Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer Pei-Xiang Li, Duc Ngo, Anthony M. Brade, and Henry J. Klamut Division of Experimental Therapeutics, Ontario Cancer Institute, Princess Margaret Hospital, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirusmediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents. Key words: Herpes simplex virus thymidine kinase; ganciclovir treatment; breast cancer; apoptosis; p53 tumor suppressor gene; bystander effect; gene therapy. H erpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) (9[(1,3-dihydroxy-2-propoxy)methyl]guanine) treatment has shown good success as a tumor ablation strategy1,2 in a variety of experimental models, including brain,3 head and neck,4 skin,5 lung,6 liver,7 pancreatic,8 colon,9 prostate,10 ovarian,11 and breast cancers.12–15 Much of the interest in this treatment strategy comes from the observation of cytotoxicity in nontransduced tumor cells that lie in close proximity to HSV-tk-transduced cells. This “bystander effect” eliminates the need to transduce all cells in a target tumor cell population to achieve effecReceived November 26, 1997; accepted June 27, 1998. Address correspondence and reprint requests to Henry J. Klamut, Room 10-721, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. E-mail address: [email protected] © 1999 Stockton Press 0929-1903/99/$12.00/10 Cancer Gene Therapy, Vol 6, No 2, 1999: pp 179 –190 tive treatment.16 –21 The cytotoxic effects of HSV-tk/ GCV treatment have been related to the induction of apoptosis (programmed cell death) in a number of tumor cell types.22–24 Programmed cell death is a normal biological process used in situations in which cell deletion is required to alter tissue structure and function or to remove genetically damaged cells.25,26 It is becoming apparent that every cell has an intrinsic cell death program, and that apoptosis can be triggered by a variety of stimuli.27,28 The execution phase of apoptosis involves activation of a proteolytic cascade of cysteine proteases and results in distinctive morphological and biochemical changes that include cell volume shrinkage, plasma membrane blebbing, chromatin condensation, and DNA fragmentation.25,26 The steps that lead to activation of the execution phase of apoptosis are poorly understood. However, evidence to date suggests that death-promoting signals can be transmitted by any one of several distinct signaling pathways.29 –34 Mutations in genes 179 180 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS within these signaling pathways are thought to increase the activation threshold for apoptosis in cancer cells, leading to genetic instability and the development of resistance to radiation and chemotherapeutic agents.35–38 GCV is an acyclic nucleoside that is converted to GCV triphosphate by viral thymidine kinase and endogenous mammalian kinases. This purine analog competes with normal nucleotides during DNA synthesis and terminates DNA replication.39,40 The DNA damage that results is thought to signal apoptosis in HSV-tk/GCVtreated cell populations.22–24 The response of mammalian cells to DNA damage is complex and involves a number of genes that modulate cell cycle progression, DNA repair, and apoptosis.41– 44 The p53 tumor suppressor gene45 encodes a nuclear phosphoprotein that can activate both G1 cell cycle arrest46,47 and apoptosis35,36,48 in response to DNA damage. Mutations in the p53 gene are the most frequent genetic abnormality found in human cancer,49,50 and a loss of p53 function has been correlated with increased resistance to a wide variety of DNA-damaging agents.51–56 Previous studies have demonstrated variability in the susceptibility of individual tumor cell lines to HSV-tk/ GCV treatment.57–59 As HSV-tk/GCV cytotoxicity is mediated through DNA damage-induced apoptosis, we sought to establish whether any relationship exists between p53 gene status, p53-mediated apoptosis, and the sensitivity of human breast tumors to this treatment modality. To examine this issue, GCV cytotoxicity following adenovirus (Ad)-mediated gene transfer of recombinant HSV-tk was assessed in terms of cell viability, bystander effects, and the appearance of characteristic features of apoptotic cell death in two human breast cancer cell lines: MCF-7 cells, which express wild-type (wt) p53,60 and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His).61 Our analysis of MCF-7 cells confirmed that HSV-tk/GCV treatment can lead to a DNA damage-mediated induction of p53, p21WAF1/CIP1, and apoptosis in breast tumor cells that express wt p53. However, MDA-MB-468 cells exhibited a strong apoptotic response without an increase in p21WAF1/CIP1 protein levels, indicating that HSV-tk/GCV treatment can induce apoptosis in the absence of functional p53. This finding raised the possibility that this treatment strategy could be effective against breast tumors that express mutant p53 and that may have developed resistance to chemotherapeutic agents that require wt p53 function. Analysis of four additional human breast cancer cell lines expressing mutant p53 confirmed this result and demonstrated that chemosensitivity to HSV-tk/GCV treatment does not correlate with endogenous p53 gene status. MATERIALS AND METHODS Cells and cell culture The MDA-MB-468, MCF-7, MCF-7/Adr, BT-474, and T47D human breast cancer cell lines were kindly provided by the laboratories of Drs. R. Buick, S. Benchimol, M. Moore, and M. Rauth of the Ontario Cancer Institute, Princess Margaret Hospital. BT-20 human breast cancer cells and HS-574 cells, which were established from normal tissue from a patient with infiltrating ductal carcinoma, were obtained from the American Type Culture Collection (Manassas, Va). MCF-7, MCF-7/ Adr, MDA-MB-468, BT-20, and BT-474 breast cancer cell lines were grown in a-minimal essential medium (a-MEM). T47D cells were grown in RPMI 1640 medium, and HS-574 cells were grown in Dulbecco’s MEM. All media contained 10% fetal bovine sera and 100 U/mL penicillin/100 mg/mL streptomycin (Life Technologies, Gaithersburg, Md); all cells were incubated in the presence of 5% CO2 at 37°C. Cell lines were tested free of mycoplasma contamination by Hoechest 33258 fluorescent detection.62 Preparation of recombinant adenoviral stocks AdHSV-tk (recombinant Ad (rAd) containing a Rous sarcoma virus (RSV) promoter-HSV-tk minigene) was kindly provided by Dr. S. Woo (Baylor Medical College, Houston, Tex). AdCMVp53 (rAd containing a cytomegalovirus (CMV) wt p53 minigene), AdCMVb-gal (rAd containing a CMV-b-galactosidase (b-gal) minigene), and the 293 cell line were kindly provided by Dr. F. Graham (McMaster University, Hamilton, Ontario, Canada). Large-scale rAd stocks were prepared from single purified plaques, and all were tested free of replicationcompetent Ad essentially as described previously.63 Ad infection efficiencies Ad infection efficiencies were determined essentially as described previously.64 Briefly, cells were seeded in six-well dishes at a density of 100 cells/mm2; after 24 hours, cells were exposed to different dilutions of AdCMVb-gal in 200 mL of phosphate-buffered saline (PBS) to provide multiplicities of infection (MOIs) in the range of 0.1–100 plaque-forming units (PFU)/cell. After a 30-minute incubation at 37°C with gentle shaking, 3 mL of fresh media was added to each well, and the cells were incubated for an additional 48 hours. For b-gal histochemistry, cells were washed in cold PBS-Ca,Mg and fixed with a solution of 2% formaldehyde and 0.2% glutaraldehyde in PBS-Ca,Mg for 5 minutes at 4°C. Fixed cells were washed three times with PBS-Ca,Mg containing 2 mM MgCl2 and 0.02% Nonidet P-40 and then overlayed with b-gal staining solution (1 mg/mL 5-bromo-4-chloro-3-indolyl b-D-galactoside, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl2 in PBS-Ca,Mg) for 1–3 hours at 37°C. Stained cultures were rinsed with PBS-Ca,Mg followed by dH2O and air-dried. Transduction efficiencies, which were expressed as the percent of the total cell population expressing b-gal, were quantified by counting the number of blue-stained cells and total cells in 10 –20 randomly chosen microscopic fields for a minimum of three independent experiments. HSV-tk expression by Northern blot analysis Total cellular RNA was isolated using the RNeasy Kit (Qiagen, Chatsworth, Calif) according to the manufacturer’s instructions. Total RNA (10 mg) was separated by electrophoresis in 1.2% agarose-formaldehyde gels,65 transferred to zetaprobe nitrocellulose membranes (Bio-Rad, Hercules, Calif), and hybridized to the following 32P-labeled cDNA probes prepared by random priming:66 (a) a 2556-base pair HSV-tk cDNA fragment, and (b) a 1.3-kilobase glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA fragment. Hybridizations were performed overnight at 42°C in 40% formamide, 0.12 M Na2PO4 (pH 7.2), 0.25 M NaCl, 0.5% sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic Cancer Gene Therapy, Vol 6, No 2, 1999 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS acid (EDTA). Filters were washed in 23 standard saline citrate (SSC)/0.1% SDS at 42°C for 20 minutes followed by washes in 0.53 SSC/0.1% SDS and 0.13 SSC/0.1% SDS at room temperature for 20 minutes each. Filters were exposed to Kodak autoradiography film overnight at 270°C. Autoradiographs were analyzed densitometrically (Molecular Dynamics, Sunnyvale, Calif) using ImageQuant software. Cell viability assays Cells were seeded in either 6- or 24-well plates at a density of 100 cells/mm2; after 24 hours, each well was infected with either AdHSV-tk at 0.1, 1, 10, 25, 50, or 100 PFU/cell or AdCMVb-gal at 100 PFU/cell followed by GCV (Hoffman-La Roche, Nutley, NJ) at concentrations ranging from 3 to 18 mg/mL. Cell viability was determined at days 5 and 7 after treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St. Louis, Mo). Briefly, MTT (in a-MEM minus phenol red) solution was added to each well to give a final concentration of 1 mg/mL. After a 4-hour incubation at 37°C, the formazan product was solubilized by overlaying cells with 0.01 N HCl in isopropanol for 4 hours at 37°C. Under our experimental conditions, there was a direct correlation between formazan production as detected by measuring the absorbance of the overlay solution at 570 nm and total viable cell numbers per well. The results are expressed as the percentage of cell viability relative to uninfected control cultures. Bystander effects were measured by mixing cells infected with AdHSV-tk (100 PFU/cell) with uninfected cells at ratios (infected to uninfected) of 1:0, 3:1, 2:1, 1:1, 1:3, 1:5, 1:10, 1:100, and 0:1 and plating the cells onto 24-well plates at 105 cells/well. Control infected (AdCMVb-gal) and uninfected cells plated at the same ratios and densities were included in each study. Cells were exposed to GCV at 10 mg/mL at 24 hours after plating, and cell viability was measured at day 7 after GCV treatment using the MTT assay. The results are expressed as the percentage of cell viability relative to corresponding uninfected control cultures. Acridine orange and ethidium bromide (EB) staining Cells treated with either control virus (AdCMVb-gal) or AdHSV-tk/GCV were harvested at several timepoints posttreatment, washed with PBS, and resuspended in 1 mL of PBS containing 5 mg/mL acridine orange (Sigma) and 5 mg/mL EB (Sigma). After a 5-minute incubation at room temperature, cells were pelleted by centrifugation and resuspended in 25 mL of PBS containing 10% glycerol. Stained cell samples were examined and photographed by fluorescence microscopy. DNA fragmentation assays Control and treated cultures were harvested at several timepoints post-treatment and tested for DNA fragmentation essentially as described previously.67 Briefly, cells were scraped, harvested, and pelleted in ice-cold PBS; next, 5 3 106 cells were resuspended in 200 mL of guanidine thiocyanate containing 100 mM 2-mercaptoethanol and stored overnight at 270°C. DNA was precipitated overnight at 220°C after the addition of 100 mL of 7.5 M ammonium acetate and 600 mL of absolute ethanol and pelleted by centrifugation at 14,000 3 g for 20 minutes at 4°C. DNA was resuspended in TE (10 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, 1 mM EDTA; pH 8.0) containing 20 mg/mL ribonuclease A, electrophoresed on 2% agarose gels, and visualized by EB staining. Controls included cells infected with AdHSV-tk alone (minus GCV Cancer Gene Therapy, Vol 6, No 2, 1999 181 treatment) and uninfected cells exposed to GCV at a concentration of 10 mg/mL. Western blot analysis of p53 and p21WAF1/CIP1 expression Cells were infected with AdHSV-tk or AdCMVb-gal (control) at 100 PFU/cell and exposed to GCV at a concentration of 10 mg/mL. Cells were harvested at various timepoints post-treatment by washing and scraping into ice-cold PBS-Ca,-Mg. Cells were pelleted by centrifugation and solubilized (25 mL per 106 cells) in protein solubilization buffer (0.1 M Tris/HCl (pH 8.0), 1% SDS, 10 mM EDTA, and 2 mM dithiothreitol). Protein concentrations were measured using the bicinchoninic acid assay (Pierce, Rockford, Ill) according to the manufacturer’s instructions. Cell lysates were loaded onto 10% SDS-polyacrylamide gels at a protein concentration of 50 mg/lane, separated electrophoretically, and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using a semidry gel transfer apparatus (Bio-Rad).68 Transfer efficiency was monitored by staining nitrocellulose membranes with Ponceau red (Sigma) and post-transferred gels with Coomassie blue R250. Membranes were blocked with a solution of 5% skim milk powder and 1% heat-inactivated sera in Tris-buffered saline (TBS) (0.01 M Tris/HCl (pH 7.5) and 0.15 M NaCl) for 2 hours at 4°C, washed three times with TBS containing 0.05% Tween 20, and incubated with antibody (Ab) binding solution (1% skim milk powder and 1% heat-inactivated sera in TBS) containing either a 1/100 dilution of monoclonal p53 Ab (Ab-6, Oncogene Science, Uniondale, NY) or a 1/100 dilution of a monoclonal p21WAF1/CIP1 Ab (Ab-1, Oncogene Science) for 60 minutes at room temperature. Filters were washed with three changes in TBS containing 0.05% Tween 20 for 10 minutes each at room temperature and incubated with horseradish peroxidase-conjugated donkey anti-mouse Ab (1:10,000, Jackson Laboratories, West Grove, Penn) in Ab binding solution for 60 minutes at room temperature. Membranes were washed again as described previously, and bands were visualized using enhanced chemiluminescence detection reagents (Amersham, Arlington Heights, Ill) and autoradiography according to the manufacturer’s instructions. RESULTS Ad-mediated gene transfer results in high levels of HSV-tk mRNA expression To provide a basis for comparison of the cytotoxic effects of AdHSV-tk/GCV treatment of MDA-MB-468 and MCF-7 cells, we first examined Ad infection efficiencies and HSV-tk mRNA expression levels in these two cell lines. Ad infection efficiencies were evaluated by infecting MDA-MB-468 and MCF-7 cells with AdCMVb-gal at MOIs ranging from 0.1 to 100 infectious particles (PFU)/cell and determining the percentage of the total target cell population staining positive for b-gal. The results are summarized in Figure 1. Ad infection efficiencies were ;2-fold higher in MDA-MB-468 cells at MOIs of 1 and 10 PFU/cell. However, in cells infected at 100 PFU/cell, .95% of both MDA-MB-468 and MCF-7 cells tested positive for b-gal expression. Northern blot analysis indicated that HSV-tk mRNA is expressed as early as 24 hours and peaks by 48 –72 hours postinfection in both MDA-MB-468 and MCF-7 cells infected with 182 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS Figure 1. Recombinant adenoviral vectors transduce MDA-MB-468 and MCF-7 cells at efficiencies approaching 100%. Cells were transduced with a rAd expressing either b-gal (AdCMVb-gal) or luciferase (AdCMVluc) at an MOI ranging from 0.1 to 100 infectious particles (PFU)/cell. At 48 hours after infection, cells were stained for b-gal activity. Transduction efficiency was determined as the percentage of cells staining blue in the treated cell population. Values represent the mean 6 SD for $10 random fields, each representing 2 an area of 0.39 mm and containing a minimum of 100 cells. AdHSV-tk at 100 PFU/cell (Fig 2a). Densitometric analysis using endogenous GAPDH gene expression as an internal reference (HSV-tk/GAPDH ratio; Fig 2b) indicated that HSV-tk mRNA levels in MDA-MB-468 cells were 1.5-fold higher than in MCF-7 cells at 24 and 48 hours postinfection but had reached equivalent levels in the two cell lines by 72 hours. These results indicated that the transduction of MDA-MB-468 and MCF-7 cells with AdHSV-tk at an MOI that provides for an infection of .95% of the target cell population results in the expression of HSV-tk mRNA at levels that differed by no more than 1.5-fold over a period of 72 hours after infection. AdHSV-tk/GCV treatment results in induction of p53 and p21WAF1/CIP1 in MCF-7 cells HSV-tk/GCV cytotoxicity is thought to be mediated by the induction of programmed cell death in response to DNA damage, which leads to the induction of endogenous p53 protein levels and p21WAF1/CIP1-mediated G1 cell cycle arrest.47,69,70 To establish that HSV-tk/GCV treatment leads to the induction of p53 and p21WAF1/CIP1 protein levels in MCF-7 cells, and that mutant p53 gene expression precludes the induction of p21WAF1/CIP1 in MDA-MB-468 cells, total cell extracts isolated at 12, 24, and 36 hours after treatment with AdHSV-tk/GCV were examined for increases in endogenous p53 and Figure 2. HSV-tk MRNA levels in MDA-MB-468 and MCF-7 cells transduced with AdHSV-tk at 100 PFU/cell. a: Northern blot analysis of total RNA isolated from MDA-MB-468 and MCF-7 cells at 0 –72 hours after infection with AdHSV-tk at 100 PFU/cell. Blots were hybridized sequentially with 32P-labeled HSV-tk and GAPDH cDNA probes and autoradiographed for 24 (MDA-MB-468) to 72 (MCF-7) hours at 270°C. b: Densitometric analysis of HSV-tk and GAPDH band intensities. Normalization of HSV-tk band intensities to GAPDH (HSV-tk/GAPDH ratio) demonstrated that recombinant HSV-tk mRNA levels did not vary by .1.4-fold (48-hour timepoint) between MDA-MB-468 and MCF-7 cells over the time period studied. p21WAF1/CIP1 protein levels by Western blot analysis. Results are shown in Figure 3. As expected, high levels of mutant p53 expression were evident in cell extracts derived from AdHSV-tk (100 PFU/cell)-infected MDAMB-468 cells in both the absence (time 0) and presence of GCV (9 mg/mL) for #36 hours postinfection. However, no increase in endogenous p21WAF1/CIP1 protein was observed at any of the timepoints studied. A cell extract prepared from MDA-MB-468 cells transduced with a rAd expressing wt p53 (AdCMVp53) was included as a positive control for the induction of endogenous p21WAF1/CIP1 protein expression in this cell line. Endogenous p53 and p21WAF1/CIP1 protein levels were low to undetectable in AdHSV-tk (100 PFU/cell)-infected MCF-7 cell cultures at time 0. Treatment with GCV (9 mg/mL) resulted in a significant increase in Cancer Gene Therapy, Vol 6, No 2, 1999 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS Figure 3. AdHSV-tk/GCV treatment induces endogenous p53 and p21WAF1/CIP1 protein levels in MCF-7 cells. Western blot analysis of cell lysates prepared from MDA-MB-468 and MCF-7 cells at 0 –36 hours after treatment with AdHSV-tk (100 PFU/cell) and GCV (9 mg/mL). Blots were incubated with Abs to either p53 (ap53) or p21WAF1/CIP1 (ap21); reactive bands were visualized using horseradish peroxidase-conjugated secondary Abs, enhanced chemiluminescence reagents, and autoradiography as described in Materials and Methods. Included as positive controls for p53 and p21WAF1/CIP1 were cell lysates prepared from MDA-MB-468 and MCF-7 cells infected with a rAd expressing wt p53 (Adp53; 100 PFU/cell). Untreated (0 hours) MDA-MB-468 cells express high levels of endogenous mutant p53 and undetectable levels of endogenous p21WAF1/CIP1. Endogenous p21WAF1/CIP1 protein is induced in response to recombinant wt p53 overexpression (Adp53) but not in response to AdHSV-tk/GCV treatment (12–36 hours). Endogenous p53 (wt) and p21WAF1/CIP1 protein is undetectable in untreated MCF-7 cells. Both are increased in cells transduced with an adenoviral vector that expresses recombinant p53 (Adp53). Treatment with AdHSV-tk/GCV also results in the induction of endogenous p53 (12–36 hours) and p21WAF1/CIP1 (36 hours) protein levels. p53 protein levels by 12 hours afer infection and in p21WAF1/CIP1 protein levels by 36 hours after infection. A cell extract prepared from MCF-7 cells transduced with AdCMVp53 was included as a positive control for both wt p53 and the induction of endogenous p21WAF1/CIP1 protein expression. These results substantiate the notion that HSV-tk/GCV treatment leads to an induction of p53 and p21WAF1/CIP1 protein levels in tumor cells expressing wt p53, and establish a clear difference in the p53 response of these two cell lines to this treatment strategy. MDA-MB-468 and MCF-7 cell lines display differential sensitivities to AdHSV-tk/GCV treatment These experiments were designed to examine the relationship between HSV-tk transgene dosage, GCV concentration, and cytotoxicity in MDA-MB-468 and MCF-7 cell cultures. Target cell populations were infected with AdHSV-tk over a range of 1.0 –100 PFU/cell and exposed to GCV concentrations of 3, 9 (clinically achievable dose),3 and 18 mg/mL. Cell viability was determined at 7 days after treatment relative to control cultures using the MTT assay, which measures the number of viable cells in each group at the end of the treatment period. Controls included mock-infected and AdCMVb-gal (100 MOI)-infected cultures treated with Cancer Gene Therapy, Vol 6, No 2, 1999 183 Figure 4. MDA-MB-468 and MCF-7 cells display differential sensitivities to AdHSV-tk/GCV treatment. Cells were infected with AdHSV-tk at MOIs ranging from 1 to 100 PFU/cell and treated with GCV at concentrations of 3, 9 (clinically achievable dose), and 18 mg/mL. Cell viability was determined using the MTT assay as described in Materials and Methods. Solid symbols, MDA-MB-468 cells; shaded symbols, MCF-7 cells. GCV concentrations are represented by: triangles, 3 ug/mL; circles, 9 ug/mL; and squares, 18 mg/mL. Results represent the mean 6 SD of a minimum of three independent determinations and are expressed as the percentage of viability relative to mock-infected controls. GCV at 9 mg/mL. GCV in the range of 3–18 mg/mL of culture medium was determined to have no significant effect on the viability of uninfected controls. As shown in Figure 4, treating AdHSV-tk-infected MDA-MB-468 cells with GCV at 9 mg/mL resulted in a decrease in cell viability to 75% of controls at 1 PFU/cell, to 25% of controls at 10 PFU/cell, and to ,5% of controls at 25–100 PFU/cell. Differences in MDA-MB-468 cell viability in AdHSV-tk-infected cells treated with GCV at 3 and 18 mg/mL ranged from 10 –20% at MOIs of 1–25 PFU/cell and decreased to ,10% at an MOI of $50. Treatment of AdHSV-tk-infected MCF-7 cells with GCV at 9 mg/mL decreased cell viability to 85% of controls at 1 PFU/cell and to 70% of controls at 100 PFU/cell. Differences in cell viability in cultures treated with GCV at 3 and 18 mg/mL averaged 22% over the range of MOIs tested. These results demonstrate a clear difference in the sensitivity of these two breast cancer cell lines to AdHSV-tk/GCV treatment. At the highest doses of AdHSV-tk and GCV used in this study, MDAMB-468 cell viability was reduced to ,1% of controls. In comparison, MCF-7 cell viability declined to only 60% of controls under conditions that provide for similar levels of Ad infection and HSV-tk mRNA expression in the two cell lines. 184 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS portion of cells infected with AdHSV-tk and total cell viability demonstrated that this treatment strategy results in a significant bystander effect in the MDA-MB468 cell line. In contrast, identical mixing experiments with MCF-7 cells resulted in only a 20 –30% decline in cell viability over the range of infected to uninfected cell ratios studied (Fig 5). AdHSV-tk/GCV treatment leads to apoptosis of both MDA-MB-468 and MCF-7 cells Figure 5. Bystander effects in MDA-MB-468 and MCF-7 cells treated with AdHSV-tk/GCV. Bystander effects were examined by mixing cells infected with AdHSV-tk at 100 PFU/cell with uninfected cells at ratios (infected to uninfected) ranging from 1:10 to 1:0. Cells were seeded onto 24-well plates at a density of 105 cells/well and exposed to GCV at a concentration of 9 mg/mL for 7 days. Cell viability was determined by MTT assay, and results are expressed as the percentage of viability relative to untreated controls. Filled bars, MDA-MB-468 cells; shaded bars, MCF-7 cells. Values represent the mean 6 SD of a minimum of three independent observations. Bystander effects are more pronounced in AdHSV-tktreated MDA-MB-468 cells Infecting MDA-MB-468 cells with AdCMVb-gal at 1 and 10 PFU/cell resulted in b-gal staining of 4.9% and 49% of cells, respectively (Fig 1). However, MDA-MB468 cells infected with AdHSV-tk at 1 and 10 PFU/cell displayed 28% and 90% declines, respectively, in cell viability at a GCV concentration of 18 mg/mL (Fig 4). These results suggested that a bystander effect occurs under these treatment conditions. To examine this phenomenon in more detail, a series of experiments were performed in which cells infected with AdHSV-tk at 100 PFU/cell were mixed at different ratios with uninfected cells and treated with GCV at a concentration of 9 mg/mL. Cell viability was determined at 7 days after treatment using the MTT assay, and results were expressed relative to controls containing equivalent mockinfected to uninfected cell numbers. As shown in Figure 5, no significant decrease in cell viability was observed in either MDA-MB-468 or MCF-7 cell cultures seeded at ratios of infected to uninfected cells of 1:10 or lower. At an infected to uninfected cell ratio of 1:5, MDA-MB-468 cell viability decreased to 30% of controls. At ratios of 1:5–1:1, viability decreased further to 10 –15% of controls; at ratios of $2:1, a decrease was observed to ,5% of control cultures. The discrepancies between the pro- To examine the mechanism of AdHSV-tk/GCV cytotoxicity in MDA-MB-468 and MCF-7 cells, treated cells were examined for morphological (chromatin condensation, nuclear fragmentation) and biochemical (DNA fragmentation) features associated with apoptotic cell death. As shown in Figure 6, no cytotoxic effects were observed in either MDA-MB-468 or MCF-7 cells treated with AdHSV-tk alone (Fig 6, B and E) compared with mock-infected controls (Fig 6, A and D). Treating MDA-MB-468 cells with AdHSV-tk at 100 PFU/cell and GCV at 9 mg/mL (Fig 6C) led to detachment of the majority of cells from the culture dish by 36 hours after treatment. Acridine orange and EB staining indicated that this treated cell population contained large numbers of condensed, fragmented nuclei and apoptotic bodies characteristic of apoptosis. At 36 hours after treatment, no significant morphological changes were observed in MCF-7 cells treated under the same conditions (data not shown). However, at 72 hours after treatment, morphological changes characteristic of apoptosis began to appear in the treated MCF-7 cell population (Fig 6F) and steadily increased to include 30 – 40% of the total cell population by 7 days after treatment. Agarose gel electrophoretic analysis of genomic DNA laddering (Fig 7) confirmed that HSV-tk/GCV treatment leads to the induction of apoptosis in both MDA-MB-468 and MCF-7 cells. However, the apoptotic response was significantly delayed in MCF-7 cells (72 hours after treatment) compared with MDA-MB-468 cells (36 hours after treatment). It should be noted that in these experiments, the extent of DNA laddering is not representative of the proportion of the total cell population undergoing apoptosis. DNA fragmentation was not observed in mock-infected controls or in cells treated with AdHSV-tk or GCV alone. Taken together, these results demonstrate that AdHSV-tk/GCV treatment results in the induction of apoptosis in both MDA-MB-468 and MCF-7 cells. However, the apoptotic response of MCF-7 cells was significantly delayed and involved a significantly lower percentage of the total cell population relative to MDA-MB-468 cells treated under the same conditions. Breast cancer cell lines display differential chemosensitivities to GCV treatment following HSV-tk gene transfer Our studies of MDA-MB-468 cells established that AdHSV-tk/GCV treatment can lead to the activation of Cancer Gene Therapy, Vol 6, No 2, 1999 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS 185 Figure 6. MDA-MB-468 and MCF-7 cells display morphological features consistent with the induction of apoptosis in response to AdHSV-tk/GCV treatment. Phase contrast and fluorescence microscopy of MDA-MB-468 (A–C) and MCF-7 cells (D–F) stained with acridine orange/EB revealed morphological features characteristic of apoptosis in AdHSV-tk/GCV-treated cells. A and D: mock-infected controls. B and E: AdHSV-tk (100 PFU/cell) alone. C and F: AdHSV-tk (100 PFU/cell) and GCV (9 mg/mL). The apoptotic bodies (arrow, panel C) and chromatin condensation (arrow, panel F) observed in AdHSV-tk/GCV-treated cells are characteristic features of apoptosis. Phase contrast: Bar, 50 mm. Acridine orange/EB staining: Bar, 18 mm. a strong, p53-independent apoptotic response in human breast cancer cells that lack functional p53. As this observation has important implications for the treatment of breast cancer cells that have developed resistance to genotoxic therapeutic agents, we examined AdHSV-tk/GCV cytotoxicity in four additional human breast cancer cell lines that have been reported to express mutant p53 (MCF-7/Adr, BT-20, BT-474, and T47D).61,71–74 The results, shown in Figure 8, are expressed as the percentage of viability relative to untreated controls. The percentages of cell viability measured for MCF-7 and MDA-MB-468 cells, as well as a normal human control (HS-574), were also included for comparative purposes. Whereas a significant variation in chemosensitivity to AdHSV-tk/GCV treatment could be seen among the five breast cancer cell lines expressing mutant p53, all displayed a greater sensitivity to this therapeutic modality than either MCF-7 cells or normal HS-574 control cells. Interestingly, the multidrug-resistant subline of MCF-7 cells (MCF-7/Adr)71 showed at least a 50-fold greater chemosensitivity to AdHSV-tk/ GCV treatment than the parental line. Although the mechanism(s) responsible for the differential sensitivities of these cell lines to AdHSV-tk/GCV treatment are not clear, the results suggest that endogenous p53 status is not predictive of chemosensitivity to this treatment modality, and point to a potential application of this Cancer Gene Therapy, Vol 6, No 2, 1999 treatment strategy to tumor cells that express mutant p53 and have developed resistance to conventional genotoxic therapeutic agents. DISCUSSION Prodrug activation strategies involving HSV-tk gene transfer followed by GCV treatment have shown considerable potential for cancer therapy.1,2 The bystander effect, which refers to cytotoxic effects seen in neighboring cells that have not been transduced with HSV-tk, makes this approach particularly attractive, as it obviates the need to deliver a therapeutic gene to all cells in the target tumor.16 –21 GCV cytotoxicity is thought to be mediated through the induction of apoptosis (programmed cell death) in response to DNA damage.22–24 The p53 tumor suppressor gene product plays a major role in regulating the response of mammalian cells to DNA-damaging agents,51–53,55,56,75 and abnormalities in p53-dependent apoptosis arising from mutations in the p53 gene have been shown to contribute to the development of chemotherapeutic drug resistance.51–56,75 The relationship between HSV-tk/GCV cytotoxicity and p53dependent apoptosis has not been firmly established in human tumor models. In view of the high incidence of p53 gene mutations in breast cancer49 and their associ- 186 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS Figure 7. DNA laddering in MDA-MB-468 and MCF-7 cells treated with AdHSV-tk/GCV. Agarose gel electrophoresis and EB staining of genomic DNA extracted from MDA-MB-468 and MCF-7 cells infected with AdHSV-tk at 100 PFU/cell and treated with GCV at a concentration of 9 mg/mL for 24 – 48 hours (MDA-MB-468) or 48 –96 hours (MCF-7). Controls include untreated cells and cells treated with either AdHSV-tk (100 PFU/cell) alone or GCV (9 mg/mL) alone. The DNA laddering that is characteristic of apoptotic cell death was observed in both MDA-MB-468 and MCF-7 cells treated with AdHSV-tk/GCV. ation with increased resistance to drug and radiation treatments,51–56,75 we sought to establish whether HSVtk/GCV treatment results in the predicted increase in p53 protein levels and in the induction of apoptotic cell death, as well as whether mutations in the p53 gene correlate with increased resistance to this treatment modality. Our observation in MCF-7 cells of increases in endogenous p53 and p21WAF1/CIP1 protein levels, along with the appearance of morphological and biochemical features characteristic of apoptotic cell death, is consistent with the notion that DNA damage arising from AdHSVtk/GCV treatment triggers a p53-dependent apoptotic cell death pathway.22,24,35 The relative resistance of MCF-7 cells to this treatment strategy is also consistent with previous studies showing that this cell line does not readily undergo apoptosis following DNA damage.76 However, the robust apoptotic response observed in MDA-MB-468 cells, which overexpress mutant p53,61 also points to a significant role for p53-independent apoptosis in the cytotoxic response of tumor cells to AdHSV-tk/GCV treatment. Furthermore, cell viability studies demonstrated that breast tumor cells expressing mutant p53 can be highly sensitive to the cytotoxic Figure 8. Comparisons of the relative chemosensitivities of different human breast cancer cell lines with AdHSV-tk/GCV treatment. Cell viability was determined in a series of six human breast cancer cell lines expressing either wt (MCF-7) or mutant (MCF-7/Adr, MDAMB-468, BT-20, BT-474, and T47D) p53 as well as in one normal control (HS-574). Measurements were taken at 5 days after treatment with AdHSV-tk at an MOI of 100 PFU/cell and 10 mg/mL GCV. A significant degree of variation in the chemosensitivities of the different cell lines to GCV treatment was observed. However, cell lines expressing mutant p53 consistently showed greater chemosensitivity than cells expressing wt p53, and no direct correlation could be made between chemosensitivity and endogenous p53 gene status. Values represent the mean 6 SD of a minimum of three independent observations. effects of AdHSV-tk/GCV treatment. These results are consistent with a recent study of HSV-tk/GCV cytotoxicity in mouse thyrocytes,23 and serve to expand the predicted effective range of this treatment strategy to breast tumors that express mutant p53 and that may have developed resistance to other genotoxic therapeutic modalities. This latter point is illustrated by our observation of a 50-fold increase in the sensitivity of MCF-7/Adr cells to HSV-tk/GCV treatment. MCF-7/ Adr cells display cross-resistance to a number of chemotherapeutic agents, including vinca alkaloids, anthracyclines, colchicine, taxol, and actinomycin D71,77,78 as well as g radiation.79 Although the mechanism by which the development of multidrug resistance increases the sensitivity of MCF-7 cells to HSV-tk/GCV treatment is not clear, similar results have been observed with multidrug (vincristine)-resistant mouse leukemia P388 cells.80 The apparent lack of a correlation between cytotoxicity and endogenous p53 gene status points to the existence of alternative molecular and biochemical factors responsible for the range of chemosensitivities to AdHSV-tk/GCV treatment seen among the six breast cancer cell lines studied. Possibilities include differences in HSV-tk gene dosage, the rate of GCV uptake, the strength of the bystander effect, and defects in one or Cancer Gene Therapy, Vol 6, No 2, 1999 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS more downstream regulators of apoptosis. In this study, we undertook to examine some of these issues in MCF-7 and MDA-MB-468 cells, two human breast cancer cell lines that were observed to exhibit widely different chemosensitivities to AdHSV-tk/GCV treatment. HSV-tk gene dosage Variable sensitivities to GCV treatment following gene transfer of a recombinant HSV-tk gene to mesothelioma, non-small cell carcinoma, and ovarian carcinoma cell lines have been attributed to differences in adenoviral infection efficiencies as demonstrated by AdCMVb-gal histochemistry.81 However, our observation that both MDA-MB-468 and MCF-7 cells are infected at efficiencies approaching 100% when exposed to AdCMVb-gal at 100 PFU/cell, and that comparable levels of HSV-tk mRNA are expressed in each of these cell lines under these conditions, suggests that the differential chemosensitivities of MCF-7 and MDA-MB-468 cells to GCV are not related to differential HSV-tk gene dosage effects. GCV uptake Because most nucleosides and nucleoside analogs are highly hydrophillic, they do not readily cross the lipid bilayer of biological membranes in the absence of functional nucleoside transport processes.82 The permeation of nucleosides across the plasma membrane of mammalian cells is complex and is mediated by at least five distinct transporters that differ in their sensitivity to inhibitors and in their specificity for individual nucleosides.83 Differences in the distribution of these transporters could account for the differential GCV cytotoxicities seen in MCF-7 and MDA-MB-468 cells. However, our observation of similar decreases in cell viability (15–20%) as GCV concentrations were increased from 3 to 18 mg/mL suggests that GCV transport is not significantly different in these two cell lines. Bystander effects The absence of a significant bystander effect could contribute to resistance to AdHSV-tk/GCV treatment at MOIs that provide for transgene expression in ,100% of the target cell population. Gap junctions have been shown to mediate the cell-to-cell transfer of phosphorylated nucleotides,84 and gap junction-mediated transfer of phosphorylated GCV molecules from HSV-tk-expressing cells to neighboring nontransduced cells has been proposed as a mechanism for the bystander effect.18 –20 In this study, MDA-MB-468 cells were seen to exhibit a significant bystander effect, but no bystander effect was seen in MCF-7 cells treated under the same conditions. The absence of a bystander effect in MCF-7 cells has been independently confirmed and has been attributed to a deficiency in gap-junctional intercellular communication.59 Interestingly, significant bystander effects have been observed in MCF-7 cells treated with either a human thymidine phosphorylase gene and 59deoxy-5-fluorouridine59,85 or a liver cytochrome p450 Cancer Gene Therapy, Vol 6, No 2, 1999 187 gene in combination with cyclophosphamide.86 These results are consistent with the view that tumors can exhibit differential chemosensitivities to individual prodrug activation strategies.59 However, a deficiency in the HSV-tk/GCV-mediated bystander effect does not account for the relative resistance of MCF-7 cells treated under conditions used in this study that provide for the transduction of .95% of the target cell population. Bcl-2 family proteins and apoptotic thresholds The molecular mechanisms that establish the relative sensitivity of tumor cells to different apoptotic agents remain to be fully elucidated. However, results to date indicate that members of the Bcl-2 family of proteins play a prominent role in regulating intracellular thresholds to apoptotic cell death-promoting stimuli.87 Bcl-2 functions as a negative regulator of apoptosis and has been reported to be overexpressed in #70% of breast cancers.88 Furthermore, high levels of bcl-2 gene expression have been correlated with an increased resistance to therapeutic agents that activate both p53-dependent and -independent apoptotic signaling pathways.89,90 Bax functions as a dominant repressor of Bcl-2 activity and promotes apoptosis, and the ratio of Bcl-2 to Bax is considered to be an important determinant of the threshold of a cell to death-promoting stimuli.91 Other related genes implicated in the regulation of apoptotic thresholds include Bcl-xL92 and BAG-1,93 both of which are genetic homologs of bcl-2 that also function as negative regulators of apoptosis. Like Bax, Bad and Bcl-xS function as dominant repressors of Bcl-2 and Bcl-xL to promote apoptosis,94,95 and the MCL1 gene has been implicated as a mediator of p53-independent apoptosis in human cell lines defective in p53 function.96,97 Recently, monospecific Abs, immunoblotting, and immunohistochemical methods were used to analyze the relative levels of the Bcl-2, Bcl-xL, MCL1, Bax, Bak, and BAG-1 proteins in a series of human breast cancer cell lines that included each of the breast cancer cell lines used in this study.74 MCF-7 cells were shown to express Bcl-2 at levels that were 8-fold higher than in MDA-MB-468 cells, and MCF-7/Adr cells have been reported to express decreased levels of Bcl-2 relative to the parental cell line.98 Thus, Bcl-2 levels do seem to correlate with the relative sensitivities of MCF-7, MDAMB-468, and MCF-7/Adr cells to AdHSV-tk/GCV treatment. However, high levels of Bcl-2 expression in BT474 cells and undetectable levels of Bcl-2 in BT-20 cells are inconsistent with the relative sensitivities of these cell lines to AdHSV-tk/GCV treatment. In fact, no direct correlation could be made between the levels of expression of any one of the Bcl-2 family of proteins and the sensitivity of individual breast cancer cell lines to AdHSV-tk/GCV treatment. This finding raises the possibility that multiple genetic and biochemical factors contribute to the sensitivity of individual tumors to this treatment strategy, and the relative contribution of each of these factors to HSV-tk/GCV cytotoxicity will need to be established if prognostic assays for the responsiveness 188 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS of individual tumors to this treatment modality are to be developed. In summary, we have demonstrated that AdHSV-tk/ GCV treatment of breast cancer cells can lead to increases in endogenous p53 levels and to the induction of apoptotic cell death. However, this cytotoxic response does not seem to depend upon the expression of a functional p53 gene, and tumor cell lines expressing mutant p53 exhibit a high degree of variability in their sensitivity to this treatment modality. Evidence for a p53-independent apoptotic response to AdHSV-tk/GCV treatment serves to extend the potential of this treatment strategy to breast tumors that express mutant p53. However, further studies will be needed to elucidate the precise molecular and biochemical mechanisms responsible for HSV-tk/GCV cytotoxicity and the development of predictive assays for the responsiveness of individual tumors to this treatment strategy. 10. 11. 12. 13. 14. ACKNOWLEDGMENTS We thank S. Woo for the AdHSV-tk construct and F. Graham for the AdCMVb-gal and AdCMVp53 constructs. This work was supported in part by the Medical Research Council of Canada, the Foundation for Gene and Cell Therapy, and an Amgen predoctoral fellowship (to P.-X.L). 15. 16. REFERENCES 1. Moolten FL. Drug sensitivity (“suicide”) genes for selective cancer chemotherapy. Cancer Gene Ther. 1994;1:279 –287. 2. Freeman SM, Whartenby KA, Freeman JL, Abboud CN, Marrogi AJ. In situ use of suicide genes for cancer therapy. Semin Oncol. 1996;23:31– 45. 3. Culver KW, Van Gilder J, Link CJ, et al. Gene therapy for the treatment of malignant brain tumors with in vivo tumor transduction with the herpes simplex thymidine kinase gene/ganciclovir system. Hum Gene Ther. 1994;5:343–379. 4. O’Malley BW, Jr, Chen SH, Schwartz MR, Woo SL. Adenovirus-mediated gene therapy for human head and neck squamous cell cancer in a nude mouse model. Cancer Res. 1995;55:1080 –1085. 5. Bonnekoh B, Greenhalgh DA, Bundman DS, et al. Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma. J Invest Dermatol. 1996;106:1163–1168. 6. Osaki T, Tanio Y, Tachibana I, et al. Gene therapy for carcinoembryonic antigen-producing human lung cancer cells by cell type-specific expression of herpes simplex virus thymidine kinase gene. Cancer Res. 1994;54:5258 –5261. 7. Wills KN, Huang WM, Harris MP, Machemer T, Maneval DC, Gregory RJ. Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene. Cancer Gene Ther. 1995;2:191–197. 8. Aoki K, Yoshida T, Matsumoto N, et al. Gene therapy for peritoneal dissemination of pancreatic cancer by liposomemediated transfer of herpes simplex virus thymidine kinase gene. Hum Gene Ther. 1997;8:1105–1113. 9. Coll JL, Mesnil M, Lefebvre MF, Lancon A, Favrot MC. Long-term survival of immunocompetent rats with intraperitoneal colon carcinoma tumors using herpes simplex 17. 18. 19. 20. 21. 22. 23. 24. 25. thymidine kinase/ganciclovir and IL-2 treatments. Gene Ther. 1997;4:1160 –1166. Eastham JA, Chen SH, Sehgal I, et al. Prostate cancer gene therapy: herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models. Hum Gene Ther. 1996;7:515–523. Link CJ, Jr, Moorman D, Seregina T, Levy JP, Schabold KJ. A phase I trial of in vivo gene therapy with the herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer. Hum Gene Ther. 1996;7:1161–1179. Manome Y, Abe M, Hagen MF, Fine HA, Kufe DW. Enhancer sequences of the DF3 gene regulate expression of the herpes simplex virus thymidine kinase gene and confer sensitivity of human breast cancer cells to ganciclovir. Cancer Res. 1994;54:5408 –5413. Colak A, Goodman JC, Chen SH, Woo SL, Grossman RG, Shine HD. Adenovirus-mediated gene therapy in an experimental model of breast cancer metastatic to the brain. Hum Gene Ther. 1995;6:1317–1322. Moolten FL, Vonderhaar BK, Mroz PJ. Transduction of the herpes thymidine kinase gene into premalignant murine mammary epithelial cells renders subsequent breast cancers responsive to ganciclovir therapy. Hum Gene Ther. 1996;7:1197–1204. Yee D, McGuire SE, Brunner N, et al. Adenovirusmediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer. Hum Gene Ther. 1996;7:1251–1257. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM. In vivo gene transfer with retroviral vectorproducer cells for treatment of experimental brain tumors. Science. 1992;256:1550 –1552. Freeman SM, Abboud CN, Whartenby KA, et al. The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res. 1993;53: 5274 –5283. Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther. 1993;4:725–731. Fick J, Barker FG, Dazin P, Westphale EM, Beyer EC, Israel MA. The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro. Proc Natl Acad Sci USA. 1995;92: 11071–11075. Mesnil M, Piccoli C, Tiraby G, Willecke K, Yamasaki H. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc Natl Acad Sci USA. 1996;93:1831–1835. Ishii-Morita H, Agbaria R, Mullen CA, et al. Mechanism of “bystander effect” killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment. Gene Ther. 1997;4:244 –251. Samejima Y, Meruelo D. “Bystander killing” induces apoptosis and is inhibited by forskolin. Gene Ther. 1995;2: 50 –58. Wallace H, Clarke AR, Harrison DJ, Hooper ML, Bishop JO. Ganciclovir-induced ablation non-proliferating thyrocytes expressing herpesvirus thymidine kinase occurs by p53-independent apoptosis. Oncogene. 1996;13:55– 61. Hamel W, Magnelli L, Chiarugi VP, Israel MA. Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic death of bystander cells. Cancer Res. 1996;56:2697– 2702. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic Cancer Gene Therapy, Vol 6, No 2, 1999 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239 –257. White E. Life, death, and the pursuit of apoptosis. Genes Dev. 1996;10:1–15. Wyllie AH. The genetic regulation of apoptosis. Curr Opin Genet Dev. 1995;5:97–104. Lund LR, Romer J, Thomasset N, et al. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development. 1996;122:181–193. Clarke AR, Purdie CA, Harrison DJ, et al. Thymocyte apoptosis induced by p53-dependent and -independent pathways. Nature. 1993;362:849 – 852. Symonds H, Krall L, Remington L, et al. p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell. 1994;78:703–711. Liebermann DA, Hoffman B, Steinman RA. Molecular controls of growth arrest and apoptosis: p53-dependent and independent pathways. Oncogene. 1995;11:199 –210. Vaux DL, Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci USA. 1996;93:2239 –2244. Nagata S. Apoptosis by death factor. Cell. 1997;88:355–365. Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature. 1997;389: 300 –305. Lowe SW, Ruley HE, Jacks T, Housman DE. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 1993;74:957–967. Lowe SW, Jacks T, Housman DE, Ruley HE. Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells. Proc Natl Acad Sci USA. 1994;91:2026 – 2030. Smith ML, Fornace AJ, Jr. Genomic instability and the role of p53 mutations in cancer cells. Curr Opin Oncol. 1995;7:69 –75. Anthoney DA, McIlwrath AJ, Gallagher WM, Edlin AR, Brown R. Microsatellite instability, apoptosis, and loss of p53 function in drug-resistant tumor cells. Cancer Res. 1996;56:1374 –1381. St Clair MH, Lambe CU, Furman PA. Inhibition by ganciclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Antimicrob Agents Chemother. 1987;31:844 – 849. Foti M, Marshalko S, Schurter E, Kumar S, Beardsley GP, Schweitzer BI. Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement. Biochemistry. 1997;36:5336 –5345. Hartwell LH, Kastan MB. Cell cycle control and cancer. Science. 1994;266:1821–1828. Murray AW. Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 1992;359:599 – 604. O’Connor PM, Kohn KW. A fundamental role for cell cycle regulation in the chemosensitivity of cancer cells? Semin Cancer Biol. 1992;3:409 – 416. Hickman JA. Apoptosis and chemotherapy resistance. Eur J Cancer. 1996;32A:921–926. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature. 1991;351:453– 456. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB. Wildtype p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA. 1992;89:7491–7495. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 1995;55:5187–5190. Cancer Gene Therapy, Vol 6, No 2, 1999 189 48. Bellamy CO. p53 and apoptosis. Br Med Bull. 1997;53: 522–538. 49. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science. 1991;253:49 –53. 50. Levine AJ, Wu MC, Chang A, et al. The spectrum of mutations at the p53 locus: evidence for tissue-specific mutagenesis, selection of mutant alleles, and a “gain of function” phenotype. Ann N Y Acad Sci. 1995;768:111–128. 51. Lee JM, Bernstein A. p53 mutations increase resistance to ionizing radiation. Proc Natl Acad Sci USA. 1993;90:5742– 5746. 52. Riou G, Le MG, Travagli JP, Levine AJ, Moll UM. Poor prognosis of p53 gene mutation and nuclear overexpression of p53 protein in inflammatory breast carcinoma. J Natl Cancer Inst. 1993;85:1765–1767. 53. Lowe SW, Bodis S, McClatchey A, et al. p53 status and the efficacy of cancer therapy in vivo. Science. 1994;266: 807– 810. 54. Fan S, Smith ML, Rivet DJ, et al. Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res. 1995;55:1649 –1654. 55. McIlwrath AJ, Vasey PA, Ross GM, Brown R. Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity. Cancer Res. 1994;54:3718 –3722. 56. O’Connor PM, Jackman J, Bae I, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res. 1997;57:4285– 4300. 57. Beck C, Cayeux S, Lupton SD, Dorken B, Blankenstein T. The thymidine kinase/ganciclovir-mediated “suicide” effect is variable in different tumor cells. Hum Gene Ther. 1995;6:1525–1530. 58. Sturtz FG, Waddell K, Shulok J, et al. Variable efficiency of the thymidine kinase/ganciclovir system in human glioblastoma cell lines: implications for gene therapy. Hum Gene Ther. 1997;8:1945–1953. 59. Denning C, Pitts JD. Bystander effects of different enzymeprodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes. Hum Gene Ther. 1997;8:1825–1835. 60. Wosikowski K, Regis JT, Robey RW, et al. Normal p53 status and function despite the development of drug resistance in human breast cancer cells. Cell Growth Differ. 1995;6:1395–1403. 61. Nigro JM, Baker SJ, Preisinger AC, et al. Mutations in the p53 gene occur in diverse human tumour types. Nature. 1989;342:705–708. 62. Celis A, Celis JE. General procedures for tissue culture. In: JE Celis, ed. Cell Biology: A Laboratory Handbook. San Diego: Academic Press; 1994. 63. Zhang WW, Alemany R, Wang J, Koch PE, Ordonez NG, Roth JA. Safety evaluation of Ad5CMV-p53 in vitro and in vivo. Hum Gene Ther. 1995;6:155–164. 64. Li P, Bui T, Gray D, Klamut HJ. Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53. Breast Cancer Res Treat. 1998;48:273–286. 65. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1989. 66. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983;132:6 –13. 190 LI, NGO, BRADE, ET AL: AdHSV-TK/GCV TREATMENT OF BREAST CANCER CELLS 67. Brezden CB, Rauth AM. Differential cell death in immortalized and non-immortalized cells at confluency. Oncogene. 1996;12:201–206. 68. Harlow E, Lane D. Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1988. 69. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991;51:6304 – 6311. 70. Lane DP. Cancer: p53, guardian of the genome. Nature. 1992;358:15–16. 71. Ogretmen B, Safa AR. Expression of the mutated p53 tumor suppressor protein and its molecular and biochemical characterization in multidrug-resistant MCF-7/Adr human breast cancer cells. Oncogene. 1997;14:499 –506. 72. Wosikowski K, Regis JT, Robey RW, et al. Normal p53 status and function despite the development of drug resistance in human breast cancer cells. Cell Growth Differ. 1995;6:1395–1403. 73. Elstner E, Linker-Israeli M, Said J, et al. 20-epi-vitamin D3 analogues: a novel class of potent inhibitors of proliferation and inducers of differentiation of human breast cancer cell lines. Cancer Res. 1995;55:2822–2830. 74. Zapata JM, Krajewska M, Krajewski S, et al. Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors. Breast Cancer Res Treat. 1998;47:129 –140. 75. O’Connor PM, Jackman J, Jondle D, Bhatia K, Magrath I, Kohn KW. Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of Burkitt’s lymphoma cell lines. Cancer Res. 1993;53:4776 – 4780. 76. Zhan Q, Fan S, Bae I, et al. Induction of bax by genotoxic stress in human cells correlates with normal p53 status and apoptosis [published erratum appears in Oncogene. 1995; 10:1259]. Oncogene 1994;9:3743–3751. 77. Fairchild CR, Ivy SP, Kao-Shan CS, et al. Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells. Cancer Res. 1987;47:5141–5148. 78. Sinha BK, Haim N, Dusre L, Kerrigan D, Pommier Y. DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action. Cancer Res. 1988;48: 5096 –5100. 79. Alaoui-Jamali MA, Batist G, Lehnert S. Radiation-induced damage to DNA in drug- and radiation-resistant sublines of a human breast cancer cell line. Radiat Res. 1992;129:37– 42. 80. Takenaga K, Tagawa M, Sakiyama S. Therapeutic potency of transduction with herpes simplex virus thymidine kinase gene against multidrug-resistant mouse leukemia cells. Anticancer Res. 1996;16:681– 685. 81. Smythe WR, Hwang HC, Elshami AA, Amin KM, Albelda SM, Kaiser LR. Differential sensitivity of thoracic malignant tumors to adenovirus-mediated drug sensitization gene therapy. J Thorac Cardiovasc Surg. 1995;109:626 – 630. 82. Fang X, Parkinson FE, Mowles DA, Young JD, Cass CE. Functional characterization of a recombinant sodiumdependent nucleoside transporter with selectivity for py- 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. rimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells. Biochem J. 1996;317:457– 465. Belt JA, Marina NM, Phelps DA, Crawford CR. Nucleoside transport in normal and neoplastic cells. Adv Enzyme Regul. 1993;33:235–252. Hooper ML, Subak-Sharpe JH. Metabolic cooperation between cells. Int Rev Cytol. 1981;69:45–104. Patterson AV, Zhang H, Moghaddam A, et al. Increased sensitivity to the prodrug 59-deoxy-5-fluorouridine and modulation of 5-fluoro-29-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase. Br J Cancer. 1995;72:669 – 675. Chen L, Waxman DJ, Chen D, Kufe DW. Sensitization of human breast cancer cells to cyclophosphamide and ifosfamide by transfer of a liver cytochrome P450 gene. Cancer Res. 1996;56:1331–1340. Reed JC, Miyashita T, Takayama S, et al. BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem. 1996;60:23–32. Silvestrini R, Benini E, Veneroni S, et al. p53 and bcl-2 expression correlates with clinical outcome in a series of node-positive breast cancer patients. J Clin Oncol. 1996; 14:1604 –1610. Chiou SK, Rao L, White E. Bcl-2 blocks p53-dependent apoptosis [published erratum appears in Mol Cell Biol. 1994;14:4333]. Mol Cell Biol. 1994;14:2556 –2563. Strasser A, Harris AW, Jacks T, Cory S. DNA damage can induce apoptosis in proliferating lymphoid cells via p53independent mechanisms inhibitable by Bcl-2. Cell. 1994; 79:329 –339. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609 – 619. Cheng EH, Levine B, Boise LH, Thompson CB, Hardwick JM. Bax-independent inhibition of apoptosis by Bcl-xL. Nature. 1996;379:554 –556. Takayama S, Sato T, Krajewski S, et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell. 1995;80:279 –284. Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995; 80:285–291. Minn AJ, Boise LH, Thompson CB. Bcl-xS antagonizes the protective effects of Bcl-xL. J Biol Chem. 1996;271:6306 – 6312. Kozopas KM, Yang T, Buchan HL, Zhou P, Craig RW. MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci USA. 1993;90:3516 –3520. Zhan Q, Bieszczad CK, Bae I, Fornace AJ, Jr, Craig RW. Induction of BCL2 family member MCL1 as an early response to DNA damage. Oncogene. 1997;14:1031–1039. Ogretmen B, Safa AR. Down-regulation of apoptosisrelated bcl-2 but not bcl-xL or bax proteins in multidrugresistant MCF-7/Adr human breast cancer cells. Int J Cancer. 1996;67:608 – 614. Cancer Gene Therapy, Vol 6, No 2, 1999