The Nonapoptotic Pathway Mediating Thymidine Kinase/Ganciclovir ...

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doi:10.1006/mthe.2002.0521, available online at http://www.idealibrary.com on IDEAL

The Nonapoptotic Pathway Mediating Thymidine Kinase/Ganciclovir Toxicity Is Reduced by Signal from Adenovirus Type 5 Early Region 4 Maha Katabi,1,* Shala Yuan,2,* Helen Chan,2 Jacques Galipeau,2 and Gerald Batist1,† 1 McGill Center for Translational Research in Cancer, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, H3T 1E2, Canada 2 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, H3T 1E2 Canada

*These authors contributed equally to this work. †

To whom correspondence and reprint requests should be addressed. Fax: (514) 340-7916. E-mail: [email protected].

Suicide gene therapy using thymidine kinase/ganciclovir (Tk/GCV) yields highly variable results, in vitro and in vivo. To determine the reasons for such variations, we examined cellular mechanisms mediating its cytotoxicity in view of their interaction with adenoviral vectors (Ad) used for gene delivery. Here we report that the presence of adenovirus early region 4 (AdE4)-encoded viral proteins significantly decreases toxicity of Tk/GCV. The E4 region-encoded proteins exerted this effect when found on the adenoviral delivery vector and when provided in trans in Tk retrovirally transduced cells. The apoptotic response was assessed in GCV-treated cells. The decrease in toxicity caused by AdE4 proteins was not correlated with apoptotic response, as measured by internucleosomal DNA degradation and TUNEL assays. Our results indicate that apoptosis is not the only mechanism of Tk/GCV-induced cell death and that other mechanisms equally important in determining the success of such a gene therapy strategy should be considered when optimizing treatment conditions. Key Words: adenovirus early region 4, suicide gene therapy, thymidine kinase, ganciclovir, adenoviral vectors, apoptosis

INTRODUCTION The combination of thymidine kinase and ganciclovir (Tk/GCV) has been studied in several tumor models using a variety of promoters and delivery systems. Although in highly selected circumstances it is effective at cell killing, a number of reports have directly or indirectly identified a spectrum of cellular sensitivity to activated GCV. In one case, in a transgenic mouse model of breast tumors, resistance was attributed to lack of Tk expression in residual tumors that were originally retrovirally transduced [1]. The importance of Tk expression levels on sensitization to GCV was also confirmed by other groups [2]. However, in other reports, the dependence of the resistance phenotype on Tk levels was ruled out by establishing equivalent levels in cells under study that were stably transfected with Tk [3]. The mechanism by which the Tk/GCV combination kills cancer cells has not been fully elucidated. Several reports have recently demonstrated the activation of apoptotic pathways. Apoptosis involves an initiation and an execution phase. The p53-dependent apoptosis induction through

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ligand-independent death receptor aggregation provides a partial explanation for the levels of cell death observed in a retrovirally transduced human neuroblastoma cell line [4]. Apoptosis independent of p53 was also reported in several human mammary carcinoma cell lines transduced with adenoviruses for transfer of the Tk gene, implying the importance of several induction pathways, depending on the cell types evaluated. The execution phase of apoptosis is mainly mediated through the activation of the effector caspase-3. Several apoptosis triggers converge on the mitochondria, which have an important role in stimulating the execution phase of apoptosis, as well as in its initiation in response to triggers such as cytotoxic agents. Recent results demonstrate the importance of mitochondrially amplified death cascades in the mediation of Tk/GCV toxicity [5]. Three generations of adenoviral vectors (Ad) for gene transfer have been described. The first- and second-generation vectors harbor deletions in the E1/E3 and E1/E4 regions, respectively. The third generation has most adenovirally encoded proteins eliminated and is also called

MOLECULAR THERAPY Vol. 5, No. 2, February 2002 Copyright © The American Society of Gene Therapy 1525-0016/02 $35.00


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FIG. 1. Vector dependence of the Tk/GCV resistance phenotype. (A) Percentage survival in different cell lines exposed to GCV at 10 g/ml. T47D and ZR75 were infected with AdRSVTk at an MOI of 10. Control condition is AdCMVLacZ for adenoviral infections at the same MOI. ZR75-AP were transduced with AP3 (Tk/GFP bicistronic retrovirus). Control condition is AP-2 infections (GFP-only retrovirus) for retroviral transduction. ZR75-AP-Ad are the same as ZR75-AP, but they were infected with AdCMVLacZ at an MOI of 50 preceding GCV exposure. (B) Tk/GCV resistance. Cell-killing curves in several cell lines showing toxicity over a range of GCV doses in Tk-infected cells, under various conditions. Results are the average of three independent experiments with each condition carried out in quadruplicate. Error bars represent SD. Asterisks represent a statistically significant result (P < 0.05) using t-tests to compare test and control conditions.

“gutless.” A series of limitations with the first-generation adenoviral vectors has led to the synthesis of newer models, including but not limited to finite gene persistence, potential hepatotoxicity, and contamination with replication-competent adenovirus. In addition, nonnegligible levels of adenoviral proteins are expressed from these replication-deficient viruses [6]. Here, we investigated the effect of E4 region-encoded proteins on the efficacy of Tk/GCV-mediated toxicity. We found that the presence of E4-encoded proteins reduces Tk/GCV toxicity. However, this resistance to Tk/GCV was not due to alteration in the apoptotic response. Rather, the induction of resistance to killing by AdE4 open reading frames (ORFs) is mediated through a nonapoptotic pathway, demonstrating the limited function of apoptosis in determining sensitivity to Tk/GCV.

RESULTS Cytotoxicity studies with the Tk/GCV suicide gene system have demonstrated the existence of a spectrum of sensitivity to the activated drug regardless of promoter activation status in the expression cassette, in our laboratory and in those of others [3]. We have examined the phenomenon of resistance to viral Tk (vTk)/GCV killing, observed most strikingly in ZR75 cells, by looking at several parameters that are likely to have a role in this suicide gene strategy. Our goal was to determine whether this resistance was dependent on interference of the viral delivery vehicle with the cell death response. Spectrum of Sensitivity Is Vector-Dependent ZR75 and T47D, two human breast cancer cell lines, were selected as a system to study Tk/GCV sensitivity and

MOLECULAR THERAPY Vol. 5, No. 2, February 2002 Copyright © The American Society of Gene Therapy

identify parameters that are important in determining sensitivity. This choice was based on the observation reported in Fig. 1A. We infected cells with AdRSVTk at a multiplicity of infection (MOI) of 10 to express the Tk gene under the control of a strong constitutive promoter of the Rous sarcoma virus (RSV). When we treated cells with GCV at 10 g/ml, percentage survival was decreased by half in T47D cells (P = 0.005), whereas there was only a 20% decrease in survival in ZR75 cells, relative to control cells infected with AdCMVLacZ. To confirm our observation concerning the resistance of ZR75 cells to Tk/GCV combination, we tested it in cells retrovirally transduced with the Tk gene. The recombinant retrovirus AP-3 was used to stably transduce ZR75 cells with the Tk gene, and AP-2 was used to generate a control population expressing the green fluorescent protein (GFP) alone. Cytotoxicity assays demonstrate a significant decrease of 40% in percentage survival (P = 0.035) at the same dose of GCV. One reason for vector dependence of Tk/GCV sensitivity in ZR75 cells could be that adenoviral proteins that are being expressed in these cells along with the transgene are interfering with ZR75’s sensitivity to GCV. To test this hypothesis, we infected each group of ZR75 cells, ZR75/AP-2 and ZR75/AP3, with AdCMVLacZ at an MOI of 50 (ZR75-AP-Ad), as a means of introducing back adenoviral proteins into the stably transfected cells. The infected cell population was then exposed to increasing concentrations of GCV. At a dose of 10 g/ml, decrease in survival was only 20% in the AP-3-transduced cells. Sensitization of these cells to GCV was now reversed. We carried out dose–response experiments on the cells just described. The concentration of ganciclovir that results in 50% cell killing (IC50) values for these cell lines reflected the same changes in sensitivities for ZR75,

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depending on the context of A Tk gene delivery. After a 5day exposure to GCV, cell survival was determined with a methyl thiazolyl tetrazolium (MTT) assay. The results shown in Fig. 1B demonstrate a 10- to 100-fold difference in GCV dose required to decrease survival by 50% between the sensitive and resistant populations. T47D cells infected with AdRSVTk at an MOI of 10 had an IC50 of 10–20 mg/ml, whereas ZR75 cells infected with AdRSVTk under similar conditions had an IC50 of ~ 500 g/ml. ZR75 cells transduced with a retrovirus-expressing Tk gene (ZR75-AP3) had an IC50 of 10–20 g/ml, whereas ZR75-AP3 cells infected with AdCMVLacZ at an MOI of 50 before GCV exposure were resistant to GCV toxicity. GCV Uptake To measure differences in prodrug accumulation, if any, when ZR75/AP3 cells are infected with various adenoviruses, we carried out GCV uptake studies (data not shown). Although GCV is known to be a highly lipophilic drug, an active uptake mechanism was shown to play a role in increasing the intracellular concentration of this nucleoside [7]. ZR75/AP3 cells infected with AdE1E3, AdE1E4, AdCMVLacZ, or AdRSVTk were exposed to 3H-GCV for increasing time periods, and GCV content was measured and normalized for cell number (reflected by protein amounts). There was no significant difference in 3H-GCV content per mg of protein between the different infections and conditions, until up to 24 hours of exposure. Therefore, GCV uptake was not a limiting factor in the phenotype observed. Thymidine Kinase Expression Levels The level of suicide gene expressed in target cells can have an influence on the level of sensitization observed. We made comparisons at the RNA level for viral Tk mRNA using RNase protection assays (RPAs; Fig. 2A). There was no statistically significant difference in viral Tk mRNA between ZR75/AP3 cells alone and ZR75/AP3 cells infected with AdE1E3, AdCMVLacZ, or AdRSVTk at an MOI of 10. Tk expression was normalized to -actin. The experiment was repeated at least three times. Comparisons of Tk expression at the protein level in Fig. 2B demonstrate equivalent amounts of Tk protein expressed under the different conditions as well. There was no evidence of truncated forms of Tk on the western blot.

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FIG. 2. Levels of thymidine kinase expression. (A) RNase protection assays (RPAs) show Tk RNA levels in different cell lines. Lane 1, ZR75; lane 2, ZR75/AP3; lane 3, ZR75/AP3-Ad1E3; lane 4, ZR75-AdCMVLacZ; lane 5, ZR75/AP3-AdRSVTk. Arrows to the left indicate the expected size of Tk and actin fragments. (B) Western blot analysis shows Tk protein levels under different conditions. Lane 1, ZR75; lane 2, ZR75-AdTk; lane 3, ZR75/AP3; lane 4, ZR75/AP3-AdTk; lane 5, ZR75/AP3-AdLacZ; lane 6, ZR75/AP3-AdE1E3; lane 7, ZR75/AP3-AdE1E4. Top panel, anti-Tk; bottom panel, anti-GAPDH (1:5000).

Interference of E4 Region-Encoded Adenoviral Proteins with Cytotoxic Response Adenoviral vectors used in the experiments just described are from the first-generation vectors (that is, harboring deletions in the E1 and E3 regions). Secondgeneration adenovectors are E1/E4 deleted. To test our hypothesis of adenoviral protein interference, empty (no transgene) first- and second-generation adenovectors, we infected ZR75/AP3 with AdE1E3 and AdE1E4, respectively. ZR75/AP3-AdE1E3 cells treated with GCV showed a statistically significant decrease in sensitivity relative to AR75/AP3 cells, with IC50 increasing from 3.31 g/ml to nearly 120 g/ml (Fig. 3A). In contrast to the decrease in sensitivity observed in ZR75/AP3AdE1E3, there was no change in IC50 between GCVtreated ZR75/AP3 cells and ZR75-AdE1E4, indicating that E4-encoded proteins may be responsible for the altered cytotoxic response. To verify the importance of the apoptotic response in the cytotoxic effect of GCV to ZR75/AP3 cells, we used antimycin, a known blocker of the mitochondrial respiratory chain that causes a concentration-dependent inhibition of apoptosis as assessed by phosphatidylserine exposure and Hoechst staining [8]. We observed a dose-dependent decrease in sensitivity of ZR75/AP3 cells to GCV (Fig. 3B). There is a 100-fold shift in IC50 when cells are exposed to 10 M of antimycin for 5 minutes before plating with GCV. This demonstrates that antimycin blocks the Tk/GCV effect in a manner similar to AdE1E3. Apoptosis was measured in antimycin-treated cells by looking at internucleosomal DNA degradation (Fig. 4C). There was a dose-dependent inhibition of DNA degradation with antimycin. Adenoviral proteins encoded by the E4 region could therefore be altering the apoptotic response to decrease response to Tk/GCV.



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FIG. 3. Effect of E4 on cell death. (A) E4-region-encoded adenoviral proteins interfere with cytotoxic response. Cell-killing curves in several cell lines show toxicity over a range of GCV doses in Tk-infected cells, under various conditions. (B) Importance of the apoptotic response in the cytotoxic effect of GCV to ZR75/AP3 cells. Antimycin causes a dose-dependent decrease in sensitivity of ZR75/AP3 cells to GCV. For (A) and (B), results are the average of three independent experiments with each condition done in quadruplicate. Error bars represent SD. (C) Internucleosomal DNA degradation in ZR75/AP3 cells under different treatments. Lane 1, marker DNA-HindIII; lane 2, ZR75/AP3; lane 3, ZR75/AP3-GCV; lane 4, ZR75/AP3-GCV-antimycin 0.1 M; lane 5, ZR75/AP3GCV-antimycin 1 M; lane 6, ZR75/AP3-GCV-antimycin 10 M; lane 7, ZR75/AP3-GCV-antimycin 30 M.

differences in protein levels (Fig. 4A, compare lane 4 with lanes 6 and 7), indicating that this antiapoptotic pathway is not affected by E4-encoded ORFs in our model system. We evaluated the apoptotic response in the model system using internucleosomal DNA degradation and TUNEL assays (Figs. 4B and 4C). Both assays indicate no changes or inhibition of the apoptotic pathway to explain the variation in sensitivity. Apoptosis is still taking place through nuclear mechanisms, yet there is a resistance phenotype observed. There was no significant difference in apoptosis levels in the sensitive and resistant cells. Taken together, our results indicate that AdE4 may be interfering with another apoptotic pathway that was not detected by any of the measurements we used, or alternatively that nonapoptotic mechanisms play an important role in determining Tk/GCV toxicity response and they are also influenced by the expression of adenoviral proteins from AdE4.

DISCUSSION

Adenoviral Proteins Interference Mechanism The antiapoptotic protein Bcl-2 and its family members have been shown to reside and participate in mitochondrial orchestration of apoptosis [9]. Mitochondrially amplified death signals were also shown to be important determinants of Tk/GCV apoptosis [5]. In addition, a recent report demonstrates increased Bcl-2 expression in AdE1–E4–-infected endothelial cells, thus resulting in prolonged survival under serum deprivation conditions [10]. However, our evaluation of Bcl-2 in ZR75/AP3 cells coinfected with AdE1E3 or AdE1E4 showed no significant


Our results on the use of adenoviral or retroviral vectors for transgene expression have shown that viral gene delivery systems can interfere with treatment outcome. GCV sensitivity was measured through a MTT cell viability assay. To eliminate the possibility of a shortcoming of the MTT assay in ZR75 cells, results were confirmed using the sulforhodamine B staining cell viability assay (data not shown). Identical survival curves were obtained. The possibility of observing an effect of infectivity on proliferation rate was eliminated, in that the control condition in which ZR75 cells were infected with different adenoviral constructs did not show the same survival profile (Fig. 3A). Adenoviral vectors that were used in our study are from the so-called first generation of recombinant adenoviruses. A common observation with these E1/E3-deleted, replication-defective vectors is leakiness of viral protein expression in host cells. This was reported in several in vivo studies with these vectors. Short-lived transgene expression and strong inflammatory responses in injected organs support the idea of the immune system recognizing and reacting to certain viral proteins or transgene-derived ones expressed in host cells. While immune responses to viral proteins may not be problematic in the context of in vitro

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FIG. 4. Indicators of apoptosis. (A) Western blot analysis shows Bcl-2 protein levels under different conditions: lane 1, ZR75; lane 2, ZR75-AdTk; lane 3, ZR75/AP3; lane 4, ZR75/AP3-AdTk; lane 5, ZR75/AP3-AdLacZ; lane 6, ZR75/AP3-AdE1E3; lane 7, ZR75/AP3-AdE1E4. (B) Internucleosomal DNA degradation in ZR75/AP3 cells under different treatments. Lane 1, ZR75/AP3; lane 2, ZR75/AP3-cisplatin 6 M; lane 3, ZR75/AP3-GCV; lane 4, ZR75/AP3GCV-AdRSVTk; lane 5, ZR75/AP3/GCV-AdCMVLacZ; lane 6, ZR75/AP3/GCVAdE1E3; lane 7, ZR75/AP3/GCV-AdE1E4. (C) Apoptosis induction by GCV (10 g/ml) on ZR75 cells transduced with AP3. Control condition is untreated with GCV. The number of apoptotic cells is represented as a percentage and is the mean of three separate experiments.

studies we were doing, it is conceivable that interactions between cellular and viral proteins are affecting the final sensitivity of a cell population. Interactions of these proteins with cellular factors can affect sensitivity to activated drug or interfere with cell death pathways. The adenoviral vectors we used were deleted between 339 and 3533 bp, corresponding roughly to map units 1–9.5, and between 27,865 and 30,995 bp, or map units 76–85. This region encompasses the E1A and E1B regions, as well as a nearly complete E3 deletion. The E1A region encodes several proteins that were demonstrated to have a role in inducing cellular DNA synthesis and promoting apoptosis by overactivating p53 [11–13]. This apoptosisinducing action is inhibited by E1B genes, which interact with p53 to prevent premature cell death. Therefore both E1A and E1B are needed for viral replication. E1B genes also interact with proteins other than p53, such as CED5, to inhibit caspase activation and the ensuing apoptosis [11,14]. E3 genes are implicated in control of host cell activation of inflammatory responses that lead to cell death and that help the virus evade immune surveillance in vivo. It was shown to interact with cellular proteins to inhibit NF-B activity, thereby inducing apoptosis, and prevent tumor necrosis factor- (TNF)-induced cytolysis [15]. These actions may seem contradictory, but not all functions of E3 proteins have been characterized yet to give a global idea on the outcome of such interactions with host defense mechanisms. The E4 region of adenoviral vectors has also been shown to produce proteins that interact with cellular proteins to inhibit apoptosis. Seven proteins are made from E4 genes, but E4orf6 effects are the most

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documented. E4orf6 or E4 34 kd was first thought to inhibit apoptosis through interactions with E1B 55 kd, but it has also recently been shown to interact independently with p73 [16]. p73 is a p53-related protein that has similar actions in relation to cell death. E4 34 kd inhibits p73 transcriptional activation and cell killing. Moreover, E4 34 kd has been shown to interact with several other cellular proteins whose normal function has not yet been determined [17]. More studies are required to identify the ORF involved in promoting death and survival and to characterize the mechanism of these effects. This will allow the design of better and safer gene delivery vectors. In view of these results and others, the E1/E4-deleted Ads are more adequate as “neutral” gene delivery vehicles. The importance of apoptosis in Tk/GCV-mediated toxicity has been emphasized in several reports. Recent efforts have focused on dissecting the mechanism involved in Tk/GCV apoptosis. In a human neuroblastoma cell model,



the role of death receptors signaling caspases [4] was thought to be crucial. However, according to the same report, > 50% of cells were still dying by apoptosis as measured by DNA fragmentation in the presence of the caspase inhibitor zVADfmk. In a second report by the same group, the central role played by mitochondria in this apoptosis is highlighted. Inhibition of cytochrome c release through Bcl-2 overexpression totally reversed apoptosis. However, our results point to the presence of other death pathways that are nonapoptotic and that can play an important role in determining cellular sensitivity to Tk/GCV treatment. Alternatively, another apoptotic pathway may be functioning that was undetected by the traditional measures of apoptosis we used. In either case, it is important to understand the mechanism of action of Tk/GCV killing and to define it in each tumor model. This will allow a reassessment of the value of Tk/GCV as a suicide system in gene therapy strategies, and perhaps a shift in focus to better alternatives.

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Cell culture and reagents. All media and sera were bought from Mediatech (Herndon, VA). The human mammary carcinoma cell lines, T47D (T-47D) and ZR75 (ZR-75-1), from the American Type Culture Collection (ATCC, Rockville, MD) were maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS (RPMI 10%). Ad5E1-transformed 293 human embryonic kidney cells (Microbix Biosystems Inc., Toronto, ON, Canada), were used for recombinant adenovirus production and maintained in Dulbecco’s modified Eagle’s medium (DMEM) 10%. 293 GPG cells [18] were grown in DMEM 10% supplemented with 2 g/ml puromycin (Sigma), 0.3 g/ml G418 (Canadian Life Technologies, Burlington, ON, Canada), and 1 g/ml tetracycline (Sigma) and were used for production of vesicular stomatitis virus G (VSV-G)-pseudotyped recombinant retroviruses. This packaging cell line was a gift from Richard Mulligan (Howard Hughes Medical Institute, Boston, MA). All culture media were supplemented with 50 units/ml of penicillin and streptomycin. Cells were maintained at 37C in a humidified atmosphere with 5% CO2. Antimycin A was bought from Sigma. Recombinant plasmids and viruses. All first-generation adenoviral vectors used were deleted between 339 and 3533 bp, corresponding roughly to map units 1–9.5, and between 27,865 and 30,995 bp, or map units 76–85. This region encompasses the E1A and E1B regions, as well as a nearly complete E3 deletion. AdCMVLacZ, where the cytomegalovirus (CMV) promoter is driving the expression of the -galactosidase gene, was a gift from Bernard Massie (Biotechnology Research Institute, Montreal, PQ, Canada); AdE1E3 was a gift from Jack Gauldie (McMaster University, Hamilton, ON, Canada); AdRSVTk was provided by Savio Woo (Baylor University, Houston, TX). The second-generation vector AdE1E4 was a gift from Philip Branton (McGill University, Montreal, PQ, Canada). It is deleted in the E1 and E4 regions as described [19] based on the H5d11014 clone. AP-2 and AP-3 are retroviral vectors that respectively encode for IRES/EGFP and vTk/IRES/EGFP [20]. Cell viability assays, viral infections, and prodrug treatments. Cell viability was determined, after the various treatments described below, using a colorimetric assay that measures the ability of viable cells to reduce MTT (Sigma), a soluble yellow tetrazolium salt, to an insoluble purple formazan precipitate. The MTT assay was carried out as described [21], and readings were done in a Bio-Rad plate reader, model 450, at 570 nm. The sulforhodamine B assay [22] was also carried out to confirm findings. GCV was obtained from Hoffman–La Roche (Cytovene). Cells were seeded in T 25cm2 flasks on day 1. On day 2, they were infected at an MOI of 10 or 50 with appropriate viruses. On day 3, they were trypsinized, counted, and


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plated at 5 103 cells/well in 96-well plates. When needed, antimycin A treatment was done for 5 minutes. GCV was added at concentrations ranging from 0 to 5000 g/ml. On day 5, the culture medium was changed and a fresh dose of GCV was added. On day 7, the MTT assay was done and cell survival percentages derived as described. Each condition was carried out in quadruplicate, and each cell line was tested at least three times. Cells stably expressing Tk were seeded in 96-well plates at 5 103 cells/well and treated with GCV as described [23]. Ribonuclease protection assay (RPA). RNA was extracted from cells using RNAzol (TEL-TESTinc., Friendswood, TX), as described by the manufacturer. Contaminating DNA was digested with 5–10 units of DNase I (Pharmacia Biotech, Baie d’Urfe, PQ, Canada), in appropriate buffer solution. The viral Tk (0.1 fmol) and human -actin (2 fmol) probes were simultaneously hybridized to 20–30 g of total RNA using the RPA II kit (Ambion, Austin, TX) according to the manufacturer’s recommendations. Probes were prepared by in vitro transcription using the MAXIscript kit (Ambion), and [-32P]UTP, 6000 Ci/mmol, 40 mCi/ml. To generate the vTk probe template, the pMC1Tk plasmid (Mario Cappechi, NCI, Bethesda, MD) was cut with EcoRV. The resulting 104-bp fragment was aligned with the human Tk sequence and found to have no homology at 80% and 90%. This alignment was done to demonstrate that the probe used to hybridize RNA in the RPA was recognizing the transduced viral Tk and not the endogenous one present in human cells. The human -actin template pTRI-actin human was purchased from Ambion. Samples were electrophoresed in 5% acrylamide/8 M urea gels. Immunoblotting. A 10-g aliquot of protein from each cell sample was electrophoresed on a 10% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were probed with anti-HSV-Tk rabbit antiserum (1:1000), anti-Bcl-2 monoclonal mouse IgG (1:200; Calbiochem) or anti-GAPDH. Antigen–antibody complexes were detected using ECL western blot detection reagents (Amersham) according to the manufacturer’s instructions. GCV uptake studies. We seeded cells in 24-well plates at 8 104 cells/well. Cells were washed 48 hours later with PBS, and the medium was replaced with [8-3H]-GCV (Moravek Biochemicals Inc., Brea, CA), at a concentration of 0.5 g/ml. Cells were harvested by trypsinization at 60 minutes and 24 hours. GCV content was determined by counting samples in a Wallace scintillation counter. Replicate wells were seeded under the same conditions and harvested at the same time points to determine protein concentration. Radioactivity measurements were normalized to protein contents. The experiment was repeated three times. Apoptosis assays. For DNA laddering, after applying various treatments as described above, cellular DNA fragments were prepared using DNAzol (Canadian Life Technologies), separated in 1.7% agarose gel by electrophoresis, and stained with ethidium bromide. For the TUNEL assay, cells were plated in 100-mm dishes and incubated for 24 hours. They were infected with adenovirus at MOI of 50 and treated with 10 g/ml of GCV as described above. The samples were prepared by using a TUNEL reactionbased in situ cell death detection kit, according to the manufacturer’s protocol (Roche Diagnostics, Indianapolis, IN). Specific detection and quantification of apoptosis was based on labeling of DNA strand breaks of fixed cells with fluorescein dUTP, and analysis was performed by flow cytometry. The experiment was repeated three times. Statistical analysis. One-factor analysis of variance (ANOVA), t-tests with Bonferroni’s correction for multiple comparisons, P values, and standard deviation were calculated for the appropriate experiments using Microsoft Excel. RECEIVED FOR PUBLICATION MARCH 26; ACCEPTED NOVEMBER 28, 2001.

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