extfieldsofstudy: Medicine Biology

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Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells

Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) to drive production of an intracellular photosensitiser, protoporphyrin IX (PpIX), is a promising cancer treatment. However, ALA-PDT is still suboptimal for thick or refractory tumours. Searching for new approaches, we tested a known inducer of cellular differentiation, methotrexate (MTX), in combination with ALA-PDT in LNCaP cells. Methotrexate alone promoted growth arrest, differentiation, and apoptosis. Methotrexate pretreatment (1 mg l−1, 72 h) followed by ALA (0.3 mM, 4 h) resulted in a three-fold increase in intracellular PpIX, by biochemical and confocal analyses. After exposure to 512 nm light, killing was significantly enhanced in MTX-preconditioned cells. The reverse order of treatments, ALA-PDT followed by MTX, yielded no enhancement. Methotrexate caused a similar relative increase in PpIX, whether cells were incubated with ALA, methyl-ALA, or hexyl-ALA, arguing against a major effect upon ALA transport. Searching for an effect among porphyrin synthetic enzymes, we found that coproporphyrinogen oxidase (CPO) was increased three-fold by MTX at the mRNA and protein levels. Transfection of LNCaP cells with a CPO-expressing vector stimulated the accumulation of PpIX. Our data suggest that MTX, when used to modulate intracellular production of endogenous PpIX, may provide a new combination PDT approach for certain cancers.

Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) as a prodrug that is converted to an intracellular photosensitiser, protoporphyrin IX (PpIX), represents an increasingly accepted treatment for superficial epithelial neoplasias (Kennedy et al, 1990;Marmur et al, 2004) and for diagnostic imaging of internal malignancies at locations where light can be delivered through an optical fibre (Kennedy et al, 1996;Eker et al, 1999;Landry et al, 2003;Schmidbauer et al, 2004). However, ALA-mediated PDT (ALA-PDT) remains suboptimal for the treatment of deeper tumours, such as nodular basal cell and squamous cell carcinoma of the skin, because recurrence rates after single treatments are unacceptably high as compared to the current standard of care, surgical excision (Marmur et al, 2004). For internal malignancies in organs such as the bladder, GU tract, and oesophagus, ALA-PDT has been used for palliative treatment of these cancers (Kennedy et al, 1996;Waidelich et al, 2001;Friesen et al, 2002). However, ALA-PDT in its current form seldom achieves a definitive cure of deep or refractory tumours in any location. Clearly, new approaches are needed, including ways to increase the clinical responsiveness of tumour cells to ALA-PDT.
Methotrexate (MTX) is a widely used and highly successful anticancer agent. A structural analogue of folic acid and a potent inhibitor of dihydrofolate reductase, MTX interferes with the synthesis of thymidylate and purine nucleotides (Hatse et al, 1999), and blocks DNA synthesis, thereby inhibiting tumour growth. However, another important property of MTX is its ability to trigger cellular differentiation in a number of tumour cells, including human and rat choriocarcinoma cells (Friedman and Skehan, 1979;Hatse et al, 1998), human promyelocytic HL-60 cells (Bodner et al, 1981), human neuroblastoma LA-N-1 cells (Ross et al, 1995), as well as normal human keratinocytes (Schwartz et al, 1992). This differentiation-promoting property of MTX is of particular interest to us. Malignant cells typically display an uncoupling of the balance between cellular proliferation and differentiation, in which cellular proliferation is favoured owing to a block in maturation (Marks and Rifkind, 1991). However, in certain cases, differentiation-inducing agents can reactivate a tumour cell's differentiation programme and bypass this defective growth regulation, forcing cells into a more differentiated state that has a more favourable clinical prognosis. One major clinical example of this is the use of all-trans retinoic acid for differentiation therapy of acute promyelocytic leukaemia (Douer, 2000). Another well-known example is the successful treatment of highly aggressive human choriocarcinoma tumours using MTX, which was shown to induce cytodifferentiation in human and rat choriocarcinoma cells in culture (Berkowitz et al, 1986).
Because MTX can induce cellular differentiation in some types of tumour cells, and because we have recently shown that agents capable of inducing cellular differentiation will also cause an elevation in the level of PpIX photosensitiser in epithelial cells of the skin (Ortel et al, 1998) and prostate (Ortel et al, 2002), we became interested in the notion of combination therapy involving MTX and ALA-PDT. Specifically, we reasoned that if MTX could promote differentiation and PpIX accumulation in carcinoma cells, then MTX might serve as a means to augment the efficacy of ALA-PDT for tumour cell killing. The data described in this paper, obtained using a carcinoma cell line, suggest that MTX may indeed be a useful adjunct to ALA-PDT when administered before the phototherapy.

Source of reagents
ALA and MTX were from Sigma-Aldrich (St Louis, MO, USA). Methyl-ALA and hexyl-ALA were obtained from ORGANIX (Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK). R1881 was obtained from Perkin-Elmer Life Sciences (Boston, MA, USA).

Culture of LNCaP cells
LNCaP human prostate carcinoma cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at 371C in a humidified CO 2 incubator in Rosewell Park Memorial Institute (RPMI) 1640 medium with L-glutamine (Mediatech Inc., Herndon, VA, USA) supplemented with 10% foetal bovine serum (Invitrogen Corp., Carlsbad, CA, USA) and 1% penicillin -streptomycin and 10 mM HEPES buffer. To maintain reproducible behaviour of the LNCaP cells, we found it important to keep the cells subconfluent. The original vial of cells was thawed and plated in the medium in a 100 mm dish, and at 24 h, the medium was replaced and left on for another 3 -4 days. When nearly confluent, the cells were released with 0.25% trypsin (noting under the microscope when cells had just detached, usually o3 min), and were replated in RPMI medium at a 1 : 3 dilution. Subsequently, cells were passaged at 1 : 3 dilution every 3 -4 days, always before they reached full confluence.

Measurement of proliferation, differentiation and apoptosis
Control LNCaP cells were plated in 35 mm dishes at an initial density of B100 000 cells dish À1 , and after 72 h of growth, were trypsin-released and counted. Methotrexate-treated dishes were plated at a higher initial density of B250 000 cells dish À1 , to give a final cell density that was roughly equal to the controls after 72 h. Proliferation was estimated as the ratio (percentage) of the final trypsinised cell number divided by the number of cells plated. Growth arrest was assessed by upregulation of p27/kip-1, and differentiation by upregulation of E-cadherin in MTX-treated LNCaP cells, as described (Ortel et al, 2002).
Apoptosis was measured by a fluorescein isothiocyanate (FITC)annexin V/propidum iodide assay (Vybrant Apoptosis Assay, Molecular Probes Inc., Eugene, OR, USA). Supernatants from each culture dish were collected in 15 ml Falcon tubes, cells were trypsin-released (0.5 ml of trypsin) and pooled with the respective supernatant, and cells pelleted for 10 min at 1000 r.p.m. in a refrigerated centrifuge (201C). The supernatant was discarded, the cell pellet was washed by gentle resuspension in phosphatebuffered saline, repelleted, and the cell pellet was resuspended in 100 ml of 1 : 5 diluted buffer supplied in the kit. In a fresh microcentrifuge tube, 20 ml of the cell suspension was mixed with 80 ml of 1 : 5 diluted buffer, then 5 ml of annexin-FITC and 1 ml of 1 : 10 diluted propidium iodide (supplied in the kit) was added in each tube. After 15 min at room temperature, 400 ml of 1 : 5 diluted buffer was added and the cell suspension was transferred to a fluorescent-activated cell sorting (FACS) tube on ice (Becton Dickinson Labware, Bedford, MA, USA) and measured by fluorescent-activated cell analysis (FACSCalibur, Becton Dickinson, CA, USA).

Pretreatment of cells with MTX or R1881, and measurement of PpIX in cell lysates
In all cases, cells were plated at B80 -100k for untreated dishes and B180 -250k for MTX-treated dishes, in order to yield an approximately equal cell density at the end of the experiment. Methotrexate (1 mg l À1 in media) was added on the second day after plating and incubated for an additional 72 h, after which the medium was aspirated and ALA in fresh medium was added. In the experiment shown in Figure 3A, the androgen analogue R1881 (0.1 mM) was added for 72 h to some of the dishes. For the last 4 h of all experiments, ALA (at 0.3 mM concentration, unless otherwise noted) was added to the culture medium, the cells trypsin-released, and final cell counts were determined with a haemocytometer. The cells were lysed and vortexed in 2 ml of 1% sodium dodecyl sulphate in 0.1 N NaOH. The amount of PpIX per cell was determined as follows. An aliquot of cell lysate was measured in a scanning spectrophotometer (Horiba Instruments SA Inc., Jobin Yvon/Spex Division, Edison, NJ, USA) and the peak area between 580 and 720 nm was determined. The PpIX concentration of each sample was calculated by comparison to a calibration curve constructed from measuring PpIX standards, run on the same day. Based upon the known volume of the lysate and the cell count before lysis, the PpIX concentration was converted to total PpIX (in pmol) per 1 million cells.

Fluorescence analysis of PpIX in living cells
Protoporphyrin IX-specific fluorescence in living LNCaP cells was analysed by fluorescence microscopy on a confocal laserscanning microscope (Leica Microscopy Systems GmbH, Wetzlar, Germany). LNCaP cells were plated on microscope coverslips in 35 mm dishes at 50 000 cells dish À1 , then 24 h later the culture media were replaced either with media alone or with media containing MTX (1 mg l À1 ). The cells were cultured for an additional 72 h, then ALA (0.3 mM) was added to the wells for a final 4 h of incubation. Protoporphyrin IX-specific fluorescence in the living cells was analysed on the confocal microscope. Using excitation at 488 nm, images were collected in the red channel, through a 590 nm long-pass filter.

Transient transfection of LNCaP cells for analysis of PpIX fluorescence
LNCaP cells (3 Â 10 5 cells dish À1 ) were plated on a 22 Â 22 mm, No. 1 cover glass in a 35 mm dish. At 24 h, cells were transfected with variable quantities of pcDNA3.1(À) mouse coproporphyrinogen oxidase (CPO) expression plasmid (gift of Dr Shigeru Taketani (Kohno et al, 1993)) using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN, USA), exactly as described by the manufacturer. At 24 h post-transfection, the medium was replaced with fresh medium containing 1 mM ALA and incubated for 4 h before analysis of PpIX fluorescence by confocal microscopy. Controls for the analysis included dishes with no treatment, FuGENE only, and ALA only. Fluorescence images along with their corresponding phase-contrast images were captured from regions representing monolayered (not clustered) cells, using a digital camera. Protoporphyrin IX-positive cells were counted from fluorescence images using Adobe Photoshop software, by setting the brightness to a fixed value that rendered approximately half of the cells visible on the ALA-only control slide. The same brightness threshold was then applied to all images and kept constant throughout the analysis. Cells above the threshold were counted for each slide and expressed as the relative number of PpIX-positive cells (% of total cells).

Cell survival assays
For experiments to measure the effects of MTX upon phototoxicity, sets of 35 mm dishes were plated at a cell number of 8 Â 10 4 (control) or 2 Â 10 5 (MTX treatment), and incubated for 24 h. Then, the control or MTX plates received fresh medium alone, or MTX in the medium, respectively. This procedure assured similar cell densities for both sets of cells at the time of photosensitisation, which was confirmed by actual cell counts in parallel dishes. After 72 h in MTX, cells were incubated with 0.3 mM ALA (for most experiments, unless otherwise noted) for 4 h, and then exposed to graduated doses of light delivered by a 514 nm argon laser (Coherent Inc., Santa Clara, CA, USA) for the clonogenic experiments. Dosimetry was performed using a Coherent Lasermate power meter (Coherent Inc., Santa Clara, CA, USA).
For quantifying long-term survival, a colony formation assay was used. After performing ALA incubation and light exposures as detailed above, LNCaP cells were detached using trypsin/ethylenediaminetetraacetic acid and resuspended in complete medium. Serially diluted suspensions of the photosensitised cells (range 1 : 5 -1 : 1375) were plated on 100 mm dishes and incubated for 13 days. Cells were fixed with 0.2% buffered formalin in methanol and stained with 0.1% aqueous crystal violet. Colonies of more than 50 cells were counted under a dissecting microscope.
To assess if the order of MTX and ALA-PDT is important for efficacy of the combined therapy, colony formation was measured in experiments using a reverse order of administration, that is, ALA-PDT given first, followed by MTX treatment. Cells were exposed for 4 h to ALA (0.3 mM) and irradiated with graduated light doses. Duplicate sets of diluted samples (see above) were then plated on 100 mm dishes in medium either with or without MTX. After 72 h, the medium was replaced by fresh medium in all dishes. After incubation for an additional 10 days, colonies were stained and counted as described above.
A murine CPO cDNA sequence (Kohno et al, 1993) was amplified with specific PCR primers and cloned into the TAcloning vector PCRII-TOPO, then transferred via restriction enzymes SpeI/XhoI followed by ligation into the NheI/XhoI sites of pcDNA3.1(À), both vectors were from Invitrogen Inc. This mCPO construct was used as a standard for the RT -PCR analyses.

Epitope-targeted antibody for CPO
A rabbit polyclonal antiserum against CPO was generated from a peptide comprising the last 12 amino acids (LEVLRHPKDWVH) of murine CPO, as per the sequence reported by Kohno et al (1993). This peptide has 83% homology between mouse and human at the amino-acid level. The peptide was synthesised with an extra Nterminal cysteine for conjugation to Keyhole limpet antigen in the Biopolymers Core facility of the Massachusetts General Hospital, and sent to HTI Bio-Products Inc. (Ramona, CA, USA) for rabbit immunisation, blood collection, and affinity purification of the antiserum. Specificity was confirmed by Western blots of recombinant mCPO protein expressed in a pcDNA3.1(À) vector transfected into cos-7 cells (see Figure 7C).

Statistics
Two-sample t-tests were used to compare the amount of PpIX accumulation and cytotoxicity between treated and untreated controls (Figures 3, 4, 6, and 7). In addition, analyses of variance (ANOVA) were performed to determine whether there was an additive vs synergistic effect in Figure 4 by testing the interaction term between MTX and ALA-PDT. A P-value of 0.05 or less was considered statistically significant.

RESULTS
Methotrexate stimulates accumulation of intracellular PpIX while promoting growth arrest, differentiation, and apoptosis in LNCaP cells.
We had previously described a strong increase in PpIX accumulation in epithelial keratinocytes that were differentiating in response to high calcium (Ortel et al, 1998), or in LNCaP cells in response to androgens (Ortel et al, 2002). To establish whether such a correlation might exist if MTX were used as the prodifferentiating agent, LNCaP cells were pretreated for 72 h with a cytostatic dose of MTX (1 mg l À1 ; see details below), then exposed to ALA for the last 4 h, and analysed for the amount of PpIX synthesised within the cells. Qualitatively, MTX pretreatment led to a significantly higher PpIX accumulation as compared to non-pretreated controls (Figure 1). The same MTX treatment led to growth arrest, as measured by a complete block on cell proliferation (Figure 2A), and by the accumulation of the cellcycle inhibitor, p27/kip-1 ( Figure 2B, upper), whose upregulation correlates with growth arrest in LNCaP cells (Campbell et al, 1997). Methotrexate also promoted cellular differentiation, as measured by a significant rise in E-cadherin ( Figure 2B, middle), a marker of differentiation in the LNCaP cells (Campbell et al, 1997). The induction of the differentiation marker was MTX dosedependent ( Figure 2B, lower). At the 1 mg l À1 concentration, MTX also triggered classical apoptosis, as measured by fluorescent-activated cell analysis with annexin V ( Figure 2C). The net effect of growth arrest and apoptosis was to reduce the total number of cells in MTX-treated dishes to B25% of the number in untreated control dishes at the end of the 72 h period ( Figure 2A).

The mechanism by which MTX enhances accumulation of PpIX involves the haeme synthesis pathway
We examined the phenomenon of MTX-mediated enhancement of intracellular PpIX levels in LNCaP cells in detail. Enhancement occurred consistently in eight out of eight experiments, with some variability in magnitude between experiments; typically, a threefold increase in PpIX was observed in MTX-conditioned vs nonconditioned cells ( Figure 3A). The PpIX-elevating effect of MTX was only slightly less robust than that of a positive control, the androgen R1881 shown previously to induce PpIX levels in this cell line (Ortel et al, 2002).
To study the effect of ALA concentration upon PpIX accumulation, ALA dose-ranging experiments in the presence or absence of MTX were performed. Protoporphyrin IX content was seen to rise monotonically with increasing ALA concentration ( Figure 3B), and the slope of the curve was markedly higher in the presence of MTX (open circles) than in its absence (closed circles). Protoporphyrin IX elevation was appreciated even at the lowest ALA concentrations tested (see Figure 3B, inset).
To rule out the possibility that PpIX accumulation in MTXpreconditioned cells might be owing to increased membrane transport of ALA precursors, we used ALA and two of its alkyl esters, 5-methyl-ALA (m-ALA) and 5-hexyl-ALA (h-ALA), in comparative experiments ( Figure 3C). Our approach was based on reports that show distinct membrane transport mechanisms for ALA and m-ALA. 5-Aminolaevulinic acid enters cells via membrane pores that also transport b-amino acids and GABA, whereas m-ALA uses a different route (Rud et al, 2000;Gederaas et al, 2001). Although details of the transport mechanism for hexyl-ALA uptake are not known, it also appears to be distinct from b-amino-acid transport (Rud et al, 2000). To examine whether MTX might preferentially alter PpIX accumulation depending upon whether the prodrug was in the form of free ALA or one of its esters, LNCaP cells were pretreated with MTX or media (control) for 72 h, and then exposed to ALA, m-ALA, or h-ALA for 4 h. Cellular production of PpIX was assessed by quantitative spectrofluorometry ( Figure 3C). Because the hexyl ester of ALA is significantly more permeable than ALA itself, it was necessary to use 300 times less h-ALA (1 mM) than ALA (0.3 mM) to achieve a comparable baseline level of PpIX ( Figure 3C, fifth vs first bar). However, under conditions in which ALA and its esters give similar PpIX levels at baseline, the addition of MTX caused a similar degree of PpIX accumulation (three-to five-fold), regardless of which form of ALA was used. Because it seems highly unlikely that MTX could affect three different membrane ALA-transporting mechanisms to exactly the same extent, the PpIX-elevating action of MTX does not occur primarily through an effect upon precursor uptake. Methotrexate enhances the lethal effects of ALA-based photodynamic treatment We next tested whether the elevated intracellular levels of PpIX, observed in MTX-preconditioned cells, can have functional consequences in terms of cell survival after exposure to visible light. LNCaP cells were pretreated with MTX for 72 h, exposed to ALA for 4 h, and then irradiated with 512 nm light and analysed for cytotoxicity using two different assays, MTT dye conversion ( Figure 4A and B), and colony formation ( Figure 4C and D). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide conversion at 24 h is a short-term cytotoxicity assay; MTT serves as an indicator of intact cellular metabolism. In a typical experiment ( Figure 4A), MTX caused an initial B75% decrease in viability as compared to nonconditioned cells; the subsequent administration of light caused an additional 41 log of cell kill, considerably more than in nonconditioned cells. Because the absolute magnitude of viability data often varies between experiments, we expressed the data as per cent survival (relative to nonirradiated controls) in order to allow the normalisation and pooling of multiple experiments. These aggregate results demonstrated that the enhancement of cytotoxicity in the MTX-preconditioned group was statistically significant ( Figure 4B). To assess long-term survival, clonogenic growth of the LNCaP cells at B2 weeks after irradiation was plotted against increasing doses of light ( Figure 4C). Again, MTX significantly enhanced cell killing in the photodynamic regimen, and when the results of multiple experiments were pooled and compared, the improvement with MTX was highly significant ( Figure 4D). To begin to determine whether MTX and ALA-PDT were working independently to promote cell killing, we subjected the data to an ANOVA. In our experiments, the combination of MTX pretreatment and ALA-PDT were synergistic for cell killing only at the lowest dose of light (4 J cm À2 , Po0.001), but in general the lethal effects of MTX and ALA-PDT were additive.

The MTX effect upon ALA-mediated cell killing is order-dependent
Although we hypothesised that enhanced cytotoxicity with MTX occurs because PpIX production is increased in the MTX-preconditioned cells, other possible mechanisms warranted consideration. For example, because MTX inhibits cell proliferation, lower cell densities in the cultures at the time of PDT could hypothetically lead to differences in cellular cytotoxic responses. Therefore, we were careful to eliminate cell density as a variable by adjusting the number of cells plated so as to yield an equivalent cell density at the time of irradiation (see Materials and Methods). An equally important possibility was that MTX might affect cell behaviour after photodynamic treatment, for example, by accelerating cell death pathways. To test this possibility, we reversed the order of MTX and ALA-plus-light treatments ( Figure 5). Cells were subjected to ALA and light exposure first, followed by a 72 h exposure to MTX. Under these conditions, no significant differences in long-term cell survival between MTX-vs vehicle-treated cells were observed ( Figure 5). These data indicate that the presence of MTX does not appreciably alter the mechanisms of cell lethality, once initiated by ALA-PDT.

Methotrexate at noncytotoxic concentrations induces PpIX levels
Whereas the majority of the preceding data were obtained with a relatively high concentration of MTX (1 mg l À1 ¼ 2. approximate the therapeutic levels typically sought in the clinic, MTX at that concentration has antiproliferative and cytotoxic effects in addition to the prodifferentiating effects of particular interest here. To ask whether lower MTX doses might promote accumulation of PpIX, LNCaP cells were exposed to MTX concentrations over a wide range (a 1000-fold, 2.2 mM -2.2 nM) and PpIX levels were assessed by semiquantitative confocal microscopy ( Figure 6A -D). The results demonstrated an MTXdependent increase in PpIX levels, seen with MTX concentrations as low as 2.2 nM ( Figure 6E). From this, we conclude that low, nontoxic concentrations of MTX can promote significant accumulation of PpIX.

Methotrexate enhances PpIX levels via an upregulation of CPO
To test for changes in the enzymes most likely to be involved in an accumulation of PpIX in the haeme synthesis pathway, we considered several potentially rate-limiting enzymes both proximal and distal to PpIX (for a schematic of this pathway, see Figure 8). Given prior evidence that CPO was involved in raising PpIX levels in differentiating epithelial cells (Ortel et al, 1998), we were especially interested in whether CPO gene expression was altered by MTX. To test for changes in mRNA expression, LNCaP cells were treated with MTX or with media alone, and RT -PCR was performed. Using gene-specific primers at the reverse transcription step, followed by a CPO-specific primer pair (see Materials and Methods), a significant and reproducible 2.5-fold increase in CPO mRNA level was observed ( Figure 7A and B). Reverse transcription -polymerase chain reaction analyses with genespecific priming for PPO, ferrochelatase, and PBGD demonstrated no changes in mRNA expression (data not shown), indicating no major effects upon gene expression for those enzymes.
To demonstrate the effect of MTX upon CPO at the protein level, we generated a rabbit antiserum to a defined epitope of the CPO enzyme (see Materials and Methods). The antiserum detected a 38 kDa complex on Western blots of lysates from cos-7 cells  Figure 5 Photodynamic therapy is not enhanced if MTX is administered after ALA and light. Cells were first subjected to ALA-PDT treatment (4 h of ALA 0.3 mM, followed by exposure to graduated light doses) and plated for colony formation assay. Subsequently, one set of plates was exposed to MTX for 72 h, the other to medium only. Colonies were counted at 13 days. (A) Colony formation assay, single experiment. (B) Colony formation assay, pooled experiments. Mean of four experiments7s.e.m. transfected with a plasmid vector expressing full-length murine CPO ( Figure 7C, left). The largest band at 38 kDa corresponds to the predicted size of mature CPO (Ortel et al, 1998), whereas the two smaller forms may represent proteolytic processing or translation occurring from internal ribosome-initiation sites. Time-course experiments, from LNCaP cells harvested at different times in MTX-containing media ( Figure 7C, right), revealed significant changes in a band with the same relative migration (38 kDa) as authentic CPO in the cos-7 lysates. Quantitation of CPO expression from several experiments showed a reproducible increase in this band, detectable by 12 h and rising three-fold by 48 h ( Figure 7D). We conclude that MTX causes a significant accumulation of CPO at both the mRNA and protein levels.
As another way to investigate the link between induced CPO expression and enhanced PpIX levels, a cDNA plasmid vector expressing murine CPO was transiently transfected into LNCaP cells and PpIX levels were observed by confocal microscopy ( Figure 7E and F). Compared to an empty-vector control, the Demonstration that MTX treatment leads to increased levels of CPO. (A) Methotrexate treatment of LNCaP cells causes accumulation of CPO mRNA. Cells were exposed for various times to MTX (1 mg l À1 ), or remained untreated (0 h). Cells were lysed, cDNA prepared, and RT -PCR performed using a CPO gene-specific primer set, as described in Materials and Methods. The resulting PCR product was confirmed by DNA sequencing and by sizesimilarity to a known plasmid cDNA (not shown). The ethidium-stained, CPO-specific bands are shown here inverted (dark bands). G3PDH, invariant control. (B) Semiquantitative analysis of CPO mRNA. Agarose gels were digitally photographed, and the integrated density of each band measured using NIH Image software. Induction (relative to untreated controls) was calculated after background subtraction and normalisation to the G3PDH bands. CPO-containing vector caused a dose-dependent increase in PpIX signal within the cells ( Figure 7E). This increase was statistically significant ( Figure 7F).

DISCUSSION
Owing to the complex nature of cancer development and progression, it is becoming increasingly clear that no single therapeutic modality is likely to be curative. It is now generally accepted that newer molecular mechanism-based therapeutics, particularly those in combination regimens, are more likely to eradicate malignant disease. In this study, we present a novel combination treatment that utilises cellular differentiation and photodynamic destruction of prostate cancer cells. When administered in the proper order, each modality enhances the other, as evidenced by an improved overall outcome in terms of cell killing. Our demonstration of a new combination therapy for ALA-PDT is important because it captures the advantages of PDT-based localised treatment, while using cellular physiology to produce an overall enhanced outcome. 5-Aminolaevulinic acid-PDT provides the targeting specificity. 5-Aminolaevulinic acid, an inert pharmacological precursor, remains inactive until it is converted into a photosensitising agent (PpIX) within the target tissue, thereby confining phototoxic chemical events to the target cells and reducing toxic side effects (Kennedy et al, 1990;Iinuma et al, 1994). However, because tumour eradication with ALA-PDT is not always obtained, a new combination that enhances ALA-PDT would be very useful. Four parameters are necessary for effective PDT: the photosensitising agent, light, oxygen, and cellular physiology. Although many laboratories are attempting to optimise the first three (physical) parameters, we are particularly interested in the cell-physiologic aspects. If higher levels of PpIX could be obtained by proper manipulation of the cellular metabolic state, then photodynamic efficacy could be improved. Methotrexate was chosen as an attractive candidate agent because MTX is already used widely in the clinical arena, has relatively low toxicity, and exerts its own antitumour effects by inhibiting proliferation, promoting apoptosis, and promoting terminal differentiation (Hatse et al, 1999). The ability to promote differentiation, in particular, suggested to us the idea of using MTX in combination with ALA-PDT. Two other agents (androgens and vitamin D) are known to promote cellular differentiation and at the same time to enhance PpIX accumulation in LNCaP cells (Ortel et al, 2002). We decided to test MTX for a similar capability. The concept is to administer MTX in a transient way, preparing the cells for PDT by promoting accumulation of the PpIX photosensitiser.
Our results show that this combined approach is indeed feasible. Preconditioning LNCaP cells with MTX sensitises them to subsequent ALA-PDT, and increases cell killing. An important finding was the fact that for MTX to affect cell lethality, it must be administered before the ALA and light, and not in the reverse order ( Figure 5). This suggests that MTX prepares the cell for death by enhancing accumulation of PpIX rather than by affecting postirradiation events. Methotrexate-related increases in PpIX accumulation are substantial (always over three-fold) and nearly as robust as PpIX induction seen with androgenic hormones. The PpIX-elevating effects of MTX occurs across a range of ALA concentrations ( Figure 3B), suggesting that despite variations in ALA concentration within tissue, sufficient PpIX might accumulate in various parts of a tumour to provide effective photosensitisation. Further, we found that PpIX elevation occurs over a wide range of MTX concentrations. Very low concentrations of MTX, 100-to 1000-fold lower than the high-dose regimen of 1.0 mg l À1 used in the cell-survival portion of our study, can significantly boost the accumulation PpIX in LNCaP cells ( Figure 6). This implies that even when MTX penetration into a tumour is relatively poor, a significant enhancement of PpIX may still be possible. The cell-survival studies performed here at MTX 1.0 mg l À1 showed a combined effect of MTX and ALA-PDT that was only additive, but this may relate to the very real possibility that the dose of MTX that we were using was (in retrospect) too high to detect a synergistic effect. Further studies are planned to ask whether very low-dose MTX combined with ALA-PDT can produce synergistic killing, as well as to validate whether the phenomenon can be reproduced in three-dimensional tissue culture and animal models.
To investigate the mechanisms of increased PpIX accumulation, we first asked whether MTX might be affecting cellular uptake of 5-ALA. Using different ALA esters as the exogenous source for the production of PpIX ( Figure 3C), we found that MTX exerted a similar PpIX-elevating effect, regardless of which ester-specific transport pathway was employed. Although not completely ruling out an effect of MTX upon ALA uptake, the data led us to search for other possible effects of MTX, such as altered PpIX synthesis and/or degradation. For this, we looked at the expression of enzymes in the haeme-synthetic pathway (see cartoon in Figure 8). No changes in mRNA expression were seen for PBGD, PPO, or ferrochelatase. However, a significant three-fold increase in the level of CPO was found to occur. To be sure of these results, we devoted considerable time toward developing an RT -PCR assay that could reliably detect small changes in CPO message levels. * NOTE: Exogenous 5-ALA is added here during ALA-PDT 1 ALAS aminolevulinic acid synthase 2 ALAD ALA dehydratase 3 PBGD porphobilinogen deaminase 4 UCS uropophyrinogen III cosynthase 5 UDC uropophyrinogen decarboxylase 6 CPO coproporphyrinogen oxidase 7 PPO protoporphyrinogen oxidase 8 FC ferrochelatase * Protogen Figure 8 Schematic of porphyrin-synthetic pathway to illustrate potential control points for increased PpIX accumulation. Enzymes, with names abbreviated and relative locations in the pathway shown in boxes, are numbered beginning with the initial condensation of glycine and succinyl-CoA. Substrates/products are shown in bold font. The intracellular location of enzymes, relative to the mitochondria and the cytoplasm, is indicated. See Bickers et al (1999) for a fully detailed version.
Pilot experiments with conventional primers for reverse transcription (oligo-dT, or random hexamers) failed to show any changes in CPO expression, despite amplification in the linear range. Potential problems can occur with the oligo-dT approach when reverse transcription starting at the 3 0 end of the RNA leads to underrepresentation of the 5 0 ends of rare transcripts, and random hexamer primers may also amplify an incorrect proportion of transcripts because of failure to produce full-length cDNAs. To resolve these uncertainties, we devised CPO gene-specific primers to direct first-strand cDNA synthesis. Under these conditions, reproducible increases in CPO mRNA from MTX-treated cells were demonstrated ( Figure 7B). To show a corresponding increase in CPO protein, it was necessary to develop our own epitope-specific antiserum to CPO. After verifying its specificity against a recombinant CPO protein expressed in cos-7 cells, we used the antiserum to show that LNCaP cells produce CPO at higher amounts in cells preconditioned with MTX ( Figure 7C and D). Relative increases in CPO mRNA and protein were modest, Bthree-fold; yet, they can fully account for the three-fold increase in PpIX levels observed in our study. Transfection studies, in which the CPO enzyme was forcibly expressed in the LNCaP cells, confirmed that an elevation in PpIX levels can result from increased expression of CPO ( Figure 7E and F). The idea of a link between terminal differentiation and the CPO enzyme, in the regulation of intracellular porphyrin levels, has some precedence in the literature. In blood, where regulation of porphyrins has been intensively studied, a relationship between CPO levels and erythroid cell differentiation was convincingly established. For example, murine erythroleukaemia MEL cells induced to differentiate by dimethylsulphoxide (DMSO), showed a large induction in mRNA for CPO within 10 h (Kohno et al, 1993). Likewise, human erythroleukaemia K562 cells undergoing differentiation in the presence of TGF-b, also displayed increased CPO expression (Taketani et al, 2001). In erythroid cells, differentiation-inducing agents induced several of the haeme-synthetic enzymes simultaneously (Taketani et al, 1995), but even under such circumstances, CPO may be rate-limiting and therefore important in the regulation of haeme and PpIX production (Conder et al, 1991). Our own demonstrations of CPO upregulation in differentiating keratinocytes (Ortel et al, 1998) and LNCaP cells (Ortel et al, 2002) were among the first to demonstrate such a link in nonhaematopoietic cells. How MTX causes an increase in CPO expression in these situations remains an open question, but possibilities for transcriptional regulation of the CPO promoter are intriguing. For example, MTX regulates expression of three transcription factors, Cdx2, GATA-4, and HNF-1a, that are important for epithelium differentiation in a mouse model of induced intestinal damage (de Koning et al, 2006). Putative binding sites for the above-mentioned transcription factors on the murine CPO promoter might account for increased CPO expression following MTX treatment of LNCaP cells (Takahashi et al, 1998;Maytin et al, 2006).
The notion that MTX-induced effects upon PpIX levels in the LNCaP cells might be linked to induction of a differentiated phenotype per se remains open to conjecture. We have not yet exhaustively investigated the relative effects of very low-dose MTX upon differentiation, apoptosis, and growth arrest of LNCaP (although apoptosis appears to be an insignificant fraction). In human choriocarcinoma (BeWo) cells, a well-established model of MTX-induced differentiation (Friedman and Skehan, 1979); treatment with MTX induces cytodifferentiation at concentrations between 10 À8 and 10 À7 M (Burres and Cass, 1987). Similarly in cultured human keratinocytes, MTX in the 10 À8 to 10 À 7 M range was reported to induce only 1% apoptotic cells (Heenen et al, 1998) while inducing significant numbers of differentiated (involucrin-positive) cells (Schwartz et al, 1992) in the cultures. In LNCaP cells, at this point, we can only say that MTX (at low nontoxic concentrations) clearly promotes the accumulation of PpIX, and that further studies are underway.
The current study broadens the number of situations in which epithelial-type cancer cells can be manipulated to produce higher levels of PpIX endogenously, in this case using an agent (MTX) commonly used in the clinic for cancer chemotherapy. Future studies will be needed to establish whether MTX can be effectively combined with ALA-PDT in the clinical setting.


extfieldsofstudy: Biology Medicine

text:
Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen

Background Pollen of the European white birch (Betula pendula, syn. B. verrucosa) is an important cause of hay fever. The main allergen is Bet v 1, member of the pathogenesis-related class 10 (PR-10) multigene family. To establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree, PCR amplification, cloning and sequencing of PR-10 genes was performed on two diploid B. pendula cultivars and one interspecific tetraploid Betula hybrid. Sequences were attributed to putative genes based on sequence identity and intron length. Information on transcription was derived by comparison with homologous cDNA sequences available in GenBank/EMBL/DDJB. PCR-cloning of multigene families is accompanied by a high risk for the occurrence of PCR recombination artifacts. We screened for and excluded these artifacts, and also detected putative artifact sequences among database sequences. Results Forty-four different PR-10 sequences were recovered from B. pendula and assigned to thirteen putative genes. Sequence homology suggests that three genes were transcribed in somatic tissue and seven genes in pollen. The transcription of three other genes remains unknown. In total, fourteen different Bet v 1-type isoforms were identified in the three cultivars, of which nine isoforms were entirely new. Isoforms with high and low IgE-reactivity are encoded by different genes and one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. Allergen diversity is even higher in the interspecific tetraploid hybrid, consistent with the presence of two genomes. Conclusion Isoforms of the major birch allergen Bet v 1 are encoded by multiple genes, and we propose to name them accordingly. The present characterization of the Bet v 1 genes provides a framework for the screening of specific Bet v 1 genes among other B. pendula cultivars or Betula species, and for future breeding for trees with a reduced allergenicity. Investigations towards sensitization and immunotherapy should anticipate that patients are exposed to a mixture of Bet v 1 isoforms of different IgE-reactivity, even if pollen originates from a single birch tree.

Background
Pathogenesis-related class 10 (PR-10) proteins constitute the largest group of aeroallergens and are among the four most common food allergens [1]. The main allergen is a PR-10 pollen protein from the European white birch (Betula pendula) termed Bet v 1 [2]. Birch pollen is a major cause of Type I allergies in the temperate climate zone of the northern hemisphere. Over 95% of the tree pollensensitized patients in Scandinavia display IgE binding to Bet v 1, while 60% react exclusively to this allergen [3]. Pollen of other Fagales species contains Bet v 1 homologues that share epitopes with Bet v 1 [4].
PR-10 proteins are present as a multigene family across a range of phylogenetically distant species, including Gymnosperms, Monocots, and Dicots [5][6][7]. As a consequence, several foods contain Bet v 1 homologues, including nuts, vegetables, and Rosaceae fruits [6,8,9]. Patients that are sensitized to Bet v 1 may experience mild allergic symptoms upon consumption of these foods due to IgE crossreactivity. Symptoms of this so called oral allergy syndrome (OAS) are mainly limited to the oral cavity. Crossreactivity has clearly been demonstrated by allergic responses to the celery protein Api g 1, which is initiated by sensitization to Bet v 1 [8].
The B. pendula genome contains multiple PR-10 genes with varying expression patterns. Among these, the Bet v 1 allergens are expressed in pollen. The first Bet v 1 isoform was identified by immunoscreening a pollen cDNA expression library with serum of birch pollen allergic patients [2]. Other Bet v 1 isoforms have been sequenced by various authors since then [10][11][12][13]. Pollen mixtures from multiple trees were found to contain multiple Bet v 1 isoforms [13]. Bet v 1 isoforms differ in the ability to bind IgE and in the T-cell proliferation score [14]. Two other types of PR-10 proteins were detected in birch cells that were grown in a liquid medium in the presence of microbial pathogens [15]. These proteins are expressed in roots and leaves under basal conditions or induced under various stress-related conditions [15,16]. The PR-10 genes from B. pendula form a homogeneous group, based on sequence similarities. Homogeneity is suggested to be maintained by concerted evolution [17]. Arrangements of PR-10 genes into clusters, such as found for Mal d 1 genes in apple (Malus domestica), may facilitate concerted evolution [18].
Given the prominent role of Bet v 1 allergens in the sensitization to hay fever and OAS, birch is a relevant target for development of allergy prevention strategies. Selection and breeding of hypoallergenic trees or the application of genetic modification to develop these may potentially reduce the allergenic load caused by birch. Knowledge on the diversity of PR-10 genes, their expression, and aller-genicity of the gene products is required to facilitate these strategies. In the present study, we amplified, cloned and sequenced PR-10 alleles from three B. pendula cultivars to establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree.

PCR recombination artifacts
When PCR amplification is performed on groups of closely related sequences, such as the PR-10 gene family, accurate sequences are essential to distinguish between members. When we initially determined the relationships among the recovered sequences, most clades in the Bayesian consensus tree had low posterior probabilities. Several sequences occupied intermediate positions between well-defined clusters. This suggested the possibility of recombination. Recombination could have occurred in vivo through a crossing-over or gene conversion between prior existing genes, or in vitro during the PCR through strand-switching or re-annealing of incompletely amplified fragments.
Evidence supports the view that recombinant sequences were PCR artifacts. Recombination signals were abundant in the sequences obtained after the 30-cycle PCR ( Figure  1a) and virtually absent when 22 cycles were employed ( Figure 1b). Several GenBank sequences showed clear evidence for recombination too (Figure 1c). Putative recombinants from our experiments lacked or nearly lacked unique mutations and could be separated into two or three stretches that were identical to other sequences obtained. The GenBank recombinants shared close to 100% sequence identity to combinations of other accessions.
The use of independent PCRs resolves which sequences are genuine, since the probability of isolating identical artifacts in independent PCRs is extremely low. Most sequences without recombination signal were confirmed in independent PCRs (Table 1), while those with a recombination signal were not. The only exception was the PR-10.03B02.01 allele from 'Tristis' that was found in three independent PCRs. This allele was an in vivo recombination of the first 300 bp, including the intron, from the PR-10.03D gene and 183 bp from the original PR-10.03B gene as found in 'Schneverdinger Goldbirke' and 'Long Trunk'. Putative recombination artifacts were quite abundant in the 26 and 30-cycle PCR (27-46% of the sequences), but rare in the 22 and 24-cycle PCR (2-11%) ( Table 1). We conservatively maintained the sequences that were confirmed in independent PCRs and those with at least three unique mutations for further analysis. A minimum of three unique mutations was chosen to ensure that potential base mis-incorporation artifacts were excluded.
Phylogenetic profiles for detection of recombination Figure 1 Phylogenetic profiles for detection of recombination. Phylogenetic profile of the sequences from B. pendula 'Long Trunk' obtained after a PCR of (a) 30 cycles (n = 72 sequences) and (b) 20 cycles (n = 53). (c) Phylogenetic profile of the Gen-Bank PR-10 sequences from B. pendula (n = 66). The x-axis represents the sequence position (5'-3' including only informative positions). The y-axis indicates the phylogenetic correlation. Low values are indicative for recombination [27]. Low values at the edges are artifacts of the employed method. Several of the PR-10 sequences from B. pendula that were available from GenBank/EMBL/DDBJ also showed clear evidence for recombination ( Figure 1c). However, it was not possible to do a similar check as mentioned above for the GenBank sequences, and we can only hypothesize on the presence of artifact sequences without such direct evidence. Given the regular occurrence of artifact sequences in our experiments, we maintained only those 40 Gen-Bank sequences that were not under suspicion of recombination artifacts for further analysis.

Phylogenetic analysis: newly isolated sequences
We sequenced 404 individual clones in both directions (Table 1). Fourteen different sequences were identified in the diploid cultivar 'Schneverdinger Goldbirke', 7 of which were unique for this cultivar. Fifteen different sequences (of which 10 were unique) were identified in the diploid cultivar 'Tristis'. Approximately twice as many different sequences, namely 28, were identified in the tetraploid cultivar 'Long Trunk'; of these, 16 sequences were of B. pendula origin (8 unique), and 12 sequences  were from another Betula species (all unique). In total, 32 different sequences were found.
The Open Reading Frame (ORF) of the sequences was highly conserved and the alignment was straightforward. All but one ORF contained 483 nucleotides, coding for a putative protein that is 160 amino acids long. One sequence from 'Schneverdinger Goldbirke' required the inclusion of an indel between base 388 and 389 of the consensus. This sequence was denoted as a pseudogene, since the indel introduced a stop codon at 7 codons downstream. It cannot be excluded that this pseudogene is expressed, since the stop codon was located near the 3' end. The intron position was identical in all sequences and located at codon 62, being inserted between the first and second nucleotide. Most alleles had 5' splicing sites of AG:GT, with the exception of one allele that had a GG:GT splicing site. The 3' splicing sites were AG:GC or AG:GA. This is in concordance with known motifs for plant introns. The introns were relatively AT-rich (55-65%).
We determined the relationships among the PR-10 sequences from B. pendula. For this, the 'Long Trunk' sequences that were designated to the other parental species were excluded. Excluding primer traces, 171 of the 452 aligned exon positions were variable, while 150 positions were phylogenetically informative. The consensus tree from the Bayesian analysis indicated several welldefined clusters ( Figure 2). We implemented a cut-off level of 98% identity and allowed maximally two alleles per cultivar per gene to estimate the number of genes. In this way, we putatively identified ten genes in 'Schneverdinger Goldbirke', eleven in 'Tristis', and thirteen in 'Long Trunk'. Thirteen different genes were distinguished when the information was combined. Each gene was identified as such in at least two birch cultivars and was characterized by a distinct intron, in most cases of a different size between 84 and 152 bp ( Figure 2). The similarity between different alleles of one gene ranged from 98.9 to 100% identity in the exons, which corresponds to 0-5 SNPs. Bet v 1.02A and Bet v 1.02B were not well distinguished in the coding sequences, but had distinguishable introns. The pseudogene PR-10.03B-p01 from 'Schneverdinger Goldbirke' was identical to the PR-10.03B allele from 'Long Trunk' except for its indel.
When all alleles from 'Long Trunk' were included, 182 variable positions were identified among the 452 aligned exon positions. 154 positions were phylogenetically informative. The topology of the consensus tree from the Bayesian analysis showed that seven specific 'Long Trunk' genes were clustered pair wise to the B. pendula genes and these are likely to represent orthologuous genes from the second Betula species (not shown). Given the high identity (up to 100%) to PR-10 sequences from B. ermanii, this species, or a close relative, is likely to represent the second parental species (unpublished data, Schenk et al.). Intron sequences of orthologuous genes mostly showed slight differences in length or base pair composition. Three genes were recovered only from the unknown parent species and five only from B. pendula, indicating that Betula species do not necessarily have the same (number of) genes.

Phylogenetic analysis: GenBank sequences versus newly obtained sequences
All but two GenBank sequences had an ORF of 483 base pairs that coded for 160 amino acids and was generally interrupted by a position-conserved intron. Two sequences required the inclusion of an indel, following base 354 of the consensus sequence. This resulted in a stop codon directly after the insertion. The GenBank dataset was combined with the newly obtained sequences. Primer traces were discarded, resulting in an aligned stretch of 425 bp from base 28 to 452 of the consensus. Several GenBank sequences are identical at this stretch, reducing the number of unique GenBank sequences in the analysis to thirty-three. 173 out of 425 aligned positions were variable, while 146 were phylogenetically informative. The information from the Bayesian consensus tree was added to Table 2 by indicating in which gene cluster the GenBank alleles landed. Similarly to the analysis of the newly isolated sequences we used a cut-off of 98% identity. In the resulting classification, 35 out of 40 alleles that were assigned to a particular gene showed more than 99% identity for the exons. The lowest similarity of an allele that still clustered with a particular gene in the phylogenetic analysis was 98.4% identity with the reference sequence (Table 2).
We classified the genes into five subfamilies (I-V) based on identities of the coding regions and the intron. The average identity between alleles within each subfamily was 95-100%. The GenBank sequences were in part derived from RNA extractions from specific tissues. We used this tissue information to predict the transcription of the PR-10 genes ( Table 2). For this, alleles of a single gene are assumed to have the same mode and location of transcription. Subfamily I and II consist of respectively four and three genes and include alleles that are homologous to the pollen-expressed Bet v 1 allergens [2,13]. For four genes within these subfamilies, we found alleles that were 100% identical to pollen mRNA-derived sequences previously deposited into GenBank. Alleles from two other genes were 99.8% identical (1 SNP difference) to pollen mRNA-derived sequences from GenBank, which we take to predict the location of transcription for these six genes with a very high level of confidence. For only one of the genes in subfamily II, there was no mRNA-derived homologue in the GenBank database, but the high homology (97. .2%) to the other genes in this subfamily suggests that this gene will be expressed in pollen as well.
Subfamily III consists of four genes, two of which have alleles that are 99.5% identical to homologues of the previously described ypr10a and ypr10b sequences, which are transcribed in roots and leaves [15]. Transcription of the other two genes in subfamily III is unknown, as is the transcription of the single gene in subfamily IV. Subfamily V consists of a single gene. One of the recovered alleles was 100% identical to the previously described ypr10c sequence that is also expressed in roots and leaves [15]. Given the fact that all sequences in subfamily III-V are less than 90% homologous to pollen-derived mRNAs and that there is no evidence of expression in pollen, we consider these as non-allergens.

Nomenclature
All recovered B. pendula alleles were added to    Amino acid sequences, amino acids that affect IgE-reactivity, and T-cell epitopes of the PR-10 proteins Figure 3 Amino acid sequences, amino acids that affect IgE-reactivity, and T-cell epitopes of the PR-10 proteins. Amino acid sequences of the PR-10 proteins from B. pendula 'Tristis' (Tr), 'Schneverdinger Goldbirke' (Sv), and the B. pendula alleles from 'Long Trunk' (Lt). Amino acids associated with high allergenicity are marked with grey boxes and those associated with low IgE-reactivity (located within B-cell epitopes) are marked with black boxes [12,14]. The locations of the two major T-cell activating regions are indicated above the consensus [22]. T-cell epitope   B  B B B  B  B  BB  B   10  20  30  40  50  60  70  80  90  100  110  120  130  140  150

PR-10 proteins and allergenicity
A high similarity between proteins increases the chance that they share epitopes, while on the other hand, a single amino acid change may influence allergenicity drastically. The high homogeneity among the PR-10 genes of B. pendula was reflected by a higher allelic variation at the nucleic acid level compared to the protein level. Hence, the 45 different genomic sequences encoded 32 different putative isoforms (Figure 3). This is consistent with the relatively large number of synonymous mutations compared to the number of non-synonymous mutations (K a / K s ratios) [21]. The number of synonymous (K s ) and nonsynonymous (K a ) substitutions per site were calculated from pair wise comparisons of all alleles from the three B. pendula varieties. The average value was 0.080 for K a and 0.247 for K s , resulting in an average K a /K s ratio of 0.33 (n = 45). Analysis of the occurrence of non-synonymous mutations per codon indicated two interesting regions. The region between codon Asn 42 and Ile 56 lacked non-synonymous mutations and is characterized by a phosphatebinding loop with the sequence motive GxGGxGx ( Figure  3). Relatively many amino-acid differences were present beyond codon 125, especially between isoforms of the genes PR-10.04/PR-10.05 and PR-10.01 to PR-10.03 (Figure 3).
Previous research has identified isoforms with varying IgE reactivity within mixtures of pollen. In fact, this is also true for pollen from a single tree. Ferreira et al. [14] [12,14]. These are marked in Figure 3. The tetraploid 'Long Trunk' contained several isoforms with unique amino acid substitutions due to its putative hybrid origin. This greatly enlarges the variation in putative IgEreactivity among isoforms of this cultivar.

PCR artifacts
When PCR amplification is performed on groups of closely related sequences, such as the PR-10 gene family, accurate sequences are essential to distinguish between members. We used Pfu polymerase, which has proofreading functionality and reduces the number of base substitution error rates. However, previous research has shown that Pfu polymerase may generate more and more complex recombination artifacts than Taq polymerase [23,24] through incomplete primer extension and re-annealing to a different template [25], or strand switching between different templates [26]. Reducing the number of cycles [24] was an efficient solution to lower the amount of artifacts.
We identified PCR recombination artifacts in several of our sequences using the computer program Phylpro [27]. The comparison of independent PCRs enabled sequence validation and exclusion of both recombination and base substitution artifacts. The high occurrence of recombination artifacts in our experiments (27-46% after 30 PCR cycles) is not uncommon. For example, Wang and Wang [28] report 32% recombination artifacts after 30 cycles of PCR amplification for 16S rRNA genes. The occurrence of recombinant sequences within a mixture of approximately 15 different sequences is expected to be high, because almost all recombinations are detected. Half of the recombinations would, for example, remain undetected if only two templates are present. The presence of 13 genes also increases the amount of template compared to a single copy gene.
Using the same analysis to detect recombination in Gen-Bank/EMBL/DDJB accessions, we observed a recombination signal in 22 out of 62 (35%) accessions. These are not necessarily all PCR artifacts, as some recombinations may have occurred in vivo during evolution of the genes. However, we found only one true recombinant in our dataset and no evidence of past recombination events in the comparison between sequences from two different species (within the hybrid). This indicates that the occurrence of in vivo recombination is probably rare.
Base substitution error rates for mixtures of non-proofreading Taq and proofreading Pfu are approximately 5.6 × 10 -6 under optimal conditions [29]. This error rate is ~2-4 fold higher when only Taq is used [29,30]. Without confirmation in independent PCRs these errors can not be excluded and it is very likely that Bet v 1 sequences with base substitution errors have been deposited into Gen-Bank/EMBL/DDJB. Therefore, not all published isoforms will be clinically relevant. In addition, the clinical relevance of the isoforms will be influenced by their expression levels. If multiple allergen isoforms exist, there is a risk for selecting a recombinant isoform with low IgEreactivity as a diagnostic tool, or even selecting isoforms that resulted from PCR artefacts. We therefore strongly suggest the use of primers that are highly specific for one gene [18] or, preferentially, the application of multiple independent PCRs to facilitate sequence validation in future sequencing work on allergens.

PR-10 and Bet v 1 genes
The PR-10 gene family of Betula pendula was shown to encompass at least thirteen genes. This is a conservative estimate since we used strict inclusion criteria. The distinction between genes is supported by the presence of a distinct intron. Each gene was identified as such in at least two birch cultivars. We attributed previously described GenBank sequences to these genes. Alleles from ten identified genes had previously been described [2,[10][11][12][13], while we identified three new genes. The genes are grouped into five subfamilies, based on sequence homologies in the ORF and intron. Differences in transcription coincide with the division between subfamilies. An organization of PR-10 genes into subfamilies was also reported for Malus domestica [18] and for Pinus monticola [5].
A striking feature of the PR-10 isoforms in B. pendula is their homogeneity, which may extend to other Fagales species, such as alder (Alnus glutinosa) and hazel (Corylus avellana). The intron has conserved 3' and 5' splicing sites and is always located at codon 62, as is e.g. reported for C. avellana and M. domestica [11]. High homogeneity may result from strong purifying selection or from concerted evolution. The presence of low K a /K s ratios among the isoforms suggests the occurrence of purifying selection. Evidence for concerted evolution is present in the overall gene tree of the PR-10 family [17]. Concerted evolution causes genes to evolve as a single unit with members exchanging genetic information through gene conversion and unequal crossing-over. Tandemly arranged genes may have high conversion rates [31], while this is a prerequisite for the occurrence of unequal crossing-over. Most PR-10 genes in apple map to two loci and are arranged in a duplicated cluster [18]. This organization may be a common feature for PR-10 genes. However, as pointed out by Nei and Rooney [32], the molecular mechanism of gene conversion is not well understood, and the model of birth-and-death evolution of genes may also explain the evolution of the PR-10 gene family. The presence of pseudogenes, although at a low frequency, is therefore of particular interest. An analysis of a species at an intermediate evolutionary distance, such as C. avellana, would be useful to clarify which mechanisms determine the evolution of PR-10 genes and to investigate a possible recent radiation of PR-10 genes.
The birch genome contains at least seven pollen-expressed genes that encode a mixture of Bet v 1 isoforms with varying IgE-reactivity. Swoboda et al. [13] found that pollen mixtures from multiple trees contain multiple Bet v 1 isoforms. We identified 14 different Bet v 1 isoforms in the three cultivars, nine of which are entirely new. The IgEreactivity has been tested for several isoforms using recombinant proteins [14]. The allergenicity of the new isoforms can be examined in the future by expressing the isoforms as recombinant proteins and use these in a SPT or T-cell activation tests. Ferreira et al. [14] divided the Bet v 1 isoforms into three groups according to their IgE-reactivity and confirmed the division between high, moderate, or low IgE-reactivity in a Skin Prick Test (SPT). One high and one low IgE-reactive isoform from their analysis were 100% identical to isoforms that we have obtained from a single tree, while two intermediate IgE-reactive isoforms differed only by one amino acid from the alleles of two other identified genes. This strongly suggests that isoforms of different IgE-reactivity are in fact alleles encoded by different genes. Thus, each examined cultivar has the genetic background to express a mixture of isoforms with a high, moderate, and low IgE-reactivity. We plan to confirm this at the protein level in the near future.

Nomenclature
The nomenclature of Bet v 1 raises several issues. The first isoforms were termed Bet v 1a to Bet v 1n by Swoboda et al. [13], but these have subsequently been renamed and incorporated into the official database of the allergen nomenclature committee [19]. This database currently lists 37 allergen isoforms that have been termed Bet v 1.0101 to 1.3101. However, as can be seen from the list of known isoforms in Table 2, not all published isoforms have been added to this list, even though several of these isoforms were obtained from pollen mRNA. On the other hand, isoforms which have been recovered only from mRNA from roots and leaves are included as allergens (Bet v 1.1101 to Bet v 1.1301). Also, several of the described isoforms are highly suspicious as we observed clear recombination signals. As a result, the list is a random series of alleles that belong to different genes and has no biological basis. Similar problems were described for the Mal d 1 and Mal d 3 allergens by Gao et al. [18,20]. To allow differentiation between alleles from different genes for the Bet v 1 alleles we have adopted their system.

Allergenicity of birch trees
The exact isoform composition of the three cultivars differed due to allelic variations. This may result in differ-ences in allergenicity between cultivars. However, an exact copy of the most allergenic allele, Bet v 1a, was present in all three cultivars. Quantity measurements on expression indicate that Bet v 1a is the dominant isoform in pollen [13]. Given that the diversity of Bet v 1 isoforms within a single tree is larger than the diversity between the examined B. pendula cultivars, a characterization of Bet v 1-type isoforms should be done in other Betula species as well.
Investigations towards sensitization and immunotherapy should anticipate that patients are exposed to a mixture of Bet v 1 isoforms of different IgE-reactivity, even if pollen originates from a single birch tree. Differences in allergenicity between birch trees may also result from variation in allergen content. Variation in allergen content has e.g. been shown for apple [33] and olive pollen [34].
Many Betula species and Betula hybrids have higher ploidy levels (tetraploid, hexaploid, and even octaploid) than B. pendula and are likely to contain increased numbers of allergen isoforms, as we found in the tetraploid cultivar 'Long Trunk'. For example, the tetraploid B. pubescens is dispersed throughout Europe, while other exotic birch species are increasingly introduced as cultivars, contributing to a larger allergen pool. However, interspecific Betula hybrids, which have a higher ploidy level, may also pose a potential source of hypoallergenic trees. Especially hybrids between less related species often display a reduced fertility, which may result in a reduced or aborted pollen production.
If breeding for hypoallergenic trees is implemented, approaches should take into account that the Bet v 1/PR-10 genes may be clustered and differences in allergenicity between clusters of genes may be used to guide breeding efforts. Clustering may be determined by mapping studies [18] and by screening and partial sequencing of a genomic library. In the genome sequence of Populus [35] we indeed can observe an organization of PR-10 genes into clusters. Other approaches to generate hypoallergenic trees may include the search for sterile or low pollen producing trees, or the application of RNA interference technology, which proved to be successful to silence the Mal d 1 allergens in apple [36] without phenotypic abnormalities. One issue that needs the be resolved is that although birch PR-10/Bet v 1 proteins have been suggested to act as plant steroid carriers [37], the exact conditions under which transcription is induced are still unknown. Given the abundance of Bet v 1 in birch pollen, silencing may affect pollen viability. However, for breeding of hypoallergenic cultivars that are propagated vegetatively this would be considered a welcome side-effect.

Conclusion
We have shown that the PR-10 gene family of Betula pendula encompasses at least thirteen genes that can be grouped into five distinct subfamilies. Differences in expression coincide with the division between subfamilies. Genes from two subfamilies were shown to be transcribed in pollen, based on a high (99.8-100%) homology with cDNA sequences available in GenBank/ EMBL/DDJB. The seven genes that belong to these subfamilies encode a mixture of Bet v 1 isoforms of varying IgE-reactivity. The present characterization of the PR-10 family in birch provides a framework for the screening of Bet v 1 genes among other Betula species or B. pendula cultivars and for potential breeding approaches for birch trees with a reduced allergenicity.

Plant material
The natural distribution range of B. pendula Roth (syn. B. verrucosa) covers almost the whole of Europe. Several B. pendula cultivars have been bred, including interspecific hybrids (also referred to as interspecies hybrids) between B. pendula and other Betula species. We collected young leaves from three B. pendula cultivars in the collection of PPO Boskoop (WUR, the Netherlands), namely 'Long Trunk', 'Schneverdinger Goldbirke', and 'Tristis'. Fresh leaf samples were sent to Plant Cytometry Services (Schijndel, The Netherlands) and screened by flow cytometry to estimate the ploidy level. Diploid (B. pendula) and tetraploid (B. pubescens) controls were included. The cultivars 'Schneverdinger Goldbirke' and 'Tristis' were diploid, while the cultivar 'Long Trunk' was tetraploid. The latter is likely to be an interspecific hybrid between B. pendula and a second, unknown, Betula species. The alleles recovered from 'Long Trunk' were either assigned to B. pendula based on sequence and intron similarity or were considered specific for this cultivar. The specific 'Long Trunk' alleles were analyzed separately. DNA was extracted using the DNeasy Plant Mini kit (Qiagen) according to the manufacturer's instructions. and Taq polymerase (Goldstar)(5U/μl), and 20-80 ng template DNA. H 2 O was added to obtain a total volume of 20 μl. PCR mixtures were subjected to the following conditions: initial heating step at 95°C for 15 minutes, denaturation at 94°C for 30 s, annealing at 50°C for 45 s, and extension at 72°C for 60s. A final extension step of 10 min at 72°C was added after 22-30 cycles. Given the observation of recombination among the recovered sequences, we subsequently varied the number of PCR cycles at intervals of 2 cycles. The minimum number of cycles was established by visual inspection of the amplification products on agarose gel at 22 for the BpI primer pair and at 24 for the BpII primer pair. Originally, 30 PCR cycles had been used. We repeated the experiment at 22-24 cycles to ensure that amplification was in its linear phase.

PCR, cloning, and sequencing
To obtain the A-tailing that facilitates the ligation procedure, five additional cycles were run on 1-4 μl of PCR product with Taq polymerase (Goldstar). PCR conditions were similar as described above. PCR products were purified with the MinElute PCR Purification Kit (Qiagen). Purified samples were ligated into the pGEM-T easy Vector (Promega) and established in Escherichia coli XL1 Blue competent cells (Stratagene) according to the manufacturer's instructions. White colonies were picked from agar plates and grown overnight at 37°C in freeze medium. PCR-based screening was performed with vector-specific M13 primers. PCR products were purified with Sephadex G-50 (Millipore). The DYEnamic™ ET Terminator Cycle Sequencing Kit (Amersham) was used for the sequence reaction. Sequence products were analyzed on a 96-capillary system (ABI 3730xl).
Genomic B. pendula sequences have been submitted to GenBank as DQ296566-DQ296598 and DQ325525-DQ325535, and the specific 'Long Trunk' sequences as DQ296599-DQ296610.