Oct 14, 2004 - viral vectors using the herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) strategy.1,2 In this suicide gene therapy approach, the TK ...
Gene Therapy (2005) 12, 169–176 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt
RESEARCH ARTICLE
Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase V Roy1, J Qiao1,2, P de Campos-Lima and M Caruso Le Centre de Recherche en Cance´rologie de l’Universite´ Laval, L’Hoˆtel-Dieu de Que´bec, Centre Hospitalier Universitaire de Que´bec, Que´bec, Canada
The treatment of solid tumors by retroviral delivery of the herpes simplex virus thymidine kinase (TK) followed by ganciclovir (GCV) treatment has so far shown only limited success in patients. One major drawback in this approach is the lack of efficient in vivo gene delivery to cancer cells. Although, the transduction of every single tumor cell is not a requirement since the bystander effect (BE) mediated by gap junctions allows the diffusion of the toxic GCV metabolites from TK-expressing cells toward untransduced cells. To render the TK/GCV approach more potent, and independent of the level of gap junctions, we have tested the efficiency of
a TK mutant (TK30) fused to VP22, a herpes simplex protein that seems to be capable of intercellular trafficking. We failed to detect an increase in the BE with cells expressing VP22 fused to TK30 versus cells containing TK30 alone, and this result forced us to reinvestigate the trafficking properties of VP22. Using very sensitive Western blot and fluorescence assays, we were not able to detect the spread of VP22 fused either to TK30 or GFP. These results indicate that VP22 cannot be used as a cargo to translocate TK30 or GFP. Gene Therapy (2005) 12, 169–176. doi:10.1038/sj.gt.3302394 Published online 14 October 2004
Keywords: VP22; thymidine kinase; ganciclovir; cancer; GFP
Introduction The lack of gene transfer efficacy in vivo has been highlighted in gene therapy applications, and it is a major drawback for this field. In particular, it is a strong limitation for the treatment of solid tumors with retroviral vectors using the herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) strategy.1,2 In this suicide gene therapy approach, the TK enzyme transforms the nucleosidic analog GCV into GCV-monophosphate, which is subsequently converted into the di- and triphosphate forms by cellular kinases.3 Then, the GCVtriphosphate is incorporated into elongating DNA, which can lead to the death of tumor cells. The transfer of phosphorylated GCV from TK-expressing cells toward neighboring TK negative cells,4–7 a phenomenon called the bystander effect (BE) or metabolic cooperation, is an important property of this strategy. The BE is responsible for the strong antitumoral effect reported in animal models, and it has been reported that complete tumor regressions could be achieved with only 10% of the cells expressing TK.8–10 Despite very encouraging preclinical trials, this strategy has so far not shown great success in
Correspondence: Professor M Caruso, Le Centre de Recherche en Cance´rologie de l’Universite´ Laval, L’Hoˆtel-Dieu de Que´bec, 9 rue Mc Mahon, Que´bec, Canada G1R 2J6 1 These authors contributed equally to this work 2 Current address: Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905, USA Received 8 March 2004; accepted 10 August 2004; published online 14 October 2004
patients. The efficiency of TK gene transfer with retroviral vectors reported in some clinical trials was extremely low,1,2 and it could in part explain the disappointing results observed in patients.1,11 We could assume that it should be possible to increase the efficiency of gene transfer by using the latest generation of packaging cell lines that produce high-titer recombinant retroviruses.12,13 Another means to render the TK/GCV strategy more potent would be to increase the BE in order to kill more untransduced cancer cells. This goal can be achieved by combining the GCV treatment with other therapeutic molecules that would act in a synergistic or additive manner.14–16 Several studies have also shown that some TK mutants that have lost their ability to phosphorylate pyrimidine nucleosides were able to enhance the BE compared to the wildtype enzyme.17–19 Thymidine is the major competitor of GCV for the phosphorylation by TK, and its inhibitory activity is most likely attenuated with TK30 or similar mutants.20–22 It is therefore accepted that such mutants can produce more phosphorylated GCV in cells than the wild-type enzyme.19–22 We have reported that TK30expressing cells were able to kill bystander cells much more efficiently than wild-type TK-expressing cells. Furthermore, the GCV sensitivity of TK30 positive tumor cells from different origins was increased versus cells containing wild-type TK.18 The BE is mainly mediated by gap junctions that enable cells to exchange the phosphorylated forms of GCV.23–26 The gap junctions are made up of connexin molecules that are often poorly expressed in tumor cells.27–29 Therefore, pharmacological strategies aiming at
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upregulating the level of connexins have been proposed in order to increase the BE.30–35 The delivery of connexin genes together with TK has also been evaluated, and it has been shown that the BE could be induced in gap junction deficient cancer cells.26,36 A very different type of BE that is independent of the level of connexins has been recently reported.37,38 In this case, it is not the GCV metabolites but the TK enzyme that diffuses from TK positive cells toward neighboring untransduced cells. This was made possible by fusing TK to VP22 (TK/VP22), another herpes simplex virus protein that has the ability to translocate between cells.39 This property of VP22 was first demonstrated with the protein alone or fused to GFP, and it was then extended to other fusion partners that have a therapeutic potential in gene therapy.37,38,40–43 Using this very attractive strategy, an increase in the BE was reported with TK/ VP22 versus TK alone; however, the cytotoxic effect enhancement was very limited,37,38 and it was only detected when a high percentage of cells expressed the fusion protein.37 Based on these results, we hypothesized that TK30 fused to VP22 should increase the BE independently of the level of connexins. Since TK30 is a more potent enzyme than TK, a higher BE mediated by VP22 was expected. In this study, the potential use of VP22 in suicide gene therapy was tested in vitro with cells containing TK30 fused to VP22. We found that the BE in cells expressing the fusion protein was not increased as compared to cells with TK30 alone, and this result forced us to reavaluate the translocation properties of VP22 fused to heterologous proteins.
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TK30 fused to VP22 does not increase the BE in vitro Recombinant proteins formed by TK30 fused to VP22 were tested for the capacity to generate a BE that would be independent of connexin expression. Retroviral vectors containing TK30 fused in N- or in C-terminal to VP22 were constructed, and recombinant retroviruses were used to generate stable HeLa and MCF-7 cell lines expressing TK30/VP22 or VP22/TK30. The BE experiments were performed in vitro with 10% TK30-expressing cells mixed with 90% parental tumor cells, and with GCV concentrations that are nontoxic to parental cells. The GCV sensitivity of cells containing TK30 fused to VP22 was very similar to the one found in TK30 expressing cells. A BE was observed with the highest GCV concentrations, but the inhibition of proliferation of cell mixtures expressing TK30 linked to VP22 was not markedly different from the ones exhibited by cell mixtures containing TK30 alone. The BE was slightly higher at 1 mM GCV concentration with HeLa cells expressing VP22/TK30, but this difference was not observed with MCF-7 cells (Figure 1a and b). In the standard protocol of BE experiments, the cell mixtures are seeded at total confluence and cultivated for one day in the presence of GCV to allow the phosphorylated GCV produced by TK positive cells to diffuse toward neighboring cells. The next day, the cells are plated at low confluence and their proliferation in presence of GCV is measured 3 days later. To rule out a possible masking effect of the conventional BE, the experiment was repeated without incubating the cells for the first
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Figure 1 Bystander effect mediated by stable cell lines expressing TK30 and VP22/TK30. HeLa (a), (c) and MCF-7 (b), (d) cell line proliferation in the presence of GCV with mixtures of parental cells and 10% TK30 cells (K) and 10% VP22/TK30 cells (J). The proliferations of 100% TK30 cells (’) and 100% VP22/TK30 cells (&) were also measured. The cells were cultured with GCV for 4 days (a) and (b) or for 3 days (c) and (d) (see Materials and methods). Data are expressed as a proliferation percentage relative to the proliferation of cells in the absence of GCV and are the average of five values7s.d. Gene Therapy
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24-h with the drug. In this situation, there is no transfer of phosphorylated GCV when cells are in contact, and it would then be easier to observe the BE mediated by VP22. The inhibition of proliferation was less pronounced in this new experimental setting, because the cells were incubated one day less with GCV. Most importantly, the inhibition of proliferation observed in the cell mixtures containing VP22/TK30 versus TK30 was also very similar, which indicated that VP22 was not able to mediate a BE in HeLa and MCF-7 cells (Figure 1c and d). Similar results were found with TK30/VP22-expressing cells (data not shown).
VP22 fused to GFP is not detected in bystander cells by direct fluorescence unless cells are fixed with methanol To reinvestigate the intercellular trafficking properties of VP22, plasmids expressing GFP fused in N- or C-terminal to VP22 were constructed and used in flow cytometry experiments. An equal number of MCF-7 cells stably expressing GFP/VP22 were mixed with parental cells, incubated for a 24-h period, and then analyzed by flow cytometry for GFP fluorescence. Two populations were detected in live analysis: one was fluorescent and one was negative for GFP. These two populations had a very similar number of cells which indicated an absence of transfer of fluorescence from GFP/VP22-expressing cells toward parental cells (Figure 2c). On the contrary, all the cells were fluorescent when they were fixed with methanol before analysis, although two distinct populations with different fluorescence intensities were detected (Figure 2f). The cell population with the lowest GFP fluorescence intensity was most likely the parental cells that became fluorescent after GFP/VP22 transfer. A similar result was found if the cells were mixed just before the fixation procedure, which indicated that the GFP/VP22 protein diffused toward parental cells during the methanol fixation process (Figure 2g). Similar results were found with a plasmid expressing VP22/GFP (data not shown). Since two groups claimed that they could detect the transport of GFP/VP22 in live cells by direct fluorescence,44,45 this possibility was carefully investigated using tagged recipient cells. MCF-7 cells expressing GFP/VP22 were incubated for 24 h with an equal number of MCF-7 cells tagged with DsRed2, and the cell mixture was analyzed by double color fluorescence. Less than 1% of double positive cells were detected with cell mixtures containing either GFP cells or GFP/VP22 cells (Figure 3). VP22 alone or fused to a heterologous protein localizes exclusively in the cytoplasm early after transfection, but after mitosis, VP22 is mainly found in the nucleus where it binds DNA (Elliott and O’Hare46 and data not shown). This difference in localization could eventually influence the intercellular trafficking of GFP/VP22 and, therefore, similar mixing experiments were performed in transient transfections with 293T cells using the same recipient MCF-7 tagged cells. Less than 2% of double positive cells were present, and there was no significant differences between GFP alone and GFP/ VP22 (Figure 3). Thus, by measuring the intrinsic fluorescence of GFP we were not able to detect the intercellular trafficking of GFP/VP22. Similar results
Figure 2 Intercellular translocation of GFP/VP22 with methanol. Fluorescence was measured by flow cytometry in live cells (a–c) or after methanol fixation (d–g). Parental MCF-7 cells (a) and (d), MCF-7 cells stably expressing GFP/VP22 (b) and (e), and mixtures of equal numbers of MCF-7 cells expressing GFP/VP22 and parental cells (c), (f) and (g) were analyzed. Cell mixtures were incubated for 24 h (e) and (f) or analyzed without prior incubation (g).
were found with VP22/GFP, and also with transiently transfected HT-1080 cells (data not shown).
VP22 fused to GFP or TK30 does not translocate between cells It is well established that the use of anti-GFP antibodies can be more sensitive to detect GFP expression than the direct measurement of its intrinsic fluorescence. Thus, we reassessed the intercellular trafficking of GFP/VP22 by using a very sensitive assay based on Western blot detection of GFP/VP22 using an anti-GFP antibody. 293T cells were transfected with GFP/VP22 and they were incubated the following day with an equivalent amount of MCF-7 DsRed2 cells for a 24-h period. The DsRed2 cells were then sorted and analyzed by Western blot with an anti-GFP antibody for the presence of GFP/VP22 (Figure 4a). GFP/VP22 as well as the control GFP could not be detected in 105 MCF-7 DsRed2 cells that had been Gene Therapy
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in contact with the transfected cells. This Western blot assay was extremely sensitive since we could detect GFP or GFP/VP22 in as little as 103 293T transfected cells (Figure 4b). A similar experiment was carried out with TK30/VP22, and the control TK30 as well as the fusion protein could not be detected in the sorted MCF-7 DsRed2 cells that had been in contact with the transfected cells (Figure 4c). The same absence of translocation was found with the VP22 C-terminal fusion proteins VP22/GFP and VP22/TK30. These Western blot results are in agreement with our inability to detect a BE mediated by VP22, and they also confirmed the absence of translocation we observed with GFP/VP22 by measuring the intrinsic fluorescence of GFP.
Discussion
Figure 3 Lack of translocation of GFP/VP22. MCF-7 cells stably expressing DsRed2 were mixed with MCF-7 cells stably expressing GFP or GFP/VP22 (left). 293T that had been transfected 18 h before with GFP or with GFP/VP22 plasmids were mixed with MCF-7 cells stably expressing DsRed2 (right). Cells were then incubated for 24 h and analyzed live by double color fluorescence.
One way to tackle the lack of efficient in vivo suicide gene delivery to cancer cells is to develop strategies that would increase the BE. In this study, we have tested the property of TK30 fused to VP22 to generate a BE that would be independent of the level of connexin expression. Although TK30/VP22 was able to sensitize cells to the same extent of TK30 alone, it did not increase the BE in HeLa and MCF-7 cells (Figure 1). We also failed to detect any intercellular trafficking of TK30/VP22 or
Figure 4 Absence of intercellular trafficking of VP22 fused to either GFP or TK30. (a) Experimental procedure used for the Western blot experiments. pGFP/VP22, pEGFP-N3, pNC-TK30/VP22 and pNC-TK30 plasmids were transfected in 293T cells. The next day, the transfected cells were mixed with MCF-7 DsRed2 cells at a 50% ratio. On day 3, the cell mixtures were analyzed by flow cytometry, and the cells positive for DsRed2 were sorted. Western blot experiments were performed on 105 sorted MCF-7 DsRed2 cells that were in contact with 293T (b) GFP/VP22 or GFP cells and (c) TK30/VP22 or TK30 cells. Control samples with DsRed2 cells and 105, 104 and 103 293T transfected cells were analyzed to assess the sensitivity of the assay. Gene Therapy
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GFP/VP22 using very sensitive assays unless cells were fixed with methanol (Figures 2–4). VP22 is a herpes simplex virus protein that belongs to a family of positively charged proteins that can traverse cellular membranes and localize in cell nuclei.39 Among these proteins, the HIV protein Tat and the Drosophila Antennapedia homeotic transcription factor have been the most extensively studied.47 In the case of VP22, it was reported that not only VP22 could enter into cells, but it could be exported from expressing cells.39 Owing to its intercellular trafficking properties, the use of VP22 has been proposed in gene therapy to translocate therapeutic products from genetically modified cells toward untransduced neighboring cells.40 Although the intercellular trafficking capacity of VP22 alone or fused to heterologous proteins has been widely documented, some studies have challenged the transduction property of VP22. For example, it has been reported that VP22 was very inefficient to transport the diphtheria toxin A subunit into cells.48 In another study, it was suggested that VP22 is exported from cells and binds to membranes of neighboring cells, but it was also shown that the methanol fixation method was responsible for the import and the nuclear localization of VP22.49 Similar results have also been recently reported for Tat and other positively charged proteins and peptides.50–53 Data presented here on VP22 corroborate these results, but they do not support the view that VP22/GFP can be exported from expressing cells as it has been suggested by Lundberg and Johansson.49 The flow cytometry experiments performed in our study clearly indicate that the methanol permeabilization allowed VP22/GFP to spread from expressing cells toward neighboring cells (Figure 2). In one study, cells transfected with VP22/Flp recombinase were able to induce b-galactosidase expression when they were mixed to Flp-excisable LacZ recipient cells.54 The experimental setting and the transfection methods used to introduce the VP22 expressing plasmids into cells could lead to artifacts. In the Flp study and others, the translocation or the biological effect mediated by VP22 were analyzed after transient transfection which could affect the organization of the cell membrane, leading to its permeabilization. A small amount of VP22/ Flp recombinase could be picked up by recipient cells, independently of VP22 trafficking properties, and could be sufficient to induce b-galactosidase expression. Moreover, the absence of b-galactosidase expression with Flp alone could be accountable to the Flp thermolability at 371C.55 An increase in protein stability mediated by VP22 could explain these results as well as studies with shortlived proteins such as p53 and p27Kip140,41,43 and, indeed, we have found that VP22 could dramatically increase the stability of d1EGFP, a GFP version with a very short halflife (data not shown). In previous studies, the translocation of VP22 alone or fused to heterologous proteins has been shown by indirect evidence or after fixation with methanol. In this study, the diffusion of VP22 fusion proteins was studied without using fixation reagents or detergents that are prone to generate artifacts with positively charged proteins or peptides. Overall, our results clearly demonstrate that VP22/GFP and VP22/TK30 were not able to translocate efficiently between cells and, therefore, the use of VP22 as a means to increase the spread of
therapeutic products in gene therapy is very limited. We believe that these results will put an end on the controversy surrounding the use of the intercellular trafficking property of VP22 in gene therapy. VP22 fused to heterologous proteins could still be used exogenously like Tat or other positively charged proteins, but it seems that the entry and the delivery of a functional protein inside a cell by VP22 or Tat is quite inefficient.48
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Materials and methods Plasmid constructions VP22 was amplified from DNA from HSV-1 infected cells using two different sets of primers. Four restriction sites were created in the first set of primers, BamHI (underlined) and BspHI (bold) in the 50 primer 50 -CGGGATCCT CATGACCTCTCGCCGCTCC-30 and PstI (underlined) and BspHI (bold) in the 30 primer 50 -AACTGCAGTCAT GAGCTCGAC GGGCCGTCTGG-30 . In the second set of primers, a BglII restriction site was included in the 50 primer 50 -GGAGATCTATGACCTCTCGCCG-30 , and a BamHI restriction site was created in the 30 primer 50 -CCGGATCCTCACTCGACGGG-30 . The first set of primers was used to amplify VP22 without its stop codon in order to fuse it in C terminal. Both PCR products were cloned in pCITE-2a(+) (Novagen, Madison, WI, USA): VP22-C was cloned in BamHI/PstI and N-VP22 was inserted in BglII. In both plasmids, the VP22 sequence was identical to the one already published. To generate retroviral vectors with TK30 fused to VP22, we first amplified TK3020 by PCR without its stop codon with the 50 primer 50 -CATGCCATGGCTTCG TACC-30 containing a NcoI restriction site and with the 30 primer 50 -CCGGATCCACAGTTAGCC-30 containing a BamHI restriction site. The TK30 1.1-kb PCR product was cloned in pCITE-2a(+) opened in NcoI/BamHI and sequenced. The first set of vectors was derived from the MFG backbone in which the fusion gene was under the control of the 50 long terminal repeat sequence.56 The BspHI VP22 fragment was inserted in 50 of TK30 by opening MFG-TK3018 in NcoI. For the construct with VP22 located in C-terminal, TK30 was first cloned in MFG in NcoI/ BamHI, and then the VP22 BglII/BamHI fragment was inserted in BamHI. Then, a 1.4-kb BamHI fragment, which contains the neomycin resistance gene linked to the internal ribosomal entry site from encephalomyocarditis virus, was cloned in both vectors in BamHI to create MFG-TK30/VP22iNeor and MFG-VP22/TK30iNeor. In the second set of vectors, the fusion genes TK30/ VP22 and VP22/TK30 were cloned in BamHI in the pNC vector to generate pNC-TK30/VP22 and pNC-VP22/ TK30. The pNC vector is derived from MFG and it contains as an internal promoter the cytomegalovirus immediate early promoter that drives the expression of the fusion genes. The GFP fusion plasmids were constructed as follows: pGFP/VP22 was constructed by cloning VP22 digested in BglII/BamHI in the polylinker of pEGFP-C1 (Clontech, Palo Alto, CA, USA) opened in BglII/BamHI. pVP22/ GFP was constructed by cloning VP22 digested in BamHI/PstI in the polylinker of pEGFP-N3 (Clontech) opened in BglII/PstI. Gene Therapy
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Cell culture and transfections MCF-7 cells (human breast tumor), HeLa cells (human cervix adenocarcinoma, ATCC CCL-2), 293T cells (human embryonic kidney), FLYA1312 and HT-1080 cells (human fibrosarcoma, ATCC CCL-121) were cultured with Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Wisent, St-Bruno, Canada) and antibiotics. HeLa and MCF-7 cells containing TK30/VP22 and VP22/TK30 were generated by infection with MFGTK30/VP22iNeor and MFG-VP22/TK30iNeor amphotropic retroviruses produced from FLYA13 stable producers as previously described.18 The G418 concentrations (70% active) (Invitrogen) used to select the transfected and infected cell lines were 500 mg/ml for FLYA13 cells, 700 mg/ml for HeLa cells and 600 mg/ml for MCF-7 cells. Cells containing VP22/GFP, GFP/VP22, GFP and DsRed2 were transfected by the calcium phosphate procedure with 20 mg of pVP22/GFP, pGFP/VP22, pEGFP-N3 and pDsRed2-N1 (Clontech). Cells were placed in G418 selection the following day for a 15-day period and they were subsequently enriched for GFP or DsRed2 expression with a cell sorter (EPICS Elite ESP, Beckman Coulter, Miami, FL, USA). Bystander assay Cells expressing TK30/VP22, VP22/TK30 or TK30 were mixed at 10% ratios with their respective parental cell lines. To promote cell contacts, the mixed cells were plated in 24-well plates at 1.5 105 cells/well. The following day, confluent cells were treated with GCV ranging from 100 to 0.01 mM. On day 2, cells were trypsinized and a 1:100 dilution of the cells was distributed into 96-well plates in five replicates. Cells were cultured subsequently in the presence of GCV for 3 days, until cells without GCV reached confluency. The proliferation of the cells was then measured by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) assay, which consists of adding 37.5 ml of MTT (1 mg/ml) to the 150 ml of medium in each well of a 96-well plates for 4 h at 371C. After removal of the medium, 150 ml of dimethyl sulfoxide (DMSO) was added and plates were gently shaken for 10 min to dissolve the formazan blue crystals. The absorbance was then measured at 595 nm with a microplate reader (Tecan, Research Triangle Park, NC, USA). The BE experiments were also performed without adding GCV for the first 24 h when the cells are seeded in the 24-well plates. Flow cytometry For the translocation experiments, 293T or HT-1080 cells were transfected by the calcium phosphate procedure with 20 mg DNA expressing GFP fused to VP22, and the cells were mixed the day after with the MCF-7 DsRed2 cell line at a 50% ratio and at total confluence. The next day, the fluorescence of the cells was analyzed with a flow cytometer (Beckman Coulter, Brea, CA, USA). Similar experiments were performed with stable MCF-7 cell lines expressing VP22 fused to GFP. The same number of cells was mixed and cultured at total confluence for 1 day before the flow cytometry analysis. The analysis was performed with live cells or with cells fixed with methanol. Gene Therapy
Western blot analysis Translocation experiments were also analyzed by Western blot as follows (Figure 4a): cells were transfected as described above with the GFP plasmids or the TK30 plasmids (pNC vectors), and the next day 3 106 MCF-7 DsRed2 cells and 3 106 transfected cells were seeded in 10-cm dishes. In all experiments, cells were detached using a PBS buffer containing 11 mM EDTA instead of trypsin. One day after cell mixing, the MCF-7 DsRed2 cells were sorted using a flow cytometer, and proteins were extracted in lysis buffer (0.3125 M Tris pH 6.8, 10% SDS, 50% glycerol, 0.005% bromophenol blue, 25% 2mercaptoethanol). Different amounts of cell extracts were loaded on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and separated by electrophoresis. The proteins were transferred onto nitrocellulose membranes (Amersham Biosciences) that were subsequently incubated with a rabbit polyclonal antibody directed against TK (1:10 000) or against GFP (1:100) (BD Biosciences Clontech, Palo Alto, CA, USA). The reactive bands were detected using the Western Lightning Chemiluminescence Reagent Plus kit (Perkin Elmer Life Sciences, Boston, MA, USA).
Acknowledgements This study was supported by grants from the Canadian Institute of Health Research (IC074582) and Manuel Caruso is a Junior II Research Scholar of the Fonds de la Recherche en Sante´ du Que´bec. Vincent Roy holds a graduate student award from the MCETC/CIHR strategic training program. We are grateful to Margaret Black for providing us with TK30 and the rabbit polyclonal antibody directed against TK. We thank Nancy Roberge for the help in flow cytometry and the cell sorting.
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