEX technology against whole-living cells in culture, for the first time we succeeded in demonstrating that even by using complex targets as intact cells, it is possible to obtain aptamers against even rare antigens if specifically expressed on the target cell [14]. We adopted this strategy to generate nuclease resistant RNA-aptamers specific for PC12 cells expressing the human receptor tyrosine kinase, Ret and selected aptamers that bind specifically to Ret and inhibit its downstream signaling effects [14]. Herein, we have developed a whole-cell SELEX protocol with the aim to generate aptamers able to discriminate within the same tumor between two strictly related phenotypes. We used glioblastoma cell lines as model system because of the complex cellular heterogeneity of malignant gliomas and the need to find new diagnostic and therapeutic modalities for these tumors. By using a counterselection/selection approach, specifically designed to enrich for aptamers against cell surface tumor-specific targets, we have generated a panel of RNA-aptamers able to bind at high affinity to malignant U87MG cells. They do not bind the non tumorigenic T98G nor other non-related cancer cell types, but bind to glioma cell lines characterized by different malignant phenotypes at different extents. Furthermore, functional analysis revealed that some of the aptamers inhibit specific intracellular signaling pathways. Our results indicate the differential whole-cell SELEX strategy as a promising strategy to develop specific molecular probes for early diagnosis and prediction of aggressiveness and therapeutic response that is generally applicable to different strictly related cell types. Results Enrichment of Selection for a Complex Target In order to isolate cell specific ligands for a given tumor cell phenotype we decided to keep as model system stable human glioma cell lines. Even though using stable cell lines has the drawback of skipping epitopes that might be important for the in vivo cell growth, it has the obvious advantage to handle a cell population under well controlled growth conditions that remain stable all along the SELEX procedure. We used as target for the selection steps the malignant human U87MG glioma cell line and for the counterselection steps less malignant human T98G glioma cells. These two cell lines differ for the potential to form tumors in nude mice, U87MG being highly tumorigenic while the T98G are poorly tumorigenic. On the other hand, these cell lines share same cellular pathways altered, both harboring p14arf/p16 deletion and phosphatase and tensin homolog (PTEN) mutation. Major differences found between the two cell lines are the levels of ErbB2 and of phosphorylated extracellular signal-regulated protein kinase (ERK), that are higher in U87MG than in T98G, while phosphorylated Akt and neural cell adhesion molecule (NCAM) levels were similar (data not shown). The relative levels of these four molecules were monitored at each cycle of the SELEX procedure to verify and standardize the growth conditions of the cells. A library of 2'Fluoro Pyrimidines (2'F-Py), nuclease-resistant RNAs was utilized for differential SELEX against intact cells (Fig. 1). At each round the selection step on U87MG cells was preceded by one or two counterselection steps against T98G cells. During the selection process, we progressively increased the selective pressure by changing both incubation and washing conditions. During rounds 13 and 14, restriction fragment length polymorphism analysis (RFLP) profiles remain unchanged, suggesting that the population had stopped to evolve under the selection pressure (data not shown). Indeed, as assessed by comparing the in vitro binding efficiency on the two cell lines and to the naïve starting pool (not shown), after 14 rounds of selection, the pool, named G14, was enriched for aptamers that preferentially bind to U87MG cells. 10.1371/journal.pone.0007971.g001Figure 1 Selection of U87MG cell-specific aptamers. A pool of 2'F-Py RNAs was incubated with poorly tumorigenic T98G cells (Counterselection). Unbound sequences in the supernatant were recovered and incubated with tumorigenic U87MG cells for the selection step (Selection). Unbound sequences were discarded by washings and bound sequences were recovered by total RNA extraction. Sequences enriched by the selection step were amplified by RT-PCR and in vitro transcription before a new cycle of selection. Distribution of Individual Sequences In order to isolate individual aptamers that may distinguish the more malignant U87MG phenotype, a panel of 71 sequences was cloned from the pool G14, and aptamers grouped in families based on their primary sequence similarity (Fig. 2). We identified ten families of highly related aptamers that together cover more than 46% (33 aptamers) of all individual sequences obtained from the selection; one single individual sequence dominated the selection and constituted 8% of all the clones; five other sequences represented together more than 15% of the clones. The remaining 38 sequences were poorly related to each other (Fig. 2). 10.1371/journal.pone.0007971.g002Figure 2 Analysis of individual sequences similarity. Dendogram (obtained by using DNASIS software version 2.1) for visual classification of similarity among 71 individual sequences cloned after 14 rounds of selection. Aptamers are grouped in 10 families of sequences found more than once (labeled with the asterisk) or that share sequence similarity (boxed). We thus screened for those aptamers that efficiently target the U87MG cells. To this aim a panel of 21 individual aptamers, including at least one member for each family, was analysed for binding at the concentration of 500 nM. At that concentration, 8 aptamers display up to five-fold increase of binding to U87MG cells with respect to the starting pool, while the remaining 13 aptamers show no specific binding to U87MG (not shown). Using the starting pool as control of background, the binding affinity of individual aptamers to U87MG cells and T98G cells was then determined. As shown in Table 1, all 8 sequences (GL17, GL56, GL62, GL36, GL35, GL43 GL44 and GL21) bind at high affinity (with Kd ranging between 33 nM and 700 nM) to the U87MG cells and have no or low affinity for T98G (not shown). Michaelis-Menten binding curves of three aptamers are shown in Fig. 3A . Given the good specificity and high affinity for the U87MG cells we restricted our further analysis of biochemical and biological properties to these 8 sequences. 10.1371/journal.pone.0007971.g003Figure 3 Dissociation constants of GL35, GL36, GL21 and GL44-43 short aptamers. (A) Binding curve of GL35, GL36 and GL21 aptamers on U87MG. Lineweaver-Burk analysis (inset) was used for the evaluation of the binding constant (see Materials and Methods). (B) Secondary structure of a shortened sequence consisting of residues 1–39 of GL44 or GL43 aptamers (GL44-43 short). predicted by using MFOLD software version 3.1 (available at http://www.bioinfo.rpi.edu/applications/mfold/). (C) Binding curve of the GL44-43 short aptamer on U87MG cells. 10.1371/journal.pone.0007971.t001Table 1 Sequence of indicated aptamers; for simplicity fixed-primer sequences at 5′ and 3′ are not shown. Binding affinity of individual aptamers to U87MG cells Name Sequence Kd (nM) GL17 CCGUUGUUCUACAUGUCACUCAUCACGCGAGUCUUUUGUCUAA 102±12 GL21 GCCUCUCAACGAUUAAUGUUUCGUUAACAUGAUCAAUCGCCUCAA 221±25 GL62 UUCACACACUCAAUUGAACGGUGAUUCAAGUUAUUAGCAGCCUCA 710±40 GL43 ACGUUACUCUUGCAACAC–AAACUUUAAUAGCCUCUUAUAGUUC 44±4 GL44 ACGUUACUCUUGCAACACCCAAACUUUAAUAGCCUCUUAUAGUUC 38±3 GL56 UGAUUUUGCAGCACUUCUUGUUAUCUUAACGAACUGUUGAUGA 63±9 GL36 UACCAAACGCGCAAUUUUCAUCUUGUAAUAACCAAAUGCCUCUGA 190±20 GL35 UACCAAACGCGCGGUUUUCGUCUCGUAAUAACCAAAUGCCUCUGA 44±7 Residues that differ among the sequences in the couple (GL43 and GL44) or (GL36 and GL35) are in bold. Michaelis-Menten binding curves to estimate Kd (nM) were performed as described in Materials and Methods; standard deviation values were determined from at least four independent experiments. Comparison of Individual Sequences Four of the eight aptamers considered (GL21, GL17, GL56 and GL62) have unrelated primary sequences and predicted 2D folded structures. Two (GL44 and GL43) differ for the presence of two cytosines (cyt42 and cyt43) that are only present in GL44 whose presence however does not alter the affinity for the target cells (see Table 1). Comparing the predicted secondary structures defined a conserved stem-loop (residues 1–39) presents with an identical sequence in both aptamers (Fig. 3B ). Consistently the shortened sequence, constituted of the first shared 39 residues (herein named GL44-43 short), is sufficient to bind to the U87MG cells displaying a Kd of 30 nM (Fig. 3C ) and discriminate them from T98G cells (not shown). The opposite situation was found in another couple of aptamers (GL35 and GL36) that even if poorly differ in their primary structures, have binding affinities that differ of around 4 times (Table 1). Binding Specificity The identification of a small set of aptamers that may distinguish the U87MG cells from the T98G cells raises the obvious question of whether these aptamers may bind as well other cell types. To this aim we determined the relative binding potential of each aptamer to several cell lines. We first determined the cell type specificity by measuring at the same concentration of 50 nM the binding of each aptamer on a panel of unrelated cell lines. We found that any of the eight aptamers did not bind to other human cancer cell types analyzed including neuroblastoma (SK-N-BE and SH-SY5Y), lung (H460 and Calu1) and breast (MCF7 and SKBR3) cancer cells nor to murine fibroblast NIH3T3 cells, as assessed by comparison with the unspecific binding of the starting pool, G0 data not shown). On the other hand, even if at different extents they bind to various glioma cell lines (U251MG, TB10, LN-18 and LN-229) and a U87MG derivative, U87MGΔEGFR, but not the T98G cells used for counterselection. These cell lines are characterized by different tumor type derivation and malignant phenotypes and have different genetic backgrounds. As shown in Fig. 4A, at that concentration each aptamer has a distinct pattern of binding on different glioma cell lines (see Legend). At these experimental conditions, all aptamers have good binding with the highly tumorigenic cell lines (U87MG, LN-229, U87MGΔEGFR and TB10), the aptamers GL56 binds to all cell lines except to the non tumorigenic T98G, and GL17 binds only the four highly tumorigenic cell lines. Thus the pattern of binding of five of these aptamers (for example, GL44, GL17, GL56, GL36 and GL35) is sufficient to distinguish two cell lines. Further we confirmed that aptamers can distinguish tumorigenic from non tumorigenic glioma cells by determining binding of four of them (GL21, GL35, GL36 and GL44-43 short) to highly tumorigenic cell lines (U87MG, Gli36, and Gli36ΔEGFR) as compared to two cell lines (A172 and T98G) that are unable to form tumors in nude mice (Fig. 4B ). Furthermore, the eight aptamers bind also to primary cell cultures of malignant glioblastomas and discriminate them from a not-related meningeoma cell line, thus excluding the recognition of possible unwanted epitopes enriched upon immortalization of a stabilized cell line (Fig. 4C ). For each aptamer the differences observed in the extent of binding likely reflect the relative concentrations of the same target molecules in the different cell lines. 10.1371/journal.pone.0007971.g004Figure 4 Binding analyses of the best sequences to glioma cell lines. The indicated aptamers or the starting pool (G0) were 5′- [32P]-labeled and incubated in the same condition at 50 nM with the indicated stable glioma cell lines (A and B) or primary cultures of malignant glioma cells (C). The results are expressed relative to the background binding detected with the starting pool. In (A) and (B) the tumorigenic potential in nude mice is indicated on the basis of the time of appearance of tumor and the tumor growth rate as previously reported [15], [23], [31], [32]: high tumorigenicity is indicated as “++”; middle tumorigenicity is indicated as “+” and no tumorigenicity is indicated as “-”. In (A), the binding capacity of the aptamers to the cells is reported: high binding (more than four-fold) is indicated as “++”, middle binding (between two and four-fold) is indicated as “+” and no binding (less than two-fold) is indicated as “-”. Biological Activities of Aptamers As previously demonstrated for the anti Ret receptor tyrosine kinase D4 aptamer, high affinity aptamer binding to an extracellular receptor may inhibit activity of key downstream transducing molecules, such as ERK family members. Therefore, we first determined whether treating the U87MG cells with any of these aptamers may affect the phosphorylation of either Akt and ERK 1/2. Treating cells with five aptamers (GL36, GL35, GL44, GL43 and GL21) at 200 nM inhibited ERK phosphorylation of at least two-fold, as compared to the control starting pool and to the other aptamers (GL17, GL56, GL62) (not shown). On the other hand, no aptamer had any relevant effect on the phosphorylation of Akt and of the 3-Phosphoinositide-dependent protein kinase-1 (PDK1) (not shown), most likely because the U87MG harbor a mutated inactive phosphatase and tensin homolog (PTEN), a phosphatase that dephosphorylates the phosphatidylinositol (3,4,5)-trisphosphate, thus resulting in inhibition of the Akt signaling pathway [15]. To further confirm the biological activity of GL36, GL35, GL44, GL43 and GL21, we determined the extent of inhibition of expression of the cell cycle-related protein, cyclin D1 and of phosphorylation of ERK 1/2 upon treatment of U87MG cells with aptamers for increasing time periods. The cyclin D1 proto-oncogene is an important regulator of G1 to S-phase transition, causing downregulation of protein levels through its protein expression and through phosphorylation-dependent degradation, and causing inhibition of cell cycle progression by inducing a G1 arrest [16], [17]. We thus performed time-course experiments with the five inhibitor aptamers looking at its protein expression and phosphorylation levels. As shown in Fig. 5A , treatment with cognate aptamers either GL36 and GL35, or GL44 and GL43, inhibits at similar extents basal cyclin D1 expression and phosphorylation in a time dependent manner. Further, treating cells with the aptamer GL21 resulted as well in a stronger and more rapid inhibition of cyclin D1 reaching around 26% at 1 h. 10.1371/journal.pone.0007971.g005Figure 5 Biological activities of selected aptamers. Serum starved U87MG cells were either left untreated or treated with 200 nM of the indicated RNA aptamers or G0 for the indicated incubation times. (A) Cell lysates were immunoblotted with anti-phosphocyclin D1 and cyclin D1 antibodies. To confirm equal loading the filters were stripped and reprobed with anti-α-tubulin antibodies. (B) Cell lysates were immunoblotted with anti-phosphoERK antibodies and the filters were stripped and re-probed with anti-ERK antibodies. In (A) and (B), intensity of bands has been calculated using the NIH-Image Program on at least two different exposures to assure the linearity of each acquisition. Four independent experiments were performed. Fold values are expressed relative to the reference points, arbitrarily set to 1 (labeled with asterisk, lane 1). “C” indicates mock-treated cells. Plots of fold values corresponding to the cyclin D1 expression and to ERK activity are reported for each lane of immunoblotting shown in (A) and in (B), respectively. (C) U87MG cells were treated for 24 hs or 48 hs with the indicated aptamers or the G0 starting pool and proliferation was determined by [3H]-thymidine incorporation. In (A), (B) and (C), vertical bars indicate the standard deviation values. As shown in Fig. 5B treatment with the same five aptamers caused a similar time dependent inhibition of ERK phosphorylation, inhibition being more rapid with GL35 than GL36, thus according to their respective Kd values (see Table 1), and, as expected, at comparable extents treating with the highly related GL44 and GL43 aptamers. In agreement with its Kd value, the GL44-43 short aptamer, inhibited cyclin D1 expression and ERK phosphorylation at a similar extent of the GL44 aptamer (not shown). To further demonstrate the biological activity of these aptamers we treated growing U87MG cells with either GL44-43 short or the GL21 aptamer and monitored the inhibitory effect on cell proliferation by pulse labeling thymidine incorporation. According with inhibition of ERK activity, treating cells with aptamers for 24 and 48 hs strongly reduces [3H]thymidine incorporation (Fig. 5C ). Taken together the results indicate that these five aptamers harbor intrinsic biological activity and may act as inhibitory ligands of critical cell surface molecules. Discussion Cancer cell surface epitopes that may discriminate a specific cell phenotype whilst embedded in a heterogeneous cell population are highly promising targets for developing personalized innovative therapeutics. Human gliomas are highly heterogeneous tumors whose sensitivity to therapeutic approaches is hard to predict. Several glioma derived cell lines have been shown to differ in the pattern of expression of receptor tyrosine kinases and in their involvement in cell proliferation [18], thus gliomas may constitute a model of choice in which to demonstrate the possibility to obtain a panel of aptamers that may distinguish a given glioma phenotype. Here we adopted a differential SELEX protocol to target whole living human glioma derived cells, the U87MG cell line, and we obtained a panel of nuclease-resistant RNA ligands capable of binding at high affinity to the surface of target cells and to distinguish them from other glioma cell lines. Furthermore, because of the importance of several membrane bound proteins as regulators of cancer cell proliferation we determined in U87MG cells whether any of such aptamers may interfere with the transmission of intracellular signaling and demonstrated that five of these molecules inhibited the activity of critical molecules in cell proliferation, ERK 1/2 and cyclin D1, but not Akt. Lack of inhibition of Akt likely rely on the fact that these cells bear an inactive PTEN, a negative regulator of Akt, and thus the levels of Akt phosphorylation are only poorly regulated by extracellular stimuli [15]. The use of living cells as targets for SELEX has been already described by us and by others [19]. Indeed, we have already demonstrated that it is possible to obtain aptamers for a given transmembrane receptor tyrosine kinase if an appropriate protocol is adopted thus providing the first evidence of the possibility to use SELEX to distinguish between cells that differ for a single (or few) membrane epitope [14], [20]. Here we adopted a similar approach to distinguish between closely related tumor cell lines by targeting unknown epitopes on the cell surface. To surmount the possible drawback of obtaining aptamers targeting few highly represented epitopes we monitored the evolution process by RFLP and stopped the SELEX rounds as soon as the enrichment of the library was visible, i.e. at round 14. This strategy allowed us to obtain aptamers against multiple surface epitopes with Kd values in the nanomolar range, a further increase in affinity is expected by applying further SELEX rounds to each individual aptamers (work in progress). As a first attempt to identify those aptamers that better discriminate the U87MG cells, we screened 21 individual aptamers for their binding at 500 nM to U87MG cells over the naïve G0 random pool. Even if we identified eight aptamers that specifically bind cells at that concentration, given that in this assay we used an unique aptamers concentration we cannot conclude on the specificity of the binding properties of the remaining thirteen aptamers on either U87MG or T98G cells. Our strategy allowed us to obtain eight, of which six are unrelated, aptamers that bind bona fide distinct epitopes present on the cell surface. Further, by this approach we generated a panel of ligands that bind cells used for selection but neither those used for counterselection nor unrelated cancer cells, from neuroblastoma, breast, and lung cancer cell lines. Most importantly, by combining their pattern of binding allows to identify a given glioma cell line among the seven analyzed. Further, the differences in binding are not randomly distributed but in contrast they fit well with important growth properties of each cell line, as tumorigenicity in nude mice, thus suggesting that the extent of binding of an aptamer (see for example GL17) may associate with at least one biological property. We measured binding efficiency on each cell line using the same concentration of 50 nM for all aptamers. This strategy even if it doesn't exactly reflect the differences in Kd between aptamers (calculated on the U87MG cells) revealed as a simple and direct way to obtain a specific pattern of binding for each cell line. As expected the two cell lines used for selection and counterselection have the best and worst binding for all the aptamers, respectively. Aptamers have been shown to frequently inhibit the function of their target molecules, presumably by interfering with binding of the cognate ligand [21]. As mentioned above the strategy adopted should allow to enrich for aptamers that target molecules even present at low abundance on the cell surface as for example receptors and other transducing molecules, as already shown for ligands to the receptor tyrosine kinase Ret [14]. The effectiveness of the strategy adopted is well highlighted by the fact that out of eight aptamers that bind to the U87MG, five interfere with the activity of critical molecules for intracellular signaling and cell proliferation likely binding to and/or inhibiting a membrane bound receptor in its active conformation. In conclusion, we developed a differential SELEX-based procedure that allowed us to generate a highly informative panel of few specific and selective aptamers for malignant glioma cells. The identification of cellular targets for each of these aptamers is in progress. Major advantages of SELEX are the relative rapidity of the entire process and to be only based on artificial molecules. Therefore, the concrete prospective that by such a procedure it is possible to identify and validate important surface target molecules will certainly reveal this as a rather unique tool to discover new molecular targets for antibodies or short peptides. Materials and Methods Ethics Statement Primary tumor cultures were derived from surgical biopsies. The study protocol was approved by the local Ethics Committee of the University of Cologne. Written informed consent was acquired prior to surgery from every patient for further studies on primary glioma cultures. Cell Culture and Immunoblotting Human glioma U87MG (American Type Culture Collection, ATCC no. HTB-14), T98G (ATCC no. CRL-1690), A172 (ATCC no. CRL-1620), U251MG (kindly provided by A. Porcellini), TB10 (kindly provided by A. Porcellini), Gli36 [22] cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Human glioma, LN-18 (ATCC no. CRL-2610), LN-229 (ATCC no. CRL-2611) were grown in Advanced DMEM supplemented with 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen, Carlsbad, CA). U87MGΔEGFR and Gli36ΔEGFR, expressing a truncated mutant EGFR receptor due to an in-frame deletion of exons 2–7 from the extracellular domain (ΔEGFR) [23], were grown in DMEM supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 500 µg/ml gentamycin (Invitrogen, Carlsbad, CA). Growth conditions for cell lines used were previously reported: human neuroblastoma SH-SY5Y and SK-N-BE cells [24], human breast MCF7 and SKBR3 cells [25], human NSCLC H460 and Calu1 cells [26] and NIH3T3 cells [14]. Primary tumor cultures from surgical biopsies of patients with brain tumors were derived as described previously [27]. Each tumor specimen was cut into small pieces, removing blood vessels, then resuspended in trypsin solution (4% in 0.01 M PBS, pH 7.2). After incubation (37°C, 10 min), DMEM (Life Technologies, Karlsruhe, Germany) supplemented with 20% FBS (Roche Diagnostics, Mannheim, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin (P/S; Life Technologies) and 1% amphotericin (SIGMA, Steinheim, Germany) was added and the cell suspension was centrifuged (210 rcf, 6 min). The pellet was resuspended in DMEM with 20% FBS, 1% P/S and 1% amphotericin and grown at 37°C in a 5% CO2/95% air atmosphere. When primary tumor cell cultures formed nearly confluent monolayers, they were frozen and stored in liquid nitrogen for further use. To assess the functional effects of aptamers, U87MG (300 000 cells per 3.5-cm plate) were serum starved for 2 hs and then treated with the RNA aptamers or the starting RNA G0 pool prior subjected to a short denaturation-renaturation step. Cell extracts preparation and immunoblotting analysis were performed as described [28]. The primary antibodies used were: anti-ERK1 (C-16) (Santa Cruz Biotechnology, Santa Cruz, California, United States) and anti-phospho-44/42 MAP kinase (indicated as anti-pErk), anti-Akt, anti-phospho-Akt (Ser473, indicated as anti-pAkt), anti-PDK1, anti-phospho-PDK1 (Ser241, indicated as anti-pPDK1), anti-phospho-cyclin D1 (Thr286, indicated as anti-pcyclin D1) and anti-cyclin D1 (all from Cell Signaling, Beverly, MA), anti-α-tubulin (DM 1A) (Sigma, St. Louis, MO). Whole-Cell SELEX The SELEX cycle was performed essentially as described [29]. Transcription was performed in the presence of 1 mM 2′-F pyrimidines and a mutant form of T7 RNA polymerase (2.5 u/µl T7 R&DNA polymerase, Epicentre Biotechnologies, Madison, WI.) was used to improve yields. 2'F-Py RNAs were used because of their increased resistance to degradation by seric nucleases. 2'F-Py RNAs (800-300 pmol) were heated at 85°C for 5 min in 1.5 ml of DMEM serum free, snap-cooled on ice for 2 min, and allowed to warm up to 37°C. Before incubation with the cells, 13.5 ml of medium were added to RNA to reach a final volume of 15 ml. Counterselection against T98G cells. To avoid selecting for aptamers non-specifically recognizing the U87MG cell surface, the pool was first incubated for 30 min (up to round 9) or for 15 min (for the following rounds) at 37°C with 107 T98G cells (150-mm cell plate), and unbound sequences were recovered for the selection phase. This step was meant to select sequences recognizing specifically the U87MG cells. Selection against U87MG cells. The recovered sequences were incubated with 107 U87MG cells for 30 min at 37°C and recovered after several washings with 5 ml of DMEM serum free by total RNA extraction (Ambion, Austin, TX). During the selection process, we progressively increased the selective pressure by increasing the number of washings (from one for the first cycle up to five for the last cycles) and by decreasing the incubation time (from 30 to 15 min from round 9). To follow the evolution of the pool we monitored the appearance of four-base restriction sites in the population by RFLP as previously described [30]. Binding Analysis Binding of individual aptamers (or the starting pool as a control) to glioma cells was performed in 24-well plates in triplicate with 5′-[32P]-labeled RNA. 3.5×104 cells per well were incubated with various concentrations of individual aptamers in 200 µl of DMEM serum free for 20 min at RT in the presence of 100 µg/ml polyinosine as a nonspecific competitor (Sigma, St. Louis, MO). After five washings of 500 µl DMEM, bound sequences were recovered in 300 µl of SDS 1%, and the amount of radioactivity recovered was counted. The background values obtained with the starting pool were subtracted from the values obtained with the specific aptamers. Apparent Kd values for each aptamer were determined by Lineweaver-Burk analysis according to the equation: [3H]-Thymidine Incorporation Assay U87MG cells were plated in 24-well dishes (2×104 cells/well) and treated for 24 hs or 48 hs with aptamer (as indicated) or the starting pool (G0) as control. During the final 4 hs, cells were pulsed with 1 µCi/ml [3H]-thymidine (45 Ci/mmol) (Amersham-Pharmacia Biosciences) added in complete growth medium and incubated at 37°C. At the end of each pulse, cells were harvested and [3H]-thymidine incorporation was analyzed by a Beckman LS 1701 Liquid Scintillation Counter. We wish to thank F. Ducongè, L. Cellai, A. Winkeler and G. Condorelli for suggestions and comments. Competing Interests: Patent has been submitted: “Method for obtaining oligonucleotide aptamers and uses thereof” #EP 08 105 194.8 Sept 1st 2008 (Inventors: V de Franciscis, Laura Cerchia, G. Condorelli). The authors confirm that this does not alter their adherence to all the PLoS ONE policies on sharing data and materials. Funding: This work was supported by funds from: C.N.R., Associazione Italiana Ricerca sul Cancro (L.C.), AIRC, MIUR-FIRB (RBIN04J4J7), EU grant EMIL (European Molecular Imaging Laboratories Network) contract No 503569. 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Hum Gene Ther 14(3) 277 97 12639307 23 Nishikawa R Ji XD Harmon RC Lazar CS Gill GN 1994 A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Nati Acad Sci USA 91 7727 7731 24 Esposito CL D'Alessio A de Franciscis V Cerchia L 2008 A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation. PLoS ONE 3 e1643 18286198 25 Buckley MF Sweeney KJ Hamilton JA Sini RL Manning DL 1993 Expression and amplification of cyclin genes in human breast cancer. Oncogene 8 2127 2133 8336939 26 Zanca C Garofalo M Quintavalle C Romano G Acunzo M 2008 PED is overexpressed and mediates TRAIL resistance in human non-small cell lung cancer. J Cell Mol Med 12 2416 2426 18284607 27 Rueger MA Winkeler A Miletic H Kaestle C Richter R 2005 Variability in infectivity of primary cell cultures of human brain tumors with HSV-1 amplicon vectors. Gene Ther 2(7) 588 596 28 Cerchia L Libri D Carlomagno MS de Franciscis V 2003 The soluble ectodomain of RetC634Y inhibits both the wild-type and the constitutively active Ret. Biochem J 372 897 903 12630912 29 Fitzwater T Polisky B 1996 A SELEX primer. Methods Enzymol 267 275 301 8743323 30 Pestourie C Cerchia L Gombert K Aissouni Y Boulay J 2006 Comparison of different strategies to select aptamers against a transmembrane protein target. Oligonucleotides 16(4) 323 335 17155908 31 Pallini R Sorrentino A Pierconti F Maggiano N Faggi R 2006 Telomerase inhibition by stable RNA interference impairs tumor growth and angiogenesis in glioblastoma xenografts. Int. J Cancer 118 2158 67 32 de Ridder LI Laerum OD Mørk SJ Bigner DD 1987 Invasiveness of human glioma cell lines in vitro: relation to tumorigenicity in athymic mice. Acta Neuropathol 72(3) 207 13 3564901	19956692	PMC2776989	CC BY	2021-01-05 16:16:56	yes	PLoS One. 2009 Nov 24; 4(11):e7971
==== Front World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central 1477-7819-7-821989570210.1186/1477-7819-7-82Case reportUrachal tumour: case report of a poorly understood carcinoma Scabini Stefano [email protected] Edoardo [email protected] Emanuele [email protected] Renato [email protected] Luigi [email protected] Veronica [email protected] Carlo [email protected] Valter [email protected] Department of Emato-Oncology, AOU San Martino Hospital, Genoa, Italy2 ASL 3 Genoa, Italy3 Department of Radiology, AOU San Martino Hospital, Genoa, Italy2009 7 11 2009 7 82 82 9 9 2009 7 11 2009 Copyright ©2009 Scabini et al; licensee BioMed Central Ltd.2009Scabini et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Urachal carcinoma is an uncommon neoplasm associated with poor prognosis. Case presentation A 45-year-old man was admitted with complaints of abdominal pain and pollakisuria. A soft mass was palpable under his navel. TC-scan revealed a 11 × 6 cm tumor, which was composed of a cystic lesion arising from the urachus and a solid mass component at the urinary bladder dome. The tumor was removed surgically. Histological examination detected poor-differentiated adenocarcinoma, which had invaded the urinary bladder. The patient has been followed up without recurrence for 6 months. Conclusion The urachus is the embryological remnant of urogenital sinus and allantois. Involution usually happens before birth and urachus is present as a median umbilical ligament. The pathogenesis of urachal tumours is not fully understood. Surgery is the treatment of choice and role of adjuvant treatment is not clearly understood. ==== Body Background Urachal carcinoma is an uncommon neoplasm associated with poor prognosis. The estimated annual incidence of urachal carcinoma in the general population is one in 5 million, or 0.01% of all cancers in adults. Urachal carcinoma has been estimated to comprise 0.17-0.34% of all bladder cancers [1]. Adenocarcinoma is common among urachal carcinomas, whereas squamous cell carcinoma (SCC) is very rare. We report a case of primary adenocarcinoma of the urachus. Case presentation A 45-year-old man was referred to our hospital with complaints of micturition pain of 5 months' duration, and lower abdominal pain and pollakisuria of 1 month's duration. A physical examination revealed a soft tender mass under his navel. Periumbilical discharge was not recognized. Laboratory data: hemoglobin, 15.9 g/dL; erythrocyte count, 4865000/mL; leukocyte count, 11600/mL; platelet count, 259000/mL. CEA: 5.1 ng/ml. Urinalysis: hemoglobin (++). Urine cytology: negative. Cystoscopy detected mucosal edema and erosion at the bladder dome. TC-scan showed a soft-tissue mass along the umbilicus, which extended from the bladder dome to the posterior wall of the bladder and the peritoneum. The size of the tumor was 11 × 6 cm (Fig. 1). Figure 1 TC-scan: a soft-tissue mass along the umbilicus, which extended from the bladder dome to the posterior wall of the bladder and the peritoneum. The size of the tumor was 11 × 6 cm It was noted that the tumor extended from the bladder dome to the umbilicus. Open laparotomy revealed bladder edema and intraoperative cytological examination was negative. The tumor was removed en bloc together with the umbilicus, lateral umbilical ligaments, adjacent peritoneum and bladder dome (Fig. 2). Figure 2 Tumor removed en bloc together with the umbilicus, lateral umbilical ligaments, adjacent peritoneum and bladder dome On gross inspection, the suprapubic mass consisted of slid and cystic lesions. Microscopically, poor-differentiated adenocarcinoma of the solid lesion was observed (Fig. 3). According to CT scan and pathologist report the tumor can be staged as Sheldon II, confined only to urachus. The grading of this tumor was impossible. Figure 3 Macroscopic aspect of tumor: the suprapubic mass consisted of slid and cystic lesions Tumor cells not invaded the bladder. Follow-up abdominal computerized tomography at 6 months showed no tumor recurrence. Discussion The urachus is the embryological remnant of urogenital sinus and allantois. Involution usually happens before birth and urachus is present as a median umbilical ligament. The pathogenesis of urachal tumours is not fully understood. It is believed that urachal carcinomas arise from malignant transformation of columnar or glandular metaplastic epithelium. They lie in the space of Retzius, between transversalis fascia anteriorly and peritoneum posteriorly, extending from the dome of the bladder to the umbilicus. Urachal cancer was first described in 1863 by Hue and Jacquin in a report translated and summarized by Sheldon [1]. Although this tumour has now become a recognisable 'neoplastic entity', its origin and pathophysiology remain unknown [2]. The estimated annual incidence of urachal carcinoma is 0.01% of all cancers in adults. The incidence of the disease ranges from 0.55 to 1.20% of bladder tumors in Japan and 0.07-0.70% of bladder tumors in Western countries. Histopathologically, adenocarcinoma accounts for 80-90% of the tumors. The 5-year cancer-specific survival rate, depending of pathologic stage [3], is 6% in Japan [4] but Ashley [5] in a 50 years of experience at Mayo Clinic reported a 5-year cancer-specific survival rate of 49%. The poor prognosis of this cancer is due to: (a) late presentation of symptoms leading to advanced stage at diagnosis; (b) a propensity for early local invasion; and (c) distal metastasis. The new Mayo staging system was less complicated than the Sheldon system, although both systems predicted cancer-specific mortality equally well. Positive surgical margins (hazard ratio [HR], 4.7), high tumor grade (HR, 3.6), positive local lymph nodes (HR, 5.1), metastases at diagnosis (HR, 3.3), advanced tumor stage (HR, 4.8), failure to perform umbilectomy (HR, 3.0), and primary radiation therapy (HR, 2.9) were all univariately associated with death (P < .05). Only grade and margins were significant in the multivariate analysis. Modern therapeutic regimens have offered minimal benefit, especially when unresectable. No survival benefit was noted for lymphadenectomy or adjuvant therapy. Salvage surgery resulted in a long-term cure for 50% of patients who had local recurrences. No effective treatment was identified for patients with metastatic UrC. The question as to whether partial or radical cystectomy is suitable for localised disease is difficult to answer since urachal tumours are rare. Furthermore, within this group, urachal adenocarcinoma is uncommon in those under 40 years. However, in order to evaluate which surgical approach is correct or whether new chemotherapeutic agents will induce objective responses and improve long-term survival requires co-operation between physicians and centres internationally so that larger studies can be conducted. This approach can only benefit patients [6-8]. Conclusion The urachus is the embryological remnant of urogenital sinus and allantois. Involution usually happens before birth and urachus is present as a median umbilical ligament. The pathogenesis of urachal tumours is not fully understood. Surgery is the treatment of choice and role of adjuvant treatment is not clearly understood. Consent Written informed consent was obtained from the patient for publication of this case report. A copy of the consent is available with editorial office Competing interests The authors declare that they have no competing interests. Authors' contributions SS, ER, ER, RS and VF are surgeons of the Unit of Surgical Oncology (Chief: VF) and have performed the operation. LV is General Psysician of the patient. VG and CF are radiologists of the Department of Radiology. All authors read and approved the final manuscript. ==== Refs Sheldon CA Clayman RV Gonzalez R Williams RD Fraley EE Malignant urachal lesions J Urol 1984 131 1 8 6361280 Mostfi FK Thomson RV Dean AL Mucinous Adenocarcinoma of the urinary bladder Cancer 1955 8 741 58 10.1002/1097-0142(1955)8:4<741::AID-CNCR2820080417>3.0.CO;2-C 13240656 Gopalan A Sharp DS Fine SW Tickoo SK Herr HW Reuter VE Olgac S Urachal carcinoma: a clinicopathologic analysis of 24 cases with outcome correlation Am J Surg Pathol 2009 33 5 659 68 10.1097/PAS.0b013e31819aa4ae 19252435 Ghazizadeh M Yamamoto S Kurokawa K Clinical features of urachal carcinoma in Japan: review of 157 patients Urol Res 1983 11 235 8 10.1007/BF00272286 6659216 Ashley RA Inman BA Sebo TJ Leibovich BC Blute ML Kwon ED Zincke H Urachal carcinoma: clinicopathologic features and long-term outcomes of an aggressive malignancy Cancer 2006 107 712 20 10.1002/cncr.22060 16826585 Tian J Ma JH Li CL Xiao ZD Urachal mass in adults: clinical analysis of 33 cases Zhonghua Yi Xue Za Zhi 2008 88 12 820 2 18756985 Paras FA JrMaclennan GT Urachal adenocarcinoma J Urol 2008 180 2 720 10.1016/j.juro.2008.05.039 18554640 Sugarbaker PH Verghese M Yan TD Brun E Managment of mucinous urachal neoplasm presenting as pseudomyxoma peritonei Tumori 2008 94 5 732 6 19112949	19895702	PMC2781004	CC BY	2021-01-04 17:49:17	yes	World J Surg Oncol. 2009 Nov 7; 7:82
==== Front Indian J Community MedIJCMIndian Journal of Community Medicine : Official Publication of Indian Association of Preventive & Social Medicine0970-02181998-3581Medknow Publications India IJCM-34-14010.4103/0970-0218.51231Original ArticleMusculoskeletal Disorders: Epidemiology and Treatment Seeking Behavior of Secondary School Students in a Nigerian Community Adegbehingbe Olayinka O Fatusi Adesegun O 1Adegbenro Caleb A 1Adeitan Opeyemi O Late1Abass Ganiyu O 1Akintunde Akintomiwa O 1Department of Orthopaedic Surgery and Traumatology, Obafemi, Awolowo University, College of Health Sciences, Faculty of Clinical Sciences, Ile Ife, Osun State, Nigeria1 Department of Community Health, Obafemi, Awolowo University, College of Health Sciences, Faculty of Clinical Sciences, Ile Ife, Osun State, NigeriaAddress for correspondence: Dr. OO Adegbehingbe, Obafemi Awolowo University, College of Health Sciences, Faculty of Clinical Sciences, Department of Orthopaedic Surgery and Traumatology, Ile-Ife, Osun State, Nigeria. E-mail: [email protected] 2009 34 2 140 144 08 3 2008 26 10 2008 © Indian Journal of Community Medicine2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Epidemiological information paucity exists on musculoskeletal disorders (MSD) among secondary school students in Nigeria. We aimed to determine the prevalence, pattern, and treatment seeking behaviors (TSB) of MSD in Southwest, Nigeria. Materials and Methods: A school-based cross-sectional study was conducted in four randomly selected secondary schools in Ile-Ife in 2007. All the students were screened for MSD using an interviewer-administered questionnaire and physical examination, which involved the use of a scoliometer and a goniometer. Affected children were recommended for follow-up treatment and a plain radiography taken. Results: A total of 133 students had 204 MSD representing a 3.0% prevalence among the 4,441students screened. Eighty-one (60.9%) students had congenital disorders and 52 (39.1%) were acquired. The lower limbs (93.1%) were most commonly affected and 87 (65.4%) students presented with a knee deformity. Other abnormalities were limb length discrepancy 6.8%, scoliosis 4.4%, Pes planus 3.9%, and poliomyelitis 2.9%. A total of 100 students (75.2%) had no form of treatment, 18.8% receive treatment in the hospital, 3.7% received treatment in a traditional healing home and 2.3% received treatment in a church. Age, family, and school type were significant factors (P<0.05) in health-seeking behavior. The factors affecting treatment outcome were the place of treatment, hospital specific treatment, and reasons for stopping treatment. Conclusion: Treatable cases constitute a large proportion of MSD among secondary school students, but TSB was generally poor. Parental socio-economic and health services factors were related to the health-seeking behavior. Strengthening of school health services and improved linkage with orthopedic services, community education on MSD, and education of all cadres of health professionals are recommended. Musculoskeletal disordersNigeriasecondary schoolschool healthorthopedics ==== Body Introduction Musculoskeletal disorders (MSD) constitute an important global health problem. Injuries and diseases of the musculoskeletal system account for more than 20% of patient visits to primary care and emergency medical practitioners in the United States of America.(1) In Africa and developing countries, poverty with its attendant malnutrition, infectious diseases, ignorance, and inadequate medical facilities are all associated with the occurrence of MSD.(2–5) Failure to diagnose and properly treat MSD in childhood has the potential to lead to significant disability later in life. Yet, studies have shown that knowledge of MSD among medical graduates even in the developed countries may be poor, creating the possibility of inadequate and/or inappropriate management.(6) Targeted screening programs during childhood offer great potential for addressing MSD.(7) Screening programs can identify most cases of previously undiagnosed orthopedic abnormalities, improve our knowledge of the prevalence and pattern of MSD, and can lead to early diagnosis, which is often beneficial in altering the natural progression of the disease.(89) The Bone and Joint Decade (2000-2010) has been launched to increase awareness and encourage research and international cooperation in the prevention and treatment of MSD.(10) In response to the declaration of the Decade, Nigeria needs to address the problem of the lack of epidemiological data regarding MSD. This study aimed at determining the prevalence, pattern of musculoskeletal disorders, and treatment seeking behavior among secondary school students in Ile-Ife, Southwest Nigeria. Materials and Methods This study was conducted in Ife Central Local Government Area (LGA) of Osun State, Nigeria. The LGA has an estimated population of 96,580 from the National Population Commission data. The LGA has its headquarters in Ile-Ife, a mainly semi-urban university town. The town has 20 secondary schools made up of 12 private and 8 public schools. The total population of students enrolled was 15,180 for the 2006/2007 academic session. A stratified sampling approach was used to select study schools to ensure that both private and public schools were represented as the socio-economic background of the students attending the two groups of schools may be significantly different. A total of 4 schools were selected: 2 public schools (Urban Day Grammar School and Oduduwa College) and 2 private schools (Ibikunle Lawal College and Adventist Grammar school). In each school, all the students were targeted for screening for MSD after the school authority had given approval for the study and informed consent was obtained from each student. The participation rate was 100%. Information on the socio-economic background and medical history of the respondents was obtained through the use of questionnaires administered by trained final year students. Physical examinations were carried out by one of the researchers, an orthopedic surgeon (AAO), and involved the use of stadiometer, weighing balance, scoliometer, and goniometer. The footprint ratio or arch index (the ratio of the middle third of the toeless footprint to the total toeless footprint area11) was used to quantify pes planus deformity. An individualized plain radiography was taken at the Obafemi Awolowo University Teaching hospitals Complex, Ile-Ife to confirm deformity and determine Cobb Meyer's angles in scoliosis. Clinical photographs were also used to document some of the significant findings. Data collection took place from July 2007 to October 2007. The sample size taken was appropriate to calculate prevalence in this population. The sample size was determined using the Armittage and Perry formula {n=p (1-p) z2 /d2} of single proportion, where the minimum sample size required was n =384.2, the prevalence (p) of MSD among Nigerian secondary students based on non available previous study data was 50%, standard normal deviation (z) was set at 1.96 corresponding to 95% confidence interval, and the degree of accuracy (d) was set at 5%. Data analysis was carried out through the use of SPSS software, Version 11.0. A Chi-square test was used to determine the association between discrete variables such as selected factors and health seeking behavior. The level of significance was at P-value < 0.05. Results A total of 4,441 secondary school students were screened, comprising of 2,449 males (55.1%) and 1,992 females (44.9%) [Table 1]. The mean age of the students was 13.5 years ± 4.4 years (range: 9–22 years). A total of 133 students (3.0%) had MSD. The total number of MSD cases detected was 204. The age range of students with MSD was 9 to 20 years old, with a mean of 14.1 years and a standard deviation of 3.6 years. There were 73 males (2.8%) and 60 females (3.0%) who had MSD. The male: female ratio of students with MSD was 6:5. Table 1 Percentage distribution of musculoskeletal disorder by selected socio-demographic factors among students screened in Ile-Ife, Nigeria MSD present (n=133) MSD absent (n=4308) Total (n=4441) Statistical significance Age group P<0.05 9-15 years old 101 3096 3197 16-22 years old 32 1212 1244 Gender P>0.05 Male 73 2346 2449 Female 60 1932 1992 School type P<0.05 Private 56 1261 1317 Public 77 3047 3124 MSD = Musculoskeletal disorder Table 1 shows the distribution of MSD among affected students. A total of 93 students (69.9%) had their deformity in the lower limbs followed by the upper limbs 27 (20.3%). The congenital cases constituted 60.9% of cases and most cases of MSD were bilateral (53.4%). The pattern of MSD is shown in Tables 2 and 3. Scoliosis was mainly non structural among 7 students (3.4%) while the remaining 2 students (1.0%) had structural scoliosis sequel to post poliomyelitis paralysis. Scoliosis was twice as common in females compared with males. Postural scoliosis was seen in 4 subjects and 3 had compensatory scoliosis due to the limb length discrepancy and pelvic inequality. The scoliosis was mild to moderate without any respiratory embarrassment reported by the students affected. Table 2 Distribution of musculoskeletal disorders among affected secondary school students in Ile-Ife, Nigeria MSD distribution Number of students with MSD (n=133) Percent Side Left 28 21.0 Right 21 15.8 Bilateral 71 53.4 Central (Spine) 13 9.8 Site Back 6 4.5 Pelvis 4 3.0 Hip 3 2.3 Upper Limb 27 20.3 Lower limb 93 69.9 Mode Congenital 81 60.9 Acquired 52 39.1 MSD = Musculoskeletal disorder Table 3 Pattern of musculoskeletal disorders among affected secondary school students in Ile-Ife, Nigeria Musculoskeletal deformity Number of MSD cases Percent Genu varum 110 53.9 Genu valgum 24 11.8 Limb length discrepancy 14 6.9 (Avascular necrosis of the femur) shortening Scoliosis 9 4.4 Knock knee 16 7.8 Pes planus 16 7.8 Poliomyelitis 6 2.9 Neglected hip dislocation (with pelvic obliquity) 3 1.5 Syndactyl 3 1.5 Congenital talipes Equino-varum deformity 1 0.5 Kyphoscoliosis 1 0.5 Erb's palsy 1 0.5 Total 204 100.0 *Many students have more than a single musculoskeletal disorder, MSD = Musculoskeletal disorder Most (80.4%) of the MSD cases affected the lower limbs while 13.2% and 6.4% were located in the upper limbs and the central spine and pelvis, respectively. Approximately two-thirds (65.4%) of the students with MSD had knee deformities (Genu varum 53.9%, Genu Valgum 11.8%, and Knock knee 7.8%). The pattern of MSD varies between students of private and public schools as shown in [Figure 1]. Painless pes planus was seen among 8 students (6.0%; males=3, females=5; age: 14 to 16 years old). The males had a higher arch index greater than the females, the difference was not significant (P>0.05). Figure 1 Pattern of musculoskeletal disorders comparing private and public schools The treatment seeking behavior among students screened in Table 4 shows that only 33 (24.8%) had previous treatment; 93 (69.9%) had no treatment, and 7 (5.3%) did not know their treatment history. Of those that had previous treatment, 25 students (18.8%) received treatment from hospitals while 5 (3.7%) received treatment from traditional healers, and 3 (2.3%) received treatment from churches. A higher proportion of students from private schools (14 of 58 students with MSD; 24.1%) compared with those in public schools (11 of 75 students with MSD; 14.7%) sought hospital treatment. Occupation of the parents did not have a significant impact on the treatment seeking behavior (P=0.685). A total of 20.6% of the civil servant children with MSD had no treatment. A total of 35.1% of the students whose mothers were traders had no form of treatment as compared with the 13.2% of those whose fathers were traders who had no treatment. A total of 18.4% of the students with orthopedic disorders who were under 16 years old sought treatment in contrast to the 6.1% in the age group of 16–20 years old (P<0.037). All 6 students (100%) with deformities whose parents were separated or divorced had no form of treatment, while 21.3% of those with parents (polygamy/monogamy) alive sought treatment. Table 4 Factors associated with treatment seeking behavior of secondary school students with musculoskeletal disorder Treatment seeking behavior Number of students with MSD frequency Students with MSD who received hospital care percent Statistical significance School type P<0.05 Private 58 14.0 (24.1) Public 75 11.0 (14.7) Parents' occupation P<0.05 Farming 16 3 (18.7) Trading 57 13 (22.8) Civil servant 43 5 (11.6) Artisan 17 4 (23.5) MSD = Musculoskeletal disorde The type of treatment varied with the pattern of MSD. The students with structural scoliosis, post poliomyelitis, and congenital non syndromic talipes equinovarum deformity received different multiple treatment. Among those who sought treatment from health facilities, 3 (12.0%) had soft tissue operations (elongation of tendo-achilles), 15 (60.0%) were given medication, 6 (24.0%) had plaster of Paris, and 9 (36.0%) received physiotherapy. The treatments were not exclusive of one another. One-third (8 students: 32%) of those who sought hospital treatment did not complete prescribed treatment. Three of the five students treated by the traditional healers said that the treatments offered were difficult and did not complete their treatment. Of those that received hospital treatment, 16 (64%) were of the opinion that their cases improved while 6 (24%) felt their condition deteriorated and 3 (12.0 %) did not notice any change. The 6 students who reported a poor outcome were cases of severe deformities from paralytic poliomyelitis (5; 19.2%) and Erb's palsy (1; 3.8%). The factors affecting the treatment of the 6 students who did not complete their treatment was mainly as a result of financial problems and the logistics that the lengthy period of treatment entailed. The other reasons were parents not having time for further treatment and that nobody was available to stay with the student at the hospital. A total of 128 of the 133 students (96.2%) with MSD indicated interest in obtaining appropriate treatment if such could be made available to them. When they were followed-up immediately, 4.0% of the students who had not received any form of treatment informed their parents and had surgery (3 corrective osteotomies; 1 triple ankle arthrodesis) with uneventful outcomes at the teaching hospital. Discussion Determination of the prevalence and pattern of musculoskeletal symptoms is the first step in the effective intervention and prevention of further chronic pain syndromes in young adults.(912) Our study has assessed the prevalence and pattern of MSD in the Nigerian population and provided evidence that could inform appropriate interventions. With the paucity of locally available work that focused on community-based secondary schools, our study is a pioneering work in the Nigerian environment and can serve as benchmark values. The characteristics of the student population screened revealed a good mix of socio-economic backgrounds as students from both private and public schools were included in the study. The preponderance of males than females may be a reflection of the entire population pattern of enrollment in the schools. The recent rapid assessment of the primary school health system in Nigeria(13) showed enrollment data and MSD prevalence to be similar to our study results. The inability to carry out genetic studies due to a lack of resources makes it impossible to rule out disorders at the microscopic level among the majority of our subjects that were not found to have macroscopic MSD. The higher rate of MSD among public schools compared with private schools is likely to be due to the effect of the socio-economic background as mostly children of parents in the lower socio-economic class attend public schools in Ile-Ife as in most parts of Nigeria. Congenital MSD is more preponderant than acquired deformity in this study, which is in agreement with the findings of Thanni and Folami in another study carried out in Southwest Nigeria,(13) although their study was hospital-based and 60% of their study population was under 5 years old. The congenital disorders identified among secondary schools may likely be a reflection of factors particularly ignorance of the conditions, lack of knowledge about treatment possibilities and appropriate treatment sources, inability to afford orthodox care, and poor health seeking behavior. Whereas MSD was found predominantly in the lower limb in our study, followed by the upper limbs and the spine, respectively, Whittfield, et al. reported that musculoskeletal symptoms were more prevalent in the neck, shoulders, upper back, and lower back among secondary schools in New Zealand.(14) The carriage of heavy schoolbags was a suspected contributory factor among the New Zealand secondary school students, which is not the case in the Nigerian environment. In our study, genu varum, genu valgum, and knock-knee accounted for a majority of MSD among the students screened. Earlier work among the Nigerian population, consisting mostly of pre-school children,(1315) had reported knee deformity as being the most prevalent MSD. The persistence of knock-knee to the secondary school level could be due to the indifference to cosmetic appearance and the fact that no mortality is associated with knee deformity in an environment where a high level of child mortality from various communicable diseases and poverty remains. Our study showed that the prevalence of pes planus and arch index is similar to the findings among Malawians.(11) This study shows an overall prevalence of spine deformities (scoliosis and/or thoracic hyperkyphosis) similar to the 7.8% of adolescents with scoliosis reported by Milenkovic,et al.(12) The prevalence of scoliosis similar to our findings was twice as high as girls compared with boys.(121617) The treatment seeking behavior was generally found to be poor in our study, with the majority of affected students found not to have had any form of treatment. This is in contrast to a finding in more developed parts of the world such as the United States, where most pupils were reported to have received a considerable amount of professional attention.(18–20) The fact that 5.3% of students did not know their treatment history reflects a low level of health communication between these students and their parents. Inclusion of parents in the study would have made it possible for us to have a more complete treatment history of the children and better insights into reasons for treatment seeking decisions and behaviors. The significant difference between treatment seeking patterns among school children in private and public schools reflects the possibility that parental socio-economic factors play a significant role in decisions for treatment seeking. It was interesting to note that none of the affected students in private schools sought treatment from churches and traditional healers. The fact that none of the children from unstable family settings had sought treatment also reflects another dimension of parental background to treatment seeking. The relevance of the socio-economic level of the parents to treatment was further reflected in the fact that an inability to afford medical bills was a major reason for the inability to complete prescribed treatment. There is an absence of functioning, effective, affordable, and accessible national health insurance schemes. Several other factors in the health system could also have been contributory to the poor treatment behavior observed. These include: inaccessibility of specialist services with the low number of orthopedic surgeons and traumatologists available in the country, ineffective school health services, and a lack of effective social medical services particularly to support those severe deformities secondary to paralytic poliomyelitis and Erb's palsy. Conclusions and Recommendations Treatable cases constitute a large proportion of MSD among secondary students in Nigeria. Parental socio-economic and health service factors were related to poor health seeking behavior. This could be improved through community education, early detection, and linkage of school health services to facility-based orthopedic services as a major approach. Source of Support: Nil Conflict of Interest: None declared. ==== Refs References 1 Schmale GA More evidence of educational inadequacies in musculoskeletal medicine Clin Orthop Relat Res 2005 437 251 9 16056057 2 Mbamali EI Badoe EA Archampong EQ da Rocha-Afodu JT Principles and practice of surgery including pathology in the tropics 2000 3rd ed 1052 3 Huckstep RL The challenge of the third world Curr Orthop 2000 14 26 33 4 Huckstep RL Appliances and operators for poliomyelitis in developing countries: In Instructional course lectures Am Acad Orthop Surg 1999 49 5 Huckstep RL Poliomyelitis - A guide for developing countries, including Appliances and Rehabilitation ELBS and French 1983 2nd ed Edinburgh Churchill living stone 6 Craton N Matheson GO Training and clinical competency in musculoskeletal medicine: Identifying the problem Sports Med 1993 15 328 37 8321946 7 Nussinovitch M Finkelstein Y Amir J Greenbaum E Volovitz B Adolescent screening for orthopedic problems in high school Public Health 2002 116 30 2 11896633 8 O'Donnell JL Smyth D Frampton C Prioritizing health-care funding Intern Med J 2005 35 409 12 15958111 9 Sugita K Epidemiological study on idiopathic scoliosis in high school students: Prevalence and relation to physique, physical strength and motor ability Nippon Koshu Eisei Zasshi 2000 47 320 5 10835893 10 International center for orthopaedic education (ICOE) news 2000 2 issue 6 11 Igbigbi PS Msamati BC The footprint ratio as a predictor of pes planus: A study of indigenous Malawians J Foot Ankle Surg 2002 41 394 7 12500791 12 Milenkovic SM Kocijancic RI Belojevic GA Left handedness and spine deformities in early adolescence Eur J Epidemiol 2004 19 969 72 15575356 13 Thanni LO Folami AO Paediatric orthopaedic disease pattern in Sagamu, Nigeria Niger Med Practitioner 2003 44 52 5 14 Whittfield J Legg SJ Hedderley DI Schoolbag weight and musculoskeletal symptoms in New Zealand secondary schools Appl Ergon 2005 36 193 8 15694073 15 Oduloju AO Oginni LM Principles of fracture management Ife Med J 1990 8 45 16 Daniels TR Alman B Wedge JH Congenital clubfoot Curr Orthop 1999 13 229 36 17 Francis RS Bryce GR screening for musculoskeletal deviations: A challenge for the physical therapist Phys Ther 1987 67 1221 5 3615591 18 Kasper MJ Robbins L Root L Peterson MG Allegrante JP A musculoskeletal outreach screening, treatment, and education program for urban minority children Arthritis Care Res 1993 6 126 33 8130288 19 Akersson K Dreinhofer KE Woolf AD Improved education in musculoskeletal conditions is necessary for all doctors Bull World Health Organ 2003 81 677 83 14710510 20 O'Hagan FJ Sandys EJ Swanson WI Educational provision, parental expectation and physical disability Child Care Health Dev 1984 10 31 8 6234106	19966961	PMC2781122	CC BY	2021-01-04 17:49:18	yes	Indian J Community Med. 2009 Apr; 34(2):140-144
==== Front BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central 1471-2474-10-1411991969310.1186/1471-2474-10-141Research articleFunctional activation of proline-rich tyrosine kinase2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus Wang Meiying [email protected] Hongsheng [email protected] Wei [email protected] Yuanchao [email protected] Department of Rheumatology and Immunology, Provincial Hospital affiliated to Shandong University, Jinan, 250021, China2 Department of Pain Management, Hospital affiliated to Medical College of Qingdao University, Qingdao, 266003, China2009 17 11 2009 10 141 141 7 5 2009 17 11 2009 Copyright ©2009 Wang et al; licensee BioMed Central Ltd.2009Wang et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease characterized by activated T cells and polyclonally activated B cells that produce autoantibodies. Activation of autoreactive T and B cells plays a pivotal role in the pathogenesis of this disease. A role of focal adhesion kinase (FAK) in the pathogenesis has been suggested. Proline-rich tyrosine kinase2 (PYK2) is structurally related to FAK, however, the functional activation of PYK2 in SLE remains unclear. In the present study, we showed that PYK2 is significantly increased and activated in peripheral blood mononuclear cells (PBMCs) of patients with SLE. In addition, we showed the involvement of PYK2 proteins in the up-regulation of CD40L and CTLA4 expression and PBMC proliferation. Methods Freshly isolated PBMCs from 48 SLE patients, 32 patients with rheumatoid arthritis(RA) and 24 healthy individuals were analyzed for the expression and activation of PYK2 by western-blotting and immunocytochemistry. The other isolated PBMCs from patients with this condition were cultured and stimulated with PMA or TyrA9, and then the expression of costimulatory molecules CD40L and CTLA4 was evaluated using flow cytometry, PBMCs proliferation was determined with [3H]-thymidine incorporation (CPM). Results Compared with RA patients and healthy donors, PBMCs from SLE patients expressed more of both the total PYK2 protein and its activated/phosphorylated form. The increase of activated PYK2 protein in SLE PBMCs was correlated with the complication of nephritis and inversly associated the level of serum complements. In active SLE patients, activation of PYK2 in PBMCs is accompanying the increased cell proliferation and the induced expression of costimulatory molecules CD40L and CTLA4. Conclusion Our findings indicate that phosphorylated PYK2 in SLE PBMCs may induce the expression of CD40L and CTLA4, and subsequently the cell proliferation. PYK2 signaling enhances the autoreactive lymphocyte activation and plays an important role in the pathogenesis of SLE. ==== Body Background Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease characterized by activated T cells and polyclonally activated B cells that produce autoantibodies. Activation of autoreactive T and B cells plays a pivotal role in the pathogenesis of this disease [1,2]. Although SLE T cells have impaired interleukin-2 (IL-2) production and proliferative response to stimulation of the T cell receptor-CD3 compound[3,4], expression of costimulatory molecules such as CD40L and CTLA4, which is essential for lymphocyte activation [5,6], is up-regulated [7-10]. These molecules are thus targets in considering effective strategies in the treatment of SLE. Lupus mice treated with antibody against CD40L or CTLA4-Ig have lower level of anti-doublestranded DNA antibodies, later development of nephritis, and prolonged survival time [11-13]. In patients with SLE, the reduced expression of CD28 costimulatory molecule on both CD4- and CD8-T cells is also well documented [14,15]. CD28-mediated costimulatory activity, following the interaction of T cells with B cells, is significantly decreased in patients with SLE [14]. Thus, it seems that costimulatory signals in SLE T cells may differ from those present in normal T cells. Lately, in SLE T cells, focal adhesion kinase (FAK) have been shown to be involved in costimulatory molecule expression and cell proliferation[16]. Similar findings regarding the involvement of FAK were also reported in other inflammation-related diseases, such as rheumatoid arthrithis (RA)[17,18], diabetes[19], chronic inflammatory bowel diseases[20] and asthma[21]. It is thus likely that FAK may represent a new molecular target for the anti-inflammatory treatment. The proline-rich tyrosine kinase 2 (PYK2) is a nonreceptor protein tyrosine kinase that is structurally related to FAK [22]. It is also known as cell adhesion kinase-β or related adhesion focal tyrosine kinase. Unlike the ubiquitous expression of prototype FAK, PYK2 is primarily expressed in populations of neuronal and hematopoetic cells[23]. PYK2 becomes activated in response to stimulation through a number of receptors, of which include integrins[24,25], cytokine receptors [26-28] and lymphocyte antigen receptors [29-31]. Numerous studies over the years have shown that PYK2 provides important signals during the activation of lymphocytes [32-35]. However, in SLE, PYK2's expression and activation in PBMCs, as well as the functional significance of PYK2 in T cell and B cell activation, remains unclear. In this study, we showed that PYK2 is significantly increased and activated in PBMCs of patients with active SLE. In addition, we showed the involvement of PYK2 proteins in the up-regulation of CD40L and CTLA4 expression and PBMCs proliferation. Methods The study protocol was approved by the Human Ethics Review Committee of Shandong Provincial Hospital, Jinan, China. A signed consent form was obtained from each subject prior to study participation. Subjects The study subjects were 24 healthy volunteers, 32 RA patients (6 men and 26 women, mean age 42.3 years [range 21-67 years]), and 48 SLE patients (7 men and 41 women, mean age 33.9 years [range 11-69 years]), of whom 12 had inactive SLE disease and 36 had active SLE disease at the time of the study. All patients fulfilled the diagnostic criteria of the American College of Rheumatology for the classification of SLE or RA [36,37]. All SLE patients were admitted to our department between 2006 and 2009, and SLE disease activity was evaluated by the SLE Disease Activity Index (SLEDAI) score[38]. Patients were classified as having inactive disease if the SLEDAI score was persistently ≤9 for at least 3 months prior to the study. Patients with active disease had a SLEDAI score of ≥10 at the time of the study. Ten patients, 9 of whom had newly diagnosed SLE, were receiving no treatment at the time of the study. Thirty-eight SLE patients were receiving low-dose steroids (≤10 mg/day prednisolone) and/or immunosuppressive agents (cyclophosphamide). None of the SLE patients were receiving prednisolone at a dosage of ≥10 mg/day. Twenty-nine patients had biopsy-proven proliferative lupus nephritis (World Health Organization [WHO] class IV [39]), 8 had WHO class II nephritis, 4 had WHO class V nephritis, 7 had WHO class I nephritis, and 6 had central nervous system (CNS) manifestations. The clinical features of patients with and without active SLE are summarized in Table 1. All enrolled patients with RA had >6 swollen joints, >6 tender joints, and an erythrocyte sedimentation rate (Westergren) of >30 mm/hour. Twenty-six RA patients were receiving low-dose steroids (≤10 mg/day prednisolone) and/or disease-modifying antirheumatic drugs (methotrexate or sulfasalazine). Table 1 Clinical features of patients with active SLE and patients with inactive SLE* Characteristic Patients with active disease Patients with inactive disease (n = 36) (n = 12) No. of men/no. of women 5/31 2/10 Age, mean ± SD (range) years 35 ± 14 (11--69) 36 ± 12 (16--59) Disease duration, mean ± SD (range) months 65 ± 101 (1--260) 118 ± 98 (5--216) Disease activity SLEDAI score (0--105), mean ± SD 16 ± 4 7 ± 4 Anti-dsDNA titer, mean ± SD IU/ml 597 ± 784 49 ± 56 CH50 level, mean ± SD units/ml 14 ± 11 26 ± 8 Clinical manifestation WHO class IV nephritis 24 5 Other than WHO class IV nephritis 12 7 CNS disease 6 0 Treatment at time of study None 10 0 ≤10 mg/day prednisolone 28 9 Immunosuppressive agents 7 1 * Except where indicated otherwise, values are the number of patients. Isolation and culture of PBMCs Peripheral blood mononuclear cells (PBMCs) from healthy volunteers, RA patients, and SLE patients were isolated from 20 ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation (Invitrogen) and then washed twice with phosphate buffered saline (PBS). The viability of PBMCs was >96% as determinded by trypan blue staining, and the purity of human T and B lymphocytes isolated was >90% as tested with anti-CD3 mAb and anti-CD19 mAb by flow cytometry. The isolated PBMCs were divided into two groups: one group was used for Western blotting and Immunocytochemistry; in the other group, the PBMCs were resuspended at 1 × 106 PBMC/ml in RPMI-1640 medium supplemented with 10% bovine fetal serum, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, then cultured with PMA (Sigma) or TyrA9 (Calbiochem) in 24-well culture dishes in 5% CO2 at 37°C to be used for further testing. Control cultures without stimulants were included in each experiment. Western blotting Freshly isolated PBMCs, 2 × 106, were lysed in 200 μl of cold lysis buffer (1%Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris [PH 7.4], 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4, 10 mM NaF, 0.2 mM PMSF, and protease inhibitors mixture) and kept on ice on a shaking platform for 30 minutes. After centrifugation at 12000 rpm for 10 minutes, supernatants were transferred to a fresh tube and stored at -80°C. Protein concentrations were determined with the BCA Protein Assay Kit (Santa Cruz Biotechnology, CA, USA). From each sample preparation, 80 ug of total proteins were mixed in Laemmli loading buffer, boiled for 4 minutes, separated by 8% SDS-PAGE and then transferred to PVDF blotting membranes (Gelman, NY, USA). Membranes were blocked with the Tris buffered saline-Tween/1% bovine serum albumin/1% nonfat dry milk and incubated with rabbit polyclonal antibodies specific for PYK2 (SC-9019), phospho-PYK2 (SC-11767-R) and β-actin (SC-1616-R) (All from Santa Cruz Biotechnology, CA, USA) overnight at 4°C (diluted according to the manufacturer's instructions). Polyclonal PYK2-specific antibody was used to detect the total protein level of PYK2 while phospho-PYK2(p- PYK2) antibody which specifically recognizes PYK2 phosphorylation on tyrosine 402 to detect the activation of PYK2. β-actin was used as a loading control to assure equal amounts of protein in all lanes. After a final incubation with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China) for two hours at room temperature, the membranes were developed with an ECL detection system (Santa Cruz CA, USA). For reprobing, the membranes were stripped in 0.2 M glycine (PH 2.5)/0.05% Tween 20 at 80°C for 20 minutes. Immunocytochemistry Single immunoenzyme staining was performed using the biotin-streptavidin peroxidase method with an LSAB-HRP kit according to the manufacturer's instructions. Briefly, the freshly isolated PBMCs suspensions were put on glass slides and air-dried for at least 2 h, and either stained immediately or stored at 80°C. After air drying, the cells were fixed/permeablized with cytofix/cytoperm solution for 20 min at 4°C, dried, and washed twice in PBS pH7.2 for 5 min. The cells were incubated with blocking solution (included in kit) for 5 min in a humid atmosphere prior to the addition of primary antibody specific for phospho-PYK2 (SC-11767-R). Phospho-PYK2 antibody was added at appropriate dilution (1:100) on slides and left for 30 min in a humid atmosphere at room temperature. The cells were subsequently rinsed three times for 3 min in PBS and then allowed to react for 10 min with biotinylated antirabbit antibody (kit component). After a 3-min rinse in PBS and incubation with streptavidin-horseradish peroxidase (LSAB kit) for 30 min, the cells were washed three times in PBS (3 min each). They were then developed in diaminobenzidine, and the reaction was stopped by dipping the slides in distilled water. The cells were counterstained with 1% hematoxylin and covered with coverslips, and then examined under a light microscope. Positive cells were counted under ×400 magnification in 10 randomly chosen fields by two independent observers in a blind fashion. The results from these two observers were averaged to obtain the percentages of positive cells per sample. Cell staining and flow cytometric analysis The cultured PBMCs of each well in suspension were stained with PE-CD40L (TRAP1, BD Biosciences) and PE-CTLA4 (BNI3, BD Biosciences) for 15 min at 4°C in the dark, PE-IgG1κ was used as a control. After staining, the cells were washed with cold PBS and were immediately analyzed using EPICS XL-4 flow cytometry along with system software (Becton Dickinson, San Jose, CA). The PE fluorescence intensity in PBMCs were measured using an argon laser with an excitation wavelength of 488 nm and emission wavelengths of 575 nm. The PBMCs were tightly gated by volume and complexity on S forward (0°) and side-light scattering (90°) mode. At least 1 × 104 cells were analyzed for each determination. Proliferation assays For assaying PBMCs proliferation by PYK2 activation, isolated human PBMCs (2 × 105 cells/well) were cultured for 24 h in 96-well flat-bottomed microtitre plates in RPMI 1640 containing 10% FCS, stimulated with PMA or TyrA9 for the indicated duration. The cultures were pulsed with [3H]-thymidine (1.0 μCi/well) 18 h before harvesting the cells, and [3H]-thymidine incorporation was measured in a Microbeta Plus liquid scintillation counter (Wallac, Gaithersburg, MD, USA). Cultures were run in triplicate, and each experiment was repeated at least three times. Statistical analysis All data were expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA) test was used for comparison of more than two groups. The differences between the groups were assessed with the Post-Hoc Bonferroni test. The analysis of correlations between clinical variables and levels of p-PYK2 was based on Pearson rank test. The level of significance was set to p < 0.05. The dataset was analyzed using the SPSS V 13.0 statistical package. Each experiment was repeated at least 3 times to assess reproducibility. Results PYK2 is increased and activated in PBMCs from patients with SLE To see the expression pattern and the activation of PYK2 in PBMCs from patients with SLE, we used Western blotting to evaluate the total amount and phosphorylated form of PYK2 in freshly isolated PBMCs from the SLE patients. As controls, PBMCs from healthy donors and RA patients were collected and processed simultaneously. As show in Figure 1A (top blot), the intensity of PYK2 band in PBMCs from SLE patients was higher than those from either heathly controls or patients with RA. Quantitative analysis shows PYK2 in PBMCs from SLE, but not RA patients (0.96 ± 0.34 in RA), was significantly up-regulated (1.13 ± 0.35 and 1.28 ± 0.30) in inactive and active SLE patients, respectively, compared with that from healthy donors (0.94 ± 0.41). Consistently, while PYK2 was weakly phosphorylated on the tyrosine 402 residue in healthy donors and RA patients, a much thicker band corresponding to p-PYK2 was seen in lanes of the SLE patients as shown in Figure 1A (middle blot). This has also been further verified by quantitative analysis in which the level of p-PYK2 was significantly higher in PBMCs from SLE patients (0.97 ± 0.31 for inactive and 1.07 ± 0.33 for active), but not in PBMCs from RA patients (0.81 ± 0.34), compared with normal PBMCs (0.77 ± 0.33) (Figure 1B). The ratio of p-PYK2/PYK2 was examined and no significant difference was found between SLE patients (0.88 ± 0.19 for inactive SLE and 0.86 ± 0.20 for active SLE), RA patients (0.85 ± 0.21) and healthy donors (0.83 ± 0.18). Figure 1 Enhanced expression and activation of PYK2 in SLE patients. (A) Representative blot of total PYK2 and p-PYK2 in PBMCs lysates proteins obtained from healthy donors (n = 24), patients with RA (n = 32), and patients with inactive (n = 12) or active (n = 36) SLE. Total proteins obtained from PBMCs lysates were examined by Western blotting analysis for PYK2 and p-PYK2 as indicated in patients and methods. Lane 1 and 2, healthy donors;Lane 3 and 4, RA patients;Lane 5 and 6, inactive SLE patients;Lane 7 and 8, active SLE patients. (B) The ratio between the optical density of specific bands and β-actin of the same sample was calculated and expressed graphically. The experiments for each sample were repeated at least three times and bars represent the mean and standard deviation of three separate experiments. Statistical comparison of all groups was made with healthy donors. The significance level was set at* = P < 0.05. To identify in which sub-population of PBMCs the activated PYK2 is expressed, we immunostained phosphorylated PYK2 in PBMCs from the samples mentioned above with an antibody against p-PYK2. As observed in Figure 2, p-PYK2 (2A and 2B) was not detectable in lymphocytes from healthy donors and RA patients, whereas in lymphocytes from SLE patients, both the intensity and the proportion of p-PYK2 immunostaining were increased (2C). These data further confirmed results obtained by Western blotting. Figure 2 Expression of p-PYK2 in PBMCs from SLE patients. Detection of p-PYK2 protein in PBMCs from healthy donors (n = 24), patients with RA (n = 32), and patients with SLE (n = 48) was performed by immunocytochemistry technique using specific anti-p-PYK2 Abs. p-PYK2 was weakly positive in PBMCs from healthy donors (A) and RA patients (B). An increase in the positive staining for p-PYK2 was observed in PBMCs from SLE patients (C). Original magnification, × 400. Experiments were repeated at least three times with similar results. p-PYK2 is exclusively upregulated in SLE patients with class IV lupus nephritis and negatively correlated with the level of serum complement We next assessed the correlation between the levels of p-PYK2 and clinical manifestation of SLE. As shown in Figure 3A and 3B, the expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis, whereas this up-regulation was not seen in either healthy donors or SLE patients with CNS disease or nephritis other than class IV. Next, we analyzed the correlation between the ratio of p-PYK2/PYK2 and lupus nephritis, and no correlation was found for class IV lupus nephritis (data not shown). Furthermore, PYK2 activation and serum complement level (CH50) showed a significant negative correlation (Figure 3C). However, PYK2 activation did not show a correlation with the SLEDAI score (data not show). These results suggest that up-regulation of p-PYK2 in PBMCs is likely to be correlated with class IV nephritis. Figure 3 (A) Activation of PYK2 in PBMCs from healthy donors (n = 24) and SLE patients with central nervous system (CNS) disease (n = 6), WHO class IV nephritis (n = 29), or nephritis other than WHO class IV (n = 19). Total proteins obtained from PBMCs lysates were examined by Western blotting analysis for p-PYK2 as indicated in patients and methods. Lane 1, healthy donors;Lane 2, SLE patients with central nervous system; Lane 3, lupus nephritis other than WHO class IV;Lane 4, lupus nephritis IV. (B) The ratio between the optical density of specific bands and β-actin of the same sample was calculated and expressed graphically. The experiments for each sample were repeated at least three times and bars represent the mean and standard deviation of three separate experiments. Statistical comparison of all groups was made with healthy donors. The significance level was set at* = P < 0.05. (C) Correlation between PYK2 activation in PBMCs and serum levels of CH50 in SLE patients (n = 36). Enhanced PYK2 phosphorylation in PBMCs in response to lymphocyte activation stimulated by PMA In vivo, lymphocytes often receive multiple stimuli and become activated to carry out their biologic functions. To understand the molecular events during lymphocyte activation and analyze the role played by PYK2, we tested the effect of PMA on phosphorylation of PYK2. PBMCs obtained from healthy donors, RA and SLE patients were treated with PMA or medium in 24-well culture dishes in 5% CO2 at 37°C for 24 h, and the status of PYK2 Y402 phosphorylation was analyzed by western boltting. As show in Figure 4A, the intensity of p-PYK2 band in PBMCs stimulated by PMA was higher than those stimulated by medium (Figure 4A). Quantitative analysis shows that p-PYK2 in PBMCs stimulated by PMA, but not by medium, was significantly up-regulated in healthy control (1.10 ± 0.32 VS 0.77 ± 0.29), RA (1.12 ± 0.35 VS 0.81 ± 0.30) and SLE (1.47 ± 0.36 VS 1.08 ± 0.34) patients, respectively (Figure 4B). These results indicate that when PBMCs were stimulated by PMA, the total level of PYK2 Y402 phosphorylation is enhanced. Figure 4 Enhanced PYK2 phosphorylation in PBMCs in response to lymphocyte activation stimulated by PMA. (A) PBMCs obtained from healthy donors (n = 24), RA patients (n = 32) and SLE patients (n = 48) were incubated in 24-well culture dishes in 5% CO2 at 37°C for 24 h for with no stimulation (medium), or stimulation with PMA (1 ng/ml). Lysates prepared from 2 × 106 cells were analyzed by western blotting for the expression of p-PYK2 as indicated in patients and methods. (B) p-PYK2 expressed relative to the value obtained with PMA stimulation. Bars represent the mean and standard deviation of three separate experiments. Statistical analysis: * = p < 0.05. p-PYK2 in PBMCs from SLE patients induces the expression of CD40L and CTLA4 To further characterize the role of p-PYK2 in lymphocyte activation, we assayed the cell surface costimulatory molecules expression by stimulating or inhibiting PYK2 phosphorylation, using PMA and PYK2 kinase inhibitor TyrA9, respectively. As expected, using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4, whereas this upregulation is not observed in PBMCs pretreated with chemical inhibitor of PYK2 kinase activity (Figure 5C). In PBMCs from normal individuals and RA patients, CD40L and CTLA4 expression were also significantly upregulated by stimulation with PMA. This effect, however, cannot be suppressed by administration of TyrA9 (Figure 5A, B). These results suggest that p-PYK2 acts as an important mediator in PMA-induced induction of CD40L and CTLA4 in PBMCs of SLE. Figure 5 Up-regulation of CD40L and CTLA-4 by PYK2 activation in PBMCs from patients with active SLE (C), but not those from normal individuals and RA patients (A and B). PBMCs were incubated for 24 h with medium, PMA (1 ng/ml) or pretreated with the PYK2 inhibitor TyrA9 (5 μM) for 1 h before the addition of PMA, and then analyzed for the expression of CD40L and CTLA4 by flow cytometric analysis. All experiments were repeated at least three times with similar results. Statistical analysis: * = p < 0.05. p-PYK2 promotes the proliferation of SLE PBMCs To explore whether upregulation of p-PYK2 may contribute to the pathogenesis of SLE, we cultured PBMCs from patients with this condition as well as from those with RA and healthy controls. Cultured cells were subjected to groups in the presence or absence of TyrA9 before stimulated with PMA and the subsequent cell proliferation assay. We found in cultures without TyrA9, the proliferation of PBMCs from all sources were enhanced by PMA. However, in the presence of TyrA9, only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA (Figure 6). These results indicate p-PYK2 transduces an activation signal for cell proliferation exclusively in PBMCs of SLE. Figure 6 Induction of cells proliferation by PYK2 activation in PBMCs from patients with active SLE, but not those from normal individuals and RA patients. Isolated human PBMCs were cultured for 24 h in 96-well flat-bottomed microtitre plates with medium, PMA (1 ng/ml) or pretreated with the PYK2 inhibitor TyrA9 (5 μM) for 1 h before the addition of PMA, and then the cultures were pulsed with [3H]-thymidine (1.0 μCi/well) 18 h before harvesting the cells, and [3H]-thymidine incorporation was measured. The mean ± SD of the counts per minute value from triplicate samples is shown. The experiments were performed more than three times, and a representative data set is presented. * = P < 0.05. Discussion In this study, we found an upregulation of PYK2 in PBMCs of SLE patients and an activation in SLE with class IV lupus nephritis. We also found the activation is negatively correlated with the level of serum complement. By isolating and culturing the PBMCs, we verified p-PYK2 a mediator specific to SLE to induce costimulatory molecules CD40L and CTLA4, and to promote the cell proliferation. This study represents the first demonstration that PYK2 expression and activation appear exclusive in SLE PBMCs and crucial for the pathogenesis of SLE. Lymphocyte activation is a fundamental component implicated in the production of autoantibodies in SLE patients. Understanding the precise molecular mechanism of SLE lymphocyte activation will be important to develop novel therapeutic strategies targeting reduction autoantibodies and to increase the sensitivity of current treatment modalities. PYK2, a nonreceptor protein tyrosine kinase which plays pivotal roles in the regulation of lymphocyte activation[33], has drawn our attention in considering the lymphocyte activation in SLE. Our studies showed no detection of p-PYK2 in PBMCs of RA and healthy controls. PYK2 in SLE PBMCs is not only increased but also phosphorylated at tyrosine 402 resides. These findings suggest that upregulation and activation of PYK2 may be implicated in the pathogenesis of SLE. PYK2 has been reported overexpressed in glomeruli but not in other tissues of human and rat crescentic glomerulonephritis, and its overexpression is closely associated with the onset of glomerulonephritis[40]. In our study, we showed a marked upregulation of p-PYK2 in PBMCs from SLE patients with class IV lupus nephritis, but not healthy donors or SLE patients with CNS disease or nephritis other than class IV. Further, we found clear negative correlation between the levels of p-PYK2 and serum CH50. It suggests that signaling pathway involving PYK2 are likely to play a role in the development and progression of nephritis. FAK, the prototype of PYK2, mediates signaling in active SLE enhancing autoreactive T cell activation by proliferation and by upregulating the expression of costimulatory molecule CD40L[16]. PYK2 also provides important signals to T and B lymphocyte activation[33,34]. Its expression and activation, however, are not seen in PBMCs from RA patients. We explored whether the similar scenario will happen in SLE. Given that costimulatory molecules are known to be a prerequisite for lymphocyte activation, we answered this question by checking the expression of costimulatory molecules and subsequent proliferation in SLE PBMCs. In contrast to what has been observed in RA, PYK2 was shown exclusively in SLE a mediator of activation signaling of proliferation. Therefore, PYK2-mediated activation of lymphocytes is not only functionally enhanced in SLE but also a process specific to SLE. Taking into account the limitations that chemical inhibitor of Pyk2 kinase activity is almost never perfect in its specificity, in subsequent experiments, we are going to use RNA-mediated interference, Pyk2-deficient PBMCs and dominant negative Pyk2 mutants to confirm the exact function of Pyk2 in regulating SLE PBMCs proliferation. Abnormal T and B lymphocytes activation and lymphocytes death underlie the pathology of SLE[41]. Potentially autoreactive T and B lymphocytes are removed by apoptosis during development and after completion of an immune response. Paradoxically, lupus T cells exhibit both enhanced spontaneous apoptosis and defective activation-induced cell death [42]. Increased spontaneous apoptosis has been linked to chronic lymphopenia in patients with SLE[42]. By contrast, defective activation-induced cell death (AICD) may be responsible for persistence of autoreactive T and B lymphocytes, leading to expansion of antigen-spectific T cell clones[42]. Indeed, in prototypical murine SLE models (i.e., lpr/lpr and gld/gld mice), the animals are defective in Fas and FasL, respectively, which are critical elements in T cell apoptosis. Our result demonstrate that in addition to increased phosphorylation of PYK2 in SLE PBMCs, phosphorylated PYK2 signaling could also enhances the autoreactive lymphocyte activation and proliferation (Fig.5 and Fig.6). However, the mechanism that phosphorylated PYK2 induce lymphocyte proliferation is not clear. It would be interesting to investigate whether phosphorylated PYK2 could promote lymphocyte activation and proliferation via inhibition of apoptosis or via enhanced hyperreactivity of lymphocyte. Our results, together with those of earlier studies, demonstrate that the PBMCs from SLE patients exhibit both increased activation and enhanced activity of PYK2. We strongly propose PYK2 a major contributor to the complex autoimmune pathogenesis of SLE. Conclusion In the present study, We found a significant increase of both the total PYK2 protein and its activated/phosphorylated form in PBMCs from patients with SLE, particularly those with the complication of nephritis (WHO IV). There is a clear inversly correlation between the activation of PYK2 and the level of serum complements. In active SLE patients, activation of PYK2 in PBMCs is accompanying the increased cell proliferation and the induced expression of costimulatory molecules CD40L and CTLA4. These results indicate that phosphorylated PYK2 may induce the expression of CD40L and CTLA4, and subsequently the cell proliferation. PYK2 signaling enhances the autoreactive lymphocyte activation and plays an important role in the pathogenesis of SLE. Abbreviations SLE: Systemic lupus erythematosus; FAK: focal adhesion kinase; PYK2: Proline-rich tyrosine kinase2; PBMC: peripheral blood mononuclear cell; RA: rheumatoid arthritis; CPM: [3H]-thymidine incorporation; IL-2: interleukin-2; SLEDAI: SLE Disease Activity Index; WHO: World Health Organization; CNS: central nervous system; PBS: phosphate buffered saline Competing interests Wang M and Zhang W work for Provincial Hospital affiliated to Shandong University. All authors declare that they have no competing interests Authors' contributions Meiying Wang has performed all the tests, has done preclinical analyses, statistics, graphics and has partially written the manuscript. Hongsheng Sun has added and operated additional experiments at the referee's suggestion, and has revised the manuscript. Wei Zhang has corrected the manuscript and has helped to write the manuscript. Yuanchao Zhang, as the last and responsible author, has initiated this study and has controlled the work. He has written and reviewed the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/10/141/prepub Acknowledgements We thank Yaoran Zhao and Chunyan Ma of Provincial Hospital affiliated to Shandong University for technical assistance. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2006603709-PONE-RA-1443110.1371/journal.pone.0008590Research ArticleMicrobiology/Medical MicrobiologyMicrobiology/Microbial Growth and DevelopmentInfectious Diseases/Bacterial InfectionsNovel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division FipA in Cell DivisionSureka Kamakshi Hossain Tofajjen Mukherjee Partha Chatterjee Paramita Datta Pratik ¤ Kundu Manikuntala Basu Joyoti * Department of Chemistry, Bose Institute, Kolkata, India Ahmed Niyaz EditorUniversity of Hyderabad, India* E-mail: [email protected] and designed the experiments: MK JB. Performed the experiments: KS TH PM PC PD. Analyzed the data: KS MK JB. Wrote the paper: MK JB. ¤ Current address: Public Health Research Institute Center, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, United States of America 2010 6 1 2010 5 1 e859023 11 2009 9 12 2009 Sureka et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress. ==== Body Introduction Cell growth and division involves exquisite temporal and spatial regulation of a succession of events which include choice of the site of division, positioning of the Z-ring at mid-cell, and formation of the septum [1]–[3]. The fidelity of cell division occurring at mid-cell depends on the assembly of a macromolecular complex at the site of division involving recruitment of proteins in a hierarchial manner [4]–[6]. In most Gram-negative and Gram-positive bacteria, genes whose products are involved in cell division, cluster in a chromosomal region termed the dcw (division and cell wall) cluster [7]. The principal player in driving cytokinesis is FtsZ, a GTP-binding protein considered to be the bacterial counterpart of eukaryotic tubulin [8], [9]. It is encoded by a gene within the dcw cluster. It forms the Z-ring [10] which serves as a cytoskeletal scaffold for the sequential recruitment and assembly of the multiprotein complex that comprises the divisome [11], [12]. Mycobacteria share many components of the cell division machinery identified in other bacteria. However, it is likely that there are several unique features in the mechanism of cell division in mycobacteria, considering that assisters of Z ring formation such as FtsA and ZipA have not been identified, nor have counterparts of the MinCD system which ensures that division occurs at midcell. For example, it is possible that direct interaction between FtsZ and FtsW stabilizes FtsZ at the membrane and links cell division to septal peptidoglycan biosynthesis [13], [14]. A family of 11 serine/threonine protein kinases (STPKs) are likely sensors of environmental signals in mycobacteria [15], [16]. In spite of an interest in this family of kinases in recent years, there is limited understanding of the signals that they sense and the consequences of such sensing. The STPKs of mycobacteria with the exception of PknG and PknK, have extracellular C-terminal sensory domains and intracellular N-terminal kinase domains. Cell wall biogenesis, cell division, central metabolic processes such as the TCA cycle and gene expression, are among the growing body of processes that are regulated by STPK-mediated phosphorylation in mycobacteria [16]–[18]. Both PknA and PknB are involved in regulation of cell shape [19], [20]. These two kinases reside in a genomic region (encompassing the ORFs Rv0014c-Rv0019c in Mycobacterium tuberculosis H37Rv) that is conserved across all species of mycobacteria for which genome sequences are available. This region also encodes PstP, the cognate phosphatase of PknA and PknB; PBPA, a penicillin-binding transpeptidase [19], and RodA, a putative player in cell shape maintenance. PknA and PknB phosphorylate Wag31[20], and PknB phosphorylates PBPA (Dasgupta et al., 2006). A phosphorylation-defective mutant of PBPA fails to localize to the site of division. In M. tuberculosis, this conserved gene cluster containing morphogenetic genes, also harbours the genes Rv0019c and Rv0020c, encoding two forkhead-associated domain (FHA) proteins. The FHA domain is a phosphopeptide recognition motif spanning 80 to 100 amino acid residues folded in an 11-stranded beta sandwich which recognizes phosphopeptide [21], [22]. PknB phosphorylates Rv0020c in vitro [23]. As of now, the functional role of Rv0020c remains unclear. Key to regulated cell division are proteins which inhibit or stabilize assembly of the divisome at mid-cell. In mycobacteria, some of the above findings have suggested that serine/threonine kinases such as PknA and PknB fine tune the process of cell division, and it is tempting to speculate that accessory proteins unique to mycobacteria, as well as dynamic protein phosphorylation could play vital roles in controlling cell septation, particularly under conditions of stress. We have tested the possibility molecular interactions among proteins encoded by genes of the dcw cluster and those encoded by the cluster Rv0014c-Rv0019c regulate cell division in mycobcteria. This report focuses on the forkhead-associated domain (FHA)-containing protein encoded by Rv0019c. A role for this gene product in cell division was tested by knocking out its counterpart in Mycobacterium smegmatis. The increased susceptibility of the mutant to oxidative stress and the altered cell morphology, further supported our contention that this particular FHA-domain-containing protein could regulate cell division. Using a variety of biochemical and genetic approaches, we demonstrate that in mycobacteria, FtsZ, FtsQ and the product of the ORF Rv0019c, form a ternary complex. In view of its ability to interact with FtsZ, we name this gene product, FtsZ-interacting protein A (FipA). The FipA-FtsZ interaction is dependent on PknA which phosphorylates FipA on amino acid residue T77 and FtsZ on T343. We demonstrate that knock down of pknA impairs the formation of the FtsZ-FtsQ-FipA complex. FtsZ fails to localize at mid-cell in fipA knock out M. smegmatis cells under oxidative stress and localization is rescued by complementation with FipAMTB. An important role of FipA is further suggested by the fact that depletion of FipA leads to reduced growth of M. tuberculosis in macrophages. Results Effect of Inactivation of FipAMSMEG and Knock Down of FipA in M. tuberculosis The genomic region encompassing the ORFs Rv0014c to Rv0019c (Figure S1A) of M. tuberculosis, is conserved across mycobacterial species. Its role in cell division and the function of some of the genes encoded by this region has been conjectured over the years. The genes Rv0014c-Rv0018c are organized in an operon. The ORF Rv0016c encodes PBPA, a transpeptidase that regulates cell morphology in a manner dependent on phosphorylation by the product of the ORF Rv0014c, PknB [19]. The product of the ORF Rv0015c, PknA phosphorylates Wag31 thereby regulating cell morphology [20]. Rv0018c encodes PstP, a phosphatase which dephosphorylates substrates such as phospho-PBPA. The function of FipA (the product of the adjacent transcriptional unit Rv0019c), a putative FHA protein, remains obscure. Computational analysis of genome-wide functional linkages, suggests that FipA has a role in cell envelope biosynthesis in M. tuberculosis [24]. Considering that FipA is conserved across pathogenic and non-pathogenic mycobacterial species, we chose M. smegmatis, the fast-growing non-pathogenic strain in order to understand the role of FipA in mycobacterial physiology. The deduced amino acid sequences of FipAMSMEG and FipAMTB (Figure S1B) show that the proteins are 88% identical in amino acid sequence. To test the role of FipA in cell growth and division, fipA was inactivated at its native locus in M. smegmatis. The absence of FipA was confirmed by Western blotting (Figure S2A). We also confirmed by Western blotting that the expression of PknB, PknA, PBPA and PstP encoded by neighboring genes, was not affected (Fig. S2A). The mutated strain will be referred to as FipA-KO. The morphology and the growth rates of wild type M. smegmatis mc2155 and FipA-KO were compared. FipA-KO exhibited reduced growth compared to the wild type in Middle Brook 7H9 broth supplemented with Tween 80. The wild type reached mid-log phase at 20 h, whereas the mutant reached mid-log phase at 24 h (Fig. 1A). At stationary phase, the growth of the mutant was lower than that of the wild type. Considering the lowered growth of the mutant particularly at stationary phase, we speculated that it was possibly less capable of handling stressful conditions than the wild type. We tested its susceptibility to conditions of stress. FipA-KO was more susceptible to H2O2 stress than the wild type (Fig. 1B). In order to test whether FipA is required for cell division under H2O2-induced oxidative stress, we determined cell lengths in the wild type and FipA-KO. In the absence of exogenous stress, cells of the wild type and FipA-KO were of similar length. However, approximately 40% of the cells were >6 µm in length in the knockout strain subjected to oxidative stress (Fig. 1C). In comparison, <5% of the wild type cells were >6 µm in length under similar oxidative stress. These results suggest that FipA is required to sustain cell division in bacteria subjected to oxidative stress. In order to assess the ability of FipA of M. tuberculosis to complement FipA-KO, a single copy of fipA of M. tuberculosis was integrated into the chromosome of FipA-KO. Complementation of FipA was confirmed by Western blotting (Fig. S2A). FipA-KO complemented with fipA MTB, was visualized by microscopy before and after subjecting to oxidative stress. Complementation with fipAMTB restored the cell length of FipA-KO to resemble that of the wild type (Fig. 1C). In addition, growth defect was rescued (Fig. 1A), and the complemented strain could withstand oxidative stress (Fig. 1B) to an extent similar to the wild type. This also confirmed that the difference in phenotypic behavior between the wild type and FipA-KO was attributable specifically to the absence of FipA. 10.1371/journal.pone.0008590.g001Figure 1 FipA regulates growth and cell division. A. Growth of M. smegmatis mc2 155 (WT), FipA-KO (KO) or FipA-KO complemented with fipA MTB (WT) or its fipA MTB(T77A) [complemented (T77A)], was followed by monitoring cfu at different periods of time. B. Cultures were treated with H2O2 as described under “Materials and Methods.” Growth was monitored by determining cfu. Percent survival was determined with respect to the cfu of untreated cells (100%). C. Cell length measurements of wild type M. smegmatis (WT), or FipA-KO (KO), or FipA-KO complemented with fipAMTB (complemented) after treatment with H2O2 as described under “Materials and Methods.” At least 100 cells from each set were measured. D. Western blotting of lysates of M. tuberculosis expressing an antisense construct of fipA under the control of the hsp60 promoter, with anti-FipA antibody. The blot was reprobed with anti-FtsW as loading control. E. THP1 cells were infected with M. tuberculosis harbouring vector alone or an antisense construct of fipA at an MOI of 2 for 90 min; washed with fresh medium and grown for different periods of time. Internalized bacteria were released by lysing the macrophages followed by determination of cfu. M. tuberculosis harbouring vector alone, -▵-; or antisense construct of fipA, -•-. F, G. THP-1 cells were infected and intracellular bacteria were obtained by lysing the macrophages, as in (E). Cell length measurements were done for intracellular M. tuberculosis harbouring vector alone (F) or expressing an antisense construct of fipA under the control of the hsp60 promoter (G) after 1, 3, 5, or 6 days of growth in THP1 cells. At least 100 cells from each set were measured. The division defect occurring after H2O2 treatment in FipA-KO could possibly be due to induction of the SOS response. RecA and LexA are two principal proteins involved in the SOS response [25], [26]. We checked the transcription levels of recA and lexA after treatment with H2O2. No increase in lexA or recA was observed in either the wild type or FipA-KO or FipA-KO complemented with fipA MTB (Figure S2B, C). O' Sullivan et al. [27] have reported that recA and lexA transcription increases after exposure of M. tuberculosis to mitomycin C, a compound that alkylates and crosslinks DNA and induces the SOS response. Mitomycin C exposure was therefore used as a positive control for induction of the SOS response. In this case, recA and lexA transcription increased in the wild type, FipA-KO and FipA-KO complemented with fipA MTB (Figure S2B, C). Taken together, this suggests that the division defect in FipA-KO cells after H2O2 treatment is not due to induction of the SOS response. In order to assess the role of FipA in M. tuberculosis growing in macrophages, we knocked down fipA of M. tuberculosis (Fig. 1D). This knock down compromised the ability of M. tuberculosis to multiply within macrophages after two days (Fig. 1E). This argued in favor of a role of FipA in mycobacterial multiplication in macrophages. A defect in cell division in the fipA-knocked down cells in macrophages was confirmed by the observation that the percentage of macrophage-grown cells over 6 µm in size was consistently higher in fipA- knocked down cells compared to the wild type over a period of 6 days (Fig. 1F, G). This suggests that FipA is required to sustain mycobacterial cell division in macrophages. FipA Interacts with FtsZ In Vivo In view of the fact that FtsZ provides the driving force of cytokinesis by forming the Z-ring at the septum [1], and that knock out of fipA is associated with a septation defect, we tested the possibility that FtsZ and FipA are interacting partners in vivo. In order to analyze the interaction of FipA with FtsZ, FtsZ was immunoprecipitated from cell lysates of M. tuberculosis or M. smegmatis, and the presence of FipA in the immunoprecipitates was tested using anti-FipA antibody. FtsZ immunoprecipitates pulled down endogenous FipA (Fig. 2A). In order to understand the role of FHA domain in the interaction of FipA with FtsZ, FipA-KO was complemented with wild type fipAMTB or fipAMTB harboring mutations in conserved amino acid residues of the FHA domain encompassing amino acid residues P82 to V132. FHA domain mutants S101A, H104A and N123A of FipA were impaired in their ability to interact with FtsZ in vivo (Fig. 2B). On the other hand, the FipA (D118A) mutant retained the ability to interact with FtsZ. These results suggest that the FHA domain of FipA plays a role in its interaction with FtsZ in vivo. 10.1371/journal.pone.0008590.g002Figure 2 The FHA domain of FipA is required for its interaction with FtsZ. A. Cell lysates of M. tuberculosis or M. smegmatis were immunoprecipitated (IP) with anti-FtsZ antibody and immunoblotted (IB) with anti-FipA antibody or antibody against an irrelevant protein (PPK1) [as a negative control]. The blot was reprobed with anti-FtsZ antibody to show equal loading. B. Lysates of M. smegmatis mc2155 or FipA-KO complemented with the wild type (WT) or different mutants of FipAMTB, were immunoprecipitated with anti-FipA antibody followed by immunoblotting with anti-FtsZ antibody and reprobing with anti-FipA antibody. Blots shown are representative of three separate experiments. FHA domains are known to interact with ser/thr kinases [21]. Therefore, we tested whether FipA of M. smegmatis (FipAMSMEG, encoded by MSMEG_0034), is phosphorylated in vivo. FipA was immunoprecipitated from cell lysates of M. tuberculosis or M. smegmatis followed by Western analysis using anti-phosphothreonine antibody. The results affirmed that FipAMTB and FipAMSMEG are phosphorylated in vivo on threonine (Fig. 3A). Incubation of immunoprecipitates with purified PstP resulted in dephosphorylation of agarose-bound FipAMTB and FipAMSMEG (Fig. 3A). In a reverse experiment, capture of proteins from lysates of M. smegmatis on phosphothreonine agarose, followed by Western analysis of the bound proteins with anti-FipA antibody, also confirmed that FipA is phosphorylated within cells (Fig. 3B). 10.1371/journal.pone.0008590.g003Figure 3 FipA is phosphorylated in vivo and localizes to the membrane. A, B. Lysates of M. tuberculosis(A) or M. smegmatis (A, B) were immunoprecipitated either with anti-FipA antibody (A) or with phosphothreonine agarose (B), left untreated (−) or treated (+) with S-tagged PstPMTB followed by immunoblotting with anti-phosphothreonine (A) or anti-FipA (B) antibody as indicated; and reprobing with anti-FipA antibody for panel A. C. Cell lysates were fractionated and membrane and cytosolic fractions were immunoblotted with anti-FipA antibody followed by reprobing with anti-FtsZ (marker for the cytosol) or with anti-FtsW (membrane marker). Blots shown are representative of three separate experiments. Subcellular Localization of FipA In order to understand the function of FipA and in view of the fact that it interacts with FtsZ, we felt it imperative to localize FipA in cells. Its topology predicted using the program HMMTOP [28] suggest that it is a transmembrane protein. Fractionation of lysates into membranes and cytosolic components, followed by Western blotting confirmed that FipA localizes in mycobacterial membranes (Fig. 3C). Crosscontamination of membrane and cytosol fractions was ruled out by the fact that membranes were positive for the membrane protein FtsW but not for the cytosolic protein FtsZ, whereas the cytosol fraction was positive for FtsZ but not for FtsW. FipA Is Phosphorylated by PknA In order to test whether FipA is phosphorylated by the neighbouring ser/thr kinase PknA, we expressed FipA in E. coli with an N-terminal hexahistidine or S-tag. Purified His-tagged FipA migrated as a protein of approximately 20 kDa (Fig. 4A). We expressed and purified the kinase domain of PknA encompassing Y13 to A273 (PknA13–273) as a GST-tagged protein (Fig. 4B). Recombinant FipA could be phosphorylated by PknA13–273 in the presence of Mg2+ and [γ-32P] ATP (Fig. 4C). The kinase-dead K42A mutant of PknA13–273 [20] was unable to phosphorylate FipA (Fig. 4C). In order to evaluate the role of FHA domain amino acid residues S101, H104, D118 and N123 on interaction of FipA with PknA and its phosphorylaton, these residues were individuallly mutated to alanine. None of the mutants other than the D118A mutant, could interact with PknA13–273 (Fig. 4D), nor were these mutants (with the exception of D118A) phosphorylated by PknA (Fig. 4E). These results confirmed the importance of the FHA domain residues S101, H104, and N123 in interaction with PknA and in PknA-mediated phosphorylation of FipA. 10.1371/journal.pone.0008590.g004Figure 4 FipA interacts with, and is phosphorylated by PknA in vitro. A, B. Coomassie blue-stained gels of uninduced (1), or induced (2) cells of E. coli expressing FipA (A), GST-PknA13-273 (B), purified FipA (A, lane 3) and GST-PknA13–273 (B, lane 3). C. Purified FipA was phosphorylated by GST-PknA13–273 or its K42A mutant in vitro in the presence of [γ-32P] ATP followed by autoradiography. D. Recombinant S-tagged FipA or its mutants were incubated with GST-PknA13–273 bound to glutathione-Sepharose beads. Proteins bound to the beads were detected by immunoblotting with anti-FipA antibody. The blot was reprobed with anti-GST antibody. E. Purified FipA or its mutants were phosphorylated by GST-PknA13–273 in vitro in the presence of [γ-32P] ATP followed by autoradiography. The first lane represents autophosphorylated GST-PknA13–273. Blots shown are representative of three separate experiments. Lower blots of panels C and E are Coomassie blue-stained gels showing the levels of FipA. The NET Phos program [29] predicted threonine residues at positions 77 and 142 as putative phosphorylatable residues. Mutation of T77 abrogated PknA13–273-dependent phosphorylation of FipA, (Fig. 4E) but not its interaction with PknA (Fig. 4D). This identified T77 as a possible site of PknA-dependent phosphorylation. Mutation of T142 had no effect in PknA13–273-mediated phosphorylation (data not shown). In order to confirm that FipA is phosphorylated on a single threonine residue by PknA13–273, mass analysis of His-FipA and the protein phosphorylated by PknA13-273, was undertaken. The increase in the molecular mass of the intact protein (Figure S3), confirmed incorporation of a single phosphate residue by PknA13–273. In order to investigate the role of phosphorylation of FipA on its interaction with FtsZ, FipA-KO was complemented with fipA MTB or fipA MTB(T77A). Wild type FipAMTB could immunoprecipitate FtsZ (Fig. 2B) while the T77A mutant could not, suggesting the requirement of FipA phosphorylation for its interaction with FtsZ. In addition, phosphorylation-defective FipA(T77A) could not restore the growth defect of the FipA-KO (Fig. 1A) or its ability to withstand H2O2 stress (Fig. 1B). This suggests that phosphorylation of FipA on T77 is required to support survival under oxidative stress. FipA Is Phosphorylated In Vivo by PknA in Mycobacteria In order to demonstrate PknA-dependent phosphorylation of FipA in vivo, we performed gradual depletion of the protein in M. smegmatis by acetamide-inducible expression of an antisense construct of pknA. Depletion of PknA was confirmed by immunoblotting of lysates with anti-PknA antibody (Fig. 5A). Gradual depletion of PknA was concomitant with gradual decrease in threonine phosphorylation of FipA (Fig. 5B), suggesting that PknA mediates phosphorylation of FipA in vivo. 10.1371/journal.pone.0008590.g005Figure 5 A, B. FipA is phosphorylated in vivo in a PknA-dependent manner. A, B. M. smegmatis was transformed with a construct carrying pknA in antisense orientation under an acetamide-inducible promoter. Transformed cells were induced with 0.2% acetamide for the indicated periods of time in order to downregulate pknA expression. Antisensing was followed by immunoblotting of cell lysates with anti-PknA antibody, followed by reprobing with anti-FtsW antibody (A); or cell lysates at different periods of time were immunoprecipitated with anti-FipA antibody followed by immunoblotting with anti-phosphothreonine antibody and reprobing with anti-FipA antibody (B). Blots shown are representative of three separate experiments. IB: immunoblotting; IP: immunoprecipitation. Interaction of FtsZ with FipA Is Required for Septal Localization of FtsZ under Oxidative Stress Considering that FipA interacts with FtsZ in vivo (Fig. 2), we attempted to understand whether this interaction is necessary for the localization of FtsZ at mid-cell in dividing cells. Immunolocalization of FtsZ in FipA-KO showed that FtsZ was compromised in its ability to localize at mid-cell in FipA-KO cells subjected to oxidative stress (Fig. 6A, B), but could localize to mid-cell in the absence of oxidative stress (data not shown). Complementation of FipA-KO with fipA MTB restored the localization of FtsZ at mid-cell even under oxidative stress (Fig. 6C,D). The frequency of Z ring formation in the absence or presence of oxidative stress, was measured by microscopy. The data presented in Table 1 clearly indicate that Z ring formation under oxidative stress requires FipA. No Z ring formation could be observed in FipA-KO under oxidative stress. However, complementation with fipA MTB restored Z ring formation in 63 out of 81 FtsZ-positive cells (Table 1). We hypothesize that FipA facilitates localization of FtsZ at mid-cell under H2O2 stress. 10.1371/journal.pone.0008590.g006Figure 6 Localization of FtsZ to mid-cell in M. smegmatis under oxidative stress requires FipA. FipA-KO (A,B) or FipA-KO complemented with fipA MTB (C, D) was subjected to stress by incubation in the presence of H2O2 as described under “Materials and Methods”. Immunolocalization of FtsZ was carried out by incubation with anti-FtsZ antibody followed by staining with Alexa 488-conjugated rabbit IgG (green). Images shown are fluorescence micrographs (A, C) and the merge of phase contrast and fluorescence micrographs (B,D). Bar, 2 µm. 10.1371/journal.pone.0008590.t001Table 1 Presence of Z ring in cells. Total septum poles others WT without stress 122 102 18 2 WT after H2O2 stress 105 79 17 9 FipA-KO without stress 101 81 10 10 FipA-KO after H2O2 stress - - - - FipA-KO complemented with fipA MTB without stress 102 75 21 6 FipA-KO complemented with fipA MTB, after H2O2 stress 81 63 14 4 FtsZ positive cells were counted. PknA-Mediated Phosphorylation of FtsZ The present study shows that FipA is phosphorylated by PknA on T77 (Fig. 4E) and phosphorylation is required for its interaction with FtsZ (Fig. 2B). Thakur and Chakraborti (2006) had earlier demonstrated that FtsZ is phosphorylated by PknA without identifying the site of PknA-dependent FtsZ phosphorylation. In order to further understand the role of PknA-dependent phosphorylations, we attempted to identify the site of phosphorylation of FtsZ. Computational analysis using the Net Phos program [29], suggested that T343 is a putative site of phosphorylation of FtsZ. Recombinant PknA13–273 could phosphorylate recombinant wild type His-FtsZMTB but not the T343A mutant in vitro (Fig. 7A). In addition, mass analysis of FtsZMTB after phosphorylation by PknA13–273 showed an increase in mass corresponding to phosphorylation on a single residue by (Figure S4). The T343A mutant, on the other hand, showed no increase in mass after treatment with PknA13-273, supporting our contention that T343 represents the only site of PknA-mediated phosphorylation of FtsZ. In order to confirm PknA-mediated threonine phosphorylation of FtsZ in vivo, pknA was partially knocked down by inducing the expression of an antisense RNA to pknA driven by the acetamidase promoter. Knock down of pknA inhibited phosphorylation of FtsZ on threonine (Fig. 7B), just as in the case of FipA. 10.1371/journal.pone.0008590.g007Figure 7 FtsZ is phosphorylated in a PknA-dependent manner. A. Purified FtsZ or its T343A mutant was phosphorylated by GST-PknA13-273 in the presence of [γ-32P] ATP followed by autoradiography. The lower blot is a Coomassie blue-stained gel showing the levels of FtsZ. B. Conditional knockdown of pknA was induced by growing M. smegmatis expressing an antisense construct of pknA (under the control of an acetamide-inducible promoter) in the absence (-) or in the presence (+) of acetamide for 12 h. Cells were lysed and lysates were immunoprecipitated with preimmune sera (first lane) or anti-FtsZ antibody, followed by immunoblotting with anti-phosphothreonine and reprobing with anti-FtsZ antibody. Blots shown are representative of three separate experiments. Formation of a Complex between FtsZ, FipA and FtsQ and the Role of PknA Considering that FipA appeared likely to play a role in cell division, we tested whether FipA influences interaction of FtsZ with any divisomal protein which follows it to the site of division. Among the proteins which have been recognized as following FtsZ and preceding FtsW to the site of division is FtsQ of E. coli (or DivIB of B. subtilis). The ORFs Rv2151c and MSMEG_4229 (Figure S5) encode M. tuberculosis and M. smegmatis FtsQ respectively [30]. Like FtsZ, both FipA and FtsQ localize to mid-cell in dividing M. smegmatis (Figure S6) as observed by immunofluorescence microscopy. Numerical data show that FipA localizes to the septum to a similar extent in untreated and H2O2-treated cells (Table 2). 10.1371/journal.pone.0008590.t002Table 2 Localization of FipA in M. smegmatis. Growth condition Septum Poles ND* Untreated 52 9 39 Subjected to H2O2 stress 48 10 42 In each set, FipA localization was counted among 100 cells. Not determined. We tested the possibility that FipA is part of a larger divisomal complex including FtsQ and FtsZ. FtsQ could be pulled down in a complex with FtsZ in wild type M. smegmatis as well as in FipA-KO (Fig. 8A) in the absence of stress. However, under H2O2 stress, FtsQ immunoprecipitated with FtsZ only in the wild type, but not in FipA-KO (Fig. 8A), suggesting that FipA is required for FtsZ to interact with FtsQ in oxidatively stressed cells, whereas FipA likely plays a redundant role in the absence of oxidative stress. In support of this contention, FipA-KO complemented with fipA MTB rescued the ability of FtsZ to interact with FtsQ under oxidative stress (Fig. 8A). FtsZ-FtsQ interaction was compromised in oxidatively stressed cells of FipA-KO complemented with fipA MTB(T77A) (i.e. phosphorylation-defective FipA) (Fig. 8B). Under oxidative stress, 67% of the FipA-KO cells showed punctate distribution of FtsQ in membranes with no visible localization at mid-cell, as opposed to 52% of the wild type cells showing septal localization of FtsQ (Table 3). Taken together, these results suggest that (a) FipA supports septal localization of FtsQ even under oxidative stress and (b) phosphorylation of FipA is required for FipA to fulfill this function. 10.1371/journal.pone.0008590.g008Figure 8 Interactions between FtsZ, FtsQ and FipA in vivo. A. M. smegmatis wild type (WT) or FipA -KO (KO) or FipA -KO complemented with fipA MTB (complemented) was left untreated (−) or treated (+) with H2O2, as described under “Materials and Methods”, lysed and immunoprecipitated with preimmune sera (first lane) or with anti-FtsZ antibody followed by immunoblotting with anti-FtsQ antibody. B. FipA-KO or FipA-KO complemented with fipA MTB(WT) or the T77A mutant, was grown, subjected to oxidative stress, lysed and immunoprecipitated with anti-FtsZ antibody followed by immunblotting with anti-FtsQ antibody. C. M. smegmatis strains mc2155, F1 or F2, were grown and subjected to the same treatment as in panel B, followed by immunoprecipitation with anti-His antibody (for pull down of FtsZ) and immunoblotting with anti-FtsQ antibody. D. M. smegmatis before or after conditional knockdown of pknA (performed as described under Fig. 7), was subjected to oxidative stress, lysed and immunoprecipitated with preimmune sera (first lane) or anti-FtsZ antibody, followed by immunblotting with anti-FtsQ antibody. E. M. smegmatis-F1 or -F2 cells subjected to oxidative stress, were lysed and immunoprecipitated with anti-His antibody followed by immunoblotting with anti-FipA antibody. Blots (A–E) were reprobed with anti-FtsZ antibody. Blots shown are representative of three separate experiments. 10.1371/journal.pone.0008590.t003Table 3 Localization of FtsQ in FipA-KO and complemented strains. Cell type and treatment punctate septum poles ND** WT (−) stress 13 64 21 2 WT (+)stress 30 52 13 5 KO (−)stress 19 58 19 4 KO (+)stress 67 0 3 30 KO with fipA MTB (−) stress 15 61 15 9 KO with fipA MTB (+) stress 35 45 12 8 Cells showing punctate distribution of FtsQ in the membrane. Not determined. In each set, FtsQ localization was scored in 100 cells. In order to understand the role of phosphorylation of FtsZ on its interaction with FtsQ, merodiploid strains lacking the chromosomal copy of ftsZ and carrying an integrated copy of the wild type His-ftsZ MTB (M. smegmatis-F1) or its T343A mutant (M. smegmatis-F2), were generated. FtsZ-FtsQ interactions were supported under oxidative stress by M. smegmatis strain F1 but not strain F2 (Fig. 8C). In M. smegmatis-F1, FtsQ showed septal localization in 67 and 55% cells in the absence of stress or under oxidative stress, respectively (Table 4). On the other hand, in M. smegmatis-F2, septal localization of FtsQ was visible in the absence of stress, but not in oxidatively stressed cells (Table 4). These observations suggest that phosphorylation of FtsZ is required for the interaction between FtsZ and FtsQ as well as for the septal localization of FtsQ. A role of PknA-mediated phosphorylation of FipA and FtsZ in sustaining FtsZ-FtsQ interactions under oxidative stress was also suggested by the observation that immunoprecipitation of FtsZ failed to pull down FtsQ in pknA-depleted cells (Fig. 8D) under oxidative stress. Further, immunoprecipitation of FtsZ from lysates of M. smegmatis-F1 or M. smegmatis-F2 strains showed that, like FtsQ, FipA could also be pulled down with wild type FtsZMTB but not with FtsZMTB(T343A) (Fig. 8E) under oxidative stress, suggesting that FtsZ phosphorylation is required for the FtsZ-FipA interaction. 10.1371/journal.pone.0008590.t004Table 4 Localization of FtsQ in M. smegmatis-F1 and M. smegmatis-F2. Cell type and treatment punctate septum poles ND** M. smegmatis-F1 (−) stress 19 67 7 7 M. smegmatis-F1 (+)stress 29 55 10 6 M. smegmatis-F2 (−)stress 26 53 13 8 M. smegmatis-F2 (+) stress 64 0 8 28 Cells showing punctate distribution of FtsQ in the membrane. *Not determined. In each set, FtsQ localization was scored in 100 cells. Immunoprecipitation of cell lysates with anti-FipA antibody, followed by immunoblotting with either anti-FtsQ or anti-FtsZ antibody showed that FipA could pull down FtsQ (Fig. 9A) or FtsZ (Fig. 9B) under oxidative stress However, FipA-FtsQ or FipA-FtsZ interaction was abrogated when pknA had been knocked down (Fig. 9A,B) supporting the contention that formation of the FtsZ-FtsQ-FipA complex under oxidative stress requires PknA. Taken together, our results suggest a role of PknA-mediated phosphorylation of FipA on T77 and FtsZ on T343 in mediating formation of a complex between FtsZ, FtsQ and FipA as well as their localization at mid-cell, under oxidative stress. 10.1371/journal.pone.0008590.g009Figure 9 FipA is a bridging partner for interactions between FtsZ and FtsQ. A, B. M. smegmatis before or after conditional knockdown of pknA, was subjected to oxidative stress, lysed and immunoprecipitated with preimmune sera (lane 1) or anti-FipA antibody (lanes 2 and 3), followed by immunblotting with anti-FtsQ (A) or –FtsZ (B) antibody and reprobing with anti-FipA antibody. C. Recombinant S-tagged FtsQMTB immobilized on S-agarose was incubated with buffer only (lane 1) or with recombinant FipAMTB followed by washing and further incubation with FtsZMTB (lane 2); or incubated with FipAMTB and FtsZMTB simultaneously (lane 3). Resin-bound proteins were separated by SDS-PAGE and immunblotted with different antibodies as indicated. For lanes 4 and 5, Ni2+ -NTA bound His-tagged FtsZMTB was incubated with (lane 4) or without (lane 5) FipAMTB followed by washing and further incubation with FtsQMTB. Bound proteins were identified by immunoblotting with the indicated antibodies. The blot was reprobed with anti-FtsZ antibody. Blots shown are representative of three separate experiments. Given the above findings, we tested the hypothesis that FtsZ, FtsQ and FipA are capable of forming a ternary complex in vitro. S-tagged FtsQMTB was immobilized on S-agarose, followed by simultaneous incubation with phosphorylated His-FtsZMTB and phosphorylated His-FipAMTB. The pulled down proteins were separated by SDS-PAGE and probed by Western blotting with anti -FtsZ and -FipA. Both FtsZMTB and FipAMTB were pulled down from lysates (Fig. 9C, lane 3). When FipAMTB was incubated with immobilized FtsQMTB, beads were washed, and FtsZMTB was subsequently added, neither FipAMTB nor FtsZMTB could be pulled down with FtsQMTB (Fig. 9C, lane 2). These observations suggest that prior interaction between FtsZ and FipA is necessary for formation of the FtsZ-FipA-FtsQ ternary complex. In support of this, FtsZ immunoprecipitates which contained FipA were able to pull down FtsQ (Fig. 9C, lane 4), whereas FtsZ immunoprecipitates alone could not pull down FtsQ (Fig. 9C, lane 5). These results suggest that FipA influences the ability of FtsZ to interact with partner divisomal proteins, and that initial interaction between FtsZ and FipA is necessary for ternary FtsZ-FipA-FtsQ complex formation. Discussion The gene cluster encompassing the products of the ORFs Rv0014c-Rv0019c is conserved throughout the genus Mycobacterium. It encompasses two transmembrane sensor kinases PknA and PknB, a phosphatase PstP, a transpeptidase PBPA and the FHA domain protein encoded by Rv0019c (FipA). There has been speculation that this gene cluster plays a role in cell division [31]. However, knowledge of the roles of the individual gene products of this cluster in the genus Mycobacterium, is scanty. The dcw cluster is conserved across the genus mycobacterium and includes genes such as ftsZ, ftsQ and ftsW whose products are central to the process of cell division. Here we establish two important findings. In the first instance we demonstrate a synergy of action between two products of the first gene cluster, (PknA and the FHA domain protein FipA), and two products of the dcw cluster (FtsZ and FtsQ) in maintaining cells in a division-competent state under oxidative stress. We uncover a novel role of phosphorylated FipA in assembly of the divisome under oxidative stress. In the second instance, we demonstrate that the network of interactions between FtsZ, FipA and FtsQ, is critically dependent on the serine/threonine kinase PknA. These findings are discussed below. M. tuberculosis resides within host macrophages following inhalation into the alveolar spaces of the lung. The bacterium is exposed to adverse environmental conditions and toxic agents such as reactive oxygen intermediates (ROI). It is logical that the bacterium must be endowed with mechanisms of withstanding the stressful conditions of its intracellular niche to an extent that allows multiplication and survival within macrophages. Control of cell division as well as maintenance of cell shape are critical factors for the survival of mycobacteria within the host. After replication, the multiprotein complex that comprises the divisome, assembles at mid-cell in order to facilitate cell division [32]. In mycobacteria, the repertoire of proteins required for cell division of mycobacteria within macrophages, remains incompletely understood. In order to test whether the FHA domain protein FipA plays a role in supporting intramacrophage growth of M. tuberculosis, fipA was knocked down. Knockdown of fipA was associated with a cell division defect of M. tuberculosis in macrophages after 5 days of infection, providing evidence that FipA is required to sustain growth of M. tuberculosis within macrophages. The role of FipA was investigated using M. smegmatis as the model. Knockout of fipA resulted in a cell division defect in M. smegmatis exposed to H2O2. In E. coli, cell division block is frequently associated with induction of recA-dependent SOS response [33]. In M. tuberculosis, ROI produced by macrophages can damage DNA and induce the SOS response. In the classical pathway, DNA damage induces recA and lexA-dependent SOS response [34]. However, in this instance we could rule out the possibility of induction of a classical SOS response at the concentrations and times of H2O2 exposure used. Consequently, we investigated other causes of FipA-linked cell division block under oxidative stress, focusing on the interaction of FipA with well-characterized proteins of the divisome. Our results indicate that FipA interacts with FtsZ in a phosphorylation-dependent manner. Earlier studies have focused on biochemical characterization of PknA [35], and identified substrates such as MurD [36]. Kang et al. [20] reported that in mycobacteria, partial knock down of pknA or pknB leads to the formation of narrow and elongated cells, suggesting a role of these protein kinases in cell division. However, the significance of PknA-dependent phosphorylation of divisomal proteins is still not clear. Unlike Thakur and Chakraborti [35] we did not observe phosphorylation-dependent inhibition of the GTPase activity of FtsZ. It is pertinent to point out that Thakur and Chakraborti [35] used GST-FtsZ, whereas we have worked with His-FtsZ. It may be mentioned that Schulz et al. [37] failed to observe PknA-dependent inhibition of polymerization of Corynebacterium glutamicum FtsZ. During infection, membrane-bound kinases are thought to transmit environmental signals inside bacteria. In fact, pknA and pknB are upregulated by more than 10-fold when THP-1 cells are infected with M. tuberculosis [38]. It therefore appears likely that the serine/threonine kinases of mycobacteria play important roles during infection. Understanding their function could in the long run provide newer directions for development of antituberculosis drugs. With this in view, we pursued investigation on the possible role of PknA in cell division. We now establish that FipA is phosphorylated on T77 in a PknA-dependent manner, and elucidate a functional role for such a phosphorylation event. Coimmunoprecipitation assays confirm that the FHA domain of FipA is necessary for its interaction with PknA. FipAMTB could complement FipA-KO with restoration of growth and survival under oxidative stress. The phosphorylation-defective T77A mutant failed to support cell division in FipA-KO under oxidative stress, underscoring the importance of PknA-dependent phosphorylation of FipA in sustaining cell division under oxidative stress. In E. coli, FtsQ interacts with a large number of divisomal proteins [39] and is required for divisome assembly [40]. Recruitment of FtsZ, FtsQ and FtsA to mid-cell is required for the septal localization of FtsW [41]. In mycobacteria, FtsQ is required for localization of FtsW to the septum in dividing cells (unpublished observations) suggesting that it plays an important role in mycobacterial cell division. We provide evidence that FipA interacts with FtsZ and FtsQ in a PknA-dependent manner. We hypothesize that FipA is required for divisomal protein assembly under oxidative stress, in a manner that is dependent on phosphorylation of FipA mediated by PknA. We show that in the absence of oxidative stress, FtsQ interacts with FtsZ in vivo both in the wild type as well as in FipA-KO, although the two proteins do not interact directly with each other (Fig. 9C). This suggests that FipA, as a member of the divisomal complex, plays a redundant role in the absence of stress. Additional, unidentified accessory protein(s) probably fulfill a bridging function (between FtsZ and FtsQ) under normal conditions. This probably suffices to sustain septation of growing cells even in the absence of FipA. However, the accessory protein(s) are probably incapable of functioning under oxidative stress. FipA assumes a critical role in these conditions. In vitro grown M. smegmatis exposed to H2O2 or M. tuberculosis growing in macrophages, are longer than cells cultured in vitro in the absence of exogenous oxidative stress. These cells nevertheless survive in the presence of functional FipA. On the other hand, there is a much more severe division block under these conditions in the absence of FipA. It therefore seems reasonable to contend that FipA supports cell division in M. tuberculosis growing in macrophages to an extent that is optimal for its survival in the stressful environment of the phagosome. The FipA proteins are unique to mycobacteria, even though FtsZ and FtsQ are present across numerous genera. It is likely that FipA fulfils a function in the process of cell division and multiplication, specifically required for survival of mycobacteria in the intracellular mileu of the host. Our observations provide knowledge for the first time that in mycobacteria (i) there is synergy between the gene cluster Rv0014c-Rv0019c and the dcw cluster; (ii) PknA-dependent phosphorylation events fine tune cell division and play a critical role under oxidative stress and (iii) the product of the ORF Rv0019c, FipA, plays an important role in supporting cell division under oxidative stress. While our work was in progress, Gupta et al. [42] reported that FipA (Rv0019c) interacts with polyketide-associated protein, PapA5 of M. tuberculosis in vitro and that it is phosphorylated by PknB on T36. However, the physiological importance of these findings is unclear in the absence of any in vivo experiments. It remains to be investigated whether PknA-mediated phosphorylation of FipA on T77 and PknB-mediated phosphorylation on T36 are interdependent, whether PknB-dependent phosphorylation is constitutive or inducible, and whether it too plays a role in cell division. It is possible that site-specific phosphorylations of FipA by two different kinases regulate interaction with different substrates. Another FHA domain containing protein, GarA [43] has been found to be phosphorylated by two kinases, PknG and PknB which differentially regulate its interaction with other proteins. Detailed investigations to resolve these unanswered question are in progress. In summary, our observations offer new insight into how targeting FtsZ-FipA interaction could block cell division to an extent that would compromise mycobacterial multiplication in macrophages. Materials and Methods Molecular Biological Procedures Standard procedures for cloning and analysis of DNA, PCR, electroporation and transformation were used. Enzymes used to manipulate DNA were from Roche Applied Science, Mannheim, Germany. All constructs made by PCR were sequenced to verify their integrity. E. coli strains were routinely grown in Luria broth (LB). Details of strains and plasmids are provided in Table S1. Construction of Suicidal Delivery Vector for Inactivation of fipA in M. smegmatis The two step homologous recombination strategy described by Parish and Stoker [44] was used to disrupt MSMEG_0034, the counterpart of Rv0019c at its native locus in M. smegmatis. Briefly, a suicide plasmid was constructed in three steps. First, an 860 bp DNA fragment containing the 5′ end of MSMEG_0034 alongwith upstream sequence was PCR amplified using the genomic DNA of wild type M. smegmatis mc2155 as template and the primer pair FipA 1F sense and FipA 1F antisense (Table S2). The amplicon was cloned between the asymmetric HindIII and BamHI sites of p2NIL to give pJB110. In the next step, a 900 bp fragment bearing the 3′ end of MSMEG_0034 and its downstream region was amplified using the primer pair FipA 2F sense and FipA 2F antisense (Table S2) and cloned between the BamHI and PacI sites of pJB110 to give pJB111 carrying a truncated fipA gene. The hygromycin cassette was excised from pUC-HY–INT (Mahenthiralingam et al., 1998) and introduced into pJB111 to give pJB112. Finally, a 6.1 kb PacI cassette carrying the lacZ, aph and sacB genes, was isolated from pGOAL17 and inserted into pJB112 to generate the suicide plasmid pJB113. Isolation of the FipA –Knockout Mutant Denatured pJB113 was electroporated into electrocompetent cells of M. smegmatis mc2155. Cells were plated on Lemco agar supplemented with hygromycin B, kanamycin and 50 µg/ml x-gal. Blue colonies were streaked onto Lemco agar without any selection to enhance the recombination process. Cells were then plated onto Lemco agar supplemented with 2% sucrose and 50 µg/ml x-gal. The white colonies were restreaked onto replica plates containing hygromycin, x-gal and sucrose, either without or with kanamycin. White, kanS, hygR, sucR colonies generated by double crossover represented the fipA –inactivated strain (FipA–KO). Expression of FipA was checked using antibody raised against FipA. Complementation of FipAMTB in FipA-KO Using an Integrating Vector An integrating vector carrying His-FipA(or its mutants) of M. tuberculosis under the control of the hsp60 promoter was constructed as described earlier [19]. Complementation with fipA MTB was achieved by electroporation of the integrating vector into FipA–KO. The presence of His-FipA was confirmed by Western blotting using anti-His antibodies. Cloning and Expression of M. tuberculosis H37Rv Genes FipA was amplified from genomic DNA of M. tuberculosis H37Rv using the sense primer “a” and antisense primers “d” (for pET28a+) and “e” (for pET29a+) depicted in Table S2. The amplicons were cloned between asymmetric BamHI and HindIII sites in pET28a or the BamHI and EcoRI sites in pET29a+ to generate pJB120 and pJB121 respectively. E. coli C41 (DE3) harbouring the respective constructs was grown to an Abs600 of 0.6 at 37oC, IPTG was added at a concentration of 0.25 mM, and growth was continued for 2–3 h. His- or S-tagged proteins were purified from cell lysates by chromatography on Ni2+-NTA- or S-agarose respectively. Mutants of FipAMTB were generated by overlap extension PCR. Initial rounds of PCR were performed using primers “a”and “b”; or primers “c” and “d” (or “e”) (Table S2). Using the products of the initial round as template, the second round of PCR was performed using primers “a” and “d” (or “e”). Products were cloned in pET28a+ or pET29a+. The T343A mutant of FtsZ was generated using pJB101 (Datta et al., 2002) as template and primers “a” and “d” (Table S2). ftsQ was amplified using the sense and antisense primers shown in Table S2, and cloned either in pET28a+ or in pET29a+ between the BamHI and HindIII sites. Expression was carried out in E. coli C41(DE3) using 0.1 mM IPTG for 4 h at 37°C. The kinase domain of pknA (pknA 13-273) was amplified using the sense and antisense primers shown in Table S2, and cloned between the BamHI and EcoRI sites of pGEX-2T to generate the plasmid pJB122. Expression was carried out in E. coli BL21 by induction with 0.1 mM IPTG at 37°C for 4 h, and the expressed protein was purified by chromatography on glutathione Sepharose. The K42A mutant was generated by overlap extension PCR using primers “b” and “c” depicted in Table S2. His-PknB1–276 was purified as described earlier [19]. The pstP (Rv0018c) gene was amplified using the sense and antisense primer pair shown in Table S2, and cloned between the BamHI and HindIII sites of pET29a+. N-terminal S-tagged PstP was obtained by induction for 3 h at 37°C with 50 µM IPTG and purified by chromatography on S-agarose Construction of ftsZ Replacement Vectors in M. smegmatis A construct for replacement of the chromosomal ftsZ of M. smegmatis was generated using the method of described above [44]. A suicide plasmid with a 488 bp internal deletion in the ftsZ gene was constructed in two steps. In the first step, a 1244 bp DNA fragment encompassing the 5′ end of ftsZ and its upstream region was PCR amplified using the primer pair FtsZ 1F sense and FtsZ 1F antisense (Table S2) and cloned in pBluescript SK between the asymmetric BamHI and EcoRI sites to give rise to pJB114. In the next step 1153 bp of DNA bearing the 3′ end of ftsZ and its downstream flanking region was amplified using the primer pair FtsZ 2F sense and FtsZ 2F antisense (Table S2) and cloned between the EcoRI and Hind III sites of pJB114 to generate pJB115. The truncated ftsZ gene was excised from pJB115 by digesting with BamHI and HindIII and cloned into p2NIL between the same sites to generate pJB116. Finally, the 6.1 kb PacI fragment was isolated from pGOAL19 and inserted into pJB116 to generate the suicide plasmid pJB117. Isolation of M. smegmatis Strains Carrying Wild Type or Mutated ftsZMTB M. smegmatis was electroporated with denatured pJB117. Single cross over (SCO)s were selected on agar plates containing kanamycin and x-gal. Kanamycin-resistant blue SCOs were isolated. In the next step, His-tagged ftsZ MTB (or its mutant) was cloned under the control of the hsp60 promoter in pUC19 using KpnI and HindIII sites as described above. The 3.757kb Hyg-integrase cassette was excised from pUC-HY-INT [45] and inserted into the above construct to generate an integrating vector. Before inactivation of ftsZ at its native location, ftsZ MTB in the integrating vector was electroporated into the SCOs. The resultant merodiploid strains carrying His-tagged ftsZ MTB (M. smegmatis-F1) or ftsZ MTB(T343A) (M. smegmatis-F2) were then screened for double cross over (DCO) as described earlier [46]. White kanS HygS and sucrose-resistant DCOs were analyzed by PCR. In Vitro Kinase Assay In vitro kinase assays were performed with the purified kinase domain of PknA13–273 (1 µg) in 25 mM Tris/HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 2 mM MnCl2, 0.1 mM sodium orthovanadate as described by Dasgupta et al. (2006). Dephosphorylation assays were carried out in 50 mM HEPES buffer pH 7.5 containing 0.1 mM EDTA, 1 mM DTT and 5 mM MnCl2 as described earlier [19]. In some experiments, proteins separated by SDS-PAGE were electrotransferred and Western blotting was performed using monoclonal anti-phospho-threonine or anti-phosphoserine (Sigma Chemical Co.) or anti-phospho-tyrosine (Cell Signaling Technology) antibody. Conditional Inactivation of pknA in Mycobacteria The pknA gene was amplified using the primer pair pknA sense and pknA antisense (Table S1) followed by cloning of the PCR product in reverse orientation in the vector pSD29 [47] between the BamHI and EcoRV sites to generate pJB118. In order to conditionally inactivate pknA, M. smegmatis was electroporated with pJB118. Transformants were grown up to mid log phase and induced with 0.2% acetamide for different periods of time. Western blotting was performed to confirm the antisensing of PknA. Knock Down of FipA in M. tuberculosis For knock down of fipA MTB, an antisense construct (pJB119) of fipA was generated by PCR amplification of the fipA gene using the primer pair FipAMTB sense and FipAMTB antisense (Table S2) followed by cloning of the PCR product in reverse orientation under the control of the hsp60 promoter using the strategy described by Sureka et al. [48]. Western blotting using anti-FipA antibody was performed to confirm the antisensing of fipA. Induction of Oxidative Stress and Treatment with Mitomycin C Cells were subjected to H2O2-mediated oxidative stress as described earlier [48]. Briefly, cells were grown to an OD600 of 0.3 to 0.4, diluted into fresh medium to an OD600 of 0.2 and treated with 50 µM hydrogen peroxide for 1 h, followed by addition of hydrogen peroxide to 5 mM and incubation for the desired period of time. Mitomycin C treatment was carried out similarly by exposing cells (suspended at an OD600 of 0.2) to mitomycin C (0.2 µg/ml) for 4 h. Preparation of Cell Lysate, Antibodies and Immunoprecipitations Cell lysates were prepared by disrupting cells in a Mini Bead Beater (Biospec Products) as described earlier [48]. Immunoprecipitations were carried out by overnight incubation of lysates with antibodies at a dilution of 1∶1,000 at 4°C, followed by precipitation with Protein A/G agarose for 3 h. Antibodies against the kinase domain of PknA, PknB, PBPA, PstP, FtsQ, FipA and FtsZ were raised by Imgenex, Bhubaneswar. Fluorescence Microscopy Immunostaining was performed as described earlier [14]. Briefly, cells were fixed by incubation for 15 minutes at room temperature followed by 45 minutes on ice in 2.5% (v/v) paraformaldehyde, 0.04% (v/v) glutaraldehyde, 30 mM sodium phosphate (pH 7.5). After washing in PBS, the cells were permeabilized by exposing to 2% toluene for 2 min, and immediately transferred to slides. The slides were washed with PBS, air-dried, dipped in methanol (−20°C) for 5 min and then in acetone (−20°C) for 30 s and allowed to dry. After rehydration with PBS, the slides were blocked for 2 h at room temperature with 2% (w/v) BSA-PBS and incubated for 1 h with appropriate dilutions of primary antibody in BSA-PBS. The slides were washed extensively with PBS and then incubated with a 1∶1,000 dilution of Alexa 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) in BSA-PBS. After extensive washing with PBS, the slides were mounted using 50% glycerol. In controls for assessing specificity of the primary antibodies, the incubation with the preimmune sera was included. Images were viewed in a Zeiss Axioimager A1 microscope. Supporting Information Figure S1 A. Schematic representation of the genomic region of M. tuberculosis encompassing Rv0014c-Rv0019c and the corresponding region in M. smegmatis. B. Alignment of the amino acid sequences of FipA of M. tuberculosis (encoded by Rv0019c) and M. smegmatis (encoded by MSMEG_0034). The FHA domain is over-scored. (0.21 MB JPG) Click here for additional data file. Figure S2 A. Expression of PknB, PknA, PBPA, PstP, FipA and FtsZ in wild type (WT) or FipA-KO (KO) or FipA-KO complemented with fipA of M. tuberculosis (com.). B,C. Transcriptional analysis of lexA and recA in M. smegmatis. Cells were treated with hydrogen peroxide or mitomycin C. RNA was isolated and subjected to q-RT-PCR for the lexA (B) and recA (C) genes. Fold increase represents the change with respect to untreated cells. Values shown are means with S.D. of three separate determinations. (0.11 MB JPG) Click here for additional data file. Figure S3 Mass analysis of FipA or FipA phosphorylated by PknA. (1.82 MB TIF) Click here for additional data file. Figure S4 Mass analysis of FtsZ (unphosphorylated), or phosphorylated by PknA or FtsZ(T343A). (0.47 MB TIF) Click here for additional data file. Figure S5 Schematic representation of the genomic region encompassing the dcw cluster of M. tuberculosis (Mtb) and M. smegmatis (Msmeg). (1.27 MB TIF) Click here for additional data file. Figure S6 Immunolocalization of FipA (A) and FtsQ (B) in M. smegmatis. Immunolocalization of was carried out by incubation with anti-FipA or anti-FtsQ antibody followed by staining with Alexa 488-conjugated rabbit IgG and visualization by fluorescence microscopy. (0.03 MB JPG) Click here for additional data file. Table S1 List of strains and plasmids used in this study. (0.04 MB DOC) Click here for additional data file. Table S2 Primers used in this study. (0.06 MB DOC) Click here for additional data file. The authors would like to thank Prof. Jaya Tyagi for genomic DNA of M. tuberculosis H37Rv, and Dr. Richard Stokes for pUC-HY-INT. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by a grant from the Council of Industrial Research, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Adams DW Errington J 2009 Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nat Rev Microbiol 7 642 653 19680248 2 Errington J Daniel RA Scheffers DJ 2003 Cytokinesis in bacteria. Microbiol Mol Biol Rev 67 52 65 12626683 3 Rothfield LI Taghbalout A Shih YL 2005 Spatial control of bacterial division-site placement. 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==== Front Indian J Community MedIJCMIndian Journal of Community Medicine : Official Publication of Indian Association of Preventive & Social Medicine0970-02181998-3581Medknow Publications India IJCM-34-22310.4103/0970-0218.55288Original ArticleProfile of Clients Tested HIV Positive in a Voluntary Counseling and Testing Center of a District Hospital, Udupi Gupta Megha Department of Community Medicine, Kasturba Medical College, Manipal - 576 104, Karnataka, IndiaAddress for correspondence: Dr. Megha Gupta, Department of Community Medicine, Kasturba Medical College, Manipal - 576 104, Karnataka, India. E-mail: [email protected] 2009 34 3 223 226 24 10 2007 02 5 2008 © Indian Journal of Community Medicine2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: The growing menace created by the HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome) has alarmed not only the public health officials but also the general community. The Voluntary Counseling and Testing Centre (VCTC) services have begun as a cost-effective intervention in reversing this epidemic. Objectives: 1) To study the sociodemographic characteristics of HIV-positive clients and their risk behaviors. 2) To elucidate the reasons for their visit to the VCTC and know the problems anticipated by the clients after revealing their HIV-positive status. Study Design: A cross-sectional record-based study. Materials and Methods: The study was conducted in August 2007 among clients who tested positive for HIV in the VCTC of a district hospital in Karnataka from January to July 2007. Results: Study included 249 individuals of whom 161 were males and rest 88 females. A high percentage of nonresponse regarding the pattern of risk behavior was noted among the subjects (males: 42.8% and females: 90.9%). Of the individuals who responded, 91 males (98.9%) and 6 females (75.0%) had multiple heterosexual sex partners, while 1 male had homosexual partner. The figures in females show that two (25%) of them had a history of blood transfusion. The reason for visiting the VCTC were cited as some form of illness (33.3%), confirmation of test results (32.9%), family members diagnosed as HIV positive (12.9%) and 11.6% were referred from Directly Observed Treatment Scheme (DOTS) center. More than one-thirds of the sample population anticipated discrimination at the time of medical treatment. Conclusion: People have begun using VCTC services, which reflects a change in their attitude toward HIV. The study provides us a clue to formulate an effective approach to educate people as well as the health personnel who are thought of as one of the important sources of discrimination. DiscriminationHIV positiverisk behaviorVCTC ==== Body Introduction The human immunodeficiency virus (HIV) infection is a global pandemic and has grown into a public health program of unprecedented magnitude. According to the acquired immunodeficiency syndrome (AIDS) epidemic update, December 2007, released by the UNAIDS and World Health Organization (WHO), approximately 33.2 million people are living with HIV/AIDS worldwide.(1) The prevalence rate of HIV in adults varies in different regions from 5% in the Sub-Saharan Africa, 0.3% (Middle East), 0.5% (Latin America), 0.9% (Eastern Europe) to 0.6% in North America (AIDS Epidemic Update 2007).(1) It is estimated that 90% of the HIV-infected persons live in the developing countries with the estimated number of Indians being 2.7 million.(2) Overall, the average prevalence rate of HIV in adults in India is approximately 0.36%, and it accounts for 10% of the global HIV burden and 65% of that in the South and South-East Asia.(3) Counseling for HIV and AIDS has become a core element of a holistic model of health care; in this model, psychological issues are recognized as integral to patient management. Both pre- and post-test counseling have become standard components of prevention-oriented HIV antibody testing programs.(4) The Voluntary Counseling and Testing Centre (VCTC) now known as the ICTC (Integrated Counseling and Testing Centre) provides a key entry point for the ‘continuum of care in HIV/AIDS’ for all segments of the population. The sentinel surveillance carried out in October 1999 revealed that the state of Karnataka has a high prevalence of HIV infection.(5) A number of factors contribute to Karnataka's vulnerability to the HIV epidemic. It is bordered by three states that have well-established and further growing HIV epidemics (Maharashatra, Tamil Nadu and Andhra Pradesh). Karnataka shares many demographic and economic ties with these neighboring states. There is an extensive migration to and from these states, and major transportation routes connect Karnataka with them. Certain economic and social factors also contribute to Karnataka's vulnerability to this epidemic. Poverty levels are high, leading to economic pressures that promote commercial sex work. The data collected in the present study from the VCTC of a district hospital in Udupi, Karnataka, may provide important clues regarding the epidemiological profile of HIV-positive individuals. Materials and Methods The present study was conducted in the VCTC of a district hospital in Udupi situated in southern Karnataka. Udupi is flanked by the verdant mountains of the Western Ghats on the east and the vast and the tranquil Arabian Sea on the west. According to the 2001 India census, the district has a population of 963548 with 51% females. The average literacy rate is 83%, higher than the national average of 59.5%. From January to July 2007, of the data of total 2586 attendees at the VCTC who were either volunteers or referred from other institutions, the study included that of 249 HIV-positive patients (9.6%). Information for all the attendees of the VCTC was available from the records maintained at the VCTC regarding variables such as age, gender, marital status, education and occupational status, residence, behavioral patterns, discrimination anticipated, support expected. In the present study, only the data from patients who tested positive for HIV at the VCTC was included. This information was recorded when the client visited the VCTC for the first time and most of them were unaware of their status of HIV infection. HIV was diagnosed by performing enzyme-linked immunosorbent assay (ELISA) by using two different antigens and a rapid test as recommended by the National AIDS Control Organization (NACO). Data was collected and analyzed using the SPSS software version 11.5. However, the current study is subject to certain limitations since it was conducted in a district hospital; therefore, the results are based on the reporting and data collection by the personnel employed in the VCTC. Information regarding certain variables such as socioeconomic status, substance abuse, counseling performed and condom use are not available. All these variables could have unmasked certain behavioral patterns and could have given new dimensions to this study. The study setting being a district hospital decreased its external validity. Results The male population constituted 64.7% (161) of the total study subjects. Table 1 clearly shows the sociodemographic profile of the attendees with positive test result. A majority of the study subjects, i.e., 221 (88.7%) belonged to age group of 15-49 years with 7 (2.8%) subjects being less than 14 years of age. The distribution according to marital status showed that 72.7% of males and 84.0% of females were married of which 11.1% of males and 44.4% of females were divorced, separated or widowed. Table 1 Sociodemographic characteristics of the study subjects Factors Male (%) N = 161 Female (%) N = 88 Age group (years) <15 2 (1.2) 5 (5.7) 15-49 143 (88.9) 78 (88.6) >50 16 (9.9) 5 (5.7) Education Illiterate 23 (14.3) 25 (28.5) Upto 4th standard 20 (12.4) 12 (13.6) Upto 8th standard 66 (41.0) 34 (38.6) Upto 12th standard 45 (28.0) 12 (13.6) College and above 7 (4.3) 5 (5.7) Occupation Unskilled 46 (28.6) 23 (26.1) Semi-skilled 80 (49.7) 17 (19.3) Skilled 19 (11.8) - Professional 6 (3.8) 3 (3.4) Housewife - 30 (34.2) Unemployed 8 (4.9) 10 (11.3) Student 2 (1.2) 5 (5.7) Marital status Married 117 (72.7) 74 (84.1) Unmarried 44 (27.3) 14 (15.9) The literacy rate among the female subjects was found to be 71.5%, while that in males was 85.7%. The most common source of income for males (48.8%) was semi-skilled occupation, such as bidi rolling and fishing. Among females, 30 (34.2%) were housewives and 23 (26.1%) were working as housemaids or laborers. The unemployment rate among the study subjects was 7.3%. All females and 149 males (93.1%) resided with their family members. Approximately half of the study subjects (50.6%) had visited the VCTC voluntarily, while almost a similar percentage (49.4%) of the subjects was referred to the VCTC by another doctor. Among the reasons cited for their visit to the VCTC, illness (medical or surgical: 33.3%) was the leading cause, followed by 32.9% who visited for the confirmation of their test result. More than one-tenth (12.9%) of the study subjects had the family members (spouse/parents) who were positive for HIV and 29 (11.6%) were referred from a Directly Observed Treatment Scheme (DOTS) centre for detection and treatment of tuberculosis. Among the total subjects, 69 males (42.8%) and 80 females (90.9%) did not respond to the question on the pattern of risk behavior followed. Of the subjects who responded, 91 males (98.9%) had multiple sex partners and 1 was involved in homosexual practices. Among the females, six (75.0%) were having multiple sex partners and two (25%) had a history of blood transfusion [Table 2]. Table 2 Risk behavior followed by the study subjects Risk behavior Male No. (%) Female No. (%) Total (%) No. (%) Heterosexual multiple partners 91 (56.4) 6 (6.8) 97 (39.0) Homosexual partner 1 (0.6) 0 1 (0.40) Blood transfusion 0 2 (2.3) 2 (0.80) No response 69 (43.0) 80 (90.9) 149 (59.8) Total 161(100.0) 88 (100.0) 249 (100) The expectation of the subjects regarding the social support after testing positive shows that 31.7% candidates were in favor of individual counseling by counselors, 23.7% preferred family counseling and 18.6% preferred one to one discussion with doctors. The views of the study subjects regarding problems they would face after disclosing their HIV status revealed that a large percentage of subjects (79.1%) believed they would be discriminated at the time of medical treatment. A small number of subjects, i.e., eight (3.2%) anticipated a disturbance in their marital life, and an almost equal number, i.e., five (2.0%) believed that they would be discriminated by their other family members. Discussion The prevalence of HIV seropositivity in VCTC clients in the present study was noted to be 9.6%, which is lower than that reported from a study conducted in a district of West Bengal (17.1%) in 2003.(6) The present study highlights the fact that males contributed to 64.7% of the case load in VCTC with 35.3% being the females. These figures are slightly lower than the national average of 38.4% for females. Such a high proportion of infection rate in females is a cause for concern since this will lead to a proportionate increase in the children being infected due to transmission from mother to child. It is believed that HIV/AIDS affects the bread winners of the society, which is also evident from the results of this study. According to the study, 88.7% of the subjects belonged to the age group of 15-49 years (the most sexually active age group), which is slightly lower than the national figure (90%) and the figure obtained from another study (92.4%) conducted at a VCTC in Darjeeling.(6) The present study clearly indicates that 93% of the infected males and 100% of the infected females are living with their families. The information regarding their disclosure of the test result to their family members is not available and hence it is difficult to say whether such a high level of acceptance by the family, especially toward females will be maintained even after the disclosure or not. The pattern of risk behavior shows that a large percentage of males (98.9%) and females (75%) of those who responded to the study, were had multiple sexual partners. However, none of the women was working as a commercial sex worker. Heterosexual contact was the commonest mode of transmission, which is supported by the findings of another study from eastern India.(7) A large part of the married women (44.4%) who were HIV positive were separated, divorced or widowed. This can be explained by the strong family ties and inhibitions that Indian females have as a part of culture. A large part of the study subjects (42.8% males, 90.8% females) did not disclose their risk status in the questionnaire. The figures for the risk status were 29.9% in males and 53.8% females in a study conducted in West Bengal (2003).(6) This can be attributed to the fear of discrimination or punishment, which still prevails in the society toward HIV-infected individuals. The major problem anticipated by the subjects was observed to be from the health personnel at the time of medical and surgical treatment. This could be a reason for only 18% subjects responding in favor of doctors as the best option for their support. More than half of the subjects were in favor of counseling since it is gaining importance in the current era as an important step toward normalizing the attitude to HIV and improving the environment for the prevention of its transmission. Another important finding of the study shows that approximately 3% of the subjects actually believed they would be discriminated by their family members, while the remaining thought that they would be easily accepted. This can be attributed to the increasing awareness among the people by the combined efforts of health care personnel and media. This assumption can also be explained by a large percentage of subjects coming to VCTC on their own without being referred by someone else. This was in contrast to the figures reported from a study in Chennai (2004–05) where only 3 of the total 89 HIV-positive patients had visited voluntarily for testing.(8) The current study highlights the existence of HIV-TB collaboration, which is evident from the study since 29 subjects (11.6%) had been referred from the DOTS centers. Conclusion The high prevalence of seropositivity in the attendees of VCTC in a district hospital in Karnataka highlights the importance of this issue for the policy makers as well as health professionals. The medical fraternity should take a stand and fight against the discrimination of sufferers, rather than ostracizing them to have a positive attitude from HIV sufferers. Increased availability and the use of VCTC services will prove to be a huge potential benefit for the society. Source of Support: Nil Conflict of Interest: None declared. ==== Refs References 1 WHO/UNAIDS AIDS Epidemic Update December 2007 Available from: www.unaids.org/en/HIV-data 2 2.5 million people living in India with HIV, according to new estimates, UNAIDS/NACO/WHO, 6 July 2007 Available from: www.who.int/mediacentre/news/releases/2007 3 HHS/CDC Global AIDS program (GAP) in India The GAP India Fact sheet Available from: www.Cdc.gov/nchstp/od/gap/countries/India.htm 4 Valdiserri RO Moore M Gerber AR Campbell CH Dillon BA Jr West GR A study of clients returning for counseling after HIV testing: implications for improving rates of return Public Health Rep 1993 108 12 8 8434087 5 Sengupta D Rewari BB Shaukat M Mishra SN Study on Clinico-epidemiological profile of HIV Patients in Eastern India J Posgrad Med 2001 15 91 8 6 Jordar GK Sarkar A Chatterjee C Bhattacharya RN Sarkar S Banerjee P Profile of attendees in the VCTC of North Bengal Medical College in Darjeeling district of West Bengal Indian J Community Med 2006 31 237 40 7 Chakravarty J Mehta H Parekh A Attili SV Agrawal NR Singh SP Study on Clinico-epidemiological profile of HIV patients in Eastern India J Assoc Physicians India 2006 54 854 7 17249252 8 Studies on HIV/AIDS Voluntary counseling and testing centre 2004-2005 Chennai National Institute of Epidemiology Available from: www.google.com	20049300	PMC2800902	CC BY	2021-01-04 17:52:11	yes	Indian J Community Med. 2009 Jul; 34(3):223-226
==== Front J Med Case ReportsJournal of Medical Case Reports1752-1947BioMed Central 1752-1947-3-932410.1186/1752-1947-3-9324Case reportMultifocal multi-organ ischaemia and infarction in a preterm baby due to maternal intravenous cocaine use: a case report Reynolds Ben C [email protected] Dawn MK [email protected] Allan G [email protected] Lesley A [email protected] Charles H [email protected] Neonatal Unit, Princess Royal Maternity Hospital, Alexandra Parade, Glasgow, UK2 Department of Paediatric Pathology, Royal Hospital for Sick Children, Dalnair Street, Glasgow, UK2009 10 12 2009 3 9324 9324 23 9 2008 10 12 2009 Copyright ©2009 Reynolds et al; licensee BioMed Central Ltd.2009Reynolds et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction Although the adverse effects of cocaine use in pregnancy are well recognised, we believe this case highlights the importance of considering the route of administration, and suggests the possibility of multifocal damage relating to intravenous use. Case presentation A Caucasian female baby of 29-weeks' gestation was spontaneously delivered and subsequently developed multi-organ failure considered unrelated to simple prematurity. Intensive care was re-orientated following the development of massive intraventricular haemorrhage. Conclusion This case illustrates the need for regular cranial ultrasound in babies of pregnancies at risk due to intravenous cocaine use and also the necessity of counselling women who misuse cocaine in the antenatal period. As such, this article will be of most interest to paediatric and obstetric staff. ==== Body Introduction Cocaine use in pregnancy has been associated with adverse fetal outcomes including congenital malformations. We report a female baby of 29 weeks' gestation whose mother had extensive polydrug misuse throughout her pregnancy, including the use of intravenous cocaine. Following spontaneous delivery, the baby died after three days of intensive support. A post-mortem examination revealed widespread ischaemic change throughout multiple organs. We hypothesise that the unusual extent of this damage is related to the route of administration and dosage of cocaine during the pregnancy. Case presentation A 29-year-old Caucasian primigravida presented at 29+0 weeks' gestation with abdominal pain and fever. A presumptive diagnosis of urinary tract infection was made with laboratory investigations demonstrating a raised C-reactive protein and peripheral leukocytosis, and treatment with intravenous cefuroxime was commenced. The expectant mother reported regular use of heroin, diazepam, 'street' methadone and cocaine. Heroin and cocaine were both smoked and injected intravenously. Frequency of use was difficult to clarify. Abdominal pain continued intermittently and antenatal betamethasone was administered. A cardiotocograph (CTG) trace was non-reassuring and necessitated an emergency Caesarean section approximately five hours after the initial dose of betamethasone. A female was delivered alive and in good condition, weighing 1530 g (75th centile). Apgar scores were 71 and 85. There were no external dysmorphic features, organomegaly, rash or bleeding. An initial cranial ultrasound scan was normal with no evidence of haemorrhage. Mean blood pressure (BP) was normal. Laboratory investigations demonstrated marked coagulopathy and abnormal liver function tests (Table 1). Aspartate transaminase (AST) was disproportionately elevated in comparison with other liver enzymes, a pattern suggesting extensive tissue injury due to the non-specificity of AST. Table 1 Temporal evolution of laboratory parameters. Laboratory parameter Age Parameter Reference Range 1 hour 12 hours 36 hours PT 10.6-16.2 secs 54 29 APTT 27.5-79.4 secs 60 41 TCT 19.2-30.4 secs 23 19 Fibrinogen 1.5-3.73 g 0.5 1.2 Urea 2.5-7.5 mmol/l 6.4 9.5 Creatinine 35-100 μmol/l 78 136 Bicarbonate 21-28 mmol/l 22.0 15.6 13.1 AST <40 U/l 1891 2168 ALT <50 U/l 200 203 Gamma-GT <55 U/l 259 227 Albumin g/l 21 17 Stated coagulation reference ranges are applicable to 30-week gestation healthy controls on the first day of life. ALT, alanine transaminase; APTT, activated partial thromboplastin time; AST, aspartate transaminase; Gamma GT, gamma glutamyl transferase; PT, prothrombin time; TCT, thrombin clotting time. Fresh frozen plasma (FFP) and cryoprecipitate were administered without improvement in the coagulopathy. Urine was noted to be pink in colour, but microscopy did not demonstrate red cells. At 16 hours of age, there was generalised seizure activity confirmed on amplitude-integrated EEG (Cerebral Function Monitoring - 'CFM'). The infant was loaded with phenobarbitone and received a half correction of sodium bicarbonate for a progressive metabolic acidosis. Morphine was infused at 10 micrograms/kg/hour. Urine output was <0.5 ml/kg/day by 24 hours of age and she was passing extremely liquid stools. Coagulopathy persisted and liver function deteriorated further on sequential monitoring (Table 1). Repeat ultrasound at 36 hours of age showed bilateral intraventricular blood with evidence of marked midline shift. It was decided that continuing care aimed at the baby's survival was inappropriate and care was re-orientated following discussion with the baby's mother. The infant was extubated one hour following baptism, and died shortly afterwards. A postmortem examination was performed and it demonstrated intraventricular haemorrhage (IVH) (Figure 1) expanding all four ventricles and extending around the brain stem and cerebellum (grade 3). Histology showed recent subarachnoid haemorrhage and cortical vascular congestion consistent with multiple small focal interstitial haemorrhages distinct from the IVH. There was hepatic necrosis (Figure 2) and evidence of colonic mucosal ischaemic injury with multiple punctate erythematous areas. The kidneys showed zonal interstitial haemorrhage involving the medullary pyramids. The bladder also contained an area of large submucosal haemorrhage. These urogenital changes probably explain the pink-coloured urine. The absence of red cells was possibly attributable to haemolysis within the urinary tract. In addition, there was ischaemia and necrosis of the islets of Langerhans with sparing of the exocrine pancreas. Thymus, heart and adrenals appeared normal. Examination of the placenta showed acute decidual haemorrhage and chronic intervillitis. Microbiological and metabolic investigations did not demonstrate any further cause for deterioration or death. Figure 1 Bilateral blood casts of cerebral ventricles. Post-mortem pathological specimen demonstrating 'cast' formed by cerebral ventricles entirely filled with blood following massive intraventricular haemorrhage. Figure 2 A clear demarcation of healthy liver on the left, ischaemic liver centrally, and necrotic areas to the right. Postmortem pathological specimen of liver demonstrating zonal multifocal necrosis, with marked macroscopic necrosis visible on right side of specimen, healthy liver on left and a 'border' of ischaemic tissue between. Discussion Cocaine has been used for recreational purposes for over 5000 years [1]. The drug can be ingested, smoked, injected or inhaled intranasally. Smoking and snorting cocaine are the most common methods of cocaine use. Intravenous use is infrequent and account for less than 10 percent of cocaine use in the USA [2]. Comparative figures for the UK are unavailable and comprehensive Department of Health public information only briefly mentions injection as a route of administration [3]. Adverse effects of cocaine on the adult user are well recognised [1]. Vasoconstrictive effects are mediated via blockage of catecholamine uptake and beta-adrenergic stimulation. Cocaine use during pregnancy and its teratogenic effects on the fetus are less well defined. Early observational reports suggested 'crack babies' could have a variety of congenital abnormalities, including gastroschisis, intraventricular haemorrhage, growth restriction, and genitourinary and renal anomalies [4]. While evidence has increased, meta-analyses [5] and larger scale studies [4] have not confirmed any of the anatomical sequelae, although behavioural effects appear true. The mode of cocaine use is rarely considered or controlled for, nor is the cumulative dose of cocaine. Polydrug use and the chaotic lifestyle associated with substance misuse are variably considered as confounding within studies. Maturity at birth is also often omitted though it is suggested that preterm babies are affected differently [6]. The role of cocaine in intraventricular haemorrhage is still unclear. A prospective study [7] comparing light and heavy cocaine users with controls demonstrated an increased incidence of subependymal haemorrhage within term babies in the heavy cocaine user group only. A subsequent retrospective review [8] found a similar finding in preterm babies. Although, the review did not stratify according to cocaine usage, it suggested that this effect may have been even more pronounced in mothers who used large quantities. A small prospective study [9] of very low birth weight (VLBW) babies showed a higher incidence of grade I to II haemorrhage, but not more severe bleeds. A further larger prospective study of VLBW babies [10] did not find any increased risk of grade III or IV intraventricular haemorrhage though it did not consider dosage for confounding or consider smaller bleeds. Widespread focal ischaemia and infarction affecting multiple organs has not previously been reported in an infant as a result of maternal cocaine use. We hypothesise that the postmortem findings are related to the vasoconstrictive effects of cocaine use. The occurrence or extent of intraventricular haemorrhage within cocaine-exposed babies may be related to dosage. Intravenous usage may aggravate this effect. This case is of particular interest due to the widespread nature of the ischaemic infarcts affecting multiple organ systems. The focal nature of the infarcts affecting multiple organs makes them highly unlikely to be attributable to either complications of prematurity or the other illicit substances taken during this pregnancy. Due to the mixed nature, another substance or a cumulative effect cannot be excluded. However, similar infarcts have not, to our knowledge, been reported with heroin, methadone, or benzodiazepine use. Conclusion We advocate early and regular coagulation screening and cranial ultrasound scans for pregnant women with significant cocaine use, particularly if taken intravenously. The risk of significant morbidity and mortality should be considered during antenatal counselling of women who use cocaine. We also suggest that there is a need for further prospective research in this area with dosage and mode of administration being considered as confounding factors. Abbreviations ALT: alanine transaminase; APTT: activated partial thromboplastin time; AST: aspartate transaminase; BP: blood pressure; CFM: cerebral function monitoring; CTG: cardiotocograph; EEG: electroencephalogram; FFP: fresh frozen plasma; GGT: gamma glutamyl transferase; IVH: intraventricular haemorrhage; PT: prothrombin time; TCT: thrombin clotting time; VLBW: very low birth weight. Consent Written informed consent was obtained from the parent of the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests. Authors' contributions BCR and LAJ were the principal contributors to the manuscript, and primarily involved in the care of the baby. DKMP and AGH performed the postmortem and kindly provided the figures. CHS revised the manuscript. All authors read and approved the final manuscript. ==== Refs Warner EA Cocaine abuse Ann Int Med 1993 119 3 226 235 8323092 Substance Abuse and Mental Health Services Administration The DASIS report: Cocaine Route of Administration Trends: 1995-2005 2007 Rockville MD: Office of Applied Studies 'Talk to Frank'. Department of Health http://www.talktofrank.com Bauer CR Langer JC Shankaran S Bada HS Lester B Wright LL Krause-Steinrauf H Smeriglio VL Finnegan LP Maza PL Verter J Acute neonatal effects of cocaine exposure during pregnancy Arch Pediatr Adolesc Med 2005 159 824 834 10.1001/archpedi.159.9.824 16143741 Addis A Moretti ME Syed FA Einarson TR Koren G Fetal effects of cocaine: an updated meta-analysis Reproductive Toxicology 2001 15 341 369 10.1016/S0890-6238(01)00136-8 11489591 Brown JV Bakeman R Coles CD Sexson WR Demi AS Maternal drug use during pregnancy: are preterm and full-term infants affected differently? Developmental Psych 1998 34 3 540 554 10.1037/0012-1649.34.3.540 Franck DA McCarten KM Robson CD Mirochnick M Cabral H Park H Zuckerman B Level of in utero cocaine exposure and neonatal ultrasound findings Pediatrics 1999 104 1101 1105 10.1542/peds.104.5.1101 10545554 Smith LM Qureshi N Renslo R Sinow RM Prenatal cocaine exposure and cranial sonographic findings in preterm infants J Clin Ultrasound 2001 29 2 72 77 10.1002/1097-0096(200102)29:2<72::AID-JCU1001>3.0.CO;2-F 11425091 Singer LT Yamashita TS Hawkins S Cairns D Baley J Kliegman R Increased incidence of intraventricular haemorrhage and developmental delay in cocaine-exposed, very low birth weight infants J Pediatr 1994 124 5 Pt 1 765 771 7513757 Dusick AM Covert RF Schreiber MD Yee GT Browne SP Moore CM Tebbett IR Risk of intracranial haemorrhage and other adverse outcomes after cocaine exposure in a cohort of 323 very low birth weight infants J Pediatr 1993 122 3 438 445 10.1016/S0022-3476(05)83438-9 8441103	20062753	PMC2803847	CC BY	2021-01-04 17:53:50	yes	J Med Case Reports. 2009 Dec 10; 3:9324
==== Front Indian J DermatolIJDIndian Journal of Dermatology0019-51541998-3611Medknow Publications India 20101332IJD-54-33010.4103/0019-5154.57607Original ArticleDERMATOLOGY LIFE QUALITY INDEX SCORES IN VITILIGO: RELIABILITY AND VALIDITY OF THE TUNISIAN VERSION Jalel Akrem Soumaya Gaigi Siala 1Hamdaoui Mohamed Hédi From the Department of Physiology, Unité de Recherche sur les Composés antioxydants, Stress Oxydant, Eléments Traces et Maladies Métaboliques, Ecole Supérieure des Sciences et Techniques de la Santé de Tunis, Tunisia.1 From the Department of Service d'anatomie-pathologie, d'Embryologie et de Foetopathologie, CHU-Centre de Maternité et de Néonatologie de Tunis, Tunisia.Address for correspondence: Dr. Akrem Jalel, Ecole Supérieure des Sciences et Techniques de la Santé de Tunis. BP 176 Bab-Souika 1006 Tunis, Tunisia. E-mail: [email protected] 2009 54 4 330 333 8 2008 8 2008 © Indian Journal of Dermatology2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Vitiligo is an important skin disease that can alter individual self-image and thus have major impact on the quality of life. Aims: The objective of this study was to translate and to test the reliability and validity of the 10-item Dermatology Life Quality Index (DLQI) questionnaire in Tunisian vitiliginous patients. Methods: Using a standard “forward-backward” translation procedure, the English language version of the questionnaire was translated into Persian (the Iranian official language) by two bilinguals. Seventy patients with vitiligo attending the Department of Dermatology, Regional Hospital, Medenine, Tunisia, were enrolled in this study. The reliability and internal consistency of the questionnaire were assessed by Cronbach's α coefficient and Spearman's correlation, respectively. Validity was performed using convergent validity. Results: In all, 70 people entered into the study. The mean age of respondents was 28.3 (SD=11.09) years. Scores on the DLQI ranged from 0 to 24 (mean ± SD, 7.05 ± 5.13). Reliability analysis showed satisfactory result (Cronbach's α coefficient=0.77). There were no statistically significant differences between daily activity (DA) and personal relationship (PR) scale mean scores in generalized versus focal-segmental involvement in sufferers (P = 0.056, P = 0.053, respectively). There were also strong differences between the mean scores of the PR scale with the involvement of covered only and covered/uncovered areas (P = 0.016) that was statistically significant in the second group. Conclusions: The study findings showed that the Tunisian version of the DLQI questionnaire has a good structural characteristic and is a reliable and valid instrument that can be used for measuring the effects of vitiligo on quality of life. Psychologic side effectsTunisian patientvitiligo ==== Body Introduction Vitiligo is an important skin disease having major impact on the quality of life of patients suffering from vitiligo. The appearance of skin can condition an individual self-image, and any pathological alteration can have psychological consequences.[12] Many vitiligo patients feel distressed and stigmatized by their condition. These patients often develop negative feelings about it, which are reinforced by their experiences over a number of years. Most patients of vitiligo report feelings of embarrassment, which can lead to a low self-esteem and social isolation.[3] The Dermatology Life Quality Index (DLQI) questionnaire is designed for use in adults, i.e. patients over the 16. It is self-explanatory and can be simply handed to the patient who is asked to fill it in without the need for detailed explanation. It is usually completed in 1-2 min. The questions were classified into six headings items: Symptoms and feelings (questions 1 and 2), daily activities (questions 3 and 4), leisure (questions 5 and 6), and personal relationships (questions 8 and 9) each item with a maximum score 6; work and school (question 7), and treatment (question10) each item with a maximum score 3.[4] The DLQI is calculated by summing the score of each question resulting in a maximum of 30 and a minimum of 0. The higher the score, the more quality of life is impaired. The DLQI can also be expressed as a percentage of the maximum possible score of 30. The scores for each of these sections can also be expressed as a percentage of either 6 or 3. Since the DLQI is a brief, simple, easy to complete, and its application in research settings as a screening tool is well documented, it was decided to translate the DLQI into Persian (the Iranian official language) and to examine reliability and validity of this questionnaire in an Iranian population with vitiligo. Materials and Methods The standard “forward–backward” procedure was applied to translate the questionnaire from English into Persian. Two independent bilinguals translated the items and two others translated the response categories and after cultural adaptation, a provisional version was provided. Subsequently, it was back translated into English and then the final version was provided. The cultural adaptation was done by the translation of the “partner” to the “spouse” in Persian language and adding of it in questions 8 and 9, respectively. The final draft of the Persian version was administered to a sample of patients with vitiligo that referred to Department of Dermatology, Regional hospital, Medenine, Tunisia. There were no restrictions on patient selection with regard to extension of lesions. The patients were introduced to the subject of this study and informed about the personal nature of the questionnaire, and all those who gave consent were given the DLQI questionnaire to complete. The questionnaires were completed by the patients whom were referred to our clinic for psoralen and UVA (PUVA) therapy. The patients were categorized by extension of lesions into covered only (vitiligo patches are covered by cloths) and covered/uncovered involvement and by severity of disease to focal-segmental (focal is defined as a single or a few depigmented macules that are located in a discrete area, segmental is the unilateral localization of one or more macules to one area of the body),[5] which in this study were settled into one category and generalized involvement groups (widespread distribution of numerous macules over the integument in a random pattern).[6] Age of patients, marital status, and the number of treatment sessions were recorded. Responses on the DLQI were scored according to the guidelines of Finlay and Khan.[4] All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS 11.0 for Windows). To test the reliability, the internal consistency of the questionnaire was assessed by Cronbach's α coefficient and α equal to or greater than 0.70 was considered satisfactory.[7] Validity was performed using convergent validity to demonstrate the extent to which the DLQI correlated with global quality of life. Construct validity was checked by factor analysis. Results Seventy patients aged 18-68 (mean ± SD, 28.3 ± 11.09) years completed the questionnaire. We did not have any incomplete questionnaires; therefore, we included all questionnaires in our study. Scores on the DLQI ranged from 0 to 24 (mean ± SD, 7.05 ± 5.13). The reliability of the questionnaire was obtained by Cronbach's α coefficient (α = 0.77). There were no statistically significant differences between the sex, item scores, and mean DLQI score. Table 1 shows the results of item convergent validity tests. The scaling success rates were 100% for convergent validity of each scale. Table 1 Item scaling tests: Convergent validity for DLQI scales Scale No. of items per scale Convergent validity (range of correlation) Scaling success1 Scaling success rate2 Internal consistency (Cronbach's α) SF 2 0.69-0.78 2/2 100 0.70 DA 2 0.71-0.78 2/2 100 0.76 L 2 0.67-0.88 2/2 100 0.71 WS 1 1.00 1/1 100 1.00 PR 2 0.50-0.99 2/2 100 0.79 T 1 1.00 1/1 100 1.00 SF-Symptoms and feelings; DA-Daily activities; L-Leisure; WS-Work and School; PR-Personal relationships; T-Treatment 1 Number of correlation between items and hypothesized scale corrected for overlap ≥ 0.4/total number of convergent validity tests 2 Scaling success rate is the previous column as a percentage Cronbach's α coefficient by gender, marital status, severity, and extension of disease are shown in Table 2. Table 2 Cronbach's coefficient by gender, marital status, severity, and extension of disease Variable Cronbach's coefficient (n1) Gender Male 0.60 (27) Female 0.80 (43) Marital status Single 0.79 (42) Married 0.75 (28) Severity Focal/segmental 0.58 (18) Generalized 0.79 (52) Extension Covered/uncovered 0.78 (54) Covered 0.67 (16) 1 The number of patients in each category There were no statistically significant differences between item and mean DLQI scores of males versus females and married versus single cases. Cronbach's α reliability coefficients ranged from 0.69 to 0.78 for symptoms and feelings (SF) scale, 0.71 to 0.78 for daily activities (DA) scale, 0.67 to 0.88 for leisure (L) scale, and 0.50 to 0.99 for personal relationships (PR) scale. The reliability coefficient for work and school (WS) scale was equal to treatment (T) scale that was 0.100. Table 2 shows comparison of Cronbach's α a in each scale separately. The DA scale was found to have a strong association with gender (female scores were greater than male ones). The L scale was found to have significant relationship with the severity of the disease (generalized versus focal/segmental) (P value = 0.018). The DA and PR scales, also had no statistical association with severity factor (P = 0.056 and P = 0.053, respectively). The PR scale had strong correlation with the type of the extension of lesions (covered only versus covered/uncovered type) (P value = 0.016). There was no association between the numbers of treatment sessions and the type of disease (generalized versus focal/segmental). The number of treatment sessions and mean DLQI score was found to have a positive correlation coefficient (P value = 0.02, r = 0.28), but this correlation was statistically significant in the generalized type only (P value = 0.008, r = 0.37). Spearman's correlation coefficient of severity of the disease with questions 5, 6, and 8 were 0.25, 0.24, and 0.26, respectively. For question 8 and the extension of disease and also for question 1 and the stage of disease it was equal to 0.26. The result in question 4 and gender status was statistically significant (0.008); there was no statistically significance correlation between the age and each of questions. The result of question 9 and marital status was statistically significant (P = 0.002) and it was higher in married patients. The range of the paired correlations between the items was 0.17–0.68. Factor analysis is performed to determine the Persian version is a two-dimensional measure including social and psychological parameters [Table 3]. Table 3 Factor loadings (rotated)1 of two-factor solution DLQI items Social factor Psychological factor Q 1 0.086 0.485 Q 2 0.545 0.305 Q 3 0.535 0.374 Q 4 0.470 0.285 Q 5 0.564 0.473 Q 6 0.190 0.468 Q 7 0.088 0.619 Q 8 0.813 0.186 Q 9 0.681 2 0.195 Q 10 0.099 0.500 1 Varimax Discussion The DLQI questionnaire is a well-known instrument for measuring dermatological distress and has been translated into a variety of languages.[8–10] The translation process set by the international quality of life assessment (IQOLA) project was built on lessons from cross-cultural psychology and other health survey projects to develop protocols for translating, validating, and forming health status questionnaires, entails forward translation by at least two translators who were native speakers of the target language, rating of translation equality by two other bilinguals, and back translation by two translators who were native speakers of American-English or British-English.[11] Because native English speakers were unavailable, we did not fully adhere to this strategy. Two independent Tunisian health professionals translated the items, and subsequently, it was back translated into English by two others and then the final version was provided. Vitiligo is an acquired depigmentation disorder of great cosmetic importance affecting 1-4% of the world's population. The disease has a major impact on quality of life of patients, many of whom feel stigmatized by their condition.[12] Porter et al. studied the effect of vitiligo on sexual relationships and found that embarrassment during sexual relationships was especially frequent for men with vitiligo.[13] Salzer and Schallreuter reported that 75% found their disfigurement moderately or severely intolerable.[14] Weiss et al. compared the difficulties faced by patients with vitiligo with those with leprosy in India.[15] There may be a relationship between stress and the development of vitiligo. Al-Abadie et al. indicated that psychological stress increases levels of neuroendocrine hormones, affects the immune system, and alters the level of neuropeptides, which may be the initial steps in pathogenesis of vitiligo.[16] In general, the finding of this study indicated that mental health in vitiligo patients is poor and it is strongly associated with their quality of life. Since the patients with higher DLQI scores responded less favorably to a given therapeutic modality,[12] improving quality of life in this group becomes a very important task. Severity (generalized versus focal/segmental) and extension of lesions on covered only or covered/uncovered areas have an effect on quality of life of patients. This study reports data from a validation study of the 10- item DLQI questionnaire in Iran. In general, the findings showed promising results and were comparable with other research finding throughout the world.[12] The two-dimensional Tunisian version of DLQI questionnaire assessed the social and psychological difficulties as other studies.[12] There was no relationship of DLQI score with gender, which is consistent with the study of Parsad et al.[12] The mean DLQI score in this study was 7.05 that is lower than that obtained by Finlay and Khan (mean 7.3)[4] and Parsad et al. (mean 10.67),[12] and it is higher than that found in Kent and Al-Abadie's study (mean 4.82).[17] Reliability was associated by internal consistency of the questionnaire reporting Cronbach's α coefficient and validity was examined by convergent validity showed satisfactory results (α = 0.77). Cronbach's α was < 0.7 for males, focal/segmental, and covered vitiligo that may be related to small sample size and cultural differences. The Persian version of the DLQI questionnaire proved to be acceptable to patients and it is worth noting that occasionally the questionnaire was administered by a trained nurse in face-to-face interviews. However, this was done in illiterate patients and some indicated that some questions were difficult to answer, especially question 8. Perhaps this was the reason why a weaker correlation was found for this item with its corresponding subscale. Conclusions The study finding showed that the Tunisian version of the DLQI questionnaire has a good structured characteristic and is a reliable and valid instrument that can be used for measuring the effects of the vitiligo on quality of life. Especially, the reliability of this questionnaire was high in females and patients with generalized involvement, because of the great cosmetic importance in these groups. Source of Support: Nil Conflict of Interest: Nil. ==== Refs References 1 Mattoo SK Handa S Kaur I Gupta N Malhotra R Psychiatric morbidity in vitiligo: Prevalence and correlates in India J Eur Acad Dermatol Venereol 2002 16 573 8 12482039 2 Parsad D Dogra S Kanwar AJ Quality of life in patients with vitiligo Health Qual Life Outcomes 2003 1 58 14613564 3 Savin JA The hidden face of dermatology Clin Exp Dermatol 1993 18 393 5 8252755 4 Finlay AY Khan GK Dermatology Life Quality Index (DLQI): A simple practical measure for routine clinical use Clin Exp Dermatol 1994 19 210 6 8033378 5 Ortonne JP Mosher DB Fitzpatrick TB Vitiligo and Other Hypomelanosis of Hair and Skin 1983 New York Plenum Press 6 Koga M Vitiligo: A new classification and therapy Br J Dermatol 1977 97 255 61 921895 7 Nunnally JC Bernstein IH: Psychometric Theory 1994 3 Edition New York McGraw-Hill 8 De Tiedra AG Mercadal J Badia X Mascaró JM Herdmann M Lozano R Adaptación transcultural al Español del cuestionario Dermatology Life Quality Index (DLQI): El Índice de Calidad de Vida en Dermatologia Actas dermo-sifiliográficas 1998 89 692 700 9 Schäfer T Staudt A Ring J German instrument for the assessment of quality of life in skin diseases (DIELH). Internal consistency, reliability, convergent and discriminant validity and responsiveness Hautarzt 2001 52 624 8 11475643 10 Etemesi BA Quality of life in Tanzanian adults with chronic skin disease Ann Dermatol Venereol 2002 129 1S253 11 Bullinger M Alonso J Apolone G Leplège A Sullivan M Wood-Dauphinee S Translation health status questionnaire and evaluating their quality: The IQOLA Project Approach J Clin Epidemiol 1998 51 913 23 9817108 12 Parsad D Pandhi R Dogra S Kanwar AJ Kumar B Dermatology life quality index score in vitiligo and its impact on the treatment outcome Br J Dermatol 2003 148 373 4 12588405 13 Porter JR Beuf AH Lerner AB Nordlund JJ The effect of vitiligo on sexual relationship J Am Acad Dermatol 1990 22 221 2 2312803 14 Salzer BA Schallreuter KU Investigations of the personality structure in patients with vitiligo and a possible association with catecholamine metabolism Dermatology 1995 190 109 15 7727831 15 Weiss MG Doongaji DR Siddhartha S Wypij D Pathare S Bhatawdekar M The explanatory model interview catalogue (EMIC) Br J Psychiatry 1992 160 819 30 1617366 16 Al'Abadie MS Kent GG Gawkrodger DJ The relationship between stress and the onset and exacerbation of psoriasis and other skin conditions Br J Dermatol 1994 130 199 203 8123572 17 Kent G al-Abadie M Factors affecting responses on Dermatology Life Quality Index among vitiligo sufferers Clin Exp Dermatol 1996 21 330 3 9136149	20101332	PMC2807707	CC BY	2021-01-04 19:31:59	yes	Indian J Dermatol. 2009 Oct-Dec; 54(4):330-333
==== Front Indian J DermatolIJDIndian Journal of Dermatology0019-51541998-3611Medknow Publications India 20161850IJD-54-22110.4103/0019-5154.55628Basic ResearchOXIDATIVE STRESS IN EXPERIMENTAL VITILIGO C57BL/6 MICE Jalel Akrem Yassine Mrabet Hamdaoui Mohamed Hédi Unité de Recherche sur les Composés antioxydants, Stress Oxydant, Eléments Traces et Maladies Métaboliques, Ecole Supérieure des Sciences et Techniques de la Santé de Tunis.Address for correspondence: Dr. Akrem Jalel, M H Hamdaoui, Ecole Supérieure des Sciences et Techniques de la Santé de Tunis, BP 176 Bab-Souika 1006 Tunis, Tunisia. E-mail: [email protected] 2009 54 3 221 224 6 2008 7 2008 © Indian Journal of Dermatology2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Aim: To evaluate whether oxidative stress is implicated in melanocyte damage in vitiligo. Background: Vitiligo is a complex disorder characterized by gradually enlarging areas of depigmentation. A new unifying hypothesis for the etiology of this pigment disorder is proposed, in which we postulate that the final destruction of melanocytes in vitiligo results from a cascade of reactions initiated by a disregulation of melanogenesis, as the result of a breakdown in free radical defense. Methods: We evaluated 18 vitiligo mice and 12 controls that were age matched. Parameters of oxidative stress such as catalase (CAT), superoxide dismutase (SOD), and plasma malondialdehyde (MDA) were measured by spectrophotometry. Results: MDA levels in vitiligo mice were significantly higher than in controls (P < 0.001). CAT, SOD, and glutathione peroxidase (GPx) activities in mice were significantly lower than controls (P < 0.05 and P < 0.001, respectively). Conclusion: Our results confirmed that oxidative stress plays an important role in the pathogenesis of vitiligo. Melanocyte damage in vitiligo might be linked to generalized oxidative stress. This study is the first report on antioxidant parameters in experimental vitiligo mice. Antioxidant statuscatalaseglutathione peroxydasemalondialdehydeoxidative stresssuperoxide dismutasevitiligo ==== Body Introduction Vitiligo is an acquired skin disease characterized by white areas of the skin that can be observed in 0.1-8.8% of the population. The disease may affect individuals of both sexes and is mostly characterized by loss of melanocytes.[1] Despite much research, the etiology of vitiligo and the causes of melanocyte death is not clear. At least three pathogenic mechanisms - immunological, neural, and biochemical - have been suggested, but none can completely explain the disease.[23] Some findings show that oxidative stress may be an important phenomenon in the pathophysiology of vitiligo. [3–14] Imbalances in the oxidant/antioxidant system, such as the accumulation of hydrogen peroxide (H2 O2) and low catalase (CAT) levels have recently been demonstrated in the epidermis and blood of vitiligo patients.[15–18] Recent studies have also shown antioxidant systems to play a role in the pathogenesis of generalized vitiligo.[56] Antioxidant status has also been studied in segmental and nonsegmental vitiligo.[19] However, the literature contains no information about the status of antioxidant systems in the blood of experimental vitiligo mice. The purpose of this study was to evaluate the role of oxidative stress in the pathogenesis of active localized vitiligo. We investigated the role of antioxidant systems by measuring the levels of CAT, superoxide dismutase (SOD), and the plasma levels of malondialdehyde (MDA) in vitiligo patients with active localized disease, and in healthy controls. Materials and Methods Immunization procedure Six-week old male C57BL/6 mice were purchased from Pasteur Institute, Tunis, Tunisia. They were injected intradermally on four sites at the back skin with 50 μg of tyrosinase solution prepared from mushroom and emulsified in 50 μL of Freund's complete adjuvant (Sigma, USA). Two weeks after primary immunization, the mice were injected intraperitoneallywith 50g mushroom tyrosinase in 50 μL of incomplete Freund's adjuvant (Sigma, USA). Methods CAT activity was assayed by measuring the degradation rate of H2 O2 using Beutler's method.[20] The rate of disappearance of H2 O2 was monitored spectrophotometrically at 230nm. The assay medium consisted of 50 μL 1M Tris HCl buffer (pH 8), 930 μL 10mm H2 O2, 930 μL deionized water, and 20 μL hemolysate sample. One unit of CAT activity is defined as the amount of enzyme causing about 90% destruction of the substrate in 1 min in a volume of 1ml. CAT activity in the erythrocyte was expressed as U/g proteins. SOD activity was measured according to the method described by Fridovich using adaptable kits.[21] To determine SOD activity in skin preparations, the degree of inhibition of a reaction that catalyses the generation of superoxide radical by xanthine and xanthine oxidase was monitored spectrophotometrically for 3 min. The assay medium consisted of the 50 μL 0.01 M phosphate buffer, 1.7 ml substrate solution (0.05 mm xanthine and 0.025 mm INT in 3-cyclohexilamino-1-propanesulfonicacid (CAPS) buffer pH 10.2), 250 μL 80 U/L xanthine oxidase, and 50 μL skin sample. One unit of SOD sample inhibits the reaction by approximately 50% of the initially measured xanthine oxidase reaction. The activity is given in SOD units (1 SOD unit = 50% inhibition of the xanthine oxidase reaction). SOD activity in the skin tissue was expressed as U/g proteins. The lipid peroxidation level in the skin samples was expressed in MDA. It was measured according to the procedure developed by Ohkawa et al.[22] The reaction mixture contained 0.1 ml sample, 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid, and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid. The mixture's pH was adjusted to 3.5 and the volume was then made up to 4 ml with distilled water, thereafter 5 ml of a mixture of n-butanol and pyridine (15:1, v/v) was added. The mixture was shaken vigorously. After centrifugation at 4,000 rpm for 10 min, the absorbance of the organic layer was measured at 532 nm. The rate of lipid peroxidation was expressed as nmol of thiobarbituric acid reactive substances (TBARS) formed of skin preparation using a molar extinction coefficient of 1.56 × 105 M1.cm1. Results were expressed as nmol/ml. The proteins levels were measured by the cyanomethemoglobin method with a Spectronic-UV 120 spectrophotometer.[20] Bovine serum albumin was used as a standard. Statistical analysis Results in vitiligo mice and controls were compared using the paired Student's t-test. One-way analysis of variance (ANOVA) was used to determine significant differences in antioxidant enzyme activities between the groups. A value of P < 0.05 was considered significant. Results A total of 18 vitiligo male mice and 12 controls were studied. All mice were age matched. The mean, minimum, and maximum values of the blood activities of antioxidants and MDA levels in both groups are shown in Table 1. MDA levels in vitiligo mice were significantly higher than in controls. SOD, GPx, and CAT activities in vitiligo mice were significantly lower than in controls. Table 1 Antioxidant enzyme activity and malondialdehyde level in vitiligo patients and controls CAT (U/g pt) SOd (U/g pt) GPx (U/g pt) MdA (nmol/ml) Vitiligo mice 14.8 ± 2.0 4,457 ± 930 6.1 ± 0.8 3.8 ± 0.6 (n = 18) (12-18) (2,75-6) (4.9-7.8) (2.8-5.0) Controls 16.67 ± 1.5 2,219 ± 505 9.5 ± 1.4 2.2 ± 0.3 (n = 12) (15-20) (1.5-3.1) (7.4-12) (1.9-2.7) P value* < 0.05 < 0.001 < 0.001 < 0.001 Values are expressed as mean ± SE. * P < 0.05; P < 0.02; *P < 0.01 Discussion Vitiligo is a common disease, but, unfortunately, its pathogenesis is still unclear. Oxidative stress has been proposed as the triggering event of melanocyte degeneration in vitiligo. [3–14] Some studies have also shown that melanogenesis produces significant levels of reactive oxygen species (ROS).[23] ROS and other radicals can induce oxidative stress.[24] Oxidative stress may be a good model for vitiligo pathogenesis. Many of these disorders have been reported to be associated with ROS-induced damage.[25] ROS such as O2−, H2 O2, OH are essential for life as they are involved in cell signaling, melanogenesis, and are also used by phagocytes for their bactericidal action. Nonessential production of ROS, and thus oxidative stress, can also be induced by environmental factors such as UV radiation, toxins, and stress.[2627] Prime targets of ROS are the polyunsaturated fatty acids (PUFA) in the membrane lipids. This attack causes lipid peroxidation, and further, decomposition of peroxidized lipids yield a wide variety of end products, including MDA These products can cause DNA damage and lead to cytotoxicity, mutagenicity, cell death,[28] and could be a possible pathogenic factor for vitiligo.[29] The human epidermis presents the first line of defense against invading free radicals. The major agents that can induce, or produce, ROS are most often the causes of Koebner's phenomenon in vitiligo. There are data currently supporting an impaired redox status of the epidermal melanin unit as a primary defect leading to inappropriate immune responses in vitiligo. The presence of imbalance in the antioxidant system has been reported in vitiligo melanocytes and keratinocytes. A mechanistic hypothesis supported that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. It has the role of protecting the oxidant/antioxidant balance in the cell and reducing the oxidative stress. In addition, NADPH is necessary for the formation of reduced glutathione in erythrocytes, the reduction of methemoglobin to oxyhemoglobin, and CAT activity. [30–32] CAT converts H2 O2 to H2 O and O2.[29] Some authors reported normal CAT activities in erythrocytes of vitiligo patients.[781114] However, Dell'Anna et al.,[34] found lower CAT activity in leukocytes of vitiligo patients. In addition, Shajil and Begum showed lower CAT activity in segmental vitiligo patients, whereas in nonsegmental vitiligo patients CAT activity was normal.[19] We also found significantly lower CAT activity in erythrocytes of localized vitiligo patients. Previous studies of vitiliginous melanocytes showed lower CAT activity.[1133] We believe that lower CAT activity may be associated with H2 O2 accumulation, which may further inhibit CAT activity resulting in the destruction of melanocytes.[16] SOD catalyzes the conversion of superoxide anions to O2 and H2 O2. It protects cells from the toxic effect of superoxide radicals.[34] This study found significantly higher levels of SOD activity in erythrocytes of patients with active localized vitiligo. Increased levels of erythrocyte SOD in patients with vitiligo may enhance the systemic production of H2 O2. Additionally, high SOD activities were correlated with high immune competence.[35] Previous studies were performed in patients with generalized or combined types of vitiligo. There are different reports on SOD activity in patients with vitiligo compared to the healthy controls. SOD activity in erythrocytes was found to be normal[481112] in some studies and higher in others.[57141936] On the other hand, one study[6] reported lower levels in erythrocytes. Furthermore, Dell'Anna et al.,[4] found higher SOD activity in leukocytes of vitiligo patients. Although SOD activities in the vitiliginous tissue were found to be normal in one study,[12] Maresca et al.[11] and Yildirim et al.[13] found it to be high. We hypothesized that these varied results could be related to differences in serum, leukocyte, erythrocyte, and epidermis levels, duration and activity of disease, and differences in laboratory techniques. MDA is an endproduct of a lipid peroxidation reaction and is accepted as a specific indicator of oxidative stress.[3738] Picardo et al.,[8] and Tastan et al., SUP[12] found normal serum MDA levels in erythrocytes of combined types of vitiligo. Yildirim et al.[5] and Koca et al.,[6] showed higher serum MDA levels in generalized vitiligo patients. Whereas Tastan et al.,[12] found the MDA level in vitiliginous tissue to be normal, Yildirim et al.,[13] found it to be high. In this study, we found statistically higher plasma MDA levels in localized vitiligo patients. Lipoperoxidation, the primary reaction sites of which involve membrane-associated PUFA of phospholipids, can be considered a major manifestation of oxidative stress.[9] In conclusion, our results showed that oxidative stress may play a role in the pathogenesis of vitiligo and cause the melanocyte damage in vitiligo. Published data suggest that the oxidant/antioxidant system may be affected in all types of vitiligo. The changes in oxidative stress parameters are not related to the disease types. Once the living organism is exposed to a disease, the oxidative state may be influenced in a different ways. The changed antioxidant epidermal enzyme activities in vitiligo mice might be peripheral responses of the organism to an increased oxidative stress. No study has ever investigated how the imbalance of the oxidant/antioxidant system in vitiligo affects the process of the disease. This study is the first report on some antioxidant parameters in experimental vitiligo mice. However, further larger studies are necessary to confirm our results and to verify whether antioxidant treatments may be beneficial for experimental vitiligo mice. Conclusion Today, there is no convincing theory for the etiology of vitiligo. Vitiligo as a possible paradigm of neuroendocrine immunologic skin disease wherein the final destruction of melanocytes results from disregulation of melanogenesis induced by oxidative stress and activates an autoimmune response is very agreeable, and should be explored in depth in future studies. Source of Support: Nil Conflict of Interest: Nil. ==== Refs References 1 Handa S Kaur I Vitiligo: Clinical findings in 1436 patients J Dermatol 1999 26 653 7 10554431 2 Arýcan Ö Vitiligoda etyoloji, patogenez ve klinik J Kartal Training Res Hosp 2004 15 55 60 3 Dell'Anna ML Urbanelli S Mastrofrancesco A Camera E Iacovelli P Leone G Alterations of mitochondria in peripheral blood mononuclear cells of vitiligo patients Pigment Cell Res 2003 16 553 9 12950736 4 Dell'Anna ML Maresca V Briganti S Camera E Falchi M Picardo M Mitochondrial impairment in peripheral blood mononuclear cells during the active phase of vitiligo J Invest Dermatol 2001 117 908 13 11676831 5 Yildirim M Baysal V Inaloz HS Kesici D Delibas N The role of oxidants and antioxidants in generalized vitiligo J Dermatol 2003 30 104 8 12692376 6 Koca R Armutcu F Altinyazar HC Gurel A Oxidant-antioxidant enzymes and lipid peroxidation in generalized vitiligo Clin Exp Dermatol 2004 29 406 9 15245542 7 Agrawal D Shajil EM Marfatia YS Begum R Study on the antioxidant status of vitiligo patients of different age groups in Baroda Pigment Cell Res 2004 17 289 94 15140075 8 Picardo M Passi S Morrone A Grandinetti M Di Carlo A Ippolito F Antioxidant status in the blood of patients with active vitiligo Pigment Cell Res 1994 7 110 5 8066016 9 Jimbow K Chen H Park S Thomas PD Increased sensitivity of melanocytes to oxidative stress and abnormal expression of trosinase-related protein in vitiligo Br J Dermatol 2001 144 55 65 11167683 10 Passi S Grandinetti M Maggio F Stancato A De Luca C Epidermal oxidative stress in vitiligo Pigment Cell Res 1998 11 81 5 9585244 11 Maresca V Roccella M Roccella F Increased sensitivity to peroxidative agents as a possible pathogenetic factor of melanocyte damage in vitiligo J Invest Dermatol 1997 109 310 3 9284096 12 Tastan HB Erol IE Sayal A Erbil AH Vitiligoda eser element ve attioksidan düzeyleri T Klin J Dermatol 2003 13 141 9 13 Yildirim M Baysal V Inaloz HS Can M The role of oxidants and antioxidants in generalized vitiligo at the tissue level J Eur Acad Dermatol Venereol 2004 18 683 6 15482295 14 Hazneci E Karabulut AB Ozturk C A comparative study of superoxide dismutase, catalase, and glutathione peroxidase activities and nitrate levels in vitiligo patients Int J Dermatol 2005 44 636 40 16101862 15 Schallreuter KU Moore J Wood JM Epidermal H2O2 accumulation alters tetrahyrobiopterin (6BH4) recycling in vitiligo: Identification of general mechanism in regulation of all 6BH4-dependent processes? J Invest Dermatol 2001 116 167 74 11168813 16 Schallreuter KU Moore J Wood JM In vivo and in vitro evidence for hydrogen peroxide (H2 O2 ) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase J Invest Dermatol 1999 4 91 6 17 Hasse S Gibbons NCJ Rokos H Perturbed 6-tetrahydrobopterin recycling via decreased dihydropteridine reductase in vitiligo: More evidence for H2 O2 stress J Invest Dermatol 2004 122 307 13 15009710 18 Rokos H Beazley WD Schallreuter KU Oxidative stress in vitiligo: Photo-oxidation of pterins produces H2O2 and pteridin-6-carboxylic acid Biochem Biophys Res Commun 2002 292 805 11 11944885 19 Shajil EM Begum R Antioxidant status of segmental and non-segmental vitiligo Pigment Cell Res 2006 19 179 80 16524434 20 Beutler E Red Cell Metabolism: A Manual of Biochemical Methods c1975 2nd ed New York Grune and Stratton, CAT activity measurement 261 5 21 Fridovich I Superoxide dismutases Adv Enzymol Relat Areas Mol Biol 1974 41 35 97 4371571 22 Ohkawa H Ohishi N Yagi K Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction Anal Biochem 1979 95 351 8 36810 23 Riley PA Radicals in melanin biochemistry Ann N Y Acad Sci 1988 55 111 20 3245655 24 Procter PH Reynolds ES Free radicals and disease in man Physiol Chem Physics Med NMR 1984 16 175 95 25 Grimes PE Billips MW The clinical spectrum of childhood vitiligo 1996 2 Washington, D.C Annual Meeting of the American Academy of Dermatology 10 15 26 Gadjeva V Vlajkova T Popova E Reactive oxygen species, antioxidant enzymes and human diseases: Review- I Bulgarian Med 2000 8 21 4 27 McCord J Montagnier L The importance of Oxidant - Antioxidant Balance in Oxidative Stress in Cancer, AIDS, and Neurodegenerative Diseases 1999 New York, Basel, Hong Kong Marcel Dekker, Inc 1 9 28 Cross CC Halliwell B Borish Davis conference: Oxygen radicals and human disease Ann Intern Med 1987 107 536 45 29 Marks DB Marks AD Smith CM Oxygen metabolism and oxygen toxicity in basic medical biochemistry: A clinical approach 1996 Baltimore Williams and Wilkins 327 40 30 Caglar Y Kaya M Belge E Mete U Ultrastructural evaluation of the effect of endosulfan on mice kidney Histol Histopathol 2003 18 703 8 12792881 31 Kletzien RF Harris PK Foellmi LA Glucose-6-phosphate dehydrogenase: A “housekeeping” enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress FASEB J 1994 8 174 81 8119488 32 Bainy AC Saito E Carvalho PS Junqueria VB Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site Aquat Toxicol 1996 34 151 62 33 Schallreuter KU Wood J Berger J Low catalase levels in the epidermis of patients with vitiligo J Invest Dermatol 1991 97 1081 5 1748819 34 Saha N Ahmed MA Wasfi AI El Munshid HA Distribution of serum proteins, red cell enzymes and hemoglobin in vitiligo Hum Hered 1982 32 46 8 6950922 35 Prasad T Kundu MS Serum IgG and IgM responses to sheep red blood cells (SRBC) in weaned calves fed milk supplemented with Zn and Cu Nutrition 1995 11 712 5 8748260 36 Chakraborty DP Roy S Chakraborty AK Vitiligo, psoralen, and melanogenesis: Some observations and understanding Pigment Cell Res 1996 9 107 16 8888309 37 Thomas CE Morehouse LA Aust SD Ferritin and superoxide-dependent lipid peroxidation J Biol Chem 1985 260 3275 80 2982854 38 Pugliese PT The skin's antioxidant systems Dermatol Nurs 1998 10 401 16 10670316	20161850	PMC2810685	CC BY	2021-01-04 19:31:58	yes	Indian J Dermatol. 2009 Jul-Sep; 54(3):221-224
==== Front Indian J DermatolIJDIndian Journal of Dermatology0019-51541998-3611Medknow Publications India 20161852IJD-54-22910.4103/0019-5154.55630Basic ResearchSERUM MUCOSA-ASSOCIATED EPITHELIAL CHEMOKINE IN ATOPIC DERMATITIS: A SPECIFIC MARKER FOR SEVERITY Ezzat M H M Shaheen K Y 1From the Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt.1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt.Address for correspondence: Dr. Mohamed Ezzat, 25 EL-Sebak Street, Heliopolies, Cairo, Egypt. E-mail: [email protected] 2009 54 3 229 236 1 2008 3 2008 © Indian Journal of Dermatology2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Mucosa-associated epithelial chemokine (MEC; CCL28) is considered pivotal in mediating migration of CCR3 and CCR10-expressing skin-homing memory CLA+ T cells. CCL28 is selectively and continuously expressed by epidermal keratinocytes, but highly upregulated in inflammatory skin diseases such as atopic dermatitis (AD). Aims: This controlled longitudinal study was designed to evaluate the expression of CCL28 serum levels in childhood AD and bronchial asthma (BA) and its possible relations to disease severity and activity. Methods: Serum CCL28 levels were measured in 36 children with AD, 23 children with BA, and 14 children who had both conditions as well as in 21 healthy age and gender-matched subjects serving as controls. Sixteen patients in the AD group were followed-up and re-sampled for serum CCL28 after clinical remission. Serum CCL28 levels were correlated with some AD disease activity and severity variables. Results: Serum CCL28 levels in patients with AD whether during flare (median = 1530; mean ± SD = 1590.4 ± 724.3 pg/ml) or quiescence (median = 1477; mean ± SD = 1575.2 ± 522.1 pg/ml) were significantly higher than the values in healthy children (median = 301; mean ± SD = 189.6 ± 92.8 pg/ml). However, the levels during flare and quiescence were statistically comparable. The serum levels in BA (median = 340; mean ± SD = 201.6 ± 109.5 pg/ml) were significantly lower than the AD group and comparable with the healthy control values. Serum CCL28 levels in severe AD were significantly higher as compared with mild and moderate cases and correlated positively to the calculated severity scores (LSS and SCORAD). CCL28 levels during exacerbation of AD could be positively correlated to the corresponding values during remission, the peripheral absolute eosinophil counts, and the serum lactate dehydrogenase levels. Serum CCL28 did not vary with the serum total IgE values in AD. Conclusion: Our data reinforce the concept that CCL28 might share in the pathogenesis of AD probably through selective migration and infiltration of effector/memory Th2 cells into the skin. It may also represent an objective prognostic marker for disease severity. Further studies may pave the way for CCL28 antagonism among the adjuvant therapeutic strategies. AtopyCCL28mucosa-associated epithelial chemokine ==== Body Introduction Atopic dermatitis (AD) is a common chronic inflammatory skin disorder of unknown etiology expressed early in life with peak incidence in early childhood. Disease development is primarily determined by as yet unknown genetic factors leading to the accumulation of peripheral activated T lymphocytes in the skin. It was hypothesized that AD is associated with an unbalanced establishment of the peripheral T-lymphocyte system. Cutaneous lymphocyte-associated antigen (CLA)[1] is a cell surface glycoprotein that has been postulated to play an important role in T-cell migration and homing to the skin. It has been proposed that interaction between T cells and epidermal keratinocytes plays a central role in the pathogenesis of AD.[2] Mucosa-associated Epithelial Chemokine (MEC or CCL28) is a CC chemokine that has been recently identified and characterized and is a functional ligand for CC chemokine receptor-3 and 10 (CCR3 and 10). CCL28 gene is located on chromosome 5, and the corresponding cDNA encodes a 127 amino acid (AA) precursor protein with a putative 22 AA signal sequence. The signal sequence is cleaved to generate the 105 AA mature protein. Among human chemokines, CCL28 is most similar to the cutaneous T cell-attracting chemokine (CTACK or CCL27), with which is shares 40% AA identity.[3] CCL28 has been reported to chemoattract transfectants expressing the seven-transmembrane G-protein coupled CCR3 and CCR10. Desensitization experiments had confirmed CCR10 specificity. CCR10, previously known as orphan G-protein-coupled receptor (GPR-2), has been detected on the surface of T lymphocytes, dermal microvascular endothelial cells, and dermal fibroblasts, but mRNA expression studies suggest that it may be present on additional cell types. CCL28 induces calcium mobilization in human CCR10-expressing transfectants by CCL27, indicating that these chemokines share the new receptor, CCR10.[4] CCL28 is expressed in a wide range of human cell types and tissues. It appears to be predominantly produced by epithelial cells. Its mRNA expression is most abundant in salivary and mammary glands, human saliva, and breast milk.[5] Significant mRNA expression has also been observed in other tissues associated with mucosal epithelial surfaces, including the stomach, colon, rectum, nose, and trachea. The IL-10-/- knock-out mice display increased CCL28 expression in the stomach and Peyer's patches indicating that IL-10 may be CCL28 suppressive.[6] Studies showing CCL28 production in nasal and bronchial epithelial cell lines have suggested that it might also play a role in allergic respiratory diseases such as bronchial asthma (BA). To our knowledge, there are no clinical studies on serum CCL28 levels in allergic diseases in the pediatric age groups to date. Also, no data on CCL28 levels in other biological fluid of allergic children could be cited in literature. Therefore, this study was conducted to explore the expression of CCL28 in AD and BA, hence its pathogenic role in such diseases to identify the clinical relevance of measuring serum CCL28 in terms of disease activity and/or severity. Materials and Methods This follow-up controlled study was conducted over a period of 9 months from the first of January to the end of September 2005. All patients were enrolled from the Pediatric Allergy and Immunology Unit and outpatient clinic of the Ain Shams University Children's Hospital in Cairo. Informed consent was obtained from the parents or caregivers of each child before enrollment. Children were divided into the following three groups: Atopic dermatitis This group comprised 36 children with AD diagnosed by standardized diagnostic criteria.[7] Their ages ranged between 8 and 120 months (mean ± SD = 47 ± 22 months). The group consisted of 20 males (55.5%) and 16 females (44.5%). The parents were subjected to a questionnaire concerning the precipitating factor(s). A possible cause could be traced in 18 children being exposed to food allergens [egg (6), milk (5), banana (4), and fish (3)]. In 18 children, the cause was not clear. For convenience, the severity of AD was evaluated according to two scores. First, the Leicester Sign Score (LSS; range = 0 to 108) in which severity is scored by 6 clinical features (erythema, purulence, excoriation/crusting, dryness/scaling, cracking/fissuring, and lichenification) graded at 6 defined body sites on a scale of 0 (none) to 3 (severe).[89] Second, the Scoring Atopic Dermatitis (SCORAD) index system that was used to evaluate the severity of eczema. This index includes the evaluation of extent, intensity, and subjective symptoms to a maximum score of 103 points.[10] Children with AD were classified into 3 groups (mild, moderate, and severe) according to a proposal for severity grading of AD using only objective criteria (mild AD, SCORAD score <15; moderate AD, score = 15-40; and severe AD, score >40).[11] Sixteen children (44.5%) presented with severe AD, 11 children (30.5%) presented with moderate severity, and 9 children (25%) were categorized as having mild illness. In the 16 patients with severe AD, serum CCL28 levels were measured before and after treatment with topical corticosteroids in combination with oral antihistamines. The response to treatment was scored using the Physician's Global Assessment (PGA) score. The PGA is an overall assessment from 0 to 5 of a patient's eczema, taking into consideration the quality and extent of lesions relative to the baseline assessment [0 = clear (100%), 1 = almost clear (90% to 99% improvement), 2 = marked improvement (50% to 89%), 3 = modest improvement (<50%), 4 = no change, and 5 = worse]. A total of 22 patients had AD only. The remaining 14 children had concomitant asthma (personal history and/or clinical evidences of respiratory allergy). Bronchial asthma This group comprised 23 children enrolled while presenting with an acute asthmatic exacerbation. Their ages ranged between 14 and 90 months (mean ± SD = 49 ± 16 months). The group consisted of 12 males (52%) and 11 females (48%). From the patients' medical history, the asthma exacerbations were triggered by exposure to allergens (food, animal allergens, or both) in 12 children and upper respiratory tract infections in 5 children. In 6 children, the triggering agent was not clear. Ten children (43.5%) presented with acute severe asthma and the remaining 13 children (56.5%) presented with acute exacerbations of mild to moderate severity based on clinical data and peak expiratory flowmetry (PEFR).[1213] Control group This group comprised 21 healthy children without current or family history of allergic disorders. There were 12 males (57%) and 9 females (43%) in this group. Their ages ranged between 12 and 120 months with a mean ± SD of 77 ± 26 months. They underwent clinical examination to exclude any concomitant illness particularly allergic disorders and parasitic infestations. Children with peripheral blood eosinophilia or elevated serum IgE for their age were excluded.[14] Study Measurements Blood sampling A total of 4 ml of venous blood was collected under complete aseptic conditions from each patient or control subject and was divided into 2 equal parts. One sample was mixed with EDTA as an anticoagulant for blood counting and the other was left to clot for 30 minutes for serum CCL28 assay. After coagulation, the latter sample was centrifuged for 15 minutes at 1000 × g. The separated sera were divided into 3 aliquots and stored at −20°C for the subsequent assay of serum CCL28, lactate dehydrogenase (LDH), and total IgE. Repeated thawing and freezing was avoided. Hemolyzed samples were excluded. Serum CCL28/MEC assay The kit used was a human CCL28 immunoassay (Quantikine RandD Systems, Inc. McKinley Place N.E., Minneapolis, Minnesota 55413 U.S.A.). This assay employs the quantitative sandwich enzyme immunoassay technique using E. coli-expressed recombinant human CCL28 and antibodies raised against the recombinant factor. Results obtained using natural human CCL28 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. A monoclonal antibody specific for CCL28 has been pre-coated onto a microplate. Standards and samples were pipetted into the wells and any CCL28 present was bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase-linked monoclonal antibody specific for CCL28 was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution of hydrogen peroxide-tetramethylbenzidine was added to the wells and color developed in proportion to the amount of CCL28 bound in the initial step. The color development was stopped by 2N sulfuric acid and the intensity of the color was measured at 450 nm and 540 nm. Readings at 540 nm were subtracted. For calculation of results, a standard curve was constructed on a log/log scale by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and drawing a best fit curve through the points on the graph. The minimum detectable dose (MDD) ranged from 0.58-6.05 pg/mL. The mean MDD was 2.58 pg/mL. Serum total IgE assay This was performed in all subjects by quantitative enzyme immunoassay (Medix Biotech, San Carlos, CA, U.S.A.). Results were expressed in IU/ml. Owing to the variability in serum total IgE levels with age in childhood, we calculated the percentage values from the reference ranges by dividing the subject's actual level by the highest normal for age multiplied by 100.[14] IgE levels used in the correlations were both the measured and the calculated percentage values. The serum total IgE level that exceeded the highest normal for age was considered elevated. Complete blood count A Coulter counter (Coulter MicroDiff 18, Fullerton, CA, U.S.A.) was used in this study. The differential leukocytic counts were estimated manually from the blood film and expressed in absolute count values. Infants and children whose absolute eosinophil counts (AEC) exceeded the normal reference values for age were considered to have peripheral blood eosinophilia. Blood sampling of all subjects was performed at the same time (10 am) daily to avoid diurnal variations in eosinophil counts.[14] Serum total lactate dehydrogenase assay Serum total lactate dehydrogenase (LDH) (U/L) was measured only in the AD group during flare and in the healthy subjects. LDH assay was performed on an automatic chemistry analyzer (Synchron CX5 system, Beckman, Inc., Fullerton, CA, U.S.A.). Statistical analysis All statistical analysis of the data was carried out using Statistical Package for the Social Science (SPSS), Version 9.02 for Windows®. Data were expressed as mean, standard deviation (SD), median, and interquartile ranges IQR (25th and 75th percentiles). A Student's t test was used for comparing parametric data. For non parametric comparison of serum CCL28 among different groups, a Mann-Whitney U test was used. The relation between the various numerical parameters was studied by the Pearson correlation coefficient (r) test with graphic representation using a linear regression line; r value was considered weak if < 0.5, moderate if ranged between 0.5-0.75, and strong if > 0.75. P-values below 0.05 for all tests were considered significant. Results Serum CCL28 levels of the studied groups Serum CCL28 levels for patients with AD during a flare ranged between 687 and 2256 pg/ml (median = 1530, mean ± SD = 1590.4 ± 724.3 pg/ml) [Table 1]. These values were statistically comparable with the corresponding values of the same patients during quiescence, which ranged from 970 to 1980 pg/ml (median = 1477, mean ± SD = 1575.2 ± 522.1 pg/ml) (Z = 1.1; P > 0.05). The healthy children had much lower serum CCL28 levels (range = 10 to 460 pg/ml, median = 301, mean ± SD = 189.6 ± 92.8 pg/ml) as compared with the patients' data whether during flare (Z = 4.1; P < 0.0001) or quiescence (Z = 3.7; P < 0.0001) [Figure 1]. Table 1 Serum mucosa-associated epithelial chemokine in the study groups Serum MEC/CCL28 (pg/ml) AD BA (flare) AD + BA (flare) Control group Flare Quiescence Range 687-2256 970-1980 153-620 832-2059 10-460 Median 1530 1477 340 1425 301 IQR 1032 640 240 785 220 Mean 1590.4 1575.2 201.6 1601 189.6 SD 724.3 522.1 109.5 428.3 92.8 Z1 1.1 - - - - Z2 3.1* 2.9* - - - Z3 0.7 0.9 - - - Z4 - - 2.9* - - Z5 4.1* 3.7* 0.4 3.9* - AD = Atopic dermatitis; BA = Bronchial asthma; IQR = Interquartile range; MEC = Mucosa-associated epithelial chemokine; SD = Standard deviation; Mann-Whitney test results: Z1 = AD flare versus quiescence; Z2 = AD versus BA; Z3 = AD versus (both AD and BA); Z4 = BA versus (both AD and BA); Z5 = Patients versus controls; * = Highly significant (P < 0.0001) Figure 1 Box-plot summary of serum mucosa-associated epithelial chemokine (MEC/CCL28) levels (pg/ml) in atopic dermatitis and control subjects (A Mann–Whitney test was used in the comparisons. Black squares represent the median and the boxes encompass the interquartiles (25th and 75th percentiles). The ranges are marked as maximum and minimum) Serum CCL28 levels of asthmatic patients during exacerbations ranged between 153 and 620 pg/ml (median = 340, mean ± SD = 201.6 ± 109.5 pg/ml). These values were statistically comparable to the values of controls (Z = 0.4; P > 0.05), but were significantly lower than the values of children with AD whether during flare (Z = 3.1; P < 0.0001) or quiescence (Z = 2.9; P < 0.0001) [Figure 2]. Figure 2 A box-plot summary of serum mucosa-associated epithelial chemokine (MEC/CCL28) levels (pg/ml) in the study groups (A Mann–Whitney test was used in the comparisons) Within the group of patients with AD, 14 subjects had concomitant asthma as well. Their serum CCL28 levels ranged from 832 to 2059 pg/ml (median = 1425, mean ± SD = 1601 ± 428.3 pg/ml). These values were statistically comparable with the values of the patients from the AD only group whether during flare (Z = 0.7; P > 0.05) or quiescence (Z = 0.9; P > 0.05), but were significantly higher when compared with the values of subjects with BA only (Z = 2.9; P < 0.0001) [Figure 2] and the control group (Z = 3.9; P < 0.0001). Serum CCL28 levels in relation to other clinical and laboratory variables In our study, the mean SCORAD score values ranged between 10 and 65 (mean ± SD = 24.8 ± 10.6). The median and mean ± SD of serum CCL28 levels int he severe group (1809; 1754.8 ± 1110 pg/mL) were significantly higher than the corresponding values of the mild group (759; 785.1 ± 153.3 pg/ml; Z = 2.5; P < 0.001) or the moderate group (1190; 1165.9 ± 586.5 pg/ml; Z = 1.8; P < 0.01). Similarly, moderate cases had significantly higher values than the mild cases [Figure 3]. Moreover, serum CCL28 levels correlated positively with the LSS score and the SCORAD index (r = 0.89 and 0.70 respectively, P < 0.001 for both) [Figure 4], indicating CCL28 overexpression with increased severity. Figure 3 A box-plot summary of serum mucosa-associated epithelial chemokine (MEC/CCL28) level (pg/ml) variations according to the atopic dermatitis SCORAD index of severity (A Mann–Whitney test was used in the comparisons.) Figure 4 Serum mucosa-associated epithelial chemokine (MEC/CCL28) levels (pg/ml) in relation to atopic dermatitis severity scores; (a) the LSS and (b) the SCORAD index A positive correlation could be elicited between the CCL28 serum levels during AD exacerbation and the corresponding values during remission meaning that the higher the level during acute attacks, the higher it remained after remission (r = 0.91; P < 0.0001) [Figure 5]. Figure 5 Positive correlation between the atopic dermatitis flare and remission levels of serum CCL28 (pg/ml) The mean ± SD absolute eosinophil count values were statistically comparable among subjects with AD, BA, and AD associated with BA (650 ± 235, 590 ± 197, and 660 ± 301 cells/mm3, respectively). However, each value was significantly higher when compared with the control group (220 ± 90 cells/mm3; P < 0.01 for all groups). Serum CCL28 levels correlated positively with the absolute eosinophil count in all groups during flare (r = 0.73; 0.42 and 0.65, respectively; P < 0.01). A similar correlation was found in the AD group during quiescence (r = 0.87; P < 0.01). Another positive correlation could be elicited between the SCORAD index and the absolute eosinophil count in the AD group (r = 0.46; P < 0.05). The studied patient groups (AD, BA, and AD associated with BA) were statistically comparable in terms of serum IgE levels (500.6 ± 385.6, 655 ± 492.5, and 627.9 ± 336.6 IU/ml, respectively) and each mean value was significantly higher than that of the healthy control subjects (140.6 ± 19.7 IU/ml; P < 0.001). Serum CCL28 or SCORAD index values could not be related by correlation coefficient to IgE concentrations or IgE percent values from normal in the studied groups. Serum total LDH levels of patients with AD during a flare ranged between 130 and 1450 U/L (mean ± SD = 1036.6 ± 486.2 U/L). These values were significantly higher than the control values (range = 40–110 U/L; mean ± SD = 70 ± 25 U/L, t = 1.2; P < 0.05). LDH values correlated positively with each of the serum CCL28 levels and the SCORAD indices [Figure 6]. Figure 6 Positive correlations between serum total lactate dehydrogenase (LDH) (U/L) and serum CCL28 (pg/ml) (a) and the SCORAD index (b) in atopic dermatitis during flare Serum CCL28 levels could not be correlated to age, weight centiles, and height centiles of subjects in each group. CCL28 expression was not influenced by age, gender, or family history. In the asthma group, CCL28 levels did not vary according to the PEFR values of patients. Discussion MEC or CCL28 has been implicated in the homing of lymphocytes to the inflammatory sites. It was assumed to play an important role in T-cell migration to skin in adults with AD, psoriasis vulgaris, and bullous pemphigoid;[15] however, the expression of CCL28 in both normal children and children with atopic diseases has not been extensively studied. To evaluate a possible role of CCL28 estimation in pediatric allergy, we measured serum CCL28 levels in phenotypically well-defined groups of allergic children (AD, BA, and AD associated with BA) in comparison with a group of healthy non atopic age and gender-matched control subjects. We observed that serum CCL28 levels were specifically elevated in patients with AD irrespective of allergic respiratory comorbidity. The study revealed that the levels in AD whether during a flare or exacerbation were significantly higher than the control group [Figure 1] and the asthmatic group [Figure 2]. Our results, which conform with those of Kagami, et al.,[15] probably reflect the upregulation of CCL28 in AD presenting a potentially useful marker for the presence of an atopic reaction. CCL28 regulation by allergen exposure and microbial products suggests an important role for CCL28 in the initiation and amplification of the atopic skin response. The failure to demonstrate a significant reduction in serum CCL28 levels after remission of the flare [Figure 1] could be due to the ongoing process of subclinical allergic cutaneous inflammation. Also, it could be indicative of the need for more intensive non conventional anti-inflammatory therapy. Therefore, we suggest that serum CCL28 levels be used as a marker for the ongoing inflammatory activity in the skin. Among human chemokines, CTACK (CCL27) is most similar to MEC (or CCL28) sharing 40% AA identity.[3] Like CCL28, CCL27 is also known to recruit CLA+ memory Th2 lymphocytes.[16] Dose-dependent chemotaxis of identical lymphocyte populations in response to CCL28 is nearly indistinguishable from that exhibited by CCL27; except that CCL28 has higher maximal activity.[1718] CCL27 was found to be expressed at high levels in the epidermis of lesional skin biopsy specimens from patients with AD compared with non lesional skin.[1920] Owing to the tissue-specific expression profile and receptor specificity of both chemokines (CCL27 and CCL28), a definite role of CCL28 in AD is highly suggested. CCL28 is expressed in a variety of human tissues, and it appears to be predominantly produced by epithelial cells.[5] It is strongly expressed constitutively in lesional epidermal keratinocytes of patients with AD and psoriasis vulgaris.[15] In vitro, CCL28 displays chemotactic activity for resting CD4+ CD8+ T cells.[4] These cells can be detected in a significant proportion in the peripheral blood of children with AD.[1] This may suggest a faulty maturation of the T-lymphocyte system as a basic pathophysiological change in AD, leading to skin inflammation with CD4+ CD8+ T lymphocytes resembling immature T cells.[21] This is likely to lead to the skewing of many immune reactions in those patients. CCL28 is also displayed by cutaneous venules and is strongly expressed on endothelial cells. It is thought that it triggers vascular arrest of circulating skin homing memory T cells, which uniformly express CCR10, particularly CLA+ CCR10+ memory T cells. Chronic cutaneous inflammation induces CD4+ T cells expressing E-selectin binding activity in draining lymph nodes. CLA is a ligand for E-selectin, an adhesion molecule, which is critical in recruiting T cells. These E-selectin ligand+ T cells migrate efficiently to CCL28 and to CCL27 as well.[2223] This could reinforce the hypothesis that CCL28 and CCL27 may work in a sequential way, with CCL28 acting mainly in the first steps of T-cell recruitment by inducing integrin-dependent adhesion and transendothelial migration of T cells and CCL27 playing a major role in the migration of T cells into the upper layers of the skin. Finally, T cells infiltrating the epidermis may result in the induction of apoptosis in keratinocytes, resulting in spongiosis, which is the histologic hallmark of eczema. To test the specificity of CCL28 response in AD, we sought to investigate its expression in a group of subjects with asthma during acute exacerbations. The serum CCL28 levels of patients with asthma were statistically comparable to the values of controls, but were significantly lower as compared with the values of children with AD whether during flare or quiescence. The normal values of serum CCL28 in our patients with asthma indicates that the results of studies that use bronchial cell lines and mouse models[2425] cannot simply be extrapolated to human studies in vivo. It is also possible that the local production of CCL28 in mucosal epithelial cells may be insufficient to lead to a systemic increase. In children who presented with concomitant allergic cutaneous and respiratory diseases (BA and AD), the overexpression of CCL28 can be ascribed to the presence of AD, supporting further evidence for the implication of CCL28 in atopic cutaneous responses. Our data may thus reflect the upregulation of CCL28 in AD specifically; however, our findings are still limited by the sample size. Serum CCL28 levels in subjects categorized as severe AD cases were significantly higher as compared with the moderate and mild groups with a positive correlation to the LSS and SCORAD severity scores [Figures 3 and 4]. This substantiates the usefulness of this parameter as a skin-specific objective marker of disease severity in AD. A large variety of laboratory measurements[2627] have been linked to disease activity and severity in AD, however most of them lack specificity. The high levels of IgE in AD have not been satisfactorily explained but can be ascribed to a deficiency of IgE isotype-specific “suppressor” T-cell function. It has not been established that AD is exclusively an IgE-mediated allergic disorder; and it is difficult to demonstrate consistently a role for allergens in the pathogenesis. Although patients with AD frequently possess IgE antibody specific for inhalants or food allergens, it is not generally possible to induce skin lesions or AD by intradermal injection of the suspected allergen.[2829] Moreover, typical lesions of AD may occur in individuals with X-linked agammaglobulinemia who have virtually no IgE.[7] Serum CCL28 levels and the SCORAD score results in the current study could not be correlated to the serum total IgE concentrations or percent values from normal during AD flare. Our results were not concordant with some reports.[2728] The discrepancy can be ascribed to the small number of cases with elevated IgE category (6 children only). We also recruited subjects at different stages of disease activity and serum total IgE levels are known to fluctuate throughout the disease stages. On the other hand, serum CCL28 concentrations and the SCORAD indices correlated positively with the absolute eosinophil counts both during AD flare and quiescence. Previous studies had identified eosinophil chemoattractant properties of CCL28 using human cells.[424] It is not clear whether CCL28 may have a role in regulating the constitutive numbers of eosinophils in peripheral blood by inducing eosinophil chemotaxis in a CCR3 and CCR10-dependent manner. Animal studies confirmed that CCL28 was a chemoattractant for murine eosinophils. There appears to be a temporal relationship of CCL28 production with eotaxin and other CCR3 ligands in animal models.[24] Perhaps they play an important role for the localization of eosinophils around the inflammatory sites. Interestingly, anti-CCL28-treated animals also showed reduced eosinophil accumulation.[30] Therefore, we might conclude that CCL28, a CCR3 ligand, is important for various stages of eosinophil migration and/or activation within tissues. It might be possible one day to target tissue eosinophilia in severe allergic diseases by manipulating CCL28/CCR3 interactions. The serum total LDH levels of our patients with AD during flare were significantly higher as compared with controls. Moreover, significant positive correlations could be elicited between serum LDH levels during flare and serum CCL28 concentrations and SCORAD indices [Figure 6]. Elevated levels can be attributed to its release from damaged lesional epidermal cells into peripheral blood reflecting the extent of skin involvement and the degree of epidermal injury. LDH was previously reported as useful yet it was not a specific marker for severity of skin eruption being elevated in many other diseases.[3132] In conclusion, it seems that serum CCL28 expression is upregulated in children with AD irrespective of allergic respiratory comorbidity. It might be one of the important chemokines in the lesional formation of AD and might have a distinct role in the development of atopy/allergy skin phenotypes. It could be a useful specific inflammatory marker for assessing AD severity in children. Further research using immunohistochemical staining of CCL28 in the lesional AD skin and other cutaneous hypersensitivities are warranted as well as studies addressing the relations between its expression and the conventional lines of treatment used. A potential role of CCL28 in respiratory allergy deserves further evaluation on a wider scale. Moreover, the effect of CCL28 antagonists and other immunotherapeutics, like monoclonal antibodies on the CCL28 kinetics is worth evaluation. CCL28 antagonism may provide a conceptual basis for the development of new therapeutic strategies in cases of severe AD. Source of Support: Nil Conflict of Interest: Nil. ==== Refs References 1 Olesen AB Andersen G Jeppesen DL Benn CS Juul S Thestrup-Pedersen K Thymus is enlarged in children with current atopic dermatitis: A cross-sectional study Acta Derm Venereol 2005 85 240 3 16040410 2 Vestergaard C Johansen C Otkjaer K Deleuran M Iversen L Tumor necrosis factor-alpha-induced CTACK/CCL27 (cutaneous T-cell-attracting chemokine) production in keratinocytes is controlled by nuclear factor kappaB Cytokine 2005 29 49 55 15598438 3 Hieshima K Ohtani H Shibano M Izawa D Nakayama T Kawasaki Y CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity J Immunol 2003 170 1452 61 12538707 4 Wang W Soto H Oldham ER Buchanan ME Homey B Catron D Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2) J Biol Chem 2000 275 22313 23 10781587 5 Wilson E Butcher EC CCL28 controls immunoglobulin (Ig)A plasma cell accumulation in the lactating mammary gland and IgA antibody transfer to the neonate J Exp Med 2004 200 805 9 15381732 6 Hieshima K Kawasaki Y Hanamoto H Nakayama T Nagakubo D Kanamaru A CC chemokine ligands 25 and 28 play essential roles in intestinal extravasation of IgA antibody-secreting cells J Immunol 2004 173 3668 75 15356112 7 Leung DY Behrman RE Kleigman RM Jenson HB Atopic dermatitis Nelson Textbook of Pediatrics 2004 17th ed Philadelphia WB Saunders Company 774 8 8 Sowden JM Berth-Jones J Ross JS Motley RJ Marks R Finlay AY Double-blind, controlled, crossover study of cyclosporin in adults with severe refractory atopic dermatitis Lancet 1991 338 137 40 1677063 9 Kunz B Oranje AP Labreze L Stalder JF Ring J Taieb A Clinical vali-dation and guidelines for the SCORAD index: Consensus report of the European Task Force on Atopic Dermatitis Dermatology 1997 195 10 9 9267730 10 Pucci N Novembre E Cammarata MG Bernardini R Monaco MG Calogero C Scoring atopic dermatitis in infants and young children: Distinctive features of the SCORAD index Allergy 2005 60 113 6 15575941 11 Sprikkelman AB Tupker RA Burgerhof H Schouten JP Brand PL Heymans HS Severity scoring of atopic dermatitis: A comparison of three scoring systems Allergy 1997 52 944 9 9298180 12 Liu AH Spahn JD Leung DY Behrman RE Kleigman RM Jenson HB Childhood asthma Nelson textbook of pediatrics 2004 17th ed Philadelphia WB Saunders 760 73 13 National Heart, Lung, and Blood Institute (NHLBI) National Asthma Education and Prevention Program. Expert Panel Report 2: Guidelines for the Diagnosis and Management of Asthma NIH Publication No. 97-4051A 1997 US Department of Health and Human Services, National Institute of Health Bethesda, MD 14 Geaghan SM Hoffman R Benz EJ Shattil SJ Furie B Cohen HJ Silberstein LE McGlave P Normal blood values: Selected reference values for neonatal, pediatric and adult populations Hematology basic principles and practice 2000 3rd ed New York Churchill Livingston 2520 8 15 Kagami S Kakinuma T Saeki H Tsunemi Y Fujita H Sasaki K Increased serum CCL28 levels in patients with atopic dermatitis, psoriasis vulgaris and bullous pemphigoid J Invest Dermatol 2005 124 1088 90 15854059 16 Homey B Alenius H Muller A Soto H Bowman EP Yuan W CCL27-CCR10 interactions regulate T cell-mediated skin inflammation Nat Med 2002 8 157 65 11821900 17 Morales J Homey B Vicari AP Hudak S Oldham E Hedrick J CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells Proc Natl Acad Sci USA 1999 96 14470 5 10588729 18 Romagnani S Cytokines and chemoattractants in allergic inflammation Mol Immunol 2002 38 881 5 12009564 19 Hijnen D De Bruin-Weller M Oosting B Lebre C De Jong E Bruijnzeel-Koomen C Serum thymus and activation-regulated chemokine (TARC) and cutaneous T cell-attracting chemokine (CTACK) levels in allergic diseases: TARC and CTACK are disease-specific markers for atopic dermatitis J Allergy Clin Immunol 2004 113 334 40 14767451 20 Kakinuma T Saeki H Tsunemi Y Fujita H Asano N Mitsui H Increased serum cutaneous T cell-attracting chemokine (CCL27) levels in patients with atopic dermatitis and psoriasis vulgaris J Allergy Clin Immunol 2003 111 592 7 12642842 21 Bang K Lund M Wu K Mogensen SC Thestrup-Pedersen K CD4+ CD8+ (thymocyte-like) T lymphocytes present in blood and skin from patients with atopic dermatitis suggest immune dysregulation Br J Dermatol 2001 144 1140 7 11422033 22 Giustizieri ML Mascia F Frezzolini A De Pita O Chinni LM Giannetti A Keratinocytes from patients with atopic dermatitis and psoria-sis vulgaris show a distinct chemokine production profile in response to T cell-derived cytokines J Allergy Clin Immunol 2001 107 87l 7 11149996 23 Reiss Y Proudfoot AE Power CA Campbell JJ Butcher EC CC chemokine receptor (CCR) 4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin J Exp Med 2001 194 1541 7 11714760 24 John AE Thomas MS Berlin AA Lukacs NW Temporal production of CCL28 corresponds to eosinophil accumulation and airway hyperreactivity in allergic airway inflammation Am J Pathol 2005 166 345 53 15681819 25 Humbles AA Lu B Friend DS Okinaga S Lora J Al-Garawi A The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyper responsiveness Proc Natl Acad Sci USA 2002 99 1479 84 11830666 26 Charman C Chambers C Williams H Measuring atopic dermatitis severity in randomized controlled clinical trials: What exactly are we measuring? 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==== Front BMC MicrobiolBMC Microbiology1471-2180BioMed Central 1471-2180-10-12005110710.1186/1471-2180-10-1Research articleMolecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells Takamatsu Reika [email protected] Hiromitsu [email protected] Eriko [email protected] Chie [email protected] Kunihiro [email protected] Naofumi [email protected] Jian-Dong [email protected] Klaus [email protected] Futoshi [email protected] Jiro [email protected] Naoki [email protected] Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan2 Division of Control and Prevention of Infectious Diseases, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan3 Transdisciplinary Research Organization for Subtropics and Island Studies, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0215, Japan4 Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan5 Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan6 Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, USA7 Project group 26 "Nosocomial Infections of the Elderly", Robert Koch-Institut, 20 Nordufer, Berlin 13353, Germany2010 5 1 2010 10 1 1 19 7 2009 5 1 2010 Copyright ©2010 Takamatsu et al; licensee BioMed Central Ltd.2010Takamatsu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. Results Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter. Conclusions Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease. ==== Body Background The gram-negative bacteria Legionella pneumophila is the causative pathogen of Legionnaires' disease, a potentially fatal type of pneumonia affecting both immunocompromised and immunocompetent subjects. This bacterium is a facultative intracellular pathogen of amoeba in natural and man-made aquatic environments. Infection of humans occurs after inhalation of contaminated water aerosol droplets. Dependent on its type IV secretion system Dot/Icm, L. pneumophila initiates biogenesis of a specialized vacuole that it critical for Legionella replication [1]. This Legionella-containing vacuole avoids fusion with lysosomes and acquires vesicles from the endoplasmic reticulum [2]. In addition, the bacterial flagellum with its major component flagellin is also considered to represent a virulence-associated factor [3]. For L. pneumophila pathogenesis, important results were obtained by analyzing infection of protozoans or immune cells like macrophages [4]. However, recent studies have shown that L. pneumophila replicates also in human alveolar epithelial cells [5,6]. Although Legionella less efficiently replicates within human T cells compared with macrophages [7], little is known of the consequences of T cell infection with Legionella. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. The results demonstrated that L. pneumophila interacted with and infected T cells. To investigate L. pneumophila-T cell interactions, we examined whether L. pneumophila induces production of interleukin-8 (IL-8), an inflammatory chemokine associated with immune-mediated pathology and involved in recruitment and activation of neutrophils and other immune cells. The results showed that L. pneumophila directly increased IL-8 by activation of transforming growth factor β-associated kinase 1 (TAK1), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK), leading to activation of transcription factors, NF-κB, AP-1, cyclic AMP response element (CRE) binding protein (CREB), and activating transcription factor-1 (ATF1). Results Multiplication of L. pneumophila in human T cells To investigate the interaction of L. pneumophila with T cells, we first examined intracellular growth of L. pneumophila strain AA100jm in Jurkat cells by 72-h continuous cultures. The CFU per well of AA100jm growing in Jurkat cell cultures began to increase after 24 h and then increased time-dependently (Fig. 1A). However, the CFU of the avirulent mutant strain with a knockout in dotO, encoding a protein essential for type IV secretion system, did not increase during the 72-h period (Fig. 1A). In contrast, the multiplication of flaA mutant did not change in Jurkat cells compared with the wild-type Corby (Fig. 1B). To characterize the multiplication of L. pneumophila in human T cells, intracellular growth in CD4+ T cells of L. pneumophila was examined. The CFU of the wild-type Corby increased after infection for 24 h in CD4+ T cells, although it replicated less efficiently compared with the observations with Jurkat cells (Fig. 1C). Staining of the infected Jurkat cells for L. pneumophila showed increased intracellular replication of AA100jm, Corby, and flaA mutant, but not dotO mutant after 24 h in culture (Fig. 1D and 1E). These observations suggest that L. pneumophila can replicate in human T cells and the type IV secretion system plays a role in L. pneumophila replication in human T cells. Figure 1 Intracellular growth of L. pneumophila strains in Jurkat cells and CD4+ T cells. Jurkat cells were infected with L. pneumophila strains AA100jm and dotO mutant (MOI of 100) (A) or Corby and flaA mutant (MOI of 100) (B). (C) CD4+ T cells were also infected with Corby (MOI of 50). At the indicated time points after infection, the CFU was enumerated. Data are mean ± SD of triplicate cell cultures. (D and E) Direct fluorescent antibody staining of L. pneumophila strains. Jurkat cells were infected with AA100jm and dotO mutant (MOI of 100) (D) or Corby and flaA mutant (MOI of 100) (E) for 24 h. Jurkat cells were stained with fluorescein-conjugated anti-L. pneumophila antibody. Original magnification, ×600. High serum IL-8 levels in patients with Legionella pneumonia To investigate the role of IL-8 in the pathogenesis of Legionella pneumonia, the circulating concentrations of IL-8 were measured. Serum IL-8 levels were higher in patients with Legionella pneumonia (n = 18) (189 ± 493 pg/ml) than in normal healthy controls (n = 16) (9.79 ± 15.06 pg/ml), although this difference was not statistically significant (P = 0.157). Therefore, we analyzed the signaling pathways for IL-8 activation by Legionalla infection. Infection of Jurkat and CD4+ T cells by L. pneumophila induces IL-8 expression Jurkat cells were infected with wild-type L. pneumophila strains AA100jm and Corby for up to 12 h. Total cellular RNA was isolated from these cells at 0.5, 1, 2, 4, 6, 8 and 12 h after the infection and IL-8 gene expression was analyzed by RT-PCR. IL-8 mRNA expression increased after the infection (Fig. 2A). In another series of experiments, in which Jurkat cells were infected with AA100jm and Corby at different concentrations for 4 h (Fig. 2B), both strains induced dose-dependent expression of IL-8 mRNA. Next, we examined the correlation between IL-8 expression levels and the virulence of L. pneumophila. As shown in Fig. 2A, IL-8 mRNA expression was induced after infection with the avirulent dotO mutant, but became gradually weaker from 8 to 12 h. In contrast, a flaA knockout mutant, defective in flagellin production, failed to induce IL-8 mRNA after infection (Fig. 2A). To characterize the effect of L. pneumophila infection on human T cells, IL-8 mRNA expression in CD4+ T cells in response to L. pneumophila was examined by RT-PCR. After infection for 3 h, L. pneumophila induced IL-8 mRNA expression in CD4+ T cells, similar to the observations with Jurkat cells (Fig. 2C). Figure 2 L. pneumophila-induced IL-8 mRNA expression in T cells. (A) Total RNA was extracted from Jurkat cells infected with AA100jm, dotO mutant, Corby, or flaA mutant (MOI of 100) for the indicated time intervals and used for RT-PCR. (B) Jurkat cells were infected with the indicated concentrations of L. pneumophila for 4 h. Total RNA was extracted and used for RT-PCR. (C) Total RNA was extracted from CD4+ T cells infected with Corby (MOI of 50) for 3 h and used for RT-PCR. (D) Jurkat cells were infected with live L. pneumophila Corby or flaA mutant (MOI of 100) for 4 h or incubated with L. pneumophila under the indicated treatment for 4 h. PFA, paraformaldehyde. Total RNA was extracted and used for RT-PCR. Representative examples of three experiments with similar results. To determine the correlation between IL-8 expression level and L. pneumophila bacterial proteins, heat-killed Corby was used to infect Jurkat cells at a multiplicity of infection (MOI) of 100. At 4 h, IL-8 was not expressed in Jurkat cells infected with the heat-killed strain (Fig. 2D). Furthermore, IL-8 gene expression was not induced when paraformaldehyde-fixed L. pneumophila was used (Fig. 2D). However, bacteria heated at 56°C for 30 min induced IL-8 expression. These results suggest that the surface proteins of bacteria but not lipopolysaccharide are required for IL-8 induction. Considered together, it seems that Legionella flagellin is involved in IL-8 expression in T cells. Flagellin is recognized by toll-like receptor 5 (TLR5) [8]. Thus, we also examined the expression of TLR2, TLR3, TLR4, and TLR5 mRNAs in Jurkat and CD4+ T cells. All TLR mRNAs examined were expressed in Jurkat and CD4+ T cells (Fig. 3A and 3B). Furthermore, their expression levels did not change by L. pneumophila infection in CD4+ T cells (Fig. 3B) and Jurkat cells (data not shown). Figure 3 TLR mRNA expression in T cells. (A) Expression of TLR mRNA in Jurkat cells. Total RNA was extracted from Jurkat cells and used for RT-PCR. (B) CD4+ T cells were infected without or with Corby (MOI of 50) for 3 h. Total RNA was extracted from CD4+ T cells and used for RT-PCR. Representative examples of three experiments with similar results. IL-8 production from Jurkat cells during infection with L. pneumophila We used enzyme-linked immunosorbent assay (ELISA) to determine IL-8 protein levels in culture supernatants of Jurkat cells at 8, 12, or 24 h after infection with either the parental strain Corby or flaA mutant strain at an MOI of 100. IL-8 was induced by Corby in a time-dependent manner. On the other hand, the amount of IL-8 produced by Jurkat cells infected with the flaA mutant strain was significantly less than that by cells infected with the wild-type strain (Fig. 4A). Corby-induced IL-8 production by Jurkat cells was MOI-dependent (Fig. 4B). Corby also induced a significant amount of IL-8 from CD4+ T cells (Fig. 4C). Figure 4 IL-8 production from Jurkat cells during infection with L. pneumophila strains. (A) Jurkat cells were infected with the indicated L. pneumophila strains at an MOI of 100 for the indicated time periods. (B) Jurkat cells were infected with the varying concentrations of the indicated L. pneumophila strains for 24 h. (C) CD4+ T cells were infected without or with Corby for 3 h. IL-8 concentrations in the supernatants were determined by ELISA. Data are mean ± SD values collected in three experiments. L. pneumophila induces IL-8 gene transcription via a sequence spanning positions -133 to -50 of the IL-8 gene promoter To delineate the mechanism by which L. pneumophila induces IL-8 gene transcription, we identified L. pneumophila-responsive promoter elements in the IL-8 promoter. This was achieved by transfecting Jurkat cells with various plasmid constructs containing the luciferase reporter gene driven by the IL-8 promoter. Twenty-four hours post-transfection, cells were infected with L. pneumophila strain Corby. L. pneumophila infection resulted in activation of the 5' region 1,481 bp full-length promoter in an MOI-dependent manner (Fig. 5A). These results indicate that L. pneumophila induces IL-8 expression in Jurkat cells at transcriptional level. Next, we used a deletion analysis approach to identify the essential promoter element(s) for transcriptional upregulation following a stimulus. High induction levels were observed with a reporter construct containing IL-8 5'-flanking sequence starting with position -1,481 to position -133. Deletion of sequences upstream of position -50 abolished induction of IL-8 by L. pneumophila infection (Fig. 5B). The IL-8 gene fragment spanning positions -133 to -50 bp contains three prominent DNA-protein interaction sites for the transcription factors AP-1, nuclear factor IL-6 (NF-IL-6), and NF-κB (Fig. 5B). This maps the region from -133 to -50 bp as a L. pneumophila-responsive region, which is likely to contain individual L. pneumophila-responsive regulatory elements. Figure 5 L. pneumophila infection activates IL-8 promoter in Jurkat cells. (A) Jurkat cells transfected with -1481-luc were infected with L. pneumophila Corby at the indicated MOI values for 6 h. The luciferase activities were expressed relative to cells transfected with -1481-luc followed by mock-infection. , P < 0.01, as determined by the Student t test. (B) Reporter assay using plasmid DNA containing serial deletions in 5'-flanking region of the IL-8 gene. (Left) Schematic representation of the IL-8 reporter constructs, demonstrating locations of several known binding sites for transcription factors. (Right) The indicated luciferase reporter constructs were transfected into Jurkat cells, and the cells were subsequently infected with Corby strain (MOI of 100) for 6 h. The activities are expressed relative to that of cells transfected with -50-luc followed by mock-infection, which was defined as 1. The numbers on the bars depict fold induction relative to the basal level measured in uninfected cells. (C) Relative importance of AP-1, NF-IL-6, and NF-κB binding sites in IL-8 promoter. Wild-type and mutated plasmids were transfected into Jurkat cells. The transfected cells were infected without or with Corby. The activities are expressed relative to that of cells transfected with -133-luc followed by mock-infection, which was defined as 1. Luciferase activities were normalized based on the Renilla luciferase activity from phRL-TK. The numbers on the bars depict fold induction relative to the basal level measured in uninfected cells. LUC, luciferase. Graph data are mean ± SD values of three experiments. To identify the cis-acting element(s) in the -133 to -50 bp region of the IL-8 promoter, which served as a L. pneumophila-responsive regulatory element, we prepared and tested site-directed mutant constructs (Fig. 5C). Mutation in the NF-κB site (NF-κB mut-luc) and AP-1 site (AP-1 mut-luc) suppressed L. pneumophila-induced IL-8 expression. However, mutation of the NF-IL-6 site (NF-IL-6 mut-luc) had no such effect. These results indicate that activation of the IL-8 promoter in Jurkat cells in response to L. pneumophila infection requires an intact binding site for the NF-κB and AP-1 elements. Flagellin-dependent activation of NF-κB Because the internal mutational analysis of IL-8 promoter indicated that L. pneumophila infection activated transcription through the NF-κB site, it was important to identify the nuclear factor(s) that binds to this site. The NF-κB sequence derived from the IL-8 promoter was used as a probe in electrophoretic mobility shift assay (EMSA). Jurkat cells were infected with Corby strain at different times after challenge, and nuclear protein extracts were prepared and analyzed to determine NF-κB DNA binding activity. As shown in Fig. 6A, a complex was induced in these cells within 30 min after infection with Corby and increased in a time-dependent manner. This NF-κB binding activity to IL-8 promoter was reduced by the addition of either cold probe or a typical NF-κB sequence derived from the IL-2 receptor (IL-2R) α-chain (IL-2Rα) enhancer but not by an oligonucleotide containing the AP-1 binding site (Fig. 6B, lanes 3 to 5). Next, we characterized the L. pneumophila-induced complexes identified by the IL-8 NF-κB probe. These complexes were diminished and supershifted by the addition of anti-p50 or anti-p65 antibody (Fig. 6A, lanes 6 to 10), suggesting that L. pneumophila-induced IL-8 NF-κB complexes are composed of p50 and p65. Based on these results, one can conclude that L. pneumophila infection seems to induce IL-8 gene expression at least in part through induced binding of p50 and p65 to the NF-κB site in the IL-8 promoter region. Figure 6 NF-κB signal is essential for flagellin-dependent activation of the IL-8 promoter by L. pneumophila. (A) Flagellin is required for induction of NF-κB binding activity. Nuclear extracts from Jurkat cells infected with Corby or flaA mutant were mixed with IL-8 NF-κB probe (MOI, 100:1). (B) Sequence specificity of NF-κB binding activity and characterization of proteins that bound to the NF-κB binding site. Competition assays were performed with nuclear extracts from cells infected with Corby for 2 h. 100-fold excess amounts of competitor were added (lanes 3 to 5). A supershift assay in the same nuclear extracts also was performed. Antibodies (Ab) were added (lanes 6 to 10). Arrows indicate specific complexes, while arrowheads indicate the DNA binding complexes supershifted. (C) Flagellin-induced p65 translocation. Cells were infected with Corby or flaA mutant. Nuclear extracts were subjected to immunoblotting. (D) Flagellin activates NF-κB through the classical and alternative pathways. Cells were infected with Corby or flaA mutant. Lysates were subjected to immunoblotting. (E) Overexpression of dominant negative mutants inhibits L. pneumophila-induced activation of the IL-8 promoter. Cells were transfected with -133-luc and the mutant plasmids and then infected with Corby for 6 h. The solid bar indicates luciferase activity of -133-luc and empty vector without infection. Activity is expressed relative to that of cells transfected with -133-luc with further Corby infection, which was defined as 100. Data are means ± SD values of three experiments. dn, dominant negative. , P < 0.05; **, P < 0.001 (by Student t test). As described above, the flaA mutant strain failed to induce mRNA expression and production of IL-8. Next, we determined whether the flaA mutant strain induces NF-κB DNA binding activity. As expected, NF-κB DNA binding activity was not induced by the isogenic flaA mutant, unlike the wild-type strain Corby (Fig. 6A). These results indicate that better activation of NF-κB binding by flaA-positive strain is the underlying mechanism of the observed activation of the IL-8 promoter by this bacterial strain. Considered together, these results indicate that L. pneumophila infection induces IL-8 gene expression at least in part through the induced binding of p50 and p65 NF-κB family members to the NF-κB element of the IL-8 promoter and that this effect is dependent on flagellin. Because nuclear translocation is a key step for transcriptional activity [9], we next examined whether L. pneumophila induces the nuclear translocation of NF-κB. As shown in Fig. 6C, the wild-type Corby, but not the flaA mutant, induced nuclear translocation of NF-κB. NF-κB is normally present in the cytoplasm in an inactive state and is bound to members of the IκB inhibitor protein family, chiefly IκBα. In this complex, IκBα blocks the nuclear localization signal, thus preventing nuclear translocation. Translocation of NF-κB into the nucleus requires disruption of the cytoplasmic NF-κB:IκBα complex [9]. To determine the role of IκBα phosphorylation and degradation in L. pneumophila-induced NF-κB translocation and activation, we investigated whether L. pneumophila induces phosphorylation and degradation of IκBα. The latter two processes were examined by Western blot analysis using antibodies against phosphorylated and total IκBα, respectively. Fig. 6D shows phosphorylation and degradation of IκBα in Jurkat cells infected with the wild-type Corby but not the flaA mutant for 1, 2 and 4 h. The IκBα phosphorylation became evident at 1 h and decreased thereafter. Consistent with this, Corby-induced degradation of IκBα was observed at 1 h. NF-κB signaling occurs either through the classical or alternative pathway [10]. In the classical pathway, NF-κB dimers, such as p50/p65, are maintained in the cytoplasm by interaction with IκBα. Whereas the classical NF-κB activation is IκB kinase β(IKKβ)- and IKKγ-dependent and occurs through IκBα phosphorylation and subsequent proteasomal degradation, the alternative pathway depends on IKKα homodimers and NF-κB-inducing kinase (NIK) and results in regulated processing of the p100 precursor protein to p52 via phosphorylation and degradation of its IκB-terminus [10]. Indeed, the wild-type Corby but not the flaA mutant induced phosphorylation of p65 and upstream kinase IKKβ (Fig. 6D). Next, we examined the alternative pathway, which involves the cleavage of NF-κB2/p100 to p52. The level of p52 protein increased in Jurkat cells infected with the wild-type Corby but not the flaA mutant (Fig. 6D), indicating that flagellin activates NF-κB via the alternative pathway. NF-κB signal is essential for induction of IL-8 expression by L. pneumophila To further confirm the involvement of IκBα degradation, we transfected the cells with transdominant mutant of IκBα in which two critical serine residues required for inducer-mediated phosphorylation were deleted [11]. As seen in Fig. 6E, overexpression of mutant IκBα greatly inhibited the Corby-induced IL-8 promoter activation. This observation implicates the involvement of IκBα phosphorylation and degradation in flagellin-induced IL-8 expression. To address the mechanism of flagellin-mediated IL-8 expression, we investigated the role of NIK and IKK in L. pneumophila-induced IL-8 expression. Cotransfection with the dominant-negative mutant forms of NIK, IKKα, IKKβ, and IKKγ inhibited L. pneumophila-induced IL-8 expression (Fig. 6E). MyD88 is a universal adaptor for induction of cytokines by TLR2, TLR4, TLR5, TLR7, and TLR9. It is also required for activation of NF-κB by these TLRs [12]. Likewise, overexpression of a dominant negative mutant form of MyD88 also inhibited L. pneumophila-induced IL-8 expression. Taken together, these findings clearly demonstrate that L. pneumophila induces IL-8 expression via activation of flagellin-dependent NF-κB signaling pathway. Because activation of the IL-8 promoter by L. pneumophila infection required the activation of NF-κB, we blocked NF-κB activation with Bay 11-7082, an inhibitor of IκBα phosphorylation [13]. Bay 11-7082 markedly inhibited L. pneumophila-induced phosphorylation and degradation of IκBα, as well as NF-κB DNA binding (Fig. 7A and 7B). Furthermore, Bay 11-7082 resulted in a dose-dependent reduction in L. pneumophila-induced IL-8 mRNA expression and secretion by Jurkat cells (Fig. 7C and 7D). Figure 7 Bay 11-7082 blocks L. pneumophila-induced NF-κB activation and IL-8 secretion. Jurkat cells were pretreated with or without Bay 11-7082 (20 μM) for 1 h prior to L. pneumophila Corby infection and subsequently were infected with Corby (MOI, 100:1) for the indicated times. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies (A) and nuclear extracts from the harvested cells were analyzed for NF-κB and Oct-1 (B). Jurkat cells were pretreated with the indicated concentrations of Bay 11-7082 for 1 h prior to Corby infection and subsequently infected with Corby (MOI, 100:1) for 4 h (C) and 24 h (D). IL-8 mRNA expression on the harvested cells was analyzed by RT-PCR (C) and the supernatants were subjected to ELISA to determine IL-8 secretion (D). Data in (A)-(C) are representative examples of three independent experiments with similar results. Data are mean ± SD from three experiments. Flagellin-dependent activation of AP-1 To obtain further evidence for the AP-1 site on the IL-8 promoter in response to L. pneumophila, we examined the nuclear factors that bind to this site. The AP-1 sequence derived from the IL-8 promoter was used as a probe in EMSA. Jurkat cells were infected with the wild-type Corby or the flaA mutant at different times after challenge, and nuclear protein extracts were prepared and analyzed to determine AP-1 DNA binding activity. As shown in Fig. 8A, markedly increased complexes were induced by Corby compared with that induced by the isogenic flaA mutant. These results indicate that better activation of AP-1 binding by the flagellin-positive strain is the underlying mechanism of the observed activation of the IL-8 promoter by L. pneumophila. This AP-1 binding activity to the IL-8 promoter was reduced by the addition of either cold probe or a CREB sequence but not by an NF-κB sequence derived from the IL-2Rα enhancer (Fig. 8B, lanes 2 to 4). Figure 8 L. pneumophila activates AP-1 signal through flagellin. (A) Time course of AP-1 activation in Jurkat cells infected with L. pneumophila, evaluated by EMSA. Nuclear extracts from Jurkat cells, infected with Corby or flaA mutant (MOI, 100:1), for the indicated time periods, were mixed with IL-8 AP-1 32P-labeled probe. (B) Sequence specificity of AP-1 binding activity and characterization of AP-1/CREB/ATF proteins that bound to the AP-1 binding site of the IL-8 gene. Competition assays were performed with nuclear extracts from Jurkat cells infected with Corby for 2 h. Where indicated, 100-fold excess amounts of each specific competitor oligonucleotide were added to the reaction mixture with labeled probe AP-1 (lanes 2 to 4). A supershift assay of AP-1 DNA binding complexes in the same nuclear extracts also was performed. Where indicated, appropriate antibodies (Ab) were added to the reaction mixture before the addition of the 32P-labeled probe (lanes 6 to 17 and 19). Arrows indicate specific complexes, while arrowheads indicate the DNA binding complexes supershifted by antibodies. (C) Jurkat cells were infected with Corby or flaA mutant for the indicated time periods. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. Data are representative examples of three independent experiments with similar results. Next, we characterized the L. pneumophila-induced complexes identified by the IL-8 AP-1 probe. These complexes were diminished and supershifted by the addition of anti-c-Jun, anti-JunD, anti-ATF1, or anti-CREB antibody (Fig. 8B, lanes 10, 12, 13, and 17). The addition of these four antibodies completely diminished AP-1 DNA binding (Fig. 8B, lane 19). These results suggest that flagellin-induced IL-8 AP-1 complexes are composed of c-Jun, JunD, ATF1, and CREB to the AP-1 site in the IL-8 promoter region. Next, we examined phosphorylation of these four proteins in Jurkat cells infected with Corby or the isogenic flaA mutant. Corby but not flaA mutant enhanced phosphorylation of c-Jun, JunD, ATF1, and CREB in a time-dependent manner (Fig. 8C). These transcription factors are phosphorylated by p38 MAPK, JNK, and extracellular signal-regulated kinase (ERK) [14-18]. Furthermore, activated MAPKs phosphorylate AP-1, CREB, and ATF complexes, which results in increased AP-1-dependent transcription. We investigated whether L. pneumophila Corby activates these MAPKs. The p38 MAPK pathway mediates activation of CREB and ATF1 by flagellin Phosphorylation of p38 MAPK by Corby was determined by Western blot analysis (Fig. 9A). Corby, but not the flaA mutant, phosphorylated MAPKAPK-2 and MSK1, downstream CREB/ATF kinases of p38 MAPK in Jurkat cells (Fig. 9A). Consistent with the role of p38 MAPK phosphorylation in Jurkat cells infected with Corby in IL-8 expression and release, SB203580, a p38 MAPK inhibitor, reduced Corby-induced IL-8 expression and release by Jurkat cells in a dose-dependent manner (Fig. 9B and 9C). Furthermore, SB203580 inhibited Corby-induced luciferase activity of the IL-8 promoter in a dose-dependent manner (Fig. 9D). Similarly, overexpression of a dominant-negative mutant form of either p38α or p38β also inhibited Corby-induced luciferase activity of the IL-8 promoter, confirming the involvement of p38 MAPK in flagellin-induced IL-8 expression (Fig. 9E). The finding that SB203580 prevented Corby-induced phosphorylation of CREB and ATF1, and MAPKAPK-2 and MSK1, downstream targets of p38 MAPK (Fig. 9F), suggests that MAPKAPK-2 and MSK1 seem to mediate the flagellin-induced phosphorylation of CREB and ATF1. Figure 9 MAPKs activation by L. pneumophila through flagellin and inhibition of L. pneumophila-induced CREB and ATF1 activation and IL-8 transcription by p38 inhibitor. (A) Jurkat cells were infected with Corby or flaA mutant (MOI, 100:1), and lysates were subjected to immunoblotting. Cells were pretreated with the indicated concentrations of SB203580 for 1 h prior to infection and subsequently infected with Corby (MOI, 100:1) for 4 h (B) and for 6, 8, 12, or 24 h (C). IL-8 mRNA expression on the harvested cells was analyzed by RT-PCR (B) and the supernatants were subjected to ELISA to determine IL-8 secretion (C). (D) Cells were transfected with -133-luc and then pretreated with the indicated concentrations of SB203580 for 1 h prior to infection. They were infected subsequently with Corby for 6 h. Luciferase (LUC) activity was assayed. The solid bar indicates LUC activity of -133-luc without infection. (E) Cells were transfected with -133-luc and dominant negative mutants and then infected with Corby for 6 h. The solid bar indicates LUC activity of -133-luc without infection. All values were calculated as the change (n-fold) in induction values relative to the basal level measured in uninfected cells. Data are mean ± SD of three experiments. (F) Cells were pretreated with or without SB203580 (50 μM) for 1 h prior to infection and subsequently were infected with Corby (MOI, 100:1). Lysates were subjected to immunoblotting. dn, dominant negative. Effects of JNK and ERK on flagellin-induced IL-8 expression We also examined the effect of flagellin on activation of JNK and ERK. Corby, but not the flaA mutant, markedly increased the phosphorylation of JNK and MAPK kinase 4 (MKK4), upstream activator of JNK, and ERK in Jurkat cells (Fig. 9A). In addition, SP600125, an inhibitor of JNK, suppressed Corby-induced IL-8 expression and release in a dose-dependent manner (Fig. 10A and 10B). The finding that SP600125 inhibited Corby-induced phosphorylation of c-Jun but not JunD (Fig. 10C), suggests that JNK seems to mediate the flagellin-induced phosphorylation of c-Jun. Figure 10 SP600125 inhibits L. pneumophila-induced IL-8 expression and secretion. Jurkat cells were pretreated with the indicated concentrations of SP600125 for 1 h prior to L. pneumophila Corby infection and subsequently infected with Corby (MOI, 100:1) for 4 h (A) and 24 h (B). IL-8 mRNA expression on harvested cells was analyzed by RT-PCR (A) and the supernatants were subjected to ELISA to determine IL-8 secretion (B). Data are mean ± SD of three experiments. (C) Jurkat cells were pretreated with or without SP600125 (20 μM) for 1 h prior to L. pneumophila Corby infection and subsequently infected with Corby (MOI, 100:1) for the indicated times. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. Data in (A) and (C) are representative examples of three independent experiments with similar results. To determine the direct role of ERK phosphorylation in L. pneumophila-induced IL-8 expression, Jurkat cells were infected with Corby in the absence or presence of PD98059, an inhibitor of MEK1/2, an upstream activator of ERK. RNA and supernatants were collected after 4 and 24 h of infection and assayed for IL-8 mRNA expression and release, respectively. The addition of PD98059 had no effect on L. pneumophila-induced IL-8 mRNA expression and release by Jurkat cells (Fig. 11A and 11B). The activity of this inhibitor was verified by examining the phosphorylation state of ERK in L. pneumophila-infected cells after selected incubation time periods with PD98059. Whereas ERK activity was reduced in Jurkat cells in the presence of the inhibitor, the phosphorylation of CREB, ATF1, c-Jun, and JunD was not affected (Fig. 11C). Figure 11 TAK1 but not ERK plays key roles in L. pneumophila-induced IL-8 expression. (A) Jurkat cells were pretreated with the indicated concentrations of PD98059 for 1 h prior to L. pneumophila Corby infection and subsequently infected with Corby (MOI, 100:1) for 4 h (A) and 24 h (B). IL-8 mRNA expression on harvested cells was analyzed by RT-PCR (A) and the supernatants were subjected to ELISA to determine IL-8 secretion (B). Data are mean ± SD of three experiments. (C) Jurkat cells were pretreated with or without PD98059 (50 μM) for 1 h prior to L. pneumophila Corby infection and subsequently infected with Corby (MOI, 100:1) for the indicated times. Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. (D) Jurkat cells were transfected with -133-luc and a dominant negative mutant of TAK1 or empty vector and then infected with Corby for 6 h. The solid bar indicates LUC activity of -133-luc without Corby infection. The activities are expressed relative to that of cells transfected with -133-luc and empty vector without further Corby infection, which was defined as 1. Data are mean ± SD of three experiments. Data in (A) and (C) are representative examples of three independent experiments with similar results. Effect of TAK1 on flagellin-induced IL-8 expression TAK1 is one of the most characterized MAPK kinase kinase family members and is activated by various cellular stresses including IL-1 [19,20]. TAK1 functions as an upstream stimulatory molecule of the JNK, p38 MAPK, and IKK signaling pathways. Accordingly, we investigated whether TAK1 is also involved in L. pneumophila-induced IL-8 expression. As shown in Fig. 9A, phosphorylation of TAK1 was induced in Jurkat cells infected with Corby but not with flaA mutant. Furthermore, a dominant negative mutant of TAK1 inhibited L. pneumophila-induced IL-8 activation (Fig. 11D). These data suggest that trifurcation of L. pneumophila flagellin-induced IKK-IκB, MKK4-JNK, and p38 MAPK signaling pathways occurs at TAK1. Discussion Innate immunity is essential for limiting L. pneumophila infection at cellular and microbe levels. TLRs are involved in controlling L. pneumophila infection in vivo, since mice lacking TLR2 are more susceptible to infection, and MyD88-deficient mice show defective control of L. pneumophila infection [21,22]. Knowledge about host immunoreaction against L pneumophila is mainly based on studies on macrophages. While adaptive immunity has been shown to be important for host resistance to L. pneumophila [23], the direct interaction of bacteria with adaptive immune cells such as T cells is not well known. In this study, we show that L. pneumophila stimulates Jurkat T cells. Furthermore, this stimulation of T cells is mainly provided by flagellin since the flaA mutant was deficient in stimulating T cells to produce IL-8. This difference was independent of bacterial replication, as the flaA mutant could replicate in Jurkat T cells. Although Legionella less efficiently replicates within T cells, it is possible that uninfected T cells might respond to extracellular flagellin. Whether or not T cells are infected with L. pneumophila in vivo, they might still conceivably be a source of IL-8, because extracellular flagellin could induce IL-8 expression [24] and induction of IL-8 by L. pneumophilla did not require invasion. Interestingly, TLR5-deficient mice had lower numbers of polymorphonuclear neutrophils in their broncho-alveolar lavage fluid in comparison to wild-type mice after Legionella infection [25]. Infection with flagellin-deficient L. pneumophila has been reported to induce a robust cytokine response equivalent to infection with wild-type L. pneumophila in macrophages [26]. This cytokine response requires a functional L. pneumophila Dot/Icm type IV secretion system in macrophages and dendritic cells [26-28], indicating that T cells are unique. Although bacterial lipoprotein can also stimulate T cells [29,30], stimulation with lipoprotein of L. pneumophila has not yet been shown for human T cells. In this study, we demonstrated that L. pneumophila induces IL-8 expression through flagellin and NF-κB signaling pathway modulates this induction in human T cells. Using a specific pharmacological inhibitor, we showed that IKK-NF-κB pathway augmented L. pneumophila induction of IL-8 expression. We confirmed the important role of NF-κB by showing that overexpression of dominant negative NIK, IKKs, and IκBα, potent inhibitors of NF-κB activation, inhibited IL-8 promoter activation by L. pneumophila. The alternative pathway proceeds via NIK-, IKKα, and protein synthesis-dependent processing of the p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-κB dimmers [10]. The Legionella type IV effector LegK1 has been recently reported to process p100 into p52 [31]. The dominant negative mutants of NIK and IKKα inhibited IL-8 promoter activation by L. pneumophila in Jurkat cells. Furthermore, L. pneumophila infection induced p100 processing into p52 subunit, although supershift experiments did not reveal that the NF-κB-DNA binding complexes in Jurkat cells infected with L. pneumophila involve p52 and RelB. Further basic investigations with knockout and knockdown experiments will be essential in exploring the involvement of NIK-dependent alternative NF-κB pathway in L. pneumophila flagellin-induced IL-8 expression in T cells. Recently, infection with L. pneumophila has been shown to induce a biphasic activation of NF-κB in human epithelial cells: (i) early in infection, bacterial flagellin induces signaling of TLR5 and a transient translocation of p65 into the nucleus and (ii) at later time points, an unknown factor that depends on bacterial replication and a functional Dot/Icm system induces continuous nuclear localization of p65 and permanent degradation of IκBα [32]. Certainly, IL-8 mRNA expression was induced immediately after the infection, but became gradually weaker from 8 to 12 h after infection with the dotO mutant in Jurkat cells. L. pneumophila could also induce biphasic activation of NF-κB in T cells. The Dot/Icm system was demonstrated to be necessary for NF-κB activation in infections of human macrophages [33,34]. Furthermore, the Corby strain was shown to have a severely reduced Dot/Icm-dependent NF-κB activation [32]. Therefore, the flaA mutant derived from Corby strain might be deficient in infecting T cells to produce IL-8. In addition to flagellin, the Dot/Icm system might also be necessary for NF-κB activation and subsequent upregulation of IL-8 gene in infections of T cells. In addition to NF-κB activation, MAPKs have also been implicated in the induction of IL-8 production [35]. The data presented here showing that all three MAPKs (p38, JNK, and ERK) were consistently activated upon infection with L. pneumophila in T cells, are in agreement with those published by several groups who have also reported L. pneumophila-dependent activation of these MAPKs in macrophages and lung epithelial cells [35-38]. However, p38 and JNK activation is flagellin-independent in macrophages [26]. Legionella deficient in the Dot/Icm system failed to activate p38 and JNK in macrophages [26,38]. In lung epithelial cells, deletion of the Dot/Icm did not alter IL-8 production, whereas lack of flagellin reduced IL-8 release by Legionella, although flagellin- and Dot/Icm-dependency of MAPKs activation was not analyzed [35]. It is likely that L. pneumophila flagellin provides signals to T cells as in lung epithelial cells since the flaA mutant failed to activate MAPKs in T cells. While it is clear from this report that blockade of p38 with specific inhibitors but not that of ERK, diminishes IL-8 mRNA expression and release in lung epithelial cells [35], the precise molecular mechanism underlying these inhibitions is not clear yet. We identified both NF-κB and AP-1 binding sites on the 5' flanking region of the IL-8 promoter required for maximal induction of IL-8 by L. pneumophila. Because we showed that L. pneumophila activated all three MAPKs, we also examined whether L. pneumophila triggers MAPKs-mediated IL-8 production via activation of c-Jun, JunD, CREB, and ATF1, which can bind to the AP-1 region in the IL-8 promoter, as well as its cell specificity. By using specific kinase inhibitors, we also demonstrated that IL-8 expression and production in Jurkat cells was sensitive to inhibition of p38 and JNK but not ERK. Consistent with these findings, L. pneumophila stimulated phosphorylation of c-Jun, CREB, and ATF1 was blocked by inhibitors of p38 and JNK but not ERK. Using dominant negative mutant proteins of p38α and p38β, we showed that L. pneumophila induction of IL-8 was also dependent on the p38 pathway. JunD phosphorylation can be mediated through JNK and ERK pathways [17]. Although both of these molecules were activated in response to L. pneumophila, inhibition of JNK and ERK did not reduce phosphorylation of JunD. Further studies are needed to determine the exact kinase responsible for JunD activation. Overexpression of dominant negative mutants of MyD88 and TAK1 inhibited L. pneumophila-induced IL-8 activation. Although we did not examine the effects of these dominant negative mutants on NF-κB and MAPKs activation, our results suggest that trifurcation of L. pneumophila-induced IKK-IκB, p38, and MKK4-JNK signaling pathways occurs at TAK1 (Fig. 12). Figure 12 Schematic representation of L. pneumophila-induced signal transduction pathways involved in IL-8 expression human T cells. The contributions of TLR5 and MKK3/6 are deduced. Conclusions In summary, we showed that L. pneumophila induced IL-8 expression and subsequent production through flagellin in human T cells. In addition, the study shed new light on the signaling pathways utilized by L. pneumophila in the induction of IL-8. Our findings support the role of IKK-IκB, p38, and JNK signaling pathways in L. pneumophila induction of IL-8 in human T cells. Future studies should examine these signaling pathways in T cells of animals and patients infected with L. pneumophila, and, if the pathways are found to be significant, a targeted investigation of the role they play in host defense against L. pneumophila in infected animals should be performed. Methods Antibodies and reagents Rabbit polyclonal antibodies to IκBα and NF-κB subunits p50, p65, c-Rel, p52, and RelB, AP-1 subunits c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD, ATF/CREB family ATF1, ATF2, ATF3, ATF4, and CREB, mouse monoclonal antibody to p52, and goat polyclonal antibody to Lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to actin was purchased from NeoMarkers (Fremont, CA). Mouse monoclonal antibody to phospho-IκBα (Ser-32 and Ser-36), rabbit polyclonal antibodies to p65, IKKβ, p38, phospho-p38 (Thr-180 and Tyr-182), MKK4, phospho-MKK4 (Thr-261), phospho-MAPKAPK-2 (Thr-334), phospho-MSK1 (Ser-360), phospho-JNK (Thr-183 and Tyr-185), phospho-c-Jun (Ser-73), and TAK1, and rabbit monoclonal antibodies to phospho-TAK1 (Thr-184 and Thr-187), phospho-IKKβ (Ser-180), CREB, phospho-CREB (Ser-133), ERK1/2, and phospho-ERK1/2 (Thr-202 and Tyr-204) were purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibody to phospho-p65 (Ser-536) was purchased from Applied Biological Materials (Richmond, Canada). Bay 11-7082 was purchased from Calbiochem (La Jolla, CA), respectively. p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, and MEK1/2 inhibitor PD98059 were obtained from Sigma-Aldrich (St. Louis, MO). Bacterial strains L. pneumophila serogroup 1 strain AA100jm [39] is a spontaneous streptomycin-resistant mutant of strain 130b, which is virulent in guinea pigs, macrophages, and amoebae. The avirulent dotO mutant was constructed by random transposon mutagenesis, as described previously [39]. This mutation results in severe defects in intracellular growth and evasion of the endocytic pathway [40]. The Corby flaA mutant derived from the wild-type Corby is defective in flagellin [41]. L. pneumophila strains were grown at 35°C in a humidified incubator on either buffered charcoal-yeast extract-agar medium supplemented with α-ketoglutarate (BCYE-α) or in buffered yeast extract broth supplemented with α-ketoglutarate (BYE-α). The flaA mutant was grown in an environment similar to those used for other strains, but in the presence of 20 μg/ml kanamycin. Heat-killed bacteria were prepared by heating the bacterial suspension at 56°C for 30 min or at 100°C for 1 h. Bacterial inactivation was achieved by treatment with paraformaldehyde (4%, 15 min followed by three washes in phosphate-buffered saline; PBS). Both types of treated suspensions were confirmed to contain no viable bacteria by plating them on BCYE-α agar. Cell culture Human T cells (Jurkat) were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 μg/ml streptomycin. Human peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of healthy donors using Ficoll-Hypaque gradients. PBMC were then further purified using positive selection with immunomagnetic beads specific for CD4 (Miltenyi Biotec, Auburn, CA). On the day of the experiment, cells were refed with fresh antibiotic-free medium and cocultured with L. pneumophila for the time intervals indicated below. Infection of T cells and intracellular growth kinetics experiments Jurkat or CD4+ T cells seeded in plates were inoculated with either AA100jm or dotO mutant and either Corby or flaA mutant at an MOI of 100. In some experiments, heat-killed or paraformaldehyde-fixed bacteria were inoculated in the same manner. At 2 h after infection, cells were centrifuged and the supernatant was discarded. Cells were washed three times with PBS and resuspended in fresh RPMI 1640 medium containing 100 μg/ml gentamycin for 2 h. The cells were washed three times again with PBS and were further incubated with fresh medium. The infected cells and supernatant in each well were harvested at the indicated time intervals by washing the wells three times with sterilized distilled water. These bacterial suspensions were diluted in sterilized water and plated in known volume onto BCYE-α agar. The numbers of CFU in infected cells were counted at the indicated time points after infection. Direct fluorescent antibody staining Jurkat cells were infected with bacteria for 2 h, followed by washing three times with PBS and 2 h gentamycin treatment (100 μg/ml). The infected cells were cultured in fresh antibiotics-free RPMI 1640 medium for an additional 24 h. After being harvested, the cells were fixed in 4% paraformaldehyde for 15 min. Fixed cells were washed with PBS and permeabilized with PBS containing 0.1% saponine and 1% bovine serum albumin for 45 min at room temperature. Permeabilized cells were washed and stained with fluorescein-conjugated mouse anti-L. pneumophila monoclonal antibody (PRO-LAB, Weston, FL) for 45 min at room temperature. Finally, the cells were washed and observed under a confocal laser scanning microscope (Leica, Wetzlar, Germany). Cells were stained with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). RT-PCR Total cellular RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer. First-strand cDNA was synthesized from 1 μg total cellular RNA using an RNA PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. Thereafter, cDNA was amplified using 30, 35, and 28 cycles for IL-8, TLRs, and for β-actin, respectively. The specific primers used were as follows: IL-8, forward primer 5'-ATGACTTCCAAGCTGGCCGTG -3' and reverse primer 5'-TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC-3'; TLR2, forward primer 5'-GCCAAAGTCTTGATTGATTGG-3' and reverse primer 5'-TTGAAGTTCTCCAGCTCCTG-3'; TLR3, forward primer 5'-AAGTTGGGCAAGAACTCACAGG-3' and reverse primer 5'-GTGTTTCCAGAGCCGTGCTAA-3'; TLR4, forward primer 5'-TGGATACGTTTCCTTATAAG-3' and reverse primer 5'-GAAATGGAGGCACCCCTTC-3'; TLR5, forward primer 5'-CCTCATGACCATCCTCACAGTCAC-3'and reverse primer 5'-GGCTTCAAGGCACCAGCCATCTC-3'; and for β-actin, forward primer 5'-GTGGGGCGCCCCAGGCACCA-3' and reverse primer 5'-CTCCTTAATGTCACGCACGATTTC-3'. The product sizes were 300 bp for IL-8, 347 bp for TLR2, 320 bp for TLR3, 506 bp for TLR4, 355 bp for TLR5, and 548 bp for β-actin. The thermocycling conditions for the targets were as follows: denaturing at 94°C for 30 s for IL-8, TLR5, and β-actin, and for 60 s for TLR3, and 95°C for 40 s for TLR2 and TLR4, annealing at 60°C for 30 s for IL-8 and β-actin, and for 60 s for TLR3, and 54°C for 40 s for TLR2 and TLR4, and 55°C for 30 s for TLR5, and extension at 72°C for 90 s for IL-8 and β-actin, and for 60 s for TLR2, TLR3, TLR4, and TLR5. The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining. Plasmids The IκBαΔN dominant negative mutant is IκBα deletion mutant lacking the NH2-terminal 36 amino acids [11]. The dominant negative mutants of IKKα, IKKα (K44M), IKKβ, IKKβ (K44A), IKKγ, IKKγ (1-305), NIK, NIK (KK429/430AA), MyD88, MyD88 (152-296), and TAK1, TAK1 (K63W), and the dominant negative mutant of either p38α or p38β, have been described previously [19,20,42-44]. Plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene linked to luciferase expression vectors were constructed from a firefly luciferase expression vector [45]. Site-directed mutagenesis of the IL-8 AP-1, NF-IL-6, and NF-κB sites in the -133-luc plasmid was introduced, converting the AP-1 site TGACTCA (-126 to -120 bp) to TatCTCA, the NF-IL-6 site CAGTTGCAAATCGT (-94 to -81 bp) to agcTTGCAAATCGT, and the NF-κB site GGAATTTCCT (-80 to -71 bp) to taAcTTTCCT (lower case letters indicate location of base changes). These constructs were designated as AP-1 site-mutated, NF-IL-6 site-mutated, and NF-κB site-mutated plasmids, respectively. Transfection and luciferase assay Jurkat cells were transfected with 1 μg of the appropriate reporter and 4 μg of effector plasmids using electroporation. After 24 h, L. pneumophila was infected and incubated for 6 h. The ratio of bacteria to cells (MOI) was 100. The cells were washed in PBS and lysed in reporter lysis buffer (Promega, Madison, WI). Lysates were assayed for reporter gene activity with the dual luciferase assay system (Promega). Luciferase activity was normalized relative to the Renilla luciferase activity from phRL-TK. Preparation of nuclear extracts and EMSA Cell pellets were swirled to a loose suspension and treated with lysis buffer (0.2 ml, containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM AEBSF, and 1 mM DTT) with gentle mixing at 4°C. After 10 min, NP40 was added to a final concentration of 0.6% and the solution was immediately centrifuged for 5 min at 1,000 rpm at 4°C. The supernatants were removed carefully and the nuclear pellets were diluted immediately by the addition of lysis buffer without NP40 (1 ml). The nuclei were then recovered by centrifugation for 5 min at 1,000 rpm at 4°C. Finally, the remaining pellets were suspended on ice in the following extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 2 mM AEBSF, 33 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml E-64, and 10 μg/ml pepstatin A) for 30 min to obtain the nuclear fraction. All fractions were cleared by centrifugation for 15 min at 15,000 rpm. NF-κB and AP-1 binding activities with the NF-κB and AP-1 elements were examined by EMSA as described previously [46]. To examine the specificity of the NF-κB and AP-1 element probes, we preincubated unlabeled competitor oligonucleotides with nuclear extracts for 15 min before incubation with probes. The probes or competitors used were prepared by annealing the sense and antisense synthetic oligonucleotides as follows: for the NF-κB element of the IL-8 gene, 5'-GATCCGTGGAATTTCCTCTG-3'; for the NF-κB element of the IL-2Rα gene, 5'-GATCCGGCAGGGGAATCTCCCTCTC-3'; for the AP-1 element of the IL-8 gene, 5'-GATCGTGATGACTCAGGTT-3', and for the consensus sequence of the CRE, 5'- GATCGATCTTTACCATGACGTCAATTTGAT-3'. The oligonucleotide 5'-GATCTGTCGAATGCAAATCACTAGAA-3', containing the consensus sequence of the octamer binding motif, was used to identify specific binding of transcription factor Oct-1. The above bold sequences are the NF-κB, AP-1, CREB, and Oct-1 binding sites, respectively. To identify NF-κB and AP-1 proteins in the DNA-protein complex shown by EMSA, we used antibodies specific for various NF-κB family proteins, including p50, p65, c-Rel, p52, and RelB, various AP-1 family proteins, including c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD, and various ATF/CREB family proteins, including ATF1, ATF2, ATF3, ATF4, and CREB, to elicit a supershift DNA-protein complex formation. These antibodies were incubated with the nuclear extracts for 45 min at room temperature before incubation with radiolabeled probe. Western blot analysis Cells were lysed in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 6% 2-mercaptoethanol, and 0.01% bromophenol blue. Equal amounts of protein (20 μg) were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, followed by transfer to a polyvinylidene difluoride membrane and sequential probing with the specific antibodies. The bands were visualized with an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). Measurement of IL-8 The IL-8 contents in the serum from peripheral blood and the culture supernatants were measured by ELISA (Biosource International, Camarillo, CA). Serum was obtained from healthy volunteers or each patient with Legionella pneumonia at diagnosis and stored at -80°C until use. Jurkat and CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS in 6-well plates. Cells were infected with L. pneumophila for the indicated time intervals. The supernatants were then collected after centrifugation and stored at -80°C until assayed for IL-8 by ELISA. The concentrations of IL-8 were determined using a standard curve constructed with recombinant IL-8. This study was approved by the Institutional Review Board (IRB) of the University of the Ryukyus with license number H20-12-3. Informed consent was obtained from all blood donors according to the Helsinki Declaration. Statistical analysis Values were expressed as mean ± standard deviations (SD). Differences between groups were examined for statistical significance using the Student t test. A P value less than 0.05 was considered statistically significant. Authors' contributions RT designed and performed the research, analyzed data, and wrote the manuscript. HT participated in the design of the study, performed the research, and analyzed data. ET and CI contributed to the experimental concept and provided technical support. KM, NMu, and JDL carried out the generation of plasmids. KH, FH, and JF provided bacterial strains. NMo established the research plan, supervised the project, and helped to draft the manuscript. All authors read and approved the data and final version of the manuscript. Acknowledgements We thank D. W. Ballard for providing the IκBα dominant negative mutant; R. Geleziunas for providing the NIK, IKKα, and IKKβ dominant negative mutants; K.-T. Jeang for providing the IKKγ dominant negative mutant; and M. Muzio for providing the MyD88 dominant negative mutant. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2017446809-PONE-RA-15112R110.1371/journal.pone.0009313Research ArticleNeuroscience/Neurobiology of Disease and RegenerationNeuroscience/Neuronal Signaling MechanismsNeurological Disorders/Alzheimer DiseaseNeurological Disorders/Cognitive Neurology and DementiaNeurological Disorders/Movement DisordersSelective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of α-Synucleinopathy mTor Activation in LBDCrews Leslie 1 Spencer Brian 2 Desplats Paula 2 Patrick Christina 2 Paulino Amy 2 Rockenstein Edward 2 Hansen Lawrence 1 2 Adame Anthony 2 Galasko Douglas 2 Masliah Eliezer 1 2 * 1 Department of Pathology, University of California San Diego, La Jolla, California, United States of America 2 Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America Ikezu Tsuneya EditorUniversity of Nebraska Medical Center, United States of America* E-mail: [email protected] and designed the experiments: LC BS PD ER LH DG EM. Performed the experiments: LC BS PD CP ADP ER AA EM. Analyzed the data: LC BS PD CP ADP ER LH AA DG EM. Contributed reagents/materials/analysis tools: EM. Wrote the paper: LC PD EM. Critical revision of the article: CP ADP LH DG. 2010 19 2 2010 5 2 e931322 12 2009 28 1 2010 Crews et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by α-synuclein accumulation that includes dementia with Lewy bodies (DLB) and Parkinson's Disease (PD). Recent evidence suggests that impairment of lysosomal pathways (i.e. autophagy) involved in α-synuclein clearance might play an important role. For this reason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLB and Alzheimer's Disease (AD) and in α-synuclein transgenic mice. Methodology/Principal Findings By immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor were elevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 and Beclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying α-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, and ultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations were observed in the brains of α-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injection of a lentiviral vector expressing Atg7 resulted in reduced accumulation of α-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations. Conclusions/Significance This study supports the notion that defects in the autophagy pathway and more specifically in mTor and Atg7 are associated with neurodegeneration in DLB cases and α-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects. ==== Body Introduction Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common causes of dementia and movement disorders in the elderly [1], [2]. While progressive accumulation of Aβ dimers and oligomers has been identified as one of the central toxic events in AD leading to synaptic dysfunction [3], [4], accumulation of α-synuclein (α-syn) resulting in the formation of oligomers has been linked to the pathogenesis of PD [5], [6], [7], [8], [9]. The pathology of AD and PD overlap in a heterogeneous group of conditions denominated jointly Lewy body disease (LBD) [10], [11], [12], [13], [14]. While in patients with dementia with Lewy bodies (DLB) the clinical presentation is of dementia followed by parkinsonism, in patients with PD dementia (PDD) the initial signs are of parkinsonism followed by dementia [15], [16], [17], [18]. In DLB, Aβ promotes α-syn aggregation and toxicity in vivo [19], and Aβ and α-syn might directly interact [20] to form hybrid channel like structures [21]. Alterations in the rate of synthesis, aggregation and clearance of these proteins might be responsible for the formation of toxic Aβ and α-syn oligomers in DLB [22]. Impaired clearance of the α-syn aggregates might play an important role in the pathogenesis of PD and DLB [23], [24]. Among the lysosomal pathways involved, the autophagy signaling cascade has emerged as a key mechanism for the removal of α-syn aggregates. Autophagy is the major pathway involved in the degradation of long-lived proteins and organelles, cellular remodeling, and survival during nutrient starvation [25], [26]. There are three distinct autophagic pathways [27], [28]: i) macroautophagy, ii) microautophagy and iii) chaperone-mediated autophagy (CMA). Autophagy has been linked to neuronal cell death [29], [30] and is abnormally activated in mouse models of neurodegeneration and in neurodegenerative disorders such as AD, PD and Huntington's disease (HD) [31], [32]. Macroautophagy is constitutively active and highly efficient in healthy neurons and recent studies indicate that the autophagic pathology observed in AD most likely arises from impaired clearance of autophagic vacuoles (AVs) rather than strong autophagy induction alone [33] suggesting selective alterations in molecular components of the autophagy pathway. For example, in the brains of patients with AD, levels of the AV protein Beclin-1 are severely down modulated [34]. In PD recent studies have suggested that α-syn aggregates might interfere with the autophagy mechanisms and lead to neurodegeneration [23], [35], [36], [37], [38], [39]. Mutant forms of α-syn found in familial PD patients [23] as well as oxidized forms of α-syn [40] found in sporadic PD and DLB have been shown to block autophagy, and a-syn contains a consensus sequence for CMA targeting. In neuronal cell cultures [41] and in transgenic (tg) mice, α-syn overexpression is associated with impaired autophagy and neurodegeneration that is reversed by Beclin-1 [42]. Further supporting a role for lysosomal dysfunction in LBD, previous studies have shown that in lysosomal storage disorders such as Gaucher disease [43], [44] and Niemann-Pick disease [45], there is increased susceptibility to develop parkinsonism and α-syn accumulation. Taken together, these lines of evidence suggest that in DLB and PD, specific molecular defects in the autophagy pathway might play a role in the pathogenesis of these disorders. In this context, the main objective of the present study was to investigate alterations in components of the autophagy pathway in DLB and in a-syn tg models. We found that levels of mTor were increased and Atg7 levels were reduced in the brains of patients with DLB and a-syn tg mice. Moreover, activating autophagy by rapamycin treatment or viral-mediated delivery of Atg7 ameliorated a-syn accumulation and the related neuropathology. This supports the notion that alterations in the autophagy pathway play a role in DLB/PD and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects. Materials and Methods Ethics Statement This study was conducted according to the principles expressed in the Declaration of Helsinki. For studies utilizing human tissues, all tissues were obtained from the University of California, San Diego Alzheimer's Disease Research Center (ADRC). Written informed consent for neurobehavioral evaluation, autopsy, and for the collection of samples and subsequent analysis was obtained from the patient and caregiver (usually the next of kin) before neuropsychological testing and after the procedures of the study had been fully explained. The study procedures conformed to Federal guidelines for the protection of human subjects and were reviewed and approved by the UCSD Institutional Review Board. For animal studies, all animals were handled in strict accordance with good animal practice and all procedures were completed under the specifications set forth by the UCSD Institutional Animal Care and Use Committee. Cases and Neuropathological Evaluation The study included a total of 24 cases (Table 1); of them, 6 were non-demented controls, 12 were DLB cases and the other 6 were AD cases. For the present study we chose to focus on DLB because of its frequency and widespread accumulation of a-syn in neocortical and limbic structures [46], [47], [48]. Autopsy material was obtained from patients studied neurologically and psychometrically at the UCSD ADRC. At autopsy, brains were divided sagittally, and samples from the left mid temporal cortex were fixed in 4% paraformaldehyde (PFA) and sectioned at 40 µm for immunocytochemical analysis. Frozen samples from the right were used for immunoblot analysis. The temporal cortex was selected because previous studies have shown considerable pathology and accumulation of α-syn in this region in patients with DLB [47], [48], [49]. 10.1371/journal.pone.0009313.t001Table 1 Summary of clinico-pathological characteristics. Diagnosis N = Mean Age (yrs) Mean brain weight Gender M/F Mean PMT (hrs) Mean Duration (yrs) Blessed score Frontal cortex Amyloid Plaques (0.1sq mm) Temporal cortex Lewy bodies (0.1sq mm) Control 6 79.8±2 1150±115 4/2 6.5±2 NA 0–1 0 0 AD 6 81±1 1058±97 3/3 8±1 12.8±1 19–33 50 0 DLB 12 82.7±2 1074±119 4/8 7.2±1.5 9.8± 5–32 33 12±2 For routine neuropathological diagnosis, paraffin sections from neocortical, limbic and subcortical regions were stained with haematoxylin and eosin (H&E) or thioflavine-S [50], [51], and Braak stage was assessed [52]. Based on previously published clinical and pathological findings [53], cases were subdivided into: 1) non-demented age-matched controls, 2) AD cases, and 3) DLB cases. All cases met the Consortium to Establish a Registry for AD (CERAD) and National Institute of Aging (NIA) criteria for diagnosis and displayed neuritic plaques and tangle formation in the neocortex and limbic system [12], [54]. The diagnosis of DLB was based on the clinical presentation of dementia and the pathological findings of LBs in the locus coeruleus, substantia nigra (SN), or nucleus basalis of Meynert, as well as in cortical regions. LBs were detected using H&E stain or anti-ubiquitin and anti-α-syn antibodies as recommended by the Consortium on DLB criteria for a pathologic diagnosis of DLB [12]. In addition to the presence of LBs, the great majority of these cases displayed sufficient plaques and tangles to be classified as Braak stages III–IV. Specifically, DLB cases had abundant plaques in the neocortex and limbic system but fewer tangles compared to AD cases. a-Synuclein Transgenic Mice and Treatments For this study, heterozygous tg mice (Line D) expressing human wildtype α-syn under the regulatory control of the PDGFβ promoter [55] were used. These animals were selected because they display abnormal accumulation of detergent-insoluble α-syn in the neocortex and limbic system and develop α-syn-immunoreactive inclusion-like structures in the brain [56]. Although some nuclear staining has been observed in this model, distinct cytoplasmic inclusion-like structures have been consistently identified by confocal and electron microscopy [19], [55], [56], [57]. Furthermore, these animals also display neurodegenerative and behavioral deficits that mimic certain aspects of DLB. For western blot and immunocytochemical studies levels of components of the autophagy pathway were compared to littermate non tg controls and to an APP tg model (line J9M) of AD-like pathology. These mice express human APP 770/751 and 695 under the PDGFβ promoter [58]. Additional experiments with the α-syn tg mice included treatments with the autophagy activator rapamycin (Sigma-Aldrich, St. Louis, MO). Because rapamycin poorly crosses into the CNS, it was infused intra-cerebrally into the lateral ventricle of 9-month old mice at a concentration of 20mg/kg. Briefly as previously described [59], mice were anesthetized and under sterile conditions a 26 gauge stainless steel cannula was implanted stereotaxically into the lateral ventricle using the bregma as a reference (Franklin and Paxinos, bregma 0.5mm; 1.1mm lateral; depth 3mm) and secured to the cranium using superglue. The cannula was connected via a 5 mm coil of V3 Biolab vinyl to a model 1007D osmotic minipump (Alzet, Cupertino, CA) surgically placed subcutaneously beneath the shoulder. The solutions were delivered at a flow rate of 0.5ul per hour for 2 weeks. The pump was left for an additional 2 weeks and mice were euthanized one month after the initiation of the infusions. Brains were removed and divided sagittally. One hemibrain was postfixed in phosphate-buffered 4% PFA, pH 7.4, at 4°C for 48 h and sectioned at 40 µm with a Vibratome 2000 (Leica, Nussloch, Germany) and placed in cryosolution, while the other hemibrain was snap frozen and stored at −70°C for RNA and protein analysis. Construction of Lentiviral Vectors and Injection of LV-Atg7 into α-Synuclein Transgenic Mice The mouse Atg7 cDNA (Open Biosystems, Huntsville, AL) was PCR amplified and cloned into the third generation self-inactivating lentivirus vector (LV) [60] with the CMV promoter driving expression. Lentiviruses expressing α-syn, Atg7, luciferase, shAtg7, GFP, shLuciferase, or empty vector (as controls) were prepared by transient transfection in 293T cells [60], [61]. The empty LV contained the viral genome with the CMV promoter, with no gene inserted downstream of the promoter. A total of 12 α-syn tg mice from line D (9 months old) were injected with 3 µl of the LV preparations (2.5×107 TU) into the temporal cortex and hippocampus (using a 5 µl Hamilton syringe). Briefly, as previously described [62], mice were placed under anesthesia on a Koft stereotaxic apparatus and coordinates (hippocampus: AP −2.0 mm, lateral 1.5 mm, depth 1.3 mm and cortex: AP −.5 mm, lateral 1.5 mm, depth 1.0 mm) were determined as per the Franklin and Paxinos Atlas. The LVs were delivered using a Hamilton syringe connected to a hydraulic system to inject the solution at a rate of 1 µl every 2 min. To allow diffusion of the solution into the brain tissue, the needle was left for an additional 5 min after the completion of the injection. Mice received unilateral injections (right side) to allow comparisons against the contralateral side, with either LV-Atg7 (n = 6), or LV-control (n = 6). Additional controls were performed by injecting non tg littermates with either LV-Atg7 (n = 6), or LV-control (n = 6). Mice survived for 1 month after the lentiviral injection. As an additional control for LV injection, age matched littermates were injected with LV-luciferase. Since no differences were observed between the LV-control and the LV-luciferase, all data presented here are shown with the LV-control vector. Following NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9% saline. Brains and peripheral tissues were removed and divided sagittally. The right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at −70°C for subsequent RNA and protein analysis. Cell Culture and Treatments For in vitro experiments we used the previously described rat neuroblastoma cell line B103 [63]. This model was selected because overexpression of α-syn in these cells interferes with neuronal plasticity (reduced neurite outgrowth and adhesion) but does not result in overt cell death [63], [64]. This model mimics the early pathogenic process of PD where cell death is preceded by reduced neurite outgrowth and synaptic alterations. For all experiments, cells were plated in complete media (DMEM [Invitrogen, Carlsbad, CA] supplemented with 10% FBS (Irvine Scientific, Santa Ana, CA) and infected with LVs expressing α-syn, Atg7, shAtg7, or controls at a multiplicity of infection (MOI) of 40. After infection, cells were incubated for 48 hr in a humidified 5% CO2 atmosphere at 37°C. All experiments were conducted in triplicate to ensure reproducibility. To investigate whether LC3 levels are modulated by α-syn or Atg7 over-expression or knockdown, LC3 levels were analyzed in coverslips with LC3-GFP. B103 cells were grown as described above and were then plated onto poly L-lysine coated glass coverslips at a density of 5×104 cells. Five hours after plating, cells were infected with the LV-αsyn and/or LV-Atg7 or LV-shAtg7 (or controls) and incubated for 48 hours. All coverslips were also co-infected with an LV expressing LC3-GFP at an MOI of 40. Cultures were then washed 2× with serum-free DMEM and then fed either complete media or serum-free media for 12 hours before fixation with 4% PFA. Briefly as previously described [34], coverslips were treated with Prolong Gold antifading reagent with DAPI (Invitrogen) and imaged with the LSCM to determine the number of GFP-positive granular structures consistent with autophagolysosomes using semiautomatic image analysis system and the ImageQuant software. For each condition an average of 50 cells were analyzed. Antibodies For western blot and immunohistochemical analysis of the autophagy pathway, polyclonal antibodies against mTor (1∶1000, Sigma); phosphorylated-mTor (p-mTor, 1∶1000, Cell Signaling Technology, Beverly, MA); Atg5 (1∶1000, Abcam, Cambridge, MA); Atg6 or Beclin-1 (1∶1000, Novus Biologicals, Littleton, CO); Atg7 (1∶500, Abcam); Atg8 or LC3 (1∶1000, Abcam); Atg10 (1∶500, Abcam); Atg12 (1∶1000, Abcam); Cathepsin D (1∶500, Calbiochem, San Diego, CA). Detection of α-syn was performed with a rabbit polyclonal (1∶500, Millipore, Temecula, CA) and a mouse monoclonal antibody (1∶500, clone syn211, Sigma). Immunohistochemistry, Image Analysis and Laser Scanning Confocal Microscopy Briefly, as previously described [65], free-floating 40 µm thick vibratome sections were washed with Tris buffered saline (TBS, pH 7.4), pre-treated in 3% H2O2, and blocked with 10% serum (Vector Laboratories, Burlingame, CA), 3% bovine serum albumin (Sigma), and 0.2% gelatin in TBS-Tween (TBS-T). For human brains, sections from the temporal cortex were used; for the mice sagittal sections from the complete brain were studied. Sections were incubated at 4°C overnight with the primary antibodies. Sections were then incubated in secondary antibody (1∶75, Vector), followed by Avidin D-horseradish peroxidase (HRP, ABC Elite, Vector) and reacted with diaminobenzidine (DAB, 0.2 mg/ml) in 50 mM Tris (pH 7.4) with 0.001% H2O2. Control experiments consisted of incubation with pre-immune rabbit serum. To investigate the effects of postmortem delay and fixation on the levels of mTor immunoreactivity, preliminary studies were performed in a subset of cases (n = 5) with postmortem delay ranging from 4–48 h. Immunostained sections were imaged with a digital Olympus microscope and assessment of levels of mTor, Atg7, LC3 and Cathepsin D immunoreactivity was performed utilizing the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD). For each case a total of three sections (10 images per section) were analyzed in order to estimate the average number of immunolabeled cells per unit area (mm2) and the average intensity of the immunostaining (corrected optical density). Double-immunocytochemical analyses was performed utilizing the Tyramide Signal Amplification™-Direct (Red) system (NEN Life Sciences, Boston, MA). Specificity of this system was tested by deleting each primary antibody. For this purpose, sections were double-labeled with the monoclonal antibodies against α-syn (1∶20,000, Cell Signaling) detected with Tyramide Red, and either mTor, Cathepsin D or LC3 detected with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1∶75, Vector). All sections were processed simultaneously under the same conditions and experiments were performed twice for reproducibility. Sections were imaged with a Zeiss 63X (N.A. 1.4) objective on an Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024 laser scanning confocal microscope (LSCM) system (BioRad, Hercules, CA). Analysis of Neurodegeneration To evaluate the integrity of the dendritic system, blind-coded 40µm-thick vibratome sections from mouse brains fixed in 4% PFA were immunolabeled with the mouse monoclonal antibody against microtubule associated protein 2 (MAP2) (dendritic marker, 1∶40, Millipore), as previously described [66]. After an overnight incubation with the primary antibody, sections were incubated with FITC-conjugated horse anti-mouse IgG secondary antibody (1∶75, Vector), transferred to SuperFrost slides (Fisher Scientific, Tustin, CA) and mounted under glass coverslips with anti-fading media (Vector). All sections were processed under the same standardized conditions. The immunolabeled blind-coded sections were imaged with the LSCM (MRC1024, Bio-Rad) and analyzed with the Image 1.43 program (NIH), as previously described [66], [67]. Western Blot Analysis Immunoblot analysis was performed as previously described [68]. Briefly, brain homogenates (temporal cortex for human tissues and cortex for mice) or cultured cells were solubilized in lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.4, 140 mM NaCl, 1mM EDTA, 1mM Na3VO4, 20 mM β-glycerophosphate, and proteinase inhibitor cocktails). Brain homogenates were separated into cytosolic and membrane fractions by centrifugation at 100,000×g for 1 hr at 4C. Isolation of lysosomal fractions from brain tissue was performed by differential centrifugation in a sucrose-based buffer essentially as previously described [69]. Briefly, tissues were minced with a razor blade and homogenized in ice-cold 50 mM Tris/HCl buffer, pH = 7.4, containing 0.25 M sucrose, 10 mM EDTA, 3 mM MgCl2, with protease and phosphatase inhibitor cocktails (Calbiochem) added fresh (sucrose/Tris buffer). After filtration through a mesh to remove cell debris and capsular fragments, the homogenate was centrifuged at 1000 g for 5 min to obtain the nuclear fraction (pellet 1, P1). The supernatant was centrifuged again at 10,000 g for 10 min to collect the mitochondrial fraction (pellet 2, P2). The resulting supernatant was centrifuged at 20,000 g for 10 min to obtain the lysosomal fraction (pellet 3, P3). Finally, the supernatant remaining after the removal of the lysosomal fraction was centrifuged at 180,000 g for 30 min to separate the microsomal fraction (pellet 4, P4) from the cytosol/extracellular fluid fraction (supernatant 4, S4). Pellets were gently resuspended in 0.25 mL sucrose/Tris buffer, and all fractions were stored at −80°C until further processing. For western blot analysis, 20 µg of each fraction was resolved by SDS-PAGE on 4–12% Bis-Tris gels (Invitrogen) and electroblotted onto Immobilon membranes (Millipore). The membranes were blocked with phosphate-buffered saline (PBS) with 0.2% Tween-20 (PBST) containing 3% skim milk or bovine serum albumin (BSA), followed by incubation with primary antibodies in PBST containing 5% BSA. After washing with PBS, the membranes were incubated with rabbit anti-mouse secondary antibodies (1∶5000, American Qualex, San Clemente, CA) and visualized with enhanced chemiluminescence (ECL, PerkinElmer, Wellesley, MA) and analyzed with the VersaDoc gel imaging system (BioRad). Electron Microscopy Briefly, as previously described [42], vibratome sections from control, DLB brains and α-syn tg mice were post-fixed in 1% glutaraldehyde, then treated with osmium tetraoxide and embedded in epon araldite. Once the resin hardened, blocks were sectioned with an ultramicrotome (Leica). Grids were analyzed with a Zeiss OM 10 electron microscope as previously described [70]. Micrographs from pyramidal neurons in the temporal cortex were randomly acquired from 3 grids, and electron micrographs were acquired at a magnification of 25,000 each. Statistical Analysis All experiments were conducted in triplicate on blind-coded samples. After the results were obtained, the code was broken and data were analyzed with the StatView program (SAS Institute, Inc., NC). Comparison among groups of expression levels of components of the autophagy pathway were performed by one-way ANOVA with post-hoc Dunnett's or Tukey-Kramer. All results are expressed as mean ± SEM. Results Alterations in the Levels of mTor and Atg7 Are Associated with Lysosomal Alterations the Brains of DLB Patients Recent evidence suggests that impaired functioning of the lysosomal pathways (eg: autophagy) involved in α-syn clearance might play a role in the pathogenesis of DLB [23], [24]. For this reason, expression levels of members of the autophagy pathway were analyzed in the temporal cortex of control, AD and DLB patients. By immunoblot analysis, mTor was identified as a triplet band at an estimated molecular weight of 280–290 kDa that was more abundant in the membrane than in the cytosolic fraction (Figure 1A, B). Compared to non-demented controls and AD cases, levels of mTor and phosphorylated-mTor (p-mTor) were elevated in the brains of DLB cases (Figure 1A, C). In both the membrane and cytosolic fractions Atg7 was identified as a doublet at an estimated molecular weight of 78 kDa (Figure 1A, B). In DLB cases, levels of Atg7 were moderately reduced compared to controls and AD cases (Figure 1A, D). No differences were observed between controls and AD cases (Figure 1A, D). Other components of the autophagy pathway such as Atg5 (32 kDa) and Atg12 (20kDa) were identified as single bands that were more abundant in the membrane than in the cytosolic fractions (Figure 1A, B). No differences were detected among the 3 groups in the levels of these Atg proteins (Figure 1A, D). Beclin-1 was detected as a single band at 50 kDa that was more abundant in the membrane than the cytosolic fraction (Figure 1A, B). Consistent with previous studies [34], levels of Beclin-1 in the membrane fractions were reduced in the AD cases compared to non-demented controls and DLB, however no differences in Beclin-1 levels were detected between non-demented controls and DLB cases (Figure 1A, C). 10.1371/journal.pone.0009313.g001Figure 1 Immunoblot analysis of the autophagy pathway in the brains of AD and DLB patients. Brain homogenates from the temporal cortex of non-demented controls, AD, and DLB patients were separated into membrane and cytosolic fractions, and 20 µg of each sample was subjected to gel electrophoresis. Immunoblots were probed with antibodies against mTor, phosphorylated (p) mTor, Beclin-1, Atg5, Atg7, Atg12 and Actin. (A) Representative immunoblots of membrane fractions. (B) Representative immunoblots of cytosolic fractions. (C) Semi-quantitative analysis of levels of mTor, p-mTor, and Beclin-1 in membrane fractions from the brains of control, AD and DLB patients. Levels of mTor were significantly increased in DLB patients. (D) Semi-quantitative analysis of levels of Atg5, Atg7, and Atg12 in membrane fractions from the brains of control, AD and DLB patients. Levels of Atg7 were significantly reduced in the brains of DLB patients. All semi-quantitative measurements were normalized to actin levels as a loading control. p<0.05 compared to non-demented controls by one-way ANOVA with post-hoc Dunnett's test. Immunohistochemical analysis showed moderate levels of mTor and Atg7 immunostaining in pyramidal neurons in control and AD cases (Figure 2A, B, D, E). In contrast, in DLB cases levels of neuronal mTor immunoreactivity were increased (Figure 2C, G), while the levels of neuronal Atg7 immunolabeling were reduced (Figure 2F, G). The increased levels of mTor and reduced Atg7 levels suggest there may be altered activation of the autophagy pathway in the brains of DLB patients. Consistent with this possibility, in DLB cases Cathepsin D-immunoreactive lysosomes of normal size were scant, and pyramidal cells contained enlarged lysosomes (Figure 2H–J, N) and increased levels of LC3 immunoreactivity (Figure 2K–M, O) compared to control cases. 10.1371/journal.pone.0009313.g002Figure 2 Immunohistochemical analysis of the autophagy pathway in the brains of AD and DLB patients. Vibratome sections from the temporal cortex of non-demented controls, AD, and DLB patients were immunolabeled with antibodies against mTor, Atg7, Cathepsin D, and LC3, and imaged with a digital microscope. (A–C) Representative sections from control, AD and DLB brains immunolabeled with an antibody against mTor. (D–F) Representative sections from control, AD and DLB brains immunolabeled with an antibody against Atg7. (G) Semi-quantitative image analysis reveals a significant increase in mTor levels and a reduction in Atg7 levels in DLB patients compared to controls. (H–J) Representative sections from control, AD and DLB brains immunolabeled with an antibody against Cathepsin D. Pyramidal neurons in AD and DLB cases show enlarged Cathepsin D-immunoreactive lysosomes (arrows). (K–M) Representative sections from control, AD and DLB brains immunolabeled with an antibody against LC3. (N) Increased numbers of enlarged lysosomes (>1µm) in AD and DLB brains. (O) Semi-quantitative image analysis of LC3 immunoreactivity reveals increased LC3 levels in AD and DLB brains. Scale bar in panel (C) represents 20µm in all microscopy images. p<0.05 compared to non-demented controls by one-way ANOVA with post-hoc Dunnett's test. By immunoblot analysis, in the DLB cases there was an increase in immunoreactive bands representing monomeric and aggregated α-syn both in the cytosolic and membrane fractions (Figure S1A, B). In the AD, the levels of α-syn were slightly elevated compared to controls (Figure S1C). Consistent with the immunohistochemical analysis (Figure 2), by immunoblot, levels of LC3 and Cathepsin D in the membrane fractions were reduced in the DLB cases compared to non-demented controls (Figure S1A, C). In contrast, in the AD cases levels of Cathepsin D were elevated compared to non-demented controls (Figure S1A, C). Compared to the unimpaired neurons in the control cases, in DLB brains, mTor and LC3 immunoreactivity were more abundant in neurons displaying α-syn accumulation (Figure 3A–L). In addition, in the DLB cases, LC3 immunoreactivity was occasionally associated with LBs (Figure 3J–L). Compared to control cases, these neurons also showed enlarged Cathepsin D-positive lysosomes (Figure 3M–R). Consistent with these observations, electron microscopy analysis revealed the presence of abundant and abnormal autophagosomes in these α-syn-positive neurons that were not detected in control cases (Figure 4A–D). 10.1371/journal.pone.0009313.g003Figure 3 Double-immunolabeling analysis of autophagy markers and α-syn in the brains of patients with DLB. Vibratome sections from the temporal cortex of non-demented controls and DLB patients were immunolabeled with antibodies against α-syn, and co-labeled with antibodies against mTor, LC3 or Cathepsin D, and imaged with a laser scanning confocal microscope. (A–D) Double-immunolabeling analysis showing increased mTor immunoreactivity in neurons of DLB patients showing α-syn accumulation. (G–L) Double-immunolabeling analysis showing increased LC3 immunoreactivity in neurons of DLB patients showing α-syn accumulation. LC3 immunoreactivity was occasionally associated with LBs (arrows). (M–R) Double-immunolabeling analysis showing enlarged Cathepsin D-immunoreactive lysosomes (arrows) in neurons of DLB patients showing α-syn accumulation. Scale bar in panel (C) represents 15µm in panels A–L and 8µm in panels M–R. 10.1371/journal.pone.0009313.g004Figure 4 Electron microscopic analysis of abnormal autophagosomes in patients with DLB and in α-syn tg mice. (A) Representative image from a non-demented control case showing normal neuronal lysosomes (arrow). (B–D) Abnormal autophagosomes and accumulation of electrodense deposits (arrows) in intraneuronal membrane-bound structures in the brains of patients with DLB. (E) Representative image from a non tg mouse brain showing normal neuronal lysosomes (arrow). (F–H) Abnormal autophagosome morphology and accumulation of electrodense deposits (arrows) in intraneuronal membrane-bound structures in the brains of α-syn tg mice. Scale bar in panel (C) represents 0.5µm in all panels. Alterations in Molecular Components of the Autophagy Pathway in A-Synuclein Transgenic Mice Consistent with the studies in human brains, levels of mTor and p-mTor were increased in the mebrane fractions from brains of α-syn tg mice compared to non tg controls (Figure 5A–C). Moreover, levels of a molecular initiator of autophagy, Atg7, were reduced in α-syn tg brains compared to APP tg mice and non tg controls (Figure 5A, D). In contrast, other components of the autophagy pathway such as Atg5, Atg12 and Beclin-1 were not different among the non tg and tg mouse groups (Figure 5A, C, D). 10.1371/journal.pone.0009313.g005Figure 5 Immunoblot analysis of the autophagy pathway in the brains of APP and α-syn tg mice. Brain homogenates from non tg, APP tg, and α-syn tg mice were separated into membrane and cytosolic fractions, and 20 µg of each sample was subjected to gel electrophoresis. Immunoblots were probed with antibodies against mTor, phosphorylated (p) mTor, Beclin-1, Atg5, Atg7, Atg12 and Actin. (A) Representative immunoblots of membrane fractions. (B) Representative immunoblots of cytosolic fractions. (C) Semi-quantitative analysis of levels of mTor, p-mTor, and Beclin-1 in membrane fractions from the brains of non tg, APP tg and α-syn tg mice. Levels of mTor were significantly increased in APP tg and α-syn tg brains. (D) Semi-quantitative analysis of levels of Atg5, Atg7, and Atg12 in membrane fractions from the brains of non tg, APP tg and α-syn tg mice. Levels of Atg7 were significantly reduced in the brains of α-syn tg mice. All semi-quantitative measurements were normalized to actin levels as a loading control. p<0.05 compared to non tg controls by one-way ANOVA with post-hoc Dunnett's test. In the non tg and APP tg mice, immunohistochemical analysis showed moderate levels of mTor and Atg7 immunostaining in pyramidal neurons in the neocortex (Figure 6A, B, D, E, G) and hippocampus (not shown). In contrast, in the α-syn tg mice levels of neuronal mTor immunoreactivity were increased, while the levels of neuronal Atg7 immunolabeling were reduced (Figure 6C, F, G). Compared to non tg control mice, in APP and α-syn tg mice pyramidal cells contained enlarged lysosomes (Figure 6H–J, N) and abnormal levels of LC3 immunoreactivity (Figure 6K–M, O). Abundant α-syn monomer and oligomers were detected in the membrane and cytosolic fractions of α-syn tg mice (Figure S2A, B). By immunoblot analysis, levels of LC3 in the membrane fraction were increased and Cathepsin D was reduced in the α-syn tg mice compared to non tg controls (Figure S2A, C). Compared to control mice, in the APP tg mice, levels of LC3 were elevated while levels of Cathepsin D were unchanged in the membrane fraction (Figure S2A, C). Double-labeling studies showed that mTor and LC3 were abundant in neurons displaying α-syn accumulation (Figure 7A–L). These neurons also showed the presence of enlarged Cathepsin D-positive lysosomes compared to non tg controls (Figure 7M–R). Electron microcopy analysis confirmed the presence of abundant and abnormal autophagosomes in these neurons (Figure 4E–H). 10.1371/journal.pone.0009313.g006Figure 6 Immunohistochemical analysis of the autophagy pathway in the brains of APP and α-syn tg mice. Vibratome sections from the hippocampus of non tg, APP tg and α-syn tg mice were immunolabeled with antibodies against mTor, Atg7, Cathepsin D, or LC3, and imaged with a digital microscope. All images are from the temporal cortex. (A–C) Representative sections from non tg, APP tg and α-syn tg brains immunolabeled with an antibody against mTor. (D–F) Representative sections from non tg, APP tg and α-syn tg brains immunolabeled with an antibody against Atg7. (G) Semi-quantitative image analysis reveals a significant increase in mTor levels and a reduction in Atg7 levels in α-syn tg mice compared to non tg controls. (H–J) Representative sections from non tg, APP tg and α-syn tg brains immunolabeled with an antibody against Cathepsin D. Enlarged Cathepsin D-immunoreactive lysosomes (arrows) were detected in APP tg and α-syn tg mice. (K–M) Representative sections from non tg, APP tg and α-syn tg brains immunolabeled with an antibody against LC3. (N) Increased numbers of enlarged lysosomes (>1µm) in APP tg and α-syn tg mouse brains. (O) Semi-quantitative image analysis of LC3 immunoreactivity reveals increased LC3 levels in APP tg and α-syn tg brains. Scale bar in panel (C) represents 10µm in all microscopy images. p<0.05 compared to non tg controls by one-way ANOVA with post-hoc Dunnett's test. 10.1371/journal.pone.0009313.g007Figure 7 Double-immunolabeling analysis of autophagy and α-syn in the brains of α-syn tg mice. Vibratome sections from the brains of non tg and α-syn tg mice were immunolabeled with antibodies against α-syn, and co-labeled with antibodies against mTor, LC3 or Cathepsin D, and imaged with a laser scanning confocal microscope. All images are from the temporal cortex. (A–D) Double-immunolabeling analysis showing increased mTor immunoreactivity in α-syn-positive neurons in α-syn tg mice. (G–L) Double-immunolabeling analysis showing increased LC3 immunoreactivity in neurons of α-syn tg mice showing α-syn accumulation. (M–R) Double-immunolabeling analysis showing enlarged Cathepsin D-immunoreactive lysosomes (arrows) in neurons of α-syn tg mice showing α-syn accumulation. Scale bar in panel (C) represents 20µm in panels A–L and 10µm in panels M–R. Effects of Intra-Cerebral Infusion of mTor Inhibitor in A-Synuclein Transgenic Mice Since alterations in the autophagy pathway in DLB and in α-syn tg mice might be in part related to increased levels of mTor, we investigated whether blocking mTor with rapamycin might promote α-syn clearance and be neuroprotective. Compared to vehicle-infused mice, in α-syn tg mice that received rapamycin treatment, α-syn accumulation in neuronal cell bodies and synapses was reduced and redistributed to the axons (Figure 8A–C, G). Moreover, compared to vehicle-treated α-syn tg mice, rapamycin treatment resulted in increased levels of LC3 (Figure 8D–G) and Cathepsin D (Figure 8H–J, N) immunoreactivity. Furthermore, this treatment ameliorated the dendritic pathology in the neocortex as reflected by image analysis of MAP2 immunolabeling of the neuropil (Figure 8K–N). By immunoblot analysis, compared to tg mice infused with vehicle alone, α-syn tg mice that received intra-cerebral infusions with rapamycin displayed reduced levels of α-syn in the membrane fraction with a concomitant increase in the lysosomal fraction (Figure 8O, P). In addition, rapamycin increased the levels of LC3 and Cathepsin D immunoreactivity (Figure 8O, P). 10.1371/journal.pone.0009313.g008Figure 8 Immunohistochemical and immunoblot analysis of the effects of rapamycin treatment in α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with antibodies against α-syn, LC3, Cathepsin D or MAP2 and imaged with a digital microscope. All images are from the temporal cortex. For panel O, brain homogenates from non tg and α-syn tg mice were separated into membrane and lysosomal fractions, and 20 µg of each sample was subjected to gel electrophoresis. (A–C) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against α-syn. (D–F) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against LC3. (G) Semi-quantitative image analysis showing reduced α-syn immunoreactivity and increased LC3 immunoreactivity in the hippocampus of α-syn tg mice treated with Rapamycin. (H–J) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against Cathepsin D. (K–M) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against MAP2. (N) Semi-quantitative image analysis showing increased Cathepsin D immunoreactivity in the hippocampus of α-syn tg mice treated with Rapamycin. Reduced levels of MAP2 in the hippocampus of vehicle-treated α-syn tg mice is rescued by Rapamycin treatment. (O) Representative immunoblot analysis of membrane and lysosomal fractions probed with antibodies against α-syn, LC3, and Cathepsin D. (P) Semi-quantitative image analysis of immunoblots showing redistribution of α-syn from membrane to lysosomal fractions and an associated increase in LC3 and Cathepsin D levels. All semi-quantitative measurements were normalized to actin levels as a loading control. Scale bar in panel (C) represents 40µm in panels A–C, 20µm in panels D–E and H–J, and 10µm in panels K–M. p<0.05 compared to vehicle-treated non tg controls by one-way ANOVA with post-hoc Dunnett's test. #p<0.05 compared to vehicle-treated α-syn tg mice by one-way ANOVA with post-hoc Tukey-Kramer test. Taken together, these results support the possibility that alterations in the autophagy pathway and more specifically in mTor and Atg7 are associated with accumulation of α-syn and neurodegeneration in DLB cases and α-syn tg models, and activation of autophagy with rapamycin can revert this effect. Lentivirus Delivery of Atg7 Rescues α-Synuclein Accumulation and Neuronal Deficits in Transgenic Mice Since in addition to the increase in mTor, we also observed a reduction in Atg7 levels in DLB and α-syn tg mice, we sought to determine whether viral-mediated delivery of Atg7 might also promote α-syn clearance and rescue neurodegenerative deficits in α-syn tg mice. For this purpose, we delivered the Atg7 lentivirus via stereotaxic injection to the temporal cortex and hippocampus of non tg and α-syn tg mice [55]. Compared to non tg and α-syn tg mice treated with the LV-control (Figure 9A–D, G) delivery of LV-Atg7 resulted in increased expression in Atg7 in pyramidal neurons in the hippocampus and in areas adjacent to the injection track in the neocortex (Figure 9E–G). Atg7 immunostaining was localized primarily to the neuronal perykaria surrounding the injection track with some extension to the apical dendrites (Figure 9E). Compared to non tg mice (Figure 9H, I), α-syn tg mice injected with the LV-control contained abundant intracellular aggregates of α-syn (Figure 9J, K); in contrast, following LV-Atg7 injection, there was a considerable reduction in the intra-neuronal α-syn accumulation in the areas adjacent to the injection track (Figure 9L–N). 10.1371/journal.pone.0009313.g009Figure 9 Immunohistochemical analysis of the effects of LV-Atg7 treatment in α-syn tg mice. For panels A–M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′–M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A–M. For panels O–T, effects of rapamycin treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A–F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C–F) that received injections with LV-control (A–D) or LV-Atg7 (E, F) and were immunolabeled with an antibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and α-syn tg mice show increased Atg7 levels ipsilateral to the injection sites in the brains of animals that received LV-Atg7. (H–M) Representative sections from the brains of non tg (H, I) and α-syn tg mice (J–M) that received injections with LV-control (H–K) or LV-Atg7 (L, M) and were immunolabeled with an antibody against α-syn. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (N) Semi-quantitative image analysis of α-syn immunoreactivity in non tg and α-syn tg mice show reduced α-syn levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. (O–T) Representative sections from the brains of non tg (O, P) and α-syn tg mice (Q–T) that received injections with LV-control (O–R) or LV-Atg7 (S, T) and were immunolabeled with an antibody against MAP2. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (U) Semi-quantitative image analysis of MAP2 immunoreactivity in non tg and α-syn tg mice shows a recovery of MAP2 levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. Scale bar in panel (F) represents 0.1mm in panels A–F and H–M, 20µm in panels A′–F′ and H′–M′, and 10µm in panels O–T. p<0.05 compared to LV-control-treated non tg controls by one-way ANOVA with post-hoc Dunnett's test. #p<0.05 compared to LV-control-treated α-syn tg mice by one-way ANOVA with post-hoc Tukey-Kramer test. Concomitant with the reduction in α-syn accumulation, analysis of the dendritic marker MAP2 showed an increase in the percent area of the neuropil covered by dendrites in α-syn tg mice that received the LV-Atg7 (Figure 9O–U), supporting the possibility that the reduction of α-syn accumulation in the LV-Atg7-treated animals ameliorated the structural damage to neurons of α-syn tg mice. Importantly, no significant deleterious effects on neuronal or synapto-dendritic content were observed in non tg mice that received injections with LV-Atg7 (not shown). Together, these data suggest that delivery of LV-Atg7 to the α-syn tg mice reduced the accumulation of α-syn and related neuronal pathology by inducing a physiological autophagic response. Overexpression of Atg7 in an In Vitro Model Reduces α-Synuclein Accumulation, while Atg7 Knockdown Exacerbates Autophagic Deficits and α-Synuclein Accumulation To further confirm the effects of LV-Atg7 in activating autophagy and promoting α-syn clearance, we utilized a neuronal cell model to study autophagy in the presence of α-syn accumulation [42]. For this purpose, B103 neuronal cells were infected with lentivirus expressing an LC3-GFP fusion protein and co-infected with LV-control or LV-αsyn. The LC3-GFP is a marker for autophagy activation, and increased LC3-GFP fluorescence indicates increased autophagic activity. These cells were then infected with either LV-Atg7 or a virus encoding an shRNA against Atg7 or a control shRNA against luciferase. Immunocytochemical analysis demonstrated that compared to controls (Figure S3A) levels of Atg7 were higher in LV-Atg7-infected cells (Figure S3B), while baseline levels of Atg7 were reduced by the LV-shAtg7 (Figure S3C). Consistent with the immunocytochemical analysis, western blot confirmed the expression and knockdown of Atg7 in the LV infected neuronal cell line (Figure S3D). Immunolabeling studies with an antibody against α-syn demonstrated that compared to controls (Figure 10A, F) and LV-Atg7 infected cells (Figure 10B, F), there was an increase in expression of α-syn in LV-αsyn infected cells (Figure 10C, F). Infection with LV-Atg7 resulted in increased numbers of LC3-GFP grains per cell, consistent with an activation of autophagy (Figure 10B, E). In cells co-infected with LV-αsyn, co-expression of Atg7 also resulted in significant activation of autophagy (Figure 10D, E) and a reduction in α-syn accumulation (Figure 10D, F). Similar experiments with LV-βsyn and LV-Atg7 showed no reduction in levels of β-syn, indicating that Atg7 expression specifically reduced the accumulated levels of α-syn (data not shown). 10.1371/journal.pone.0009313.g010Figure 10 Immunocytochemical analysis of the effects of Atg7 over-expression or knockdown in a neuronal cell model. For Atg7 overexpression, B103 neuronal cells on coverslips and infected with a lentivirus expressing LC3-GFP in combination with empty LV-control, LV-Atg7, or LV-α-syn. For Atg7 knockdown, B103 neuronal cells on coverslips were infected with a lentivirus expressing LC3-GFP in combination with empty LV-shControl, LV-shAtg, or LV-αsyn. Cells were fixed and immunolabled with an antibody against α-syn and imaged with a laser scanning confocal microscope. In each set of 3 panels, the upper left panel depicts GFP fluorescence, the upper right panel depicts α-syn immunolabeling, and the lower panel depicts the merged image. (A–D) Representative images showing GFP fluorescence (marker of LC3 localization) and α-syn immunoreactivity in B103 cells infected with LV-control (A), LV-Atg7 (B), LV-αsyn (C) or LV-Atg7+LV-αsyn (D). (E) Semi-quantitative analysis of LC3-GFP positive punctae shows an increase in LC3 in cultures infected with LV-Atg7 or LV-α-syn alone or in combination. (F) Semi-quantitative analysis of α-syn immunoreactivity reveals a reduction in α-syn levels in cultures co-infected with LV-Atg7 and LV-αsyn. (G–J) Representative images showing GFP fluorescence (marker of LC3 localization) and α-syn immunoreactivity in B103 cells infected with LV-shControl (G), LV-shAtg7 (H), LV-αsyn (I) or LV-shAtg7+LV-αsyn (J). (K) Semi-quantitative analysis of LC3-GFP positive punctae shows a reduction in LC3 in cultures infected with LV-shAtg7 alone or in combination with LV-αsyn. (L) Semi-quantitative analysis of α-syn immunoreactivity reveals an increase in α-syn levels in cultures co-infected with LV-shAtg7 and LV-αsyn. Scale bar in large panel in (D) represents 15µm in all small panels and 10µm in all large panels. p<0.05 compared to LV-control-treated cultures by one-way ANOVA with post-hoc Dunnett's test. #p<0.05 compared to LV-αsyn-treated cultures by one-way ANOVA with post-hoc Tukey-Kramer test. In contrast, compared to LV-shControl (Figure 10G, K), when neuronal cells were infected with the LV-shAtg7, levels of LC3-GFP were reduced (Figure 10H, K). Similar effects were observed in neuronal cells co-infected with LV-shAtg7 and LV-αsyn (Figure 10I–K). Moreover, compared LV-shControl (Figure 10G, I, L), in cells infected with LV-shAtg7, the levels of α-syn accumulation were increased (Figure 10J, L). Taken together, these results support the possibility that activating the autophagy pathway with rapamycin or with viral delivery of Atg7 might reduce the accumulation of α-syn and rescue the associated neurodegenerative alterations. Discussion Recent evidence in cell-based models of PD-like pathology indicate that alterations in lysosomal functioning and autophagy might participate in the mechanisms of α-syn-mediated neurodegeneration [23], [29], [35], [36], [37], [38], [39]. However it was unclear which molecular components of the autophagy pathway might be dysregulated in the brains of patients with DLB/PD and in α-syn tg models. For the present study we chose investigate potential alterations in components of autophagy in DLB cases (rather than pure PD) because after AD, these cases represent the most common form of dementia and movement disorders in the aging population and display widespread cortical and subcortical pathology. Remarkably, we found that in DLB cases and in α-syn tg mice levels of mTor were elevated and Atg7 expression was reduced. mTor and LC3 was co-localized with neurons displaying α-syn accumulation and neurodegenerative changes. This is of interest because it provides a potential alternative explanation for the molecular alterations in autophagy in sporadic forms of LBD. mTor and Atg7 are early initiators of the macroautophagy pathway. Inhibition of mTor by nutrient reduction or by activation of PI3K results in activation of the Atg kinase 1 that in turn phosphorylates Atgs that participate in the AV formation [71], [72], [73]. The mechanisms through which increased mTor and reduced Atg7 might participate in the neuropathology of DLB are not completely clear. However, such alterations are predicted to result in deficient initiation of the autophagy process. This in turn might result in progressive accumulation of α-syn aggregates that further interfere with the fusion of lysosomes and formation of autophagosomes, as has been suggested by other studies [23], [40], [41]. This may then lead to the formation of enlarged and atypical AV-like structures [42]. Supporting this possibility, the present study also showed that the cortical neurons in DLB cases and in α-syn tg mice contained enlarged lysosomes and autophagosomes similar to those described in AD [33]. The formation of such abnormal AV-like structures in DLB and α-syn tg mice is consistent with recent reports in neuronal cells lines overexpressing α-syn [42]. These cells show the presence of granular α-syn aggregates that co-localize with abnormally enlarged LC3-positive structures [42]. The accumulation of α-syn and the neurodegenerative phenotype in neuronal cells was reverted by activation of the autophagy pathway with a gene therapy approach delivering Beclin-1 with a lentivirus [42] or with rapamycin [42], [74], [75]. In agreement with these findings, the present study showed in vivo that infusion of rapamycin or injection of LV-Atg7 into the brains of tg mice reduced the accumulation of α-syn and was neuroprotective. This is consistent with previous in vivo studies showing that rapamycin is neuroprotective in models of neurodegeneration [76], [77], AD [78] and Huntington's Disease [79], [80]. Moreover, a recent study showed that blocking mTor by overexpression of the translation inhibitor Thor (4E-BP) can reduce the pathologic features in PD models, including degeneration of dopaminergic neurons in Drosophila [81]. Moreover, rapamycin activates in vivo 4E-BP and rapamycin is also capable of ameliorating the pathology associated with mutations in other PD associated genes such as Pink1 and parkin [81]. In familial types of parkinsonism, mutant forms of α-syn [23] have been shown to disrupt lysosomal clearance by blocking CMA. Further supporting a role for lysosomal dysfunction in DLB and PD, recent studies have shown that in lysosomal storage disorders such as Gaucher disease [43], [44] and Niemann-Pick disease [45], there is increased predisposition to develop parkinsonism and α-syn accumulation. Other animal studies in models of PD, such as in animals exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP), have also revealed autophagic dysfunction associated with alterations in signal transduction pathways [82]. In addition, increased susceptibility to develop PD appears to be associated with polymorphisms in lysosomal genes such as those associated with Gaucher disease and Niemann-Pick disease. Moreover, recent studies have shown that reduced Cathepsin D expression results in α-syn accumulation and degeneration of the dopaminergic system in experimental models and in patients with PD [83]. Cathepsin D is now considered one of the main lysosomal enzymes involved in α-syn degradation [84] and overexpression of Cathepsin D reduces the pathology associated with α-syn accumulation [85]. Selective alterations in molecular components of the autophagy pathway might result in degeneration of specific neuronal populations in neurological disorders. For example, previous studies have shown that in sporadic AD there is a profound reduction in the levels of Beclin-1 [34], while the neurodegenerative process in familial forms of fronto-temporal dementia and ALS has been linked to mutations in charged multivesicular body protein-2B (CHMP2B) [86], [87], [88], and in Huntington's Disease polyglutamate aggregates trap mTor and disrupt autophagy [80]. The mechanisms through which levels of mTor might be upregulated and Atg7 downregulated in DLB and α-syn tg mice are not completely clear. However, targeted reduction of autophagy genes including Atg7 results in behavioral defects, including abnormal limb-clasping reflexes and a reduction in coordinated movement, and died within 28 weeks of birth. Furthermore, Atg7 deficiency results in neurodegeneration of the cerebral and cerebellar cortices [89]. In conclusion, this study supports the notion that selective molecular alterations in the autophagy pathway and more specifically in mTor and Atg7 are associated with DLB and α-syn tg models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects. Supporting Information Figure S1 Immunoblot analysis of α-syn levels and lysosomal markers in the brains of AD and DLB patients. Brain homogenates from the temporal cortex of non-demented controls, AD, and DLB patients were separated into membrane and cytosolic fractions, and 20 µg of each sample was subjected to gel electrophoresis. Immunoblots were probed with antibodies against α-syn, Cathepsin D, LC3 and Actin. (A) Representative immunoblots of membrane fractions. (B) Representative immunoblots of cytosolic fractions. (C) Semi-quantitative analysis of levels of α-syn, Cathepsin D and LC3 in membrane fractions from the brains of control, AD and DLB patients. Levels of Cathepsin D were increased in the brains of AD patients but reduced in the brains of DLB patients, while levels of LC3 were increased in the brains of both AD and DLB patients. All semi-quantitative measurements were normalized to actin levels as a loading control. p<0.05 compared to non-demented controls by one-way ANOVA with post-hoc Dunnett's test. (0.41 MB TIF) Click here for additional data file. Figure S2 Immunoblot analysis of α-syn levels and lysosomal markers in the brains of APP tg and α-syn tg mice. Brain homogenates from non tg, APP tg, and α-syn tg mice were separated into membrane and cytosolic fractions, and 20 µg of each sample was subjected to gel electrophoresis. Immunoblots were probed with antibodies against α-syn, Cathepsin D, LC3 and Actin. (A) Representative immunoblots of membrane fractions. (B) Representative immunoblots of cytosolic fractions. (C) Semi-quantitative analysis of levels of α-syn, Cathepsin D and LC3 in membrane fractions from the brains of non tg, APP tg and α-syn tg mice. Levels of Cathepsin D were significantly reduced in α-syn tg brains, while levels of LC3 were increased in the brains of APP tg and α-syn tg mice. All semi-quantitative measurements were normalized to actin levels as a loading control. *p<0.05 compared to non tg controls by one-way ANOVA with post-hoc Dunnett's test. (0.33 MB TIF) Click here for additional data file. Figure S3 Immunocytochemical and immunoblot characterization of lentivirus-mediated Atg7 over-expression and knockdown in a neuronal cell line. B103 neuronal cells on coverslips were infected with LV-Atg7 or LV-shAtg7, followed by fixation and immunolabeling with an antibody against Atg7, or lysis and immunoblot analysis with antibodies against Atg7 or Actin. (A) Representative image of endogenous Atg7 immunoreactivity in cells infected with empty LV-control. (B) Representative image showing increased Atg7 immunoreactivity in cells infected with LV-Atg7. (C) Representative image showing reduced Atg7 immunoreactivity in cells infected with LV-shAtg7. (D) Representative immunoblot showing Atg7 levels in cells infected with LV-Atg7 or LV-shAtg7. Scale bar in panel (C) represents 40µm in all microscopy images. (0.80 MB TIF) Click here for additional data file. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by NIH grants AG18440 and AG5131. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Dauer W Kholodilov N Vila M Trillat AC Goodchild R 2002 Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci U S A 99 14524 14529 12376616 2 Dauer W Przedborski S 2003 Parkinson's disease: mechanisms and models. 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==== Front Cell DivCell Division1747-1028BioMed Central 1747-1028-5-410.1186/1747-1028-5-4ReviewRole of senescence and mitotic catastrophe in cancer therapy Singh Richa [email protected] Jasmine [email protected] Yogeshwer [email protected] Proteomics Laboratory, Indian Institute of Toxicology Research, (Council of Scientific & Industrial Research), PO Box 80, MG Marg, Lucknow-226001, India2010 21 1 2010 5 4 4 5 1 2010 21 1 2010 Copyright ©2010 Singh et al; licensee BioMed Central Ltd.2010Singh et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Senescence and mitotic catastrophe (MC) are two distinct crucial non-apoptotic mechanisms, often triggered in cancer cells and tissues in response to anti-cancer drugs. Chemotherapeuticals and myriad other factors induce cell eradication via these routes. While senescence drives the cells to a state of quiescence, MC drives the cells towards death during the course of mitosis. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may secrete proteins with potential tumor-promoting activities. The other anti-proliferative response of tumor cells is MC that is a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Different classes of cytotoxic agents induce MC, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. In this review, we compare the two pathways and mention that they are activated to curb the growth of tumors. Altogether, we have highlighted the possibilities of the use of senescence targeting drugs, mitotic kinases and anti-mitotic agents in fabricating novel strategies in cancer control. ==== Body Introduction The incidence of cancer worldwide is on a rise, making it only second to coronary heart disease [1]. Unifying property of cancer includes six canonical characteristics: self sufficiency in growth signals, insensitivity to growth inhibitory signals (anti-growth), evasion of programmed cell death (apoptosis), unlimited proliferation of diseased cells, sustained angiogenesis, intrusion of adjacent cells and tissues and metastasis to distant niches in the body [2]. Genetic instability associated with telomere attrition or cell cycle checkpoint dysfunction is an early event in tumorigenesis. Telomeres are guanine rich tandem nucleotide repeats flanking the ends of chromosomes in all eukaryotic cells responsible for maintaining genetic integrity and implicated in aging (senescence) and cancer [3]. Cell cycle checkpoints or mitotic kinases (MKs) are the rigorous quality control steps of mitosis [4] that function in preserving the fidelity and integrity of DNA and allow mitosis to continue with accurately functioning DNA, spindle assembly, centrosome and kinetochore thus preventing cell death via mitotic catastrophe (MC). MC therefore, refers to the process when cells attempt to divide without proper repair of DNA damage due to faulty cell cycle checkpoint functioning consequently resulting in formation of giant, multinucleated cells with condensed chromosomes, distinguishing MC morphologically from other modes of cell deaths. Abundant data amassed from several laboratories have provided innumerable instances to show that it is better to cure this dreadful disease at preventable stage by early diagnosis and consequent therapeutic intervention. Strategies for cancer treatment has generated significant interest in the recent past and therefore, the focus of research endeavors on understanding the mechanism of cell death pathways applicable in treatment of cancer which include not only apoptosis but necrosis, autophagy, MC and in context of cancer therapy, senescence has always been there [5]. This review will explore major highlights on the role of senescence and MC triggered in various cancers by chemotherapeutic intrusion and opens avenues for expanding research work by comparing the results obtained so far. Senescence: Terminal growth arrest in dividing cells The term senescence is derived from the Latin word senex, meaning "old age" or "advanced in age". Senescence at the cellular level is a physiological program of cellular growth arrest that is triggered by the shortening of telomeres or by stress [6]. This permanent growth arrest is also considered a type of cell death in the context of cancer therapy by some researchers [7,8] and some consider it similar to the programmed cell death by 'apoptosis' [9]. Senescence can be broadly categorized into two classes: accelerated or stress induced premature senescence (SIPS) and replicative senescence (RS) and both are believed to be essential anti-carcinogenic programs in normal cells. Accelerated senescence occurs in response to the activation of Ras/Raf pathways [10] and by supra-physiological mitogenic signaling [11]. The phenomenon of RS was first described in the context of normal human cells explanted in culture that failed to divide beyond a finite number of fifty divisions [12] and it is a well-known defining property of euploid mammalian cells [13]. Telomere dynamics has been shown to be a critical component of both aging and cancer [14]. Telomeres, the highly repetitive DNA (TTAGGG sequence) which camouflages chromosome ends [15] prevent nucleolytic degradation, end-to-end fusion, irregular recombination, and other events that are normally lethal to a cell [15]. With each cell division a part of telomere gets eroded [16,17] and the chromosome being passed to the progeny gets the clipped off telomere. Thus genetic integrity is gradually lost with telomeres progressively shortening after each division as a result of end-replication problems and hence, is a conspicuous feature in almost all dividing cells which do not express or maintain sufficient telomerase activity to maintain the telomeres. Telomerase reverse transcriptase (hTERT), whose amount is lessened after birth, functions by replenishing telomere by adding TTAGGG sequence at the 3'end of DNA. Telomerase activity is measured by TRAP assay or RT-PCR. Less frequently other alternative mechanism of telomere maintenance namely Alternative Lengthening of Telomeres (ALT) is opted [18]. Telomere dysfunction (short telomeres) has been associated with the initiation and progression of mouse and human intestinal neoplasia [19] and may also increase the risk of developing epithelial cancers by a process of breakage-fusion-bridge that leads to the formation of complex nonreciprocal translocations (a classical cytogenetic feature of human carcinoma) [20]. Blood relative telomere length was found to represent a strong independent prognostic indicator in patients with advanced breast cancer [21]. Similarly mean telomere length was statistically shorter in case patients with head and neck cancer as compared with control as measured with the southern blot and quantitative-fluorescent in situ hybridization assay [22]. Telomerase and p53 play critical roles in tumorigenesis and senescence. Senescent cells exhibit distinct morphology in culture. They are enlarged and flattened with increased granularity [23] exhibit SA-β-gal staining and a characteristic senescence associated heterochromatin foci (SAHF) formation [24] and comparatively less dense culture than a confluent young culture probably because they are more sensitive to cell-cell contact inhibition [12,13]. Even though they cannot divide under mitogenic stimulation yet they remain metabolically and synthetically active in in vitro conditions for several years [24] but can not resume cell growth after drug withdrawal. SA-β-gal, the most widely used surrogate marker with considerable specificity to senescent cells appears to reflect an increased lysosomal mass [23]. Another marker is clusterin/apolipopterin J, is a highly conserved ubiquitously expressed secreted glycoprotein has been implicated in many physiological processes, gets upregulated during stress induced premature senescence, in vivo aging, RS, in several age linked deformities, neuropathological disorders like Alzhiemers disease and dementia and has a direct relationship with human longevity [25]. Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb. Role of p53 Telomere-induced senescence has been proved to be as effective as apoptosis in reducing cancer incidence and is mediated by the tumor suppressor gene, p53 [26]. Mutations in the p53 gene frequently appear in human tumors conferring aggressive oncogenic properties such as exacerbated malignant transformation and metastatic phenotype when over-expressed in p53-null cells. P53 gets activated upon genotoxic and non genotoxic stresses like oxidative damage and activates p21 and ultimately culminates the cell to senescence. Mice with a point mutation (p53(R172H)) in their endogenous p53 loci act as a model for the human Li-Fraumeni syndrome. Genetic alterations at chromosomes 3p, 6p, and 1lq were frequently found early in tumor development and showed additional allelic losses at chromosome arms 6q, 17p and 18q. Genes for telomerase suppression are presumably located on chromosomes 3, 4 and 6 [27]. P53 over expression has been directly associated with unfavorable clinico-pathologic factors such as advanced stage, histologic subtype, advanced patient age and nodal metastasis in endometrial carcinomas while bcl-2 expression was related with younger age, favorable grade and PR expression by tumor cells. Patient survival is however not related to the tested biomarkers [28]. In humans, TP53 codon 72 Arginine to Proline polymorphism was found to affect both cancer incidence and longevity as well [29]. The senescence-associated signature of p53 isoform expression (that is, elevated p53beta and reduced Delta133p53) was observed in vivo in colon adenomas with senescent phenotypes. The increased Delta133p53 and decreased p53beta isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma [30]. Other tumor suppressor genes P107 is required for the initiation of accelerated cellular senescence in the absence of Rb and p130 may be required to prevent the onset of this phenomenon in un-stimulated prostate cancer cells lacking a functional Rb allele [31]. Cell cycle regulatory proteins are more sensitive to exogenous hormone treatment in postm-HBT (postmenopausal human breast tissue) than in pre-HBT (premenopausal human breast tissue) [32]. Olsson et al advocates that bfl-1 (tumor suppressor bcl-2 family member) contributes to chemo resistance and might be a therapeutic target in B-cell chronic lymphocytic leukaemia [33]. The activation of PI3K/Akt pathway is involved in the late-stage progression and metastasis of gastric cancer and attenuation of p-Akt by 2-ME suppresses metastasis [34]. Yet another tumor suppressor Promyelolytic leukemia (PML) regulates p53 acetylation in both RS as well as Ras induced accelerated senescence [35]. Senescence in cancer cells: In vitro studies A large number of in vitro studies have been reported where a wide range of chemotherapeutical antidotes induce senescence like morphological changes and SA-β-gal expression in cancer cells activating the pathway of senescence. Research into the induction of cellular senescence as cancer therapy has however, been hindered by a lack of compounds that efficiently induce this response. To overcome this, Ewald et al (2009) by using dual Hoechst 33342 and SA-β gal staining identified library compounds that induce senescence in prostate cancer cells [36]. It is well acknowledged that telomerase and the maintenance of telomeres are key players in the ability of stem and cancer cells to bypass senescence and be immortal. Proliferation of telomerase (-) pre-malignant cells leads to telomere dysfunction and increased genomic instability suggesting one possible sequence of events leading to immortalization of breast epithelial cells during cancer progression [37]. The increased h-TERT expression may be a cellular response to genomic insults by various metal toxicants like arsenic that may also act as a tumor promoter in mammalian carcinogenesis as studied in blood cells by Mumford et al hTERT-specific T cells could contribute to the immunosurveillance of breast cancer suggests novel opportunities for both therapeutic and prophylactic vaccine strategies for cancer [38]. In one of the studies, using non-small lung adenocarcinoma A549 cells, it was shown that after treatment with DNA damaging anti-tumor drugs like caffeine, cells become permanently growth-arrested as a result of so-called drug-induced premature senescence (pseudo-senescence) or SIPS. Similarly, lowered efficacy of anti-cancer doxorubicin (due to dose dependent toxicity) against breast cancer cells can be increased when used in conjunction with siRNA inhibitor of telomerase [39]. Yet another study advocated the use ofGRN163L (novel telomerase template antagonist) in the treatment of breast cancer by augmenting the effects of paclitaxel [40]. Hence clearly proposing that inhibition of telomerase is a potential treatment strategy for inducing senescence. It has also been shown that caveolin-1 targets Mdm2/p53-mediated pathway and causes senescence in breast cancer cells [41]. Another study reported that bleomycin, a widely used anti-tumor agent, causes senescence of lung cancer cells by modulating the roles of caveolin-1, a protein abundant in lung fibroblasts and smooth muscle and endothelial cells [42]. A recent study showed that the activation of the p53-p21(Cip1/WAF1) pathway acts as a major mediator of cellular senescence induced by CKII inhibition in HCT116 colon carcinoma cells [43]. A senescence-inducing effect of doxorubicin on the same cells, in another study, had a dual effect-it stopped the proliferation of the majority of the cells and led to the appearance of proliferating aneuploid cells [44]. Likewise, while characterizing ashwagandha and its molecular mechanisms Wadhwa et al provided the first example that phytochemical(s) have both anti-cancer and anti senescent activities and pointed towards the molecular link between aging and cancer using normal human fibroblasts through decreased accumulation of molecular damage, down-regulation of the SA-β gal activity and the senescence marker protein, p21(WAF-1), protection against oxidative damage, and induction of proteasomal activity [45]. In one of the studies, by silencing BRCA1 expression at different levels through RNA interference technology in a series of partially transformed (HBL100) and tumorigenic (MCF7 and T47D) breast cancer cell lines, cell models were probed by clonogenic assay for their response to several DNA-damaging agents (mitomycin C, cisplatin, doxorubicin, and etoposide) commonly used in cancer therapy [46]. The increased sensitivity to these compounds displayed by BRCA1-defective cells was correlated to an increased fraction of growth-arrested, enlarged, multinucleated SA-β-galactosidase-positive senescent cells [46]. Melanocytic nevi frequently harbor oncogenic BRAF mutations and recently it was found that a subpopulation of melanocytes possess the ability to survive BRAF induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells [47] and thus have implications in skin cancer treatment. In vitro experiments with therapeutic nucleic acids successfully inhibited E6/E7 oncogene expression and caused induction of apoptosis and/or senescence in cervical carcinoma cells. A useful assay was described by Lau et al [48] to predict the response of the patient to a set of medicines without administering them by testing the susceptibility of a sample of cancer cells in vitro and comparing it to the standard regimen. Apart from these, it has been observed that cells' passage number controls the appearance of senescence. Normal human diploid fibroblasts approach senescence near passage 64 through RNaseT2 expression, which however fails to induce senescence in SV40 immortalized cell lines [49]. Rat chondrocytes show the onset of senescence in the 4th passage [50] while human rheumatoid arthritis fibroblast-like synoviocytes exhibit ageing at 10th passage [51]. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively, suggesting that ectopic expression of hTERT, in addition to telomere length maintenance by activating telomerase, also functions in regulating senescence induction [52]. Recently, a study explored the self-renewal potential of human breast stem cells and found that it gets exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance [53]. Senescence in animal models: In vivo studies Induction of senescence upon drug administration has been proposed as a possible anti-cancer treatment in various animal models. The finite proliferative potential of normal human cells leads to RS, which is a critical barrier to tumor progression in vivo. By studying embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression in nude mice, it was postulated that they may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype [54]. β-carotene provides protection against O3-induced skin oxidative stress in female SKH-1 mice skin, which is consistent with a protective role for beta-carotene in the skin hence has implications in skin cancer and aging or senescence of skin [55]. A novel target of NESH-SH3 (TARSH), cellular senescence related gene in mouse embryonic fibroblasts may suppress tumor development in pulmonary tumorigenesis mouse model by causing an increase in SA-β-gal activity and this was attributed to p53-dependent p21(Cip1) accumulation [56]. Pituitary tumor transforming gene deletion results in pituitary p21 induction and abrogates tumor development in Rb(+/-)Pttg(-/-) mice. Senescence was evidenced by increased p21 and SA-β-galactosidase. Aneuploid pituitary cell p21 may constrain pituitary tumor growth, thus accounting for the very low incidence of pituitary carcinomas [57]. Work by Efimova et al using p38-null mice skin carcinogenesis model strongly suggests a role for p38delta (key regulator in senescence, tumorigenesis, survival, inflammation etc) in promoting cell proliferation and tumor development in epidermis and may have therapeutic implication for skin cancer [58]. Three xenograft breast cancer mouse models, 2 of them with a TP53 mutation and one without it, were studied for their immediate response to high doses of epirubicin-cyclophosphamide. TP53 wild type stained positive for SA-β-galactosidase staining and also over expressed P21 but TP53 mutant did not succumb to senescence suggesting that treatment induced senescence is mediated via functional p53 in breast cancer [59]. More in vivo studies are however, needed to elucidate the role of senescence in cancer. Although these concepts are well supported in these models, translating them to clinical oncology remains a challenge. Neosis - Achilles heel of cancer cells evading senescence The physiological phenomenon of senescence serves as a lucrative pathway to annihilate deleterious cancer cells and tissues. This program of senescence is activated upon the administration of various anti-cancer regimens. Even though this is not a universal mechanism of curbing tumor cell growth, yet a considerable number of instances of in vitro as well as in vivo studies have been cited to decipher the metabolic pathway it targets and these studies have produced useful results that have enhanced and refined our knowledge about these pathways (Figure 1) and will be helpful in delineating new treatment strategies for curtailing cancer. Several studies [60,61], however provide compelling evidence that some cancer cells which are mitotically non-viable escape cell death, due to the accumulation of some genetic and epigenetic mutations and p53/pRB/p53Ink4a-dependent senescence checkpoint malfunctioning resulting in telomerase dysfunction [61,62] and finally evade cell death via continued progression through neosis. Such cells acquire so called' immortality' and to eliminate them, different strategies need to be designed. These cells multiply by a unique route called 'neosis' that facilitates in its progression and existence thereby evading the program of senescence. It has been described as a parasexual, somatic, reduction division in cancer [62]. Although neosis-like events have been reported in the literature sporadically for more than a century [63] under different names, they have been neglected due to the lack of appreciation of the significance of this process in cancer biology. Neosis may be a fundamental step in current concept of multi step carcinogenesis. Studying the behavior of individual neotic clones has revealed the significance of their central role in cancer [64]. Non-synchronous occurrence of secondary/tertiary-neosis (Figure 2) creates the illusion of the existence of cancer stem cells and the 'mirage' of immortality of cancer cells. Figure 1 Genes involved in senescence. Figure 2 Prerequisites for the onset of neosis and step-wise depiction of primary neosis (P/neosis) and secondary and tertiary neosis (S/T neosis). When a normal diploid cell accumulates genetic mutation owing to exposure, either dies following apoptosis or necrosis or may enter mitotic crisis and after repair again re-enters cell cycle or may become tetraploid after few hours or become polyploidy and succumb to senescence or may circumvent senescence and divide by neosis. Neosis of non-viable NMCs may give rise to genetically viable daughter cells 'Raju cells' by P/neosis and further divide and re-divide by S/T neosis. The number of progenies may vary from one to infinite and differ from NMCs and other daughter cells unlike conventional mode of division, mitosis. Number of surviving progenies depends on the 'survival of the fittest'. Some of the genetic and epigenetic alterations become the achilles heel of the mutated tumors that bypass the effect of certain classes of anti-cancer agents. That is, patients whose tumors carry such defects can be stratified for respective therapy rendering some classic DNA damaging agents called neosicides into "targeted therapies." Development of novel strategies to improve current status of cancer therapy will require identification and exploitation of yet unrecognized differences between normal and tumor cells with respect to propagation, evolution and development of resistance to conventional treatments [65]. The discovery of neosis has identified novel cellular targets, against which one can identify novel neosis-specific molecular targets in order to design anti-neotic agents or neosicides that will be more specific in their action and do less harm to non diseased cells. A judicial combination of senescent drugs with efficient neosicides could further improve the status of cancer control. MC and role of MKs in cancer According to the tenets of cancer biology, tumor cells arise after about 13 mitotic divisions of the initiated cell [66]. MKs, are rigorous quality control steps of mitosis and function in preserving the fidelity and integrity of DNA and allow mitosis to continue only with accurately functioning DNA, spindle assembly, centrosome and kinetochore thus preventing MC [67]. Malfunctioning of MKs are intimately involved in the development of errors in a vast majority of solid tumors and hematological malignancies. MC is an event in which a cell is destroyed during mitosis. This is believed to be caused through apoptosis as a result of an attempt at aberrant chromosome segregation early in mitosis, or as a result of DNA damage later, during the metaphase/anaphase transition. Cells which fail to go through a MC after mitotic failures are likely to create aneuploid cells when they later reproduce, posing a risk of oncogenesis, potentially leading to cancer [67]. Hence MC is also in the league of processes which participate in prevention of cancer. MC which has been described as 'Death through a tragedy' [68] is stimulated by ionizing radiations (IR), chemotherapeutic drugs or hyperthermia and is caused by malfunctioning of cell cycle checkpoints and MKs. The normal choreography of the events in the mitotic cell cycle gets disturbed and aneuploidy follows. An aneuploid cell can be hyperaneuploid and may contribute to tumorigenesis by an enhanced expression of oncogenes or may be hypo-aneuploid and be liable for tumorigenesis by a loss of heterozygosity of various tumor suppressor genes [69]. MC shares several biochemical hallmarks of apoptosis, in particular mitochondrial membrane permeabilization and caspase activation [70] but is proposed to be fundamentally different from apoptosis [71]. Both senescence and MC are important pathways that cause cell annihilation upon chemotherapeutic intervention. The mechanism and morphology of the deceased cells is however different in both the cases. A tabular representation of the differences between MC and senescence is given in Table 1. Table 1 Comparison between senescence and mitotic catastrophe Characteristics Mitotic catastrophe Senescence Definition Synonymous with 'Terminal proliferation arrest' may proceed with apoptosis or necrosis depending on molecular profile of the cell Synonymous with 'Terminal growth arrest' Cell death in context of cancer Biomarker Multinucleated giant cells, no specific in vitro and in vivo assay available SA-β galactosidase expression, detected by X-gal staining Morphology Aneuploidy, disrupted DNA index, micronuclei formation, nuclear envelope lacking, nuclear fragmentation and uncondensed chromatin Flattened enlarged cells, granular cytoplasm, exhibit SAHF formation Genotype implicated in carcinogenesis Accelerated by G1, G2 and prophase checkpoint proteins (ATM, ATR, p53, Chk2, Cdc25A, Cdc25B, Plk1 & 3) Accelerated by telomere attrition, ras mutations, inhibited by ALT or p53 Inducing agents Hyperthermia, IR, anti-cancer drugs interfering with DNA or microtubule assembly Spontaneous as a result of cumulative divisions or challenged by oncogenic stimulus Genetic checkpoint defects lead to syndromes that demonstrate chromosomal instability, increased sensitivity to genotoxic stress and consequently cancer predisposition. The detection of persistent MK over-expression, particularly the Aurora kinase family, and centrosome amplification in precursor/pre-malignant stages, strongly correlate these molecular changes in precipitating the aneuploidy seen in many human neoplasms [72]. The sustained over-expression and activity of various members of the MK families, including Aurora kinases (A, B, C), Polo-like (Plk1-4), and Nek (NIMA1-11) in diverse human tumors strongly indicate that these entities are closely involved in the development of errors in centrosome duplication, chromosome segregation, and cytokinesis. MKs families The focus of this section is on the different MKs families. These kinases are modulated by de-novo synthesis, stability factors, phosphorylation, and ubiquitin-dependent proteolysis. They, in turn, phosphorylate innumerable centrosomal/mitotic protein substrates, and have the ability to behave as oncogenes (i.e. Aurora-A, Plk-1), providing a compelling link between errors in mitosis and oncogenic processes [73]. Additionally, dysregulation of MKs have been linked with improper cell cycle progression both in vitro and in vivo. Without getting into the basics of MKs, the main pre-clinical and clinical studies concerning MK inhibitors currently under investigation are reported and important considerations for their future development are discussed. Here is given a representation of kinases in different phases of cell cycle (Additional File 1: Table S1). Cyclin dependent kinases 1 (Cdk1) Cdk1 is vital participant in the mitotic cell cycle. Mitosis begins and ends with the activity of cdk1 with binding partner cyclin B1. First studied in fission yeast (Saccharomyces cerevesiae), Nurse [74] identified a gene that controlled mitosis and named it cdk1 or cdc2. Studies have revealed that functional p53 protein may enhance the anti-cancer activity of roscovitine (known cdk1 inhibitor) that could be beneficial for anti-cancer therapy [75]. Tumorigenecity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates consideration of the use of broad spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy [76]. Gartner et al have reported for the first time reported an association of cyclins and Cdks with the microtubule network by immunoelectron microscopy and immuno-biochemical methods. Cyclins D, E, A and B as well as Cdks 1, 2 and 4 were also found to be associated and exhibit kinase activity towards the microtubule-associated protein tau [77]. Bailet et al [78] have highlighted a new role for spleen tyrosine kinase (Syk) in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway, the inactivation of which may contribute to melanoma tumorigenicity. Study by Buchanan et al [79] on murine adenocarcinoma mammary cells provided new clues regarding the mechanism involved in the modulation of mammary tumor cell growth and survival induced by glypican-3. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib (cdk inhibitor) and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials [80]. Another Cdk inhibitor SU9516 is over expressed in HCT116 cells by the knockout of the p21WAF1/CIP1 gene which suppresses thymidylate synthase and enhances chemosensitivity to 5-Flurouracil [81]. Check point kinases 1 (Chk1) and 2 (Chk2) Chk1 and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. If DNA damage is detected after S and before G2/M transition, ATM/ATR is activated and phosphorylation of Chk1 and Chk2 occurs [82] leading to cell death during mitosis or MC. Experiments have demonstrated that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks [83]. The activation of the ATR-Chk1 pathway in response to bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment and the dependency of this response on the DNA mismatch repair capacity were investigated. Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Si-RNA knockdown of ATR also reduced Chk1 phosphorylation following exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation [84]. Polo like kinases (Plk) A family of serine/threonine kinases also designated as tubulin-associated proteins actively participate during mitosis and comprises four distinct members: Plk1 (Plk), Plk2 (Snk), Plk3 (Prk or Fnk) and Plk4 (Sak) [85] each carrying out a multitude of distinct roles. Plk1 is the most extensively characterized among the family members, suggesting that the polo box domain of it can provide an additional structural basis for discovery of new anti-cancer drugs. It was also found out that Plk1 is required for chromosomal DNA replication under stressful conditions [86] and Plk3 is more potent in inhibiting cell proliferation and inducing apoptosis [87]. Plk1 gene expression is tightly regulated with mRNA increase beginning in S phase and peak mRNA levels detected at G2-M transitions and through mitosis [88]. RNA-interference -mediated depletion of Plk1 to determine its potential for sensitizing pancreatic tumor cells to gemcitabine showed that small interfering RNA-mediated knockdown of Plk1 caused cell cycle arrest at G2/M and the reduction of cellular proliferation and decreased cell viability and increased cellular apoptosis [89]. Transcription of Plk1 is inhibited along with other G2/M specific genes like cyclin B1, cyclin B2 and cdc25B by inhibition of Nuclear Factor kappa B at G2-M phases [90]. Studies define and illuminate a late mitotic function of Plk1 that it is obligatory in the positioning and recruitment of Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle and abolishing RhoA GTPase localization to the equatorial cortex, and suppressing cleavage furrow formation and cell division [91]. Increased plk-1 gene and protein perhaps play a key role in abnormal proliferation of acute leukemia cells and correlate with the malignancy of leukemia [92] prostate carcinoma, [93] and gastric carcinoma [94]. Snk/Plk2 is transcriptionally down-regulated in B-cell neoplasms [95] and consequently provides a potential mechanistic basis underlying the strong selective pressure for abrogation of Plk2 function in B-cell neoplasia. Plk3 has been shown to catalyse the priming of Cdc25A by phosphorylated glycogen synthase kinase-3β (GSK-3β) and observations indicate that GSK-3β inactivation may account for Cdc25A overproduction in a subset of human tumors [96]. LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) has recently been identified as an inhibitor of Plks and markedly enhances the anti-cancer activity of paclitaxel [97] with anti-proliferative activity against human breast cancer [98]. Aurora kinases Aurora kinases namely, Aurora A(Aurora 2), Aurora B(Aurora 1) and Aurora C(Aurora 3) are serine/threonine kinases also known as tubulin-associated proteins [99] which are expressed only in actively dividing cells and their increase is a factor of bad prognosis in cancer. Side effects, dosing and tolerability of inhibitors have been discussed in great length by Pinel et al [100] and enzymatic characterization of GSK1070916, a potent and selective Aurora-B/Aurora-C inhibitor was done and compared with other Aurora inhibitors AZD1152 and VX-680 [101], GSK1070916 was found to exert a more prevailing inhibitory effect due to a slow rate of dissociation from the Aurora-B & C enzymes. Detailed kinetic analyses of two isogenic cell lines differing in p53 function and have been compared with the effects of ZM447439 and VE-465 to describe several mechanisms explaining how cells may evade killing by Aurora kinase inhibitors [102]. It has been proposed that perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis and also its functions have been illustrated by Parra et al [103]. Bub related kinases (Bub family) The Bub family of kinases constitutes members that are concerned with spindle assembly functioning and APC/C regulation. In one of the studies p53 was sustained to express in K562 leukemic cells after being infected by recombinant adenoviruses carrying the wt-p53 gene and it was shown that wt-p53 can suppress excessive replication of centrosomes and may contribute to the upregulation of Gadd45a and BubR1 protein expression as well as the downregulation of Aurora A protein expression [104]. A novel study reports that Ajuba, a microtubule-associated protein collaborates with Aurora B and BubR1 at the metaphase-anaphase transition and ensures proper chromosome segregation[105]. Never in mitosis A- Related kinase (NIMA, Nek, Nrk) The Nek or Nrk related kinase family are essential MKs first described in the filamentous fungus Aspergillus nidulans [106] containing 11 members (Nek1, 2, 3, 6, 7, 8, 9, and 11 are prominent) [107]. Nek1 is involved early in the DNA damage sensing/repair pathway after IR and G(1)-S-phase checkpoint control can be rescued by ectopically over-expressing wild-type Nek1. Moreover, in cells without functional Nek1, DNA is not repaired properly, double-stranded DNA breaks persist long after low dose IR, and excessive numbers of chromosome breaks are observed [108]. Recently, studies have explicated that ciliary localization of Nek8 in a subset of ureteric-bud-derived kidney tubules is essential for maintaining the integrity of those tubules in the mammalian kidney [109]. MKs and their role in cancer control- In a nutshell As current cancer therapies are still in their infancy and are not able to fulfill the expectations of cancer control, strategies targeting mitotic regulators could be a potentially pragmatic option, which may improve the therapeutic index when used either alone or in combination with current anti-cancer antidotes. The uniqueness of MKs lies in the fact that they are expressed in actively duplicating cells and not in differentiated cells further make it important targets against cancer cells. Targeting MKs would aid us in understanding the mechanism of chemo-resistance. The research efforts to examine the role of MKs and mitotic signaling pathways are, however, in its beginning. By presenting an overview of regulation of MKs in this review, we open promising avenues in designing novel therapeutic approaches in curbing cancer. Simultaneously, we also present the rationale for these kinases as an anti-cancer target. Hence, more concern needs to be laid on in vivo work to understand the role of MKs and their utility as targets before we can actually embark on translational studies in human. Conclusions and future connotations Cancer is a global health problem and various treatment strategies are premeditated for curbing this deadly biomedical manifestation. Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or IR. The cellular response to radiation or chemicals is complex and may lead to different biological outcomes. Senescence, MC, necrosis, apoptosis and autophagy are such mechanisms out of which the two former mechanisms have been discussed in this review. The physiological phenomenon of senescence is stimulated by ras/raf activity, telomere attrition and p53. Cellular senescence is a persistently growth-arrested phenotype in normal and transformed cells which may be beneficial when used to target the proliferation of tumor cells or during organogenesis or wound healing. It is well known that cancer risk rises exponentially with age fuelled by somatic mutations. Senescence leads to altered expression of genes (cell division control, cell structure and metabolism) and imparts resistance to cells towards apoptosis apart from actively secreting inflammatory cytokines, proteinases and growth factors. Keeping all these aspects about this mechanism in mind, we can design novel treatment tactics in curbing cancer. The discovery of neosis has identified novel cellular targets, against which one can identify novel neosis-specific molecular targets in order to design anti-neotic agents or neosicides that will be effective against many tumor types and theoretically be expected to have a prophylactic action against multiple primary cancer growths. Discussing the regulation of MKs, we open promising avenues in designing novel biomarkers for novel unexplored targets and present the rationale for these kinases as an anti-cancer target. More in vivo work needs to be undertaken to understand the role of MKs and their prospective as cellular targets before translational studies can be performed in humans. Several key works using clinical samples strongly suggest that point mutations of the checkpoint genes contribute to malignant transformation and genetic instability in cancer cells. However, the exact role of DNA damage checkpoints in the prevention of human carcinogenesis should be re-evaluated. The spindle checkpoint inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C), which is essential for mitotic progression, until spindles are properly attached to all kinetochores, and thus prevents precocious chromosome segregation. Because in a large proportion of tumors, cell cycle checkpoints are compromised and apoptotic pathways frequently suppressed, tumor cells preferentially execute this mitotic mode of cell death after treatment with DNA damaging regimens. A judicial combination of anti-neosicides and anti-mitotic agent may increase the therapeutic ratio under clinical settings. Moreover, results of recent important research work on senescence and MC can lay foundation of other experiments targeting different cancers for testing efficacy of already tested drugs and on some cancers for different drugs sharing similarities in chemical and physical properties with known drugs. Conflict of interests The authors declare that they have no competing interests. Authors' contributions RS composed the original manuscript. JG made extensive revisions and participated in manuscript preparation. YS edited and finalized the final manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Table S1. 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23 377 387 10.1007/s00467-007-0692-y 18189147	20205872	PMC2827387	CC BY	2021-01-04 17:58:34	yes	Cell Div. 2010 Jan 21; 5:4

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