s2fieldsofstudy: Biology Chemistry

text:
Alix, a Protein Regulating Endosomal Trafficking, Is Involved in Neuronal Death*

Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery.

A bridge between the endo-lysosomal system and cell death has been suggested by numerous observations dealing with apoptosis or with the so-called type II autophagic cell death, which is independent of caspases (1)(2)(3)(4)(5). The endo-lysosomal system is composed of a series of intracellular compartments within which endocytosed molecules and redundant cellular material are hydrolyzed (6). Endocytosed material tends to flow vectorially through the system from early endosomes, along late endosomes into lysosomes, whereas phagocytosis and autophagy provide alternative entry points.
Multivesicular bodies represent a subset of late endosomes characterized by their multiple intralumenal vesicles, which bud from the limiting membranes together with ubiquitinated transmembrane proteins destined for degradation inside lysosomes. In Saccharomyces cerevisiae, ubiquitinated membrane proteins en route to the vacuole are recognized and sorted in the multivesicular bodies by three hetero-oligomeric complexes (ESCRT-I, ESCRT-II, and ESCRT-III) composed of Vps 1 proteins (7)(8)(9). Recently, several publications have characterized mammalian homologues of Vps proteins and described their mutual interactions. They show that ESCRT-I and -III are bridged by a 97-kDa cytoplasmic protein named Alix/AIP1 (10 -13). They also demonstrate that the GAG protein from membrane-containing viruses, such as HIV, binds to Alix/AIP1, thereby recruiting the ESCRT machinery to allow budding of the virus from the cell surface (11)(12)(13). Alix has previously been shown to be highly enriched in phagosomes (14) and exosomes, the latter being extracellular vesicles secreted upon fusion of the limiting membrane of the multivesicular bodies with the plasma membrane (15). We have shown that the 150-amino acid long proline-rich domain of the C-terminal part of Alix (Alix-CT) interacts with the SH3 domain of endophilins (16), which are lysophosphatidic acid acyltransferases capable of modifying the lipid bilayer curvature during endocytosis (17). Moreover, the Alix proline-rich domain interacts with CIN85/ SETA/Ruk (18), which regulates endocytosis of ubiquitinated tyrosine kinase receptors (19,20). These different observations suggest that Alix regulates the endo-lysosomal pathway from the cell surface down to late endosomes.
Several other observations suggest a role for Alix in controlling cell death. The protein was first characterized as an interactor of the penta-EF hand calcium-binding protein ALG-2 (apoptosis-linked gene 2) (21,22). ALG-2 seems necessary for death to occur as inhibition of its expression in a T cell line blocked apoptosis (23) without impeding caspase-3 activation (24). However, death was not impaired in T cells from ALG-2deficient mice, suggesting that other proteins of similar function may replace ALG-2 (25). In addition, another binding partner of Alix, Cin85, not only regulates endocytosis but also * This work was supported in part by INSERM, the University Joseph Fourier, grants from the Association pour la Recherche contre le Cancer and the Association Française contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  influences cell death of astrocytes and neurons (18,26). A final argument for a possible role of Alix in cell death comes from our recent demonstration of its up-regulation in neurons degenerating after kainate-induced epileptic seizures (53).
Here we have used potassium depletion-induced death of cerebellar granule neurons (CGN) as a paradigm of neuronal death to test a role of Alix in this phenomenon. We demonstrate that both caspase-dependent and -independent mechanisms underlie death of these neurons because inhibition of caspases does not impair death induced by potassium deprivation. We show that Alix overexpression is sufficient to induce caspase activation and nuclear alteration leading to death of neurons cultured in high potassium. In turn, Alix-CT inhibits caspase activation after potassium deprivation; it also inhibits the caspase-independent program because transfected neurons survive in low K ϩ . Finally, we identify the region of Alix necessary for binding ALG-2 and demonstrate that this site, but not the one binding endophilins, is required for Alix to control cell death. The complex Alix/ALG-2 may thus represent a critical link between the endo-lysosomal pathway and the cell death effectors.

EXPERIMENTAL PROCEDURES
Cell Culture Media-All culture media were based on Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10 unit/ml penicillin, 10 g/ml streptomycin, 2 mM L-glutamine, and 10 mM HEPES (K5 medium). KCl was added to a final concentration of 25 mM (K25 medium) and supplemented with 10% fetal bovine serum (K25ϩS medium).
Neuronal Culture-Primary cultures of CGN were prepared from 6-day-old S/IOPS NMRI mice (Charles River Laboratories), as described previously (27,28), with some modifications. The cerebella were removed, cleared of their meninges, and cut into 1-mm pieces. They were then incubated at 37°C for 10 min in 0.25% trypsin-EDTA (Invitrogen) in DMEM. Trypsin was inactivated with an equal volume of K25ϩS medium and 3000 units/ml DNase I (Sigma) before dissociation by triturating by using flame-polished Pasteur pipettes in DMEM containing trypsin inhibitor and 300 units/ml DNase I. Dissociated cells were centrifuged for 5 min at 500 ϫ g. The pellet was resuspended in fresh K25ϩS medium and cells were plated onto poly-D-lysine (10 g/ml, Sigma) precoated 60-mm dishes or 24-well plates at a density of 3 ϫ 10 5 cells/cm 2 . The cerebellar granule neurons were grown in K25ϩS medium in a humidified incubator with 5% CO 2 /95% air at 37°C. Cytosine-␤-D-arabinoside (10 M, Sigma) was added after 1 day in vitro (DIV) to prevent the growth of non-neuronal cells. After 4 DIV, K25ϩS medium was replaced with serum-free K25 medium to eliminate the 20% of cells dying within a few hours after serum removal (28). Twentyfour hours later, cells were changed to K5 medium or maintained in K25 medium. In some experiments, 10 M BAF (boc-aspartyl(OMe)-fluoromethylketone; Bio-Rad), a general caspase inhibitor, was added directly to the medium.
Determination of Cell Viability-Survival was estimated 0, 3, 6, 12, and 24 h after switching the cultures from K25 medium to K5 medium using the Alamar blue assay (BIOSOURCE) according to the instructions of the manufacturer. In other cases, cells were fixed in 4% paraformaldehyde in PBS for 20 min at 4°C and stained with Hoechst 33342 (4 g/ml, Sigma) in PBS for 30 min at 37°C. Cell viability was then scored on the basis of nuclear morphology; condensed or fragmented nuclei were taken to indicate cell death.
Plasmids-All constructions used are based on the pCi vector (Promega) and have been described by Chatellard-Causse et al. (16). All cDNAs had a tag epitope and were coded for the following proteins: Alix, ALG-2, Alix ⌬PP14 (deleted from amino acids 748 -761), Alix ⌬PGY (deleted from amino acids 802-813), Alix-NT (N-terminal half of Alix ending at residue 434), Alix-CT (corresponds to the first nine amino acids of Alix linked to the C-terminal half of Alix starting at amino acid 468), and Alix-CT ⌬PGY (Alix-CT deprived of the PGY domain), and GFP (green fluorescent protein) cDNA as control for the toxicity of transfection.
Transfection of CGN-Transient transfection of neurons in primary culture was performed by the calcium phosphate coprecipitation method developed by Xia et al. (29) and modified by Finkbeiner and Greenberg (30). Transfection was performed on neurons cultured 4 days on 14-mm glass coverslips. K25ϩS medium was removed, and the cells were incubated for 1-2 h with fresh DMEM supplemented with 10 mM MgCl 2 in 5 mM HEPES, pH 7.4. Meanwhile, the DNA/calcium phosphate precipitate was prepared and allowed to form for 25-30 min at room temperature before addition to the cultures. Eight micrograms of total plasmid DNA were used for each coverslip. After 1 h of incubation with the DNA/calcium phosphate precipitate, the cells were "shocked" for 1 min with 2ϫ HeBS buffer (274 mM NaCl, 10 mM KCl, 1.4 mM Na 2 HPO 4 , 7H 2 O, 15 mM D-glucose, 42 mM HEPES, pH 7.6), 10 mM MgCl 2 in 5 mM HEPES, pH 7.4, and 2% Me 2 SO. The cells were then washed three times with DMEM, and K25 medium was added to each coverslip. They were then returned to the 5% CO 2 incubator at 37°C and cultured for the indicated time. The transfection efficiency was determined after immunolabeling with an anti-tag antibody; it was typically between 15 and 20%.
Immunocytochemistry-Neurons were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 4°C. Cells were permeabilized with 0.02% saponin in Tris-buffered saline for 30 min and then incubated with antibodies as follows: mouse anti-Myc (Santa Cruz Biotechnology, 1:100), mouse and rabbit anti-FLAG (Sigma, 1:100), rabbit anti-phosphoserine 63 c-Jun and rabbit anti-phospho p38 MAPK (New England Biolabs, 1:100). Primary antibodies were revealed with either goat anti-mouse or anti-rabbit immunoglobulin G coupled to fluorochrome Alexa 594 or Alexa 488 (Molecular Probes) diluted 1:1000 in saponin-Tris-buffered saline. The cultures were mounted in Mowiol (Calbiochem), and the fluorescence was observed under an Axiovert microscope (Zeiss) connected to a CDD camera or a confocal laser scanning microscope (LSM410, Zeiss). Image analysis was performed by using Metamorph TM software (Universal Imaging Corp.).
Detection of Active Caspases in Living Cells-CaspaTag TM fluorescein caspase (VAD) activity kit (Intergen) allows the detection of active caspases (caspase-1-9) in living cells through the use of a carboxyfluorescein-labeled fluoromethylketone pan-caspase inhibitor (fam-VADfmk). This probe enters the cell and binds irreversibly to activated caspases. After 24 h in either K25 or K5 medium, CGN were incubated for 1 h at 37°C with fam-VAD-fmk in accordance with the supplier's instructions and visualized under an epifluorescence microscope.
Overlay Assays-HEK293 cells were prepared as described in Chatellard-Causse et al. (16). Proteins (10 g/lane) were run in 8% SDS-PAGE and blotted to nylon membranes. Membranes were saturated in blocking solution (Tris-buffered saline, 0.1% Tween, 5% nonfat dry milk, and 2 mM CaCl 2 ) for 1 h at room temperature and incubated with Myc-ALG-2 recombinant protein in blocking solution. After 2.5 h, membranes were washed in TBS, 0.1% Tween 20, and 2 mM CaCl 2. The bound recombinant protein was revealed with an anti-Myc monoclonal antibody followed by a goat anti-mouse horseradish peroxidase-conjugated secondary antibody; then the SuperSignal detection method was employed (Pierce).

Potassium Depletion-induced Cell Death of CGN-CGN
can be grown in the absence of serum in a medium containing 25 mM KCl (K25 medium) but undergo apoptosis soon after changing to 5 mM KCl (K5 medium) (27). Cell viability, estimated by the Alamar blue assay (Fig. 1A) or by scoring pyknotic nuclei (not shown), was reduced by 50% 24 h after potassium depletion. In agreement with previous studies (31-33), addition of the general caspase inhibitor BAF (10 M) blocked nuclear fragmentation but had no effect on nuclear condensation (not shown) and cell survival (Fig. 1A).
Potassium depletion was followed rapidly by activation of caspases. Caspase-3, a central caspase executioner, was activated within 4 h, as shown by the gradual increase in the level of the active p17 form (Fig. 1B). Determination of caspase-3 proteolytic activity in cell lysates demonstrated that 6 h after K ϩ lowering, the enzymatic activity increased ϳ20 times above the control level (Fig. 1C). We also observed that PARP, a specific caspase-3 substrate, was cleaved into a characteristic 89-kDa fragment (Fig. 1D). Caspase-3 activity was blocked when BAF was added to the culture medium at the time of K ϩ depletion; indeed, the activity in cell lysates from neurons cultured in low K ϩ medium supplemented with BAF was almost identical to the activity in lysates from cells grown in K25 medium (Fig. 1C). Furthermore, besides caspase-3, K ϩ withdrawal also triggered activation of caspase-2, -6, -8, and -9, which were all blocked by BAF (data not shown). These observations, showing that caspase inhibition is not sufficient to allow survival, demonstrate that both caspase-dependent and -independent programs are at work during the death of CGN triggered by potassium depletion.
Alix Overexpression Is Sufficient to Induce Apoptosis of CGN-To evaluate a possible involvement of Alix in neuronal death, CGN were transfected with Alix and left in K25 medium. Ten hours after transfection, overexpressed Alix was homogeneously distributed throughout the cell body and neurites ( Fig. 2A). Determination of cell viability demonstrated that the transfected neurons underwent cell death; 24 h after transfection, 50% of the Alix-overexpressing neurons grown in 25 mM KCl were dead or dying, whereas neurons transfected with GFP (control vector) were unaffected (Fig. 2B). At this time, caspase activity could be detected in situ using CaspaTag TM , and nuclear condensation was clearly visible in Alix-overexpressing neurons (Fig. 2C-E). Addition to the culture medium of the general caspase inhibitor BAF blocked CGN death induced by overexpressed Alix (Fig. 2B). Thus, overexpression of Alix is sufficient to induce caspase-dependent neuronal death in CGN cultured in the presence of survival signals. C-Jun N-terminal kinases (JNK) and p38 MAPK are important regulators of the cell death program in post-mitotic neurons. In particular, the JNK pathway and one of its major downstream targets, the transcription factor c-Jun, are thought to play an essential role in the death of CGN (34 -39). We performed immunofluorescence analysis of c-Jun and p38 MAPK activation to test whether Alix overexpression launches apoptosis by activating these cascades (Fig. 3A-D). Although phospho-c-Jun and -p38 MAPK immunoreactivity were clearly detected in CGN grown in K5 medium (Fig. 3E) or treated with anisomycin, a known p38 MAPK activator (Fig. 3F), there was no evidence to suggest that JNK and p38 MAPK were activated in Alix-overexpressing neurons. Hence, Alix does not induce death of CGN by activating either JNK-or p38 MAPK-signaling pathways.
Overexpression of Alix-CT Protects CGN from Death Induced by Potassium Withdrawal-Overexpression of the C-terminal half of Alix led to the formation of numerous cytoplasmic vacuoles with a diameter ranging from 0.5 to 2 m (Fig. 4, A and C and Fig.5). These vacuoles are reminiscent of those seen in HEK293 cells overexpressing Alix-CT together with endophilins (16). Most Alix-CT-expressing neurons had a healthy nuclear appearance after K ϩ lowering, unlike nearby untransfected neurons (Fig. 4E). Twenty-four hours after potassium deprivation, neurons with a condensed nucleus represented only 15% of Alix-CT-expressing cells, in contrast to ϳ50% for untransfected, Alix, or GFP-transfected cells (Fig. 4B). The labeling with CaspaTag TM showed no detectable caspase activity in most Alix-CT neurons, in contrast to nontransfected neurons (Fig. 4D). Overexpression of Alix-CT thus protects CGN from death induced by potassium withdrawal.
Interestingly, in the few Alix-CT-overexpressing neurons showing detectable caspase activity, the CaspaTag TM staining was concentrated in spots colocalized with Alix-CT (Fig. 5A). Confocal analysis of these cells demonstrated that the CaspaTag TM fluorescence was trapped in the vacuoles delineated by Alix-CT (Fig. 5B), suggesting that active caspases were concentrated inside. We made similar observations using another CaspaTag TM (DEVD) specific for caspase-3 (not shown), demonstrating that caspase-3 is one of the active caspases found inside the vacuoles. It is noteworthy that these neurons always showed nuclear alterations typical of cells undergoing apoptosis.
ALG-2 Binds to a PGY Repeat from Alix Proline-rich Domain-We used a far Western blotting approach to determine the binding site of ALG-2 on Alix. For this, lysates from HEK293 cells overexpressing different mutants of Alix were run on SDS-PAGE and blotted onto nylon membranes. Purified Myc-tagged recombinant ALG-2 was then incubated on the membrane, and its binding was revealed with an anti-Myc antibody (Fig. 6A). The recombinant ALG-2 bound to overexpressed full-length Alix, to its C-terminal half, and to Alix lacking the site of interaction with endophilins (Alix ⌬PP14), but not to Alix-NT or to Alix ⌬PGY, which lacks residues 802-813 (Fig. 6A). The use of an anti-FLAG antibody showed that Alix ⌬PGY, Alix ⌬PP14, and Alix were all expressed at the same level (Fig. 6B). These results demonstrate that Alix amino acids 802-813 are necessary for ALG-2 binding.
Deletion of the ALG-2-binding Site Abrogates Alix Ability to Modulate CGN Survival-In cultures kept in K25 medium, neurons transfected with Alix ⌬PP14, lacking the endophilinbinding site, underwent apoptosis in the same way as Alixoverexpressing neurons, because about 50% died in less than 24 h (Fig. 7A). In contrast, the survival of CGN expressing large amounts of Alix ⌬PGY was not significantly different from control GFP-overexpressing neurons (Fig. 7A). These results indicate that apoptosis induced by overexpressed Alix is strictly dependent on the integrity of its ALG-2 interacting site. As shown in Fig. 7A, overexpression of ALG-2, in contrast to that of Alix, had no deleterious effects on neuronal survival.
We then addressed the question of whether the capacity of Alix-CT to bind ALG-2 is required for Alix-CT to block CGN death. CGN were transfected with an expression vector encoding Alix-CT lacking the ALG-2-binding domain (Alix-CT ⌬PGY) and deprived of K ϩ 24 h later. As illustrated in Fig. 7B, no significant difference in cell survival was recorded between control GFP-expressing neurons and Alix-CT ⌬PGY-expressing neurons: about 50% of the latter were dead or dying 24 h after the switch to K5 medium. Thus, these results demonstrate that the neuroprotective effect of Alix-CT on CGN apoptosis requires the region binding ALG-2.

DISCUSSION
Alix is a protein recently shown to regulate the endo-lysosomal system (10,16). This protein is also suspected to regulate neuronal death; we recently demonstrated that it is up-regulated in degenerating rat hippocampal neurons after epileptic seizures induced by kainate treatment (53). Here we show that enforced expression of Alix is sufficient to induce caspase activation and, thereby, death of cultured cerebellar granule neurons. This death, which displays all morphological features of apoptosis, is truly caspase dependent as, unlike that seen upon potassium depletion, it can be effectively blocked by the addition of a pan-caspase inhibitor to the culture medium. Neither of the stress-activated kinases tested (JNK and p38 MAPK) were activated, demonstrating that the pro-apoptotic function of Alix is not simply due to neuronal stress. Alix overexpression has recently been reported to induce detachment of cells from the substrate, and it even enhanced detachment-induced death (anoïkis) of HeLa cells (40). In our hands, Alix had no effect on neuron attachment to the substrate. Also noteworthy is the fact that the primary neurons that we transfected are post-mitotic, ruling out that the pro-apoptotic effect of Alix may be caused by its previously demonstrated capacity to induce G 1 arrest (41).
In vivo, a simple rise in intracellular Alix concentration is not sufficient to cause neuronal death, because, during avian development, the protein expression starts in motoneurons during axon outgrowth, well before the onset of programmed cell death (42). We hypothesized that ALG-2, a calcium-binding protein necessary for cell death (23) which binds in a calciumdependent way to the C-terminal proline-rich region of Alix (21,22), could regulate its pro-apoptotic activity. To test this, we first characterized the ALG-2 binding site by making Alix deletion mutants and evaluating their capacity to bind ALG-2 in a far Western. We found that ALG-2 could not bind to Alix ⌬PGY, which lacks amino acids 802-813 (PPYPTYPGYP-GYPGY), whereas it was still able to bind to Alix lacking other discrete regions of the proline-rich domain. We chose this region based on its resemblance with the PGY-biased amino acid composition of the N-terminal parts of annexins VII and XI, which also bind ALG-2 (43,44). In fact, the N-terminal region of annexin XI contains a PSYPGYPG sequence that is almost identical to the sequence found in Alix. The ALG-2 binding region is distinct from the endophilin-binding site that we previously characterized (amino acids 748 -761, deleted in Alix ⌬PP14; Ref. 16). It is also distinct from the PTPAPR sequence (amino acids 740 -745), which fulfills the PxPxPR consensus, recently described as necessary for high affinity binding to the SH3 domain of CIN85/SETA/Ruk (45) and from the PSAP (amino acids 717-720) required for Tsg101 binding (13). In overexpression experiments, Alix ⌬PGY, unlike Alix ⌬PP14, was unable to induce caspase activation, strongly suggesting that binding of ALG-2, but not binding of endophilins, is required for Alix pro-apoptotic activity. Because the binding of ALG-2 to Alix is strictly dependent on Ca 2ϩ , one may hypothesize that ALG-2 translates calcium fluctuations, induced by K ϩ deprivation, through Alix into a cell death response. The link between calcium and cell death is a matter of numerous publications (for review, see Refs. 46 and 47), and it is now clear that even non-disruptive changes in Ca 2ϩ homeostasis and calcium compartmentalization may regulate cell survival.
The Alix-ALG-2 couple may be another effector of Ca 2ϩ signaling in cell death.
It is difficult to predict a mechanism of action for Alix. It is noteworthy, however, that besides ALG-2, the proline-rich domain region interacts with CIN85, which has already been implicated in apoptosis (18,26), as its overexpression induced death of sympathetic neurons (26). In this latter case, it seems that CIN85 binds to and inhibits the type IA PI3 kinase, which is required for nerve growth factor survival activity (26). In agreement with several publications (48,49), we have observed that a potent PI3 kinase inhibitor, LY294002, induces apoptosis of cerebellar neurons kept in high K ϩ (not shown). In the near future, we will examine whether Alix deprived of its potential CIN85-binding site is still able to induce neuronal death, thereby testing the possibility that the Alix/ALG-2 complex recruits CIN85 to drive cell death.
Overexpression of Alix-CT blocked caspase activation in the majority of transfected neurons deprived of K ϩ . Alix-CT could act as a dominant-negative mutant by binding to endogenous Alix (22), which is known to interact with itself through its C-terminal half (21). Alix-CT could also directly affect endosomal function, because it possesses the site of binding to ESCRT-I but lacks the ESCRT-III interacting site (amino acids 1-176 of Alix). We have shown that overexpressing Alix-CT induces cytoplasmic vacuolization, and several Alix N-terminal deletion mutants lacking the ESCRT III-binding region were recently found to block budding of viruses at the cell surface (11)(12)(13). This latter observation was thought to reflect the impairment by truncated Alix of the cell ESCRT machinery normally recruited by viruses to bud at the cell membrane. Overexpressing Alix-CT also led to the formation of vacuoles in neurons. In potassium-depleted cultures, active caspases filled the entire soma, whereas in the few Alix-CT-expressing neurons in which we detected active caspases, these were concentrated inside the vacuoles. The vacuoles could represent a way for neurons to inactivate the proteases, which would explain why caspase activity was absent in the vast majority of Alix-CT-FIG. 6. The PGY domain of Alix is necessary for binding to ALG-2. A, 10 g of proteins from lysates of HEK293 cells transfected with FLAG-tagged Alix, Alix-NT, Alix-CT, Alix ⌬PGY, or Alix ⌬PP14 were run on SDS-PAGE gel and, after transfer to a nylon membrane, overlaid with recombinant Myc-ALG-2. Bound ALG-2 was revealed with an anti-Myc monoclonal antibody. B, expression levels of each transfected Alix mutant was controlled by using a polyclonal anti-FLAG antibody.
FIG. 7. Activation of cell death by Alix as well as the neuroprotective effect of Alix-CT requires the ALG-2 binding site. After 4 DIV, CGN were transfected with an expression vector coding for GFP, Alix, Alix lacking either endophilin (Alix ⌬PP14), ALG-2 (Alix ⌬PGY) binding domains, Alix-CT, Alix-CT deleted of its ALG-2 binding domain (Alix-CT ⌬PGY), or ALG-2. One day later, cultures were either kept in K25 medium (A) or deprived of potassium (K5 medium; B) and grown for 24 h. Cultures were then fixed, and transfected neurons were revealed by immunofluorescence using the appropriate anti-tag antibody. The transfected neurons showing condensed nuclei (Hoechst staining) were scored as dying, and the percentage of transfected dying neurons was determined. For each experiment, a total of 200 -400 transfected neurons per construct were counted in 10 random fields in two or three different wells; each experiment was performed at least three times. overexpressing neurons. We noticed that CGN containing Caspa-Tag TM inside Alix-CT vacuoles had a condensed nucleus, suggesting that, when detectable in our system, the caspases had already overwhelmed the defense system. These observations are reminiscent of those made in Alzheimer's disease and Down's syndrome brains, where neurons from affected regions show cytoplasmic granulovacuoles believed to be of autophagic origin and which are filled with activated caspase-3 (50)(51)(52).
In agreement with other reports (31-33), we have here observed that the death of CGN induced by potassium deprivation is mediated by both caspase-dependent and -independent cell death, because a pancaspase inhibitor was only able to block nuclear fragmentation without impairing cell death. Therefore, the impairment of caspase activation seen in neurons overexpressing Alix-CT cannot explain all of the beneficial rescuing effect of the truncated protein. Here again, the rescuing effect of Alix-CT was shown to be highly dependent upon the integrity of its ALG-2-binding site. Our demonstration that, besides playing a role in endosome trafficking, Alix also regulates caspase-independent neuronal death in conjunction with the calcium-binding protein ALG-2 may open new avenues toward the understanding of the mechanisms that allow cell destruction in the absence of caspase activity.


extfieldsofstudy: Chemistry Medicine

text:
Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36.

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.

Lipoproteins enable the efficient movement of lipids in an aqueous environment. The inverse correlation between high HDL 1 levels and the development of atherosclerosis and coronary artery disease has led to significant research in this field (1,2). The basis for this atheroprotective effect of HDL, at least in part, is believed to be the transport of cholesterol from peripheral vascular tissues to the liver where cholesterol is excreted in the bile or converted to bile acids.
Scavenger receptor class B, type I (SR-BI), is a well characterized HDL receptor (3)(4)(5) that is highly expressed in tissues active in cholesterol metabolism such as the liver and steroidogenic tissues (6) and is expressed to a lesser extent in macrophages and vascular endothelium (7,8). SR-BI plays a key role in systemic cholesterol transport by mediating the selective uptake of cholesteryl ester (CE) from HDL into the liver. Selective uptake is defined as the movement of CE from HDL particles into target cells without significant internalization and degradation of the HDL particle (9 -14). This mechanism is distinct from LDL receptor-mediated lipid transport, where LDL is internalized and the LDL particle is degraded (15). The selective uptake pathway of HDL CE is a major pathway for the provision of substrate for steroid production in rodents; this pathway also exists in rabbits and is active in human hepatocytes, hepatomas, and ovarian granulosa cells (10 -12, 16 -22).
Although selective HDL CE uptake is a major function of SR-BI, other activities are also attributed to this receptor. SR-BI increases the bidirectional flux of free cholesterol (FC) between cells and HDL or phospholipid vesicle acceptors (23)(24)(25)(26)(27), an activity that may be responsible for net cholesterol removal from peripheral cells as well as the rapid hepatic clearance of HDL FC and its resultant secretion into bile (28,29). Efflux of cholesterol from peripheral cells is the first step in reverse cholesterol transport, and recent findings show that SR-BI deficiency is associated with de-regulation of cholesterol homeostasis in the arterial wall (30) and that macrophage expression of SR-BI protects mice against aortic lesion development in atherosclerosis-susceptible mice (31,32). These data strongly implicate SR-BI in the vascular wall, as well as in the liver, as a significant factor in suppressing atherosclerosis.
In addition to changes in the flux of FC and CE, SR-BI has effects on plasma membrane properties that may contribute to the mechanisms of cholesterol movement into and out of the membrane (3). For example, SR-BI expression increases the fraction of plasma membrane FC susceptible to oxidation by exogenous cholesterol oxidase (23) and increases the size of the fast kinetic pool of membrane FC for efflux to cyclodextrin acceptors (33). These findings suggest that SR-BI alters the molecular packing of FC in such a way as to enhance FC desorption from the membrane. Additionally, SR-BI has significant effects on membrane morphology as evidenced by SR-BI Ϫ/Ϫ mice that have altered membrane ultrastructure and fail to form cell-surface microvillar channels on adrenal zona fasciculata cells (34). Thus, in addition to promoting cholesterol flux, SR-RI has multiple and, in some cases, dramatic effects on plasma membrane properties and structure.
In the present study SR-BI was expressed in Sf9 insect cells to determine whether the varied effects of SR-BI seen in mammalian cells also occur in a cell that has membranes with a very different lipid composition. Compared with mammalian cells (35), Sf9 cells have reduced levels of sphingomyelin and elevated levels of phosphatidylethanolamine (36,37). Additionally, even when grown in mammalian serum, plasma membranes of Sf9 cells have very low cholesterol content and a correspondingly low cholesterol to phospholipid ratio (37,38). Reaven et al. (39) showed that expression of SR-BI in insect Sf9 cells elicits the formation of double membrane structures that resemble the microvillar channels of steroidogenic tissues, indicating substantial changes in membrane morphology due to SR-BI. The present results show that, as in mammalian cells, SR-BI expression increased HDL CE-selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These findings indicate that SR-BI has fundamental effects on cholesterol flux and membrane properties in cells of evolutionarily divergent origins with different lipid compositions. In addition, analysis of phospholipids by mass spectrometry showed substantial changes in phosphatidylcholine subspecies because of expression of SR-BI or the related class B scavenger receptor CD36.

EXPERIMENTAL PROCEDURES
Plasmids and Sequencing-PCR amplifications were performed by using a DNA Thermal Cycler 9700 (PerkinElmer Life Sciences). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Plasmids used to produce the murine SR-BI and rat CD36expressing baculoviruses were constructed as follows. For construction of pFastBacI(SR-BI), primers 5Ј-GACCCAATTGGCGGCCGCCCGTCT-CCTTCAGGTCCTGAGC-3Ј and 5Ј-GACCAGATCTTCTAGAGCGGAC-AGGTGTGACATCTGG-3Ј were employed to amplify the SR-BI coding region from a previously described vector, pSG5(SR-BI) (40). The resulting PCR products were digested with MfeI and XbaI and were ligated into the pFastBacI vector (Invitrogen), which had been restricted previously with EcoRI and XbaI. For construction of pBakhis2A(CD36), the coding region of CD36 was restricted with BamHI and ligated into the BamHI site of pBakhis2A (Clontech). All plasmids were prepared using endotoxin-free maxi-prep kits (Qiagen, Valencia, CA) and sequenced throughout the coding region to confirm that no point mutations had been generated during the cloning procedures (40).
Production of Recombinant Baculoviruses in Sf9 Cells-Sf9 cells (Spodoptera frugiperda) were routinely maintained in Grace's supplemented insect medium (Invitrogen) containing 5-10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 50 units/ml penicillin, and 50 g/ml streptomycin in a 27°C incubator. Baculoviral expression of SR-BI in the Sf9 cells was accomplished using the Bac-to-Bac Baculovirus Expression System as described by the vendor (Invitrogen). Recombinant baculoviruses were harvested 72 h post-transfection and amplified three to four times by infection of Sf9 cells to produce high titer viral stocks.
Immunoblot Analysis-Baculovirus-infected Sf9 cells expressing SR-BI or CD36 (in 35-mm wells) were washed twice with phosphatebuffered saline (pH 7.4) and lysed with 300 l of Nonidet P-40 cell lysis buffer (41, 42) containing 1 g/ml pepstatin, 0.2 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 10 g/ml aprotinin. Protein con-centrations were determined by the method of Lowry et al. (43). Ten micrograms of cell lysate protein were analyzed by SDS-8% PAGE. Immunoblots were probed with antibodies directed to SR-BI (ϳ82 kDa) and CD36 (ϳ78 kDa) to confirm their expression in the Sf9 cells (44) (data not shown). Blots were also probed with antibody against the gp64 viral envelope protein (eBioscience).
HDL Cell Association, Selective CE Uptake, and Apolipoprotein Degradation-Sf9 cells (1 ϫ 10 6 ) were plated into 22-mm wells in growth medium and infected with 100 l of high titer vector, SR-BI, or CD36 baculoviruses. Baculovirus-infected Sf9 cells, 72 h post-infection, were washed once with serum-free medium, 0.5% bovine serum albumin. 125 I, 3 H-HDL particles were added at a concentration of 10 g of protein/ml in serum-free medium, 0.5% bovine serum albumin and were incubated for 1.5 h at 27°C. Values for cell-associated HDL apolipoprotein (expressed as HDL CE) and the selective uptake of HDL CE were obtained as described previously (40).
Cholesterol Efflux, Cholesterol Oxidase, and Cholesteryl Ester Hydrolysis Assays-Sf9 cells (1 ϫ 10 6 ) were plated into 22-mm wells in growth medium at 27°C and infected with 100 l of high titer vector, SR-BI, or CD36 baculoviruses. After 48 h, cells were labeled for 18 h with 3 Ci/ml [ 3 H]cholesterol (PerkinElmer Life Sciences) in Grace's insect medium containing 10% fetal calf serum. Cells were washed twice and equilibrated for 2 h in Grace's insect medium. [ 3 H]Cholesterol efflux was measured after 0.5, 1, 2, and 4 h at 27°C in triplicate using HDL as an acceptor as described previously (23). Fractional efflux values were corrected for the small amount of radioactivity released in the absence of HDL. Cholesterol oxidase assays were performed 72 h post-infection as described by Smart et al. (48), and modified by Kellner-Weibel et al. (33). For measurement of [ 3 H]CE-HDL hydrolysis, at 72 h post-infection [ 3 H]CE-HDL was added (10 g of protein/ml) in DMEM containing 0.5% bovine serum albumin. After incubation at 37°C for 4 h, the medium was removed, and the monolayers were washed three times with phosphate-buffered saline, and the distribution of 3 H between FC and CE was determined (47). Free and total cholesterol mass (and CE by difference) was measured at 72 h post-infection by gasliquid chromatography (49).
Phospholipid Analysis-Phospholipids were analyzed by nanospray electrospray ionization on a QuattroLC tandem mass spectrometer (50). Internal standards for PA (591 m/z, 28:0), PI (835 m/z, 34:1), PE (678 m/z, 28:0), PS (634 m/z, 28:0), SM (703 m/z, 32:1), and PC (678 m/z, 28:0) were purchased from Avanti. Standards (1 pM/l) were sprayed from nanovials in 2:1 methanol and chloroform with the source maintained at 80°C. A counter-current flow of nitrogen emerging around the cone (250 liters/h) was used to promote solvent evaporation from the sprayed droplets. A nanovial, loaded with 1 l of standard or sample, produced a stable spray for more than 1 h allowing both survey mass spectra and daughter ion analysis for each lipid present. For CID experiments, argon was used in the collision cell. All CID parameters were optimized for each molecular ion in the positive and negative ion modes. Sf9 cells were lysed using nitrogen decompression, centrifuged at 1000 ϫ g to remove nuclei and debris, and centrifuged again at 100,000 ϫ g to collect membranes. Internal standards were added prior to lipid extraction. The membrane lipids were extracted in chloroform/methanol and dried under nitrogen. Samples were diluted 1:5,000 in methanol/chloroform (2:1) for analysis. A survey scan from m/z 600 to m/z 1000 (4 s) was taken to establish the phospholipid peaks present in each sample. Constant neutral loss and precursor ion scans were then performed using optimized conditions determined for each phospholipid as described (50). Where appropriate, fatty acyl tail composition was determined by tandem mass spectrometry analysis in the negative ion mode.
For each phospholipid, the percent distribution of species with various acyl tail lengths was determined within each analysis based on the sum of the fatty acyl carbons in the sn-1 ϩ sn-2 positions. Percent distributions from seven independent samples were then averaged. Statistical comparisons were made by a two-tailed Student's t test between pairs of the four treatment groups with correction made for multiple comparisons as indicated in the table legends. Determinations and analyses of the total double bonds in the fatty acyl chains of each phospholipid were done in the same fashion.

HDL Binding and Selective Uptake of Cholesteryl Ester in
Sf9 Cells-To test whether binding of HDL to high affinity receptors will induce CE-selective uptake, Sf9 cells were infected with CD36, a class B scavenger receptor with 30% homology to SR-BI, or SR-BI baculovirus and binding studies were performed as described under "Experimental Procedures." Fig. 1A shows that HDL binds to both CD36-and SR-BI-expressing Sf9 cells and confirms that both receptors were expressed on the cell surface. The apparent K d values for HDL binding by CD36 and SR-BI were 37.2 and 19.2 g/ml of HDL protein, respectively. Note that in this and other comparisons of SR-BI and CD36, Sf9 cells were infected with amounts of each virus that produced equivalent levels of HDL binding activity. The ability of SR-BI-expressing Sf9 cells to mediate selective HDL COE uptake has been demonstrated previously (39). The results in Fig. 1B confirm that SR-BI-expressing cells mediate efficient and selective uptake of HDL COE and show that CD36-expressing cells do not. Most interestingly, the ability of CD36 to mediate HDL COE-selective uptake is markedly less than in mammalian cells in which CD36 has about 15% of the activity of SR-BI (40,51). After confirming SR-BI expression and function at the plasma membrane, we tested for other activities shown by SR-BI in mammalian cells.
Cholesterol Content of SR-BI-expressing Sf9 Cells-SR-BI expression in COS-7 and WI38 cells increases cellular cholesterol content of both free and esterified cholesterol (44,52). Because Sf9 cells typically have low cholesterol content, we tested whether SR-BI expression would alter cholesterol content. After 72 h of infection with either control, SR-BI, or CD36 baculovirus, cells were extracted, and their cholesterol content was determined. As shown in Fig. 2  measuring the fraction of [ 3 H]FC transferred to medium containing HDL. Without an acceptor there was virtually no efflux in 4 h (data not shown). In the presence of HDL (250 g/ml) there was significant cholesterol efflux in 4 h from Sf9 cells expressing SR-BI (Fig 4A). CD36-expressing cells showed enhanced FC efflux compared with virus-infected cells (Fig. 4A), although the effect was less than that seen with SR-BI over a range of HDL concentrations (Fig. 4B).
Cholesterol Susceptibility to Cholesterol Oxidase-In mammalian cells SR-BI alters the organization or distribution of membrane free cholesterol as judged by an increased susceptibility to exogenous cholesterol oxidase (23,33). To test whether SR-BI has this activity in insect cells, Sf9 cells were infected with control baculovirus or baculovirus encoding CD36 or SR-BI for 48 h, followed by 24 h labeling with [ 3 H]cholesterol. After exposing the cells to cholesterol oxidase for 4 h, lipids were extracted, and cholesterol oxidation was determined. Fig.  5 shows a dramatic increase in cholesterol oxidase susceptibility in Sf9 cells expressing SR-BI when compared with baculovirus-infected or CD36-expressing cells. This is similar to results in mammalian cells. Most interestingly, CD36-expressing cells also showed a substantial 2-fold increase in cholesterol oxidase susceptibility compared with baculovirus-infected cells, although this was much less than with SR-BI.
Membrane Phospholipids in SR-BI-expressing Sf9 Cells-Stable expression of SR-BI in the human lung fibroblast cell line WI38-VA13 increased both FC and phospholipid mass (52). To determine whether phospholipids were altered upon SR-BI expression in Sf9, we utilized electrospray ionization mass spectrometry to quantify and profile phospholipids. Internal standards for each phospholipid class were used to determine phospholipid/protein ratios. For each phospholipid, the percent distribution of species with various acyl tail lengths was determined within each analysis based on the sum of the fatty acyl carbons in the sn-1 ϩ sn-2 positions. Determinations of total double bonds in the fatty acyl chains of each phospholipid were done in the same fashion. To eliminate changes that could result from viral infection, Sf9 cells were infected with control baculovirus at the same level of infection as with the SR-BI virus and CD36 virus. Equivalence of infection was judged by Western blot analysis of the virus envelope protein gp64 (Fig. 6A, inset).  Functional scans were performed for all phospholipids, as described under "Experimental Procedures." The results show that total phospholipid content was changed little by SR-BI or CD36 expression or control virus infection (Fig. 6B). Fig. 6C shows that PC and PE are the most abundant phospholipids in Sf9 cells grown in calf serum with the order of abundance being PC Ͼ PE Ͼ PS Ͼ PI Ͼ PA ϭ SM. The absence of major changes in phospholipid content upon baculovirus infection is similar to results from previous studies (36 -38). The abundance of individual phospholipids in Sf9 cells (Fig. 6C) is also similar to previous results (36,37) with the exception that PI abundance was much higher in one study (37).
Although major changes in total phospholipid mass were not seen, changes within specific phospholipids were significant. For example, the data in Table I show the percent distribution of acyl tail lengths in PC species upon SR-BI or CD36 expression compared with uninfected or virus-infected cells. In this case a substantial decrease in PC with 32 fatty acid carbons was seen in SR-BI-and CD36-expressing cells. As compared with control virus-infected cells, SR-BI-expressing cells showed a 29% decrease in the relative abundance of PC 32. No change was seen in PC 34, but SR-BI expression led to significant increases in the relative abundance of PC species with 36, 38, and 40 carbons with most of the increase occurring in PC 36 that showed a 14% increase in relative abundance. Most interestingly, the changes in PC 32 and PC 36 occurred primarily in PC 32:1 and PC 36:1 although the mono-and di-unsaturated species of each were of similar abundance in virus-infected control cells (PC 32:1, 8.3% of total PC; PC 32:2, 8%; PC 36:1 14%; and PC 36:2, 16%) (data not shown). CD36-expressing cells showed similar changes. Thus, scavenger receptor expression in Sf9 cells leads to the accumulation of PC species with longer acyl tails primarily because of changes in PC 32:1 and PC 36:1. Note that as shown in Table II, SR-BI or CD36 expression had no effect on the overall distribution of PC species with 0 -4 double bonds. Furthermore, as compared with uninfected cells, baculovirus infection had no effect on double bond distribution or PC acyl tail length with the exception of a relative increase in the abundance of PC 40. No significant changes were seen in acyl tail length distribution in PE, PI, PS, PA, or sphingomyelin when SR-BI or CD36 were compared with virus-infected cells (Table III) these changes were significant compared with uninfected cells. No changes or trends were observed in PA 32 and PA 36 which were the PC acyl tail lengths showing the largest changes upon SR-BI expression. Similarly, no changes in acyl tail length distribution were seen for SR-BI cells compared with virusinfected Sf9 cells for PI, PS, or sphingomyelin (Table III).
SR-BI cells showed a relative increase in PS 36 and a decrease in SM 34 compared with uninfected Sf9 cells, but these changes were also seen as a trend (PS) or significant (SM) when comparing virus-infected with uninfected cells. The only other difference attributed to baculovirus infection was a relative decrease in the abundance of PI 34. Changes in the distribution of   (Table IV). DISCUSSION In this study we used Sf9 cells, a primitive insect cell line, to study SR-BI because these cells do not have lipoprotein receptors and, although grown in calf serum, maintain a much lower cholesterol content and have a different phospholipid profile compared with mammalian cells. Thus, these cells provide an interesting system to test the effects of SR-BI on plasma membrane properties. Previous studies showed that SR-BI-expressing Sf9 cells exhibit HDL CE-selective uptake (39), a finding confirmed in the present study. Additionally, SR-BI expression led to large increases in FC accumulation and in the susceptibility of plasma membrane FC to cholesterol oxidase. SR-BI also enhanced FC efflux to HDL. These results suggest that SR-BI facilitates FC accumulation in the membrane, organizes membrane cholesterol, and facilitates the flux of membrane FC in Sf9 cells similarly to mammalian cells. That these membrane changes occur in the evolutionarily distant Sf9 cell suggests that these are inherent properties of SR-BI that may not require interaction with other membrane proteins.
The mechanistic basis for the change in the organization of membrane cholesterol due to SR-BI is not well understood, but the present study provides new information that bears on this issue. The SR-BI-mediated increase in sensitivity of membrane FC to exogenous cholesterol oxidase is seen in both mammalian and insect cells suggesting that this effect does not depend on a particular phospholipid composition or FC content because these differ greatly between Sf9 and mammalian cells. Additionally, in mammalian cells, SR-BI-enhanced cholesterol oxidase sensitivity is not dependent on increased membrane FC content because SR-BI-expressing cells cultured in lipoproteindeficient serum have increased cholesterol oxidase sensitivity compared with control cells that have the same cholesterol content (52). Thus, it is not the absolute content of FC but how it is disposed in the membrane that is detected by cholesterol oxidase in SR-BI-expressing cells. A variety of studies with model membranes indicates that cholesterol oxidase activity reflects the ease with which cholesterol desorbs from the lipid bilayer (53,54). Recent studies with recombinant cholesterol oxidase support this view that an increase in enzymatic activity reflects an increase in the escape of cholesterol from the membrane whether this is because of altered lipid packing density or to phase changes in the membrane (55). In relation to membrane lipid organization, cholesterol oxidase shows greater reactivity with cholesterol in liquid-disordered as opposed to liquid-ordered domains because of the greater rate of cholesterol desorption from the former domain (55). This is relevant to SR-BI because this receptor appears to be present in disordered membrane domains compared with more ordered cholesterol-and sphingolipid-rich membrane rafts as judged by solubilities in nonionic detergents (56,57). The presence of SR-BI in such domains may facilitate cholesterol flux by localizing HDL particles to membrane regions with favorable energetics for cholesterol movement into and out of the membrane.
An additional feature that is expected to influence cholesterol desorption from the membrane is the interaction of cholesterol with phospholipid acyl tails. Studies in membrane vesicles measuring cholesterol exchange (58 -61) or cholesterol oxidase sensitivity (54,60) show that cholesterol interaction with phosphatidylcholine versus sphingomyelin increases cholesterol efflux from the membrane. Additionally, with PC bilayers cholesterol efflux is enhanced by longer chain acyl tails and by a greater degree of unsaturation. These features of phosphatidylcholine acyl tails are believed to weaken the interaction with cholesterol thereby facilitating cholesterol efflux from the membrane. This effect of acyl tail length is of particular interest in light of the present results. In mixed monolayer systems minimal cholesterol desorption occurred with unsat-  (58 -60). These results suggest that SR-BI expression alters the plasma membrane PC composition in a manner that is expected to facilitate cholesterol flux into and out of the membrane. How these SR-BI-mediated changes in membrane PC occur or are distributed in the membrane is unknown, but we speculate that they occur preferentially in domains on plasma membrane microvillar extensions where clusters of SR-BI are found (56).
In agreement with previous studies, we observed no significant changes in phospholipid content or phospholipid composition in Sf9 cells because of baculovirus infection (36 -38). The distributions of acyl tail lengths and double bonds among the individual phospholipids were not much affected by virus infection. Additionally, the observed changes in acyl tail lengths upon SR-BI or CD36 expression were largely limited to PC. The distributions of acyl tail lengths in PE, for example, were identical among the four groups of Sf9 cells. These results support the conclusion that the changes in PC acyl tail lengths in the scavenger receptor-expressing cells are specific to SR-BI or CD36 and are not because of baculovirus infection. Although we consider it unlikely, we cannot rule out the possibility that the changes in PC acyl tail length in SR-BI-or CD36-expressing cells require both scavenger receptor expression and baculovirus infection.
As expressed in mammalian cells (40,51), the related class B scavenger receptor CD36 binds HDL with high affinity but mediates little HDL CE-selective uptake in Sf9 cells. CD36 also fails to increase the cholesterol content of Sf9 cells. Most interestingly, CD36 expression has similar effects to SR-BI on the distribution of PC species including the decrease in PC 32 and the increases in PC 36, PC 38, and PC40. As with SR-BI these changes occur primarily in species with mono-or polyunsaturated acyl tails. These results suggest that CD36 may also alter the plasma membrane in a manner that will facilitate cholesterol flux. This suggestion is supported by the finding that CD36 expression enhanced FC efflux to HDL but less efficiently than SR-BI. Similarly, CD36 increased the cholesterol oxidase-sensitive pool of membrane FC, but much less so than SR-BI. These results indicate that CD36 has modest, but measurable, effects on FC flux but no detectable effect on HDL CE-selective uptake. One interpretation of these results is that the modest effects of CD36 on FC flux primarily reflect changes in membrane PC species, whereas the much greater effects of SR-BI are because of receptor-facilitated FC transfer that occurs in addition to the changes in membrane PC.
Do the changes in PC species because of CD36 expression have a physiological role relevant to the function of CD36? CD36 has a variety of functions including activity as a fatty acid translocase that facilitates uptake of long chain fatty acids into adipocytes, skeletal muscle, and heart (63,64). In the absence of protein-mediated transport, the rate-determining step for fatty acid movement across a phospholipid bilayer is desorption of fatty acid from the bilayer to the aqueous phase (65)(66)(67). The rate of desorption of long chain fatty acids decreases with increasing chain length and increases with the degree of unsaturation; the latter effect reflects reduced hydro-phobic interactions with phospholipid acyl tails. Similarly, an increase in the membrane fatty acid desorption rate would be expected if the degree of PC unsaturation were increased because this would also disrupt the packing of fatty acids with PC acyl tails. Although speculative, one possibility is that a CD36mediated increase in membrane PC with unsaturated acyl tails may contribute to the transport of long chain fatty acids by CD36.
Although SR-BI expression in Sf9 cells showed many similarities to mammalian cells, two effects were different. The first is that the increase in cellular cholesterol mass occurred primarily in free and not esterified cholesterol, a result likely explained by the lack of cholesterol esterification activity in Sf9 cells (Fig. 3) (37). The second is that SR-BI did not enhance the hydrolysis of HDL CE as occurs in mammalian cells (Fig. 3) (47). This suggests that SR-BI does not facilitate delivery of CE to a metabolically active pool in Sf9 cells or these cells lack a hydrolase that can interact with SR-BI. This result also supports the view that SR-BI itself does not have CE hydrolase activity (47).
In summary, this study demonstrates that SR-BI has many of the same activities in insect cells as in mammalian cells but also shows interesting differences. SR-BI increases FC accumulation in Sf9 cells and dramatically increases the susceptibility of membrane FC to cholesterol oxidase. This result as well as the demonstration of SR-BI-induced morphological changes in Sf9 cells (39) indicates that the ability of SR-BI to alter membrane organization is a highly conserved property. The accumulation of PC species with longer mono-or polyunsaturated acyl chains because of SR-BI or CD36 expression may explain some of the changes in membrane properties in Sf9 cells and may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.


How transmembranes tell of their approach

![Graphic][1]

Helical transmembrane sequences may send a message to the translocon through L17.

Johnson/ElsevierNascent transmembrane sequences (TMSs) can, while still well inside the ribosome, fold into an α-helix, according to Cheryl Woolhead, Arthur Johnson, and colleagues (Texas

Research Roundup Research Roundup
How transmembranes tell of their approach ascent transmembrane sequences (TMSs) can, while still well inside the ribosome, fold into an ␣ -helix, according to Cheryl Woolhead, Arthur Johnson, and colleagues (Texas A&M, College Station, TX). That ␣ -helix appears to send a message through the ribosome to the bound protein translocation apparatus so that it knows when to open and close its various gates.
Making a secreted protein is easy: a ribosome docks to the ER translocon, forming a tight seal, and the protein threads through the translocon tunnel. But with transmembrane proteins, the ribosome and translocon must make space for the release of both the TMS into the membrane and any following cytoplasmic loop into the cytoplasm. Previous work suggested that an ion-tight seal is maintained by the binding of BiP to the lumenal surface of the translocon.
But how does BiP know it is needed? Johnson had earlier established that BiP binding is induced if synthesis is halted just four amino acids after the completion of the TMS, with the TMS still well within the ribosome. "This threw us for a loop," says Johnson. Perhaps the nascent chain tunnel was not a passive tunnel, but a transducer of signals about TMS arrival.
Johnson now suggests that the ribosome detects a TMS as different based on the TMS's propensity to form an ␣ -helix. His evidence for ␣ -helix formation consists of fluorescence resonance energy transfer (FRET) between dyes placed at either end of a newly synthesized TMS. Other, presumably extended, sequences showed no such energy transfer.
The TMS makes two unique ribosomal contacts. The first, with L17, coincides with and may induce BiP binding. L17 is a plausible signaler, as it has one end near the site of protein synthesis and the other near the site of contact with the translocon. Further down the N All in the (network) family etworks from very different systems fall into just a few superfamilies, say Ron Milo, Uri Alon, and colleagues (Weizmann Institute of Science, Rehovot, Israel). The superfamilies are based on the relative frequencies of certain local network motifs or recurring circuit elements that have defined information processing tasks.
Alon says he wants to "break the network down into elementary circuits and building blocks. We're very much inspired by engineering. If you want to understand the device…you break the problem up." He has previously found that networks have higher than expected frequencies of certain circuit elements; such elements have functions such as detecting persistence or imposing temporal order. The team now quantifies the relative occurrence of such elements in a wide variety of networks and finds that just a few superfamilies emerge. N tunnel, the TMS contacts L39. This coincides with the later release of the ribosome-translocon seal, which may allow cytoplasmic domains to escape.
Crystallographers have suggested a very different model in which the translocon pore forms an adjustable seal. Johnson's model of sequential (ribosome then BiP) seals to the translocon is based on the changing accessibility to fluorescence-quenching ions. Resolution of the issue may have to await the tricky crystallization of a translocon with a bound nascent protein. Reference: Woolhead, C.A., et al. 2004. Cell. 116:725-736. Helical transmembrane sequences may send a message to the translocon through L17.

Johnson/Elsevier
The first includes transcription networks from microorganisms. These sensory networks must respond rapidly relative to the buildup time for each of their components, and thus feature a shallow organization. By contrast, developmental transcription networks in flies and neuron organization in worms have an output time (hours or a second) that greatly exceeds the time taken for each step

Different superfamilies are formed by microbial transcription networks (top) and developmental and neuron organization networks (bottom).
Alon/AAAS (minutes or 100 ms). Therefore, they have less bias against longer cascades and feature more feedback loops; these non-rate-limited networks form another superfamily.
The families are not restricted to biology-internet links and social networks form another superfamily, and diverse languages a fourth. All this clustering offers exciting opportunities for understanding, for example, fly development by taking lessons learned from worm neuronal architecture.
The current study covers only circuit elements with three or four components. Alon is currently pushing this number higher. "How we can ever cope with the complexity of these systems," he says, "is to work at the level of individual circuits rather than individual arrows." T EXT B Y W ILLIAM A. W ELLS WELLSW @ ROCKEFELLER . EDU

Actin as stairmaster
imultaneously holding onto and building something is no mean feat. Yingwu Xu, Michael Eck (Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA), and colleagues find that the formin homology-2 (FH2) domain is ideally suited to the task. The apparently flexible structure of an FH2 dimer is consistent with a stair-stepping model of actin filament growth in which each half of the dimer alternately dissociates to make room for an incoming actin monomer even as the other half of the dimer maintains its grip on the actin filament.
The model did not come to Eck immediately. "We stared at the structure for about a year," he says. But then the group tried to reconcile the perfect twofold symmetry of the FH2 crystal structure with the helical alignment of actin monomers in a filament. The only way the two would come together, they realized, was if the FH2 was flexible.
A prime suspect for the source of flexibility was a linker region of FH2. It was relatively unstructured in the crystal, could be clipped by proteases, and was the only significant tie between two parts of the dimer. Sure enough, a crystal of a mutant FH2 that was still functional had a completely different orientation of the linker and thus of the two parts of FH2.
Eck believes the two structures prove flexibility rather than demonstrating the two most important orientations of the FH2 regions. Eck hopes that eventually he will see oscillations in fluorescence resonance energy transfer (FRET) as the FH2 steps alternately close to and far away from the monomers of a growing actin filament.

Eck/Elsevier
Surviving to remember ot all memories live forever. Some memories may, at least in birds, die with the death of the neurons that encode them. Now, Benjamin Alvarez-Borda, Bhagwattie Haripal, and Fernando Nottebohm (Rockefeller University, New York, NY) suggest that the life expectancy of certain canary neurons, and N perhaps of their associated memories, depends on one factor.

Nottebohm/NAS
Canaries start singing and learning songs in the fall as the mating season approaches.
Neurons entering the high vocal center (HVC) during this time stick around so that song memories persist. But by spring most of the birds have found a mate and ignore singing in favor of other pursuits. Neurons born in the spring come and go more rapidly. The Rockefeller team turned the short-lived spring neurons into long-lived neurons by a short burst of brainderived neurotrophic factor (BDNF) given 14-20 days after the neurons were born, which is when differentiation is occurring. Adding BDNF one week earlier or later did not enhance survival.
BDNF may increase survival time by strengthening neural connections. But why is this regulation needed? The memory capacity of adult bird brains may be limited by the number of available nerve cells. If cell number can't be increased, survival time may need to be limited to make space for new memories. BDNF is the first handle on how canary brains assign lifetimes to neurons and perhaps memories. Similar processes may even apply to our own memory-processing hippocampus.

Snaring bugs in NETs
eutrophils spill out their DNA to ensnare and kill extracellular bacteria, according to Volker Brinkmann, Arturo Zychlinsky (Max Planck, Berlin, Germany), and colleagues.
The team figured that degradative enzymes from neutrophils, if they were to be effective, should somehow be directed to meet bacteria. When they looked at elastase localization, they discovered what they call neutrophil extracellular traps (NETs). The fibrous NETs contained degradative enzymes that could destroy bacterial virulence factors, but the overall structures break down with DNase not proteases.
NET formation does not appear to be N part of an apoptotic program, and cells with NETs are still alive: they do not release cytoplasmic proteins and continue to exclude vital dyes. (Neutrophils are, however, programmed to die within hours after entering the circulation.) Whether the cells with NETs can later phagocytose bacteria remains unclear. NETs were apparently seen before by others, but their importance was not realized. Part of their antibacterial action derives from their histones. The antibacterial action of histones was noted back in 1958 by James Hirsch. He was reportedly discouraged by others who questioned the relevance of the result, based on the idea that bacteria would never see histones anyway.
Zychlinksy is now focusing on how NETs are formed, and whether they are directed at bacteria or erected as barriers in which motile bacteria will eventually be snared.


extfieldsofstudy: Medicine Biology

text:
Activating cancer drug discovery.

Normal cells that transform into cancer cells undergo various metabolic changes, including shifts in activities of enzymes that mediate macromolecule synthesis and growth-signaling pathways. Proteomics technology now provides an elegant way to identify the enzymes that are active in processes linked with tumor progression. As demonstrated by a recent study conducted at The Burnham Institute’s Cancer Center in La Jolla, California, this approach is beginning to unveil some novel high-efficacy targets for cancer control and treatment.

Background
Colorectal cancer is the second most common cause of cancer related death in the developed world [1], in consequence advances in our understanding and treatment of colorectal cancers can potentially have a huge impact on cancer morbidity and mortality. Currently much of our understanding of cancer behaviour, including the prediction of likely patient outcomes, is based on histopathological parameters, and from this treatment is tailored to individual patients. At present TNM stage, tumour type and resection margin status are the most widely used parameters in planning adjuvant treatment. Tumour grade of differentiation, vascular invasion and more recently perineural invasion and tumour border configuration have also been used to assist the clinician in predicting colorectal tumour behaviour and hence subsequent patient management [2].
It is well recognised that clinical response and recurrence rates vary within the conventionally staged groups and that this reflects variation in the genetic and molecular make-up of these tumours. Molecular changes occur within cancer cells during tumour progression; these changes provide a potential insight into tumour development and metastasis.
Refining prognostic markers allow treatment to be more accurately tailored to individual patients, as well as suggesting potential mechanisms through which tumour progression occurs which in turn could provide targets for novel therapies.
MUC1 is a membrane bound glycoprotein which has been demonstrated to be predictive of tumour progression and worsening prognosis in both gastric [3][4][5] and colorectal cancer [6,7] including those related to HNPCC [8]. This increased expression has been seen more predominantly at the invasive tumour front [9].
MUC3 is also a trans-membrane glycoprotein which is seen in both colorectal cancers and normal colon [10]. Studies have shown an association between MUC3 expression and poor prognosis in a number of cancers including pancreatic [11], breast [12], gastric [13] and renal [14]. There is some evidence suggesting that MUC3 expression is reduced in colorectal cancers and that this varies between histological types [15]. The cellular distribution is also seen to be affected; apolar distribution is thought to reflect abnormal transport systems [16].
Whilst previous studies have suggested that tumour expression of MUC1 may be a useful prognostic factor in colorectal carcinoma [6,9] these studies have failed to include the presence or absence of vascular invasion in their analysis, this is known to be a highly significant prognostic factor in colorectal cancer [17]. We assessed the prognostic value of MUC1 on a larger set of colorectal tumours and included vascular invasion in our analysis to determine if MUC1 was truly independent as the previous studies have suggested. We also wanted to assess whether MUC3 demonstrated any prognostic influence on colorectal cancers as seen in other tumours.
Since its first description in 1998, tissue micro-array (TMA) analysis [18] has been employed for the immunohistochemical analysis of target protein expression in a wide range of primary tumour types. Initial fears that the reduced amount of individual tumour tissue analysed using this technique might not be representative of the tumour as a whole appear largely unfounded [19]. The strengths of this approach lie in its ability to provide a rapid turnover of results from very large patient cohorts, whilst reducing variability in experimental conditions and reducing costs [20]. Recently, in an attempt to overcome some of the reporting deficiencies inherent in prognostic tumour marker studies a set of guidelines, the reporting recommendations for tumour Marker prognostic studies (REMARK) have been proposed [21]. The reporting of this study therefore adheres to the REMARK guidelines. This TMA of colorectal cancer patients has previously been validated with a p53(-)/Bcl-2(+) phenotype, loss of HLA or over-expression of MICA all being independent markers of poor survival [22][23][24]. TMAs have also been utilised with MUC1 and MUC3 expression in breast cancer [12].
We have therefore used TMA technology to analyze expression of MUC1 and MUC3 in a series of 462 paraffin embedded colorectal tumour specimens, in conjunction with a detailed data base of clinicopathological variables including disease specific survival. We propose that tumours lacking expression of MUC1 and MUC3 will be more likely to metastasise, due to previously observed loss of cell-cell adhesion, and this will therefore lead to more aggressive cancers with poorer prognosis.

Patients and study design
The study population comprised a series of 462 consecutive patients undergoing elective surgical resection of a histologically proven sporadic primary colorectal cancer at the University Hospital, Nottingham, UK (table 1). These patients were treated between 1st January 1994 and 31st December 2000; this time period allowed meaningful assessment of the prognostic markers studied. All patients treated during this time-frame were considered eligible for inclusion in the study. Tumours were classified as mucinous carcinoma, when more than 50% of tumour volume consisted of mucin [25].
Only cases where the relevant pathological material was unavailable were excluded from the study. Follow-up was calculated from time of resection of the original tumour with all surviving cases being censored for data analysis at 31st December 2003, this produced a median follow up of 37 months (range 0-116) for all patients and 75 months (range 36-116) for survivors.
A prospectively maintained database was used to record relevant clinicopathological data, with data provided from the UK Office for National Statistics; this was available in more than 99% of cases. The information collected was independently validated through case note review of deceased patients. Disease specific survival was used as the primary end point; however, data was also collected on the various other relevant clinical and histopathological parameters these are summarised in table 1. There was no formal sample size calculation performed, although the inclusion of over 450 cases is in excess of most studies of prognostic tumour markers.
Adjuvant chemotherapy consisting of 5 FU and folinic acid was reserved for those patients with positive lymph nodes, although, surgical and adjuvant treatment was at the discretion of the supervising physician.
Prior ethical review of the study was conducted by the Nottingham Local Research and Ethics Committee, who granted approval for the study.
Construction of the array blocks incorporated a wide spectrum of electively resected colorectal tumours and was found to be broadly representative of the colorectal cancer population in the UK. 266 (58%) patients were male and 196 (42%) female. The median age at the time of surgery was 72 years, consistent with a median age at diagnosis of colorectal cancer of 70-74 years in the UK [26]. 69 (15%) tumours arrayed were TNM stage 1, 174 (38%) stage 2, 155 (34%) stage 3 and 54 (11%) stage 4; there were 3 cases of in-situ disease. These figures are comparable with national figures for distribution of stage 1-4 at diagnosis of 11, 35, 26 and 29% respectively [27]. The majority of tumours (392, 85%) were adenocarcinomas, and were most frequently of a moderate histological grade (353, 77%). 128 (28%) tumours were noted to have histological evidence of extramural vascular invasion, 224 (48%) had no evidence of vascular invasion, and this information was not available in 110 (24%) cases.
At the time of censoring for data analysis 228 (49%) patients had died from their disease, 64 (14%) were deceased from all other causes, and 169 (37%) were alive. The median five-year disease-specific survival for the cohort was 58 months, comparable with a national average of approximately 45% five-year survival for colorectal cancer in the UK [27].

Specimen characteristics
All tumours received following resection in the operating theatre were incised, fixed immediately in 10% neutral buffered formalin followed by standard processing through to embedding in paraffin wax, ensuring optimal tissue fixation and preservation for histological examination.
Tissue micro-arrays were constructed as described previously [18]. For each tumour, 5 µm section slides stained with haematoxylin-eosin were first used to locate representative areas of viable tumour tissue. 0.6 mm needle core-biopsies from the corresponding areas on the paraffin-embedded tumour blocks were then placed at prespecified coordinates in recipient paraffin array blocks using a manual tissue-arrayer (Beecher Instruments, Sun Prarie, WI). Array blocks were constructed with between 80-150 cores in each, with analysis of a single core from each case. Fresh 5 µm sections were obtained from each TMA block and placed on coated glass slides to allow the immunohistochemical procedures to be performed, preserving maximum tissue antigenicity.

Immunohistochemistry
Immunohistochemical analysis of MUC1 and MUC3 expression was performed using a routine streptavidinbiotin peroxidase method. Tissue array sections were first deparaffinised with xylene, rehydrated through graded alcohol and immersed in methanol containing 0.3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity. In order to retrieve antigenicity, sections were immersed in 500 mls of pH 9.0 EDTA buffer and heated for 10 min in an 800 W microwave at high power, followed by 10 min at low power. Endogenous avidin/biotin binding was blocked using an avidin/biotin blocking kit (Vector Labs, USA). In order to block nonspecific binding of the primary antibody all sections were then treated with 100 µl of 1/5 normal swine serum (NSS) in TBS for 15 min.

Evaluation of MUC1 and MUC3 staining
The tumour cores were assessed by two observers (TJD and AHA) with regard to distribution and intensity of staining, both with extensive experience in the analysis of tissue micro-arrays. Tumours were classified according to a semi-quantitative system in a coded manner and blinded to the clinical and pathological parameters of the case. In the few cases (<5%) where there was discrepancy between the classification of cores a review, using a double headed microscope, was performed and a consensus reached.
For MUC1, tumours were scored according to the proportion of viable tumour cells within the tumour core which displayed unequivocal staining and scored: None = 0, <5% = 1, 5-29% = 2, 30-59% = 3, >60% = 4 in line with previous studies using the same antibody [6,28]. As the expression of MUC 3 was more uniform throughout positive tumour cores the intensity of staining was used as the discriminator with tumours categorised as showing negative, low, moderate and high intensity of MUC3 expression. For the purposes of survival analysis tumours were further categorised as either negative or positive for marker expression. Tumours were considered positive for MUC 1 expression when at least 30% of cells demonstrated positive staining, this is in keeping with previous studies [6,28]. For MUC3 tumours displaying moderate or high intensity staining were considered positive, with the remainder considered negative

Statistical analysis
Statistical analysis of the study data was performed using the SPSS package (version 14 for Windows, SPSS Inc., Chicago, IL). Pearson χ 2 chi-square tests were used to determine the significance of associations between categorical variables. Disease-specific survival calculations included all patients whose death related to colorectal cancer. In contrast, patients whose deaths resulted from non-colorectal cancer related causes were censored at the time of death. Kaplan-Meier curves were used to assess factors which influenced survival. The statistical significance of differences in disease-specific survival between groups with differing MUC1 and MUC3 expression was estimated using the log-rank test. The Cox proportional-hazards model was used for multivariate analysis in order to determine the relative risk and independent significance of individual factors. In all cases p-values < 0.05 were considered as statistically significant.

Patient and histopathological variables and prognosis
Univariate relationships between known patient/tumour characteristics and DSS were evaluated using the log-rank test (see Table 2). There appeared to be no significant differences in DSS between patients of either gender. Similarly when patient age was considered in three groups (patients 64 years or younger at the time of surgery, patients 65-79 years, and those 80 years and over), no significant differences in DSS were noted. The strongest association of clinicopathological variables with DSS was seen with TNM staging (log-rank = 211.37, p < 0.0001), showing a progressive reduction in DSS with increasing tumour stage.

Tumour marker expression MUC1
Analysis of MUC1 expression was possible in 403 of the 462 tumours on the TMA (87%), with the remainder being lost during antigen retrieval or not demonstrating viable tumour cells within the core. This level of core loss is within the rates described by previous authors using TMAs [29,30]. The majority of staining was seen within Representative examples of positive and negative staining for each antigen are shown in figure 1.

Relationships between tumour markers and standard clinicopathological variables MUC1
For the purposes of analysis the tumours were divided into those with positive or negative expression, as described previously [6,28]. There did not appear to be Immunohistochemical staining of tissue microarray cores with MUC1 and MUC3 antibodies any relationship between any of the clinicopathological variables, including stage, and MUC1 expression (see table 3).

MUC3
As the majority of tumour cells within each core expressed a uniform staining pattern, the cores were classified according to intensity of staining as opposed to the proportion of cells staining. Cores were deemed positive if moderate or strong staining was seen. Using this system 286 (74%) tumours were positive and 101 (26%) negative. No correlation between MUC3 cytoplasmic expression and any clinicopathological variables, including stage, was seen (see table 4). Equally there was no correlation of membranous staining with any clinicopathological variables (data not shown).

Relationship between tumour markers and patient survival
Correlation between MUC1 and MUC3 expression and DSS was assessed using Kaplan-Meier plots and log rank testing (see table 2, figures 2 and 3). A significant association was seen between tumours with high MUC1 expression and a reduced DSS (mean DSS 54 months vs. 65 months; p = 0.038). In contrast, there was no correlation between MUC3 expression and DSS.
In order to determine the relative influence of MUC1 and other patient and tumour variables known to affect prognosis, a multivariate analysis was performed using the Cox proportional hazards model. We included only those variables which had been shown to be significantly related to DSS on univariate analysis i.e. intramural vascular invasion and TNM stage (see table 5). In this model, vascular invasion (p < 0.001) and TNM staging (p < 0.001) were seen to retain independent prognostic significance. High expression of MUC1 was also seen to be an independent prognostic marker of poor outcome, with a hazard ratio of 1.339 (95%CI 1.002-1.790, p = 0.048), when compared with tumour demonstrating low MUC1 expression.

Discussion
This study investigates the role of MUC1 and MUC3 as prognostic markers in colorectal cancer. Previous studies have suggested a link between MUC1 and MUC3 expression and poor prognosis both in colorectal and other tumour types [3][4][5][6][7][8]. These studies have frequently suffered from small sample sizes and/or heterogeneous methodology and study populations. The current study comprises the largest analysis of MUC1 and MUC3 expression in colorectal cancer to date; including 463 consecutively treated representative patients, who were representative of the colorectal cancer population within the UK. With a comprehensive data set of clinicopathological variables and patient outcome, over a median 3 year postoperative period, a thorough and comprehensive analysis was possible between these variables and disease specific survival.
In our study population 32% of tumours were positive for MUC1. This compares favourably with previous authors work, who also used the same semi-quantitative scoring system and found 32% and 43% MUC1 positivity in colorectal tumours respectively [6,28].
In our study population MUC1 expression was not related to any of the clinicopathological variables examined. Some previous studies demonstrated increased MUC1 expression was related to increasing TNM or Dukes stage [31][32][33]; however, a number of other studies are in line with our findings [9,34]. Variations in the findings of the current and previous studies may relate to differences in immunohistochemical protocols, antibodies used, scoring systems and area of the tumour examined e.g. Hiraga et al and Kimura et al only assessed staining at the invasion front [31,32]. A large study by Lugli et al examines the prognostic significance of MUC1 and MUC2 in relation to differing mismatch repair status in colorectal cancer, with tumours divided into three subgroups. Significant correlations were found in the "mismatch repair proficient group" between MUC1 positivity and tumour stage and grade [33]. There was no such correlation in our cohort, however our analysis did not involve sub-stratification of the population and hence may explain the dissimilar results.
Univariate and multivariate analysis of our patient population confirmed that TNM staging and vascular invasion are strong independent prognostic markers in colorectal cancer. Of particular interest was the large effect vascular invasion had on survival. Presence of vascular invasion reduced mean DSS significantly (38 vs 75 months p < 0.0001), yet no previous studies investigating the prognostic value of MUC1 have included this obviously strong predictor of survival in their analysis. Our data confirm that high expression of MUC1 in colorectal cancer confers a worse prognosis both on univariate and multivariate analysis, even when taking into account the potentially confounding influence of vascular invasion status.
The association of MUC1 with poor prognosis has been linked to effects on cell adhesion and the potential for metastasis. Regimbald et al [35] showed that MUC1 was a ligand for ICAM-1 in breast cancer and might have a pivotal role in haematogenous spread, and it has been speculated that this mechanism may occur in colorectal cancer [36]. MUC1 is also seen to have effects on the extra cellular matrix components through inhibition of kalinin and laminin [37,38].
MUC1 has been demonstrated to affect beta-catenin, a nuclear transcription factor, and its intracellular distribution has been shown to influence progression of colorectal cancer [39], it has been suggested that MUC1 exerts some of it's effects through interaction with beta-catenin, with over expression of MUC1 leading to increased levels of nuclear beta-catenin [40]. A recent study has shown that the co-expression of MUC1 and nuclear beta-catenin at the invasion front of colorectal tumours may be correlated with a worse prognosis [9].  MUC3 expression was present in moderate to high levels in 76% of tumours assessed. Some studies have suggested that MUC3 may in fact be down-regulated in colorectal cancer compared with normal colon [10,15]. We did not see any correlation between the clinicopathological variables and MUC3; in particular there was no correlation with tumour stage as is seen with gastric cancers [13]. Furthermore, MUC3 expression did not appear to correlate with prognosis, as has been reported in other tumour types [11][12][13][14]. Rakha et al demonstrated MUC3 expression in 91% of breast cancers which was associated with increased local recurrence and lymph node stage. They argued that membranous expression of MUC3 was a poor prognostic feature, which correlated with higher grade and poorer Nottingham Prognostic Index (NPI) [12]. Wang reported that increased MUC3 expression in gastric cancer worsened prognosis, with no significant differences in expression seen in relation to patient sex, tumour location, grade of differentiation, serosal invasion, or Lauren's type. However MUC3 expression was higher in those with metastasis (p < 0.01) and in clinical stage III-IV disease compared to I-II (p < 0.05). MUC3 were not detected in the normal gastric mucosa [13]. MUC3 showed a progressive increase in expression with pancreatic intraepithelial neoplasia of increasing dysplasia and was also highly expressed in ductal adenocarcinoma [11].
Normal lung tissues exhibited a distinct pattern of mucin gene expression, with high levels of MUC1 and low levels of MUC3 immunoreactivity and mRNA. In contrast, lung adenocarcinomas, especially well-differentiated cancers, exhibited increased MUC1 and MUC3 mRNA levels [41].
Copin et al found that coexpression of MUC3 and MUC1 was constant among lung adenocarcinomas [42].

Conclusion
We have demonstrated that using TMA technology and a large cohort of colorectal cancer patients with robust long term follow up data that biomarkers of prognosis can be reliably assessed. Our data clearly demonstrates a role for MUC1 in the progression of colorectal cancer, probably through its effects on cell adhesion and metastasis. MUC1 expression appears to function as an independent prognostic marker in colorectal cancer even when the conventional variables of tumour stage and vascular invasion status are included in the analysis.