Sensitization of a Human Ovarian Cancer Cell Line to Temozolomide by Simultaneous Attenuation of the Bcl-2 Antiapoptotic Protein

Molecular Cancer Therapeutics 1215

Sensitization of a human ovarian cancer cell line to temozolomide by simultaneous attenuation of the Bcl-2 antiapoptotic protein and DNA repair by O6-alkylguanine-DNA alkyltransferase

Vincent A. Barvaux,1 Paul Lorigan,2 Thus, growth of cells treated only with temozolomide Malcolm Ranson,2 Amanda M. Gillum,3 (5 Mg/mL) was 91% of control growth, whereas additional R. Stanley McElhinney,4 T. Brian H. McMurry,4 exposure to PaTrin-2 alone (10 Mmol/L) or oblimersen and Geoffrey P. Margison1 alone (33 nmol/L) reduced this to 81% and 66%, respectively, and the combination of PaTrin-2 (10 Mmol/L) and 1 Paterson Institute for Cancer Research, Manchester, United oblimersen (33 nmol/L) reduced growth to 25% of con- Kingdom; 2Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom; 3Genta Inc., Berkeley Heights, New trol. These results suggest that targeting both Bcl-2 Jersey; and 4Trinity College, Dublin, Ireland with oblimersen and MGMT with PaTrin-2 would mark- edly enhance the antitumor activity of temozolomide and merits testing in clinical trials. [Mol Cancer Ther Abstract 2004;3(10):1215–20] Temozolomide is an alkylating agent that mediates its cytotoxic effects via O 6-methylguanine (O 6-meG) adducts in DNA. O6-alkylguanine-DNA-alkyltransferase Introduction (MGMT) can repair such adducts and therefore consti- Temozolomide is an alkylating agent that mediates its tutes a major resistance mechanism to the drug. MGMT cytotoxic effect by forming O6-methylguanine (O6-meG) activity can be attenuated in vitro and in vivo by the DNA adducts, which during DNA replication pair 6 pseudosubstrate O -(4-bromothenyl)guanine (PaTrin-2, preferentially with thymidine. These O6-meG:T mispairs Patrin, Lomeguatrib), which in clinical trials is in can result in a G-to-A point mutation during a subse- combination with temozolomide. Resistance to cytotoxic quent round of DNA replication but are also substrates agents can also be mediated by the Bcl-2 protein, which for the postreplication mismatch repair pathway, which inhibits apoptosis and is frequently up-regulated in tumor after a further round of DNA replication leads to cells. Attenuation of Bcl-2 expression can be affected by apoptosis (1). O6-meG DNA adducts can be repaired by treatment of cells with the antisense oligonucleotide, the DNA repair protein O6-alkylguanine-DNA alkyltrans- oblimersen sodium (Genasense), currently in phase III ferase (MGMT), which removes adducts from the O6 clinical trials in combination with the methylating agent position of guanine by accepting them onto a cysteine dacarbazine. Using a human ovarian cancer cell line residue within its active site. Furthermore, fibroblasts and (A2780) that expresses both Bcl-2 and MGMT, we show bone marrow cells of MGMT knockout mice are signifi- that cells treated with active dose levels of either cantly more sensitive to the toxic effects of temozolomide oblimersen (but not control reverse sequence or mis- than those from MGMT wild-type mice (2). The protective match oligonucleotides) or PaTrin-2 are substantially role of MGMT against the cytotoxic effect of temozolomide sensitized to temozolomide. Furthermore, the exposure has been shown in human cell lines (3) and human of oblimersen-pretreated cells to PaTrin-2 leads to an xenograft models (4). even greater sensitization of these cells to temozolomide. MGMT can be inactivated by free guanine base deriva- tives that have alkyl groups at the O6 position, which act as ‘‘pseudosubstrates.’’ O6-benzylguanine (5) and O6-(4-bromothenyl)guanine (PaTrin-2, Patrin, Lomeguatrib, KuDOS, Cambridge, United Kingdom; ref. 6) have been identified Received 3/18/04; revised 7/21/04; accepted 8/11/04. as the most promising MGMT inactivators. Compared with Grant support: European Union Marie Curie Individual Fellowship (proposal 2000-02021), Bourse post-doctorale de recherche scientifique temozolomide used as a single agent, the combination (Mai 2000) of the Universite´Catholique de Louvain (Belgium), and Cancer PaTrin-2-temozolomide has been shown to significantly Research UK. increase tumor growth inhibition in human melanoma The costs of publication of this article were defrayed in part by the 6 payment of page charges. This article must therefore be hereby marked xenografts (7). PaTrin-2 and O -benzylguanine have re- advertisement in accordance with 18 U.S.C. Section 1734 solely to cently entered phase I/II clinical trials (8, 9). indicate this fact. The Bcl-2 family of proteins regulates the mitochondria- Requests for reprints: Geoffrey P. Margison, Paterson Institute for Cancer mediated apoptosis pathway, which includes both apo- Research, Wilmslow Road, Manchester M204BX, United Kingdom. Phone: 44-161-446-3183; Fax: 44-161-446-3109. ptosis-suppressing (e.g., Bcl-2) and apoptosis-inducing E-mail: [email protected] (e.g., Bax) proteins. Overexpression of Bcl-2 occurs Copyright C 2004 American Association for Cancer Research. frequently in human cancers (10) and is associated with

Mol Cancer Ther 2004;3(10). October 2004

tumor cell resistance to chemotherapeutic agents by Oligonucleotides preventing the apoptotic response normally induced by The following fully phosphorothioated oligonucleotides those agents (11). Oblimersen sodium (Genasense, Aventis/ were provided by Genta: oblimersen sodium (5V- Genta, Berkeley Heights, NJ) is a phosphorothioated anti- TCTCCCAGCGTGCGCCAT-3V), G3622 reversed sequence sense oligonucleotide complementary to the first six codons control (5V-TACCGCGTGCGACCCTCT-3V), and G4126 2-bp of the Bcl-2 mRNA sequence. Hybridization prevents mismatchedcontrol(5V-TCTCCCAGCATGTGCCAT-3V). translation of the mRNA by blocking ribosome binding or MGMT Assay progression, and the hybrid molecule is recognized by The preparation of cell extracts for MGMT assay, the RNase H, which degrades only the RNA portion, sparing MGMT assay itself, and protein estimation were performed the antisense oligonucleotide (12). Thus, oblimersen treat- following the protocols described by Watson and Margison ment decreases the expression of the Bcl-2 protein, an (14). Briefly, cell-free sonicates were incubated with 3H- activity that has been shown in human cancer cells and methylated DNA substrate under protein-limiting condi- tumor xenografts and in patients with hematologic or tions until the transfer reaction was complete. Excess solid malignancies (13). A randomized phase III trial substrate DNA was hydrolyzed to acid solubility, and comparing the standard therapy with the methylating radioactivity in the residual protein was measured by agent dacarbazine (DTIC) with DTIC plus oblimersen in liquid scintillation counting. patients with advanced metastatic melanoma has recently Inhibition of MGMT Activity by PaTrin-2 completed accrual. Furthermore, several studies are cur- The ability of PaTrin-2 to deplete MGMT activity in rently under way, examining oblimersen as a treatment A2780 cells was determined as follows: on day 1, an equal for a variety of cancers in addition to melanoma, and it is number (2 106) of A2780 cells was seeded in a series of hoped that, through the specific targeting of Bcl-2, T75 flasks containing 10 mL RPMI 1640 supplemented with oblimersen will avoid the undesirable side effects of 10% FCS, 200 mmol/L L-glutamine, and 10 Amol/L PaTrin- conventional chemotherapy. 2. After 2, 24, 48, 72, 96, and 120 hours, cells were rinsed Given the roles of MGMT and Bcl-2 in resistance to the twice with 10 mL EDTA (0.54 mmol/L, catalogue no. alkylating agent temozolomide, we firstly set out to 100935V, BDH, Glasgow, United Kingdom), trypsinized examine if attenuation of MGMT by PaTrin-2 or of Bcl-2 [2 mL trypsin-EDTA solution (catalogue no. T3924, Sigma by oblimersen could lead to sensitization to the cytotoxic Chemical) per flask for 2 minutes at 37jC], and removed effect of temozolomide with the human ovarian cancer cell from the culture flasks on dilution with 10 volumes of line A2780 that expresses both MGMT and Bcl-2. We then medium. The resulting cell suspension was centrifuged asked if the combination of both PaTrin-2 and oblimersen at 150 g for 5 minutes. The pellet was then washed once could further enhance the toxicity of the alkylating agent in in culture medium to remove excess trypsin, recentrifuged, this cell line. and processed (as described above) for MGMT assay. We show that attenuation of MGMT or Bcl-2 can be On day 5, the medium was removed and replaced with obtained in A2780 cells after treatment with PaTrin-2 or medium that did not contain PaTrin-2. Cells were harvest- oblimersen, respectively. We also show that A2780 cells ed for MGMT assay on days 6 to 10. treated with either PaTrin-2 or oblimersen are substantially Effect of Oblimersen on Bcl-2 Expression in A2780 sensitized to temozolomide, whereas the control reverse Cells sequence or mismatch oligonucleotides are ineffective. An equal number (0.5 106) of A2780 cells was seeded in Furthermore, the incubation of oblimersen-pretreated cells T75 flasks containing 10 mL RPMI 1640 supplemented with with PaTrin-2 leads to an even greater sensitization of these 10% FCS and 200 mmol/L L-glutamine on day 1. On day 2, cells to temozolomide. cells were rinsed twice with 10 mL 0.54 mmol/L EDTA and the medium was changed to one containing 17 or 33 nmol/L Materials and Methods oblimersen and 15 or 30 Amol/L jetPEI for oligonucleotides transfection reagent (catalogue no. GDSP10130, Cell Line Qbiogene, Carlsbad, CA), respectively, or the transfection A2780 cells (kindly provided by Dr. Robert Brown, Beatson Laboratories, Glasgow, United Kingdom) were reagent alone as negative control. This was repeated on cultured in T75 flasks (catalogue no. 353135, Falcon, days 3 to 6 (following the clinical trial protocol for the Becton Dickinson, Franklin Lakes, NJ) containing 10 mL phase III melanoma study). On day 7, the culture medium RPMI 1640 (catalogue no. E15-039, PAA, Pasching, was removed and cell pellets were obtained as described Austria) supplemented with 10% FCS (catalogue no. above. Cells were fixed by the addition of 70% ethanol and A15-043, PAA, Pasching, Austria) and 200 mmol/L L- left overnight at room temperature. Ethanol was then glutamine (catalogue no. G-5763, Sigma Chemical Co., St. removed, the samples were embedded in paraffin wax, and Louis, MO). A2780 cells were screened on a weekly basis 4 Am sections were cut and mounted on slides. Fixed cells throughout the experiment for Mycoplasma by PCR were immunostained for Bcl-2 using an immunoperoxidase (VenorGeM PCR-Mycoplasma Detection Kit, protocol procedure as described previously (15): monoclonal anti- detailed in the user manual, pp. 9–12) and always bodies against human Bcl-2 (sc-7382, Santa Cruz Biotech- proven to be contamination free. nology, Santa Cruz, CA) were used as the primary

Mol Cancer Ther 2004;3(10). October 2004

antibody. The secondary antibody was a biotinylated rabbit treated cells and 0–100 Ag/mL DMSO for the vehicle- anti-mouse (catalogue no. E0413, DAKO, Carpinteria, CA), treated cells). On day 11, cells were rinsed twice with 10 mL which was then followed by streptavidin–horseradish 0.54 mmol/L EDTA, harvested, and counted. An equal 3 peroxidase incubation (StreptABComplex/HRP, catalogue number (10 ) of cells was seeded in Petri dishes (catalogue no. 353004, Falcon, three dishes per experiment, i.e., 90 no. K0337, DAKO). The samples were counterstained with dishes in total) with 5 mL medium (no drug addition). On hematoxylin, dehydrated, mounted, and examined using day 18, the medium was removed and 5 mL gentian violet optical microscopy. [10% w/v methyl violet 2B (Sigma-Aldrich Co. Ltd., Poole, Effect of Oblimersen, Reversed Sequence, and 2-bp United Kingdom) in 70% ethanol (Prolabo, Manchester, Mismatch Control Oligonucleotides on Sensitization of United Kingdom)] was added to each dish for 5 minutes A2780 Cells toTemozolomide 6 (cell staining). The dye was then poured off; the plates were An equal number (0.5 10 ) of A2780 cells was seeded rinsed with tap water, inverted, and allowed to dry; and in T75 flasks containing 10 mL RPMI 1640 supplemented colonies were counted. with 10% FCS and 200 mmol/L L-glutamine on day 1. On Statistical Analysis day 2, cells were rinsed twice with 10 mL 0.54 mmol/L In each experiment and for each concentration of EDTA and the medium was changed to one containing temozolomide, a series of nine absorbance data measure- 33 nmol/L oblimersen or 33 nmol/L reverse sequence con- ments (MTT assay) or three cell colony counts (clonogenic trol oligonucleotide or 33 nmol/L 2-bp mismatch control assay) was obtained. The mean was taken and expressed oligonucleotide and 30 Amol/L jetPEI transfection reagent as a percentage of the appropriate control. Each set of or the transfection reagent alone as negative control. This experimental data was compared using Student’s t test. was repeated on days 3 to 6. On day 7, an equal number (250) of vehicle-treated or oligonucleotide-treated A2780 Results cells was seeded in 96-well plates (catalogue no. 353072, Effect of PaTrin-2 on MGMT Activity in A2780 Cells Falcon) with 100 AL medium. On day 8, 100 AL medium The MGMT activity of A2780 cells was 447.2 F 45.3 containing temozolomide at increasing concentrations (0–30 fmol/mg protein. The MGMT activity became undetectable Ag/mL DMSO, catalogue no. D2650, Sigma Chemical) was 2 hours after the addition of PaTrin-2 and remained added to each well. On day 14, 50 AL 3-(4,5-dimethylth- undetectable for a total of 7 days. When the medium was iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 3 mg/ replaced with medium not containing PaTrin-2, the MGMT mL H O, catalogue no. M5655, Sigma Chemical) was 2 activity remained undetectable for 48 hours. Five days after added to the cells and incubated at 37jC for 3 hours. Each medium replacement, the MGMT activity of the cells had well was then emptied, 200 AL DMSO was added, and after recovered but only to 53% of its initial value (Fig. 1). 10 minutes the plates were read at 540 and 690 nm on a Effect of Oblimersen on Bcl-2 Expression in A2780 multiscan plate reader. Cells Cell Growth Assays Immunocytochemical analysis of untreated cells showed MTT Assay. Cells were seeded in T75 flasks and treated extensive expression of Bcl-2 in the majority of the A2780 with oblimersen and jetPEI transfection reagent or the cells. As expected, expression was predominantly cytoplas- transfection reagent alone as described above. On day 7, an mic. After 5 days of 17 or 33 nmol/L oblimersen treatment, equal number (250) of vehicle-treated or oblimersen-treated expression of Bcl-2 in the cells was extensively decreased, (17 or 33 nmol/L) cells were seeded in 96-well plates, with 100 AL medium containing 10 Amol/L PaTrin-2 or the equivalent concentration of the vehicle (0.05% DMSO) as negative control. On day 8, 100 AL medium containing temozolomide at increasing concentrations (0–10 Ag/mL DMSO) was added to each well. On day 14, the cells were processed as above. Clonogenic Assay. The clonogenic assay was performed following the protocol described by Plumb (16). Cells were seeded in T75 flasks and treated with oblimersen and jetPEI transfection reagent or the transfection reagent alone as described above. On day 7, an equal number (8 104)of vehicle-treated or oblimersen-treated (17 or 33 nmol/L) cells was seeded in T25 flasks (catalogue no. 353014, Falcon), with 5 mL medium containing 10 Amol/L PaTrin-2 or the equivalent concentration of the vehicle as negative control. On day 9, the medium was replaced by Figure 1. Effect of PaTrin-2 on MGMT activities in A2780 cells. MGMT one containing 10 Amol/L PaTrin-2 or the equivalent activity was measured before the addition of 10 Amol/L PaTrin-2 and 2, 24, 48, 72, 96, and 120 hours after the addition of 10 Amol/L PaTrin-2. concentration of the vehicle and temozolomide at increas- Cell culture medium was changed on day 5 (arrow) for one not containing ing concentrations (0–10 Ag/mL DMSO for the oblimersen- PaTrin-2. MGMT activity was measured on days 6 to 10.

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Figure 2. Representative photomicrographs of Bcl-2-stained A2780 cells before oblimersen (A) and after 5-day exposure to 17 (B)or33(C) nmol/L oblimersen.

the effect being greater with the higher dose (Fig. 2). In further enhanced the sensitivity of A2780 cells to temozo- these experiments, residual faint cytoplasmic staining lomide (P = 0.01; Figs. 4 and 5; Table 1). It is noteworthy could be seen, but in the majority of the cells, no staining that PaTrin-2 given alone did not affect cell survival unlike was evident. 17 or 33 nmol/L oblimersen given alone, which reduced Effect of Oblimersen and Control Oligonucleotides cell survival to 66.4 F 1.5% or 40.8 F 1.7%, respectively, on Sensitization of A2780 Cells toTemozolomide compared with controls. A2780 cells were significantly more sensitive (P = 0.003) to the growth inhibition effect of increasing concentrations Discussion of temozolomide after 5 days of treatment with 33 nmol/L Our hypothesis was that simultaneous MGMT and Bcl-2 oblimersen compared with the reverse or mismatch control attenuation would increase temozolomide sensitivity in oligonucleotides. The sensitivity of A2780 cells to temozo- the ovarian cancer cell line A2780. To test this, we firstly lomide after treatment with the reverse or mismatch control showed that PaTrin-2 would inactivate MGMT and oligonucleotides was not significantly different from the determined that the level of inactivation would remain vehicle control (P = 0.9; Fig. 3; Table 1). adequate during the experiment. Having achieved this, Effect of Combined Oblimersen and PaTrin-2 on Sen- we then showed, as anticipated, that MGMT inactivation sitization of A2780 Cells toTemozolomide PaTrin-2-treated or oblimersen-treated A2780 cells were Table 1. Effect of PaTrin-2, oblimersen, reverse, and mismatch significantly more sensitive (P = 0.02) to the cytotoxic effect oligonucleotides on sensitization of A2780 cells to temozolomide of temozolomide than the control cells (Figs. 4 and 5; (5 Ag/mL) Table 1). The combination of oblimersen and PaTrin-2 MTT-Based Assay Clonogenic Assay

Cell Growth SD Cell Survival SD (% of Control) (%) (% of Control) (%)

Control 91.9 2.6 92.3 (E) ND 33 nmol/L reverse 90.9 0.6 91.1 (E) ND oligonucleotide 33 nmol/L mismatch 89.7 1.8 89.9 (E) ND oligonucleotide 10 Amol/L 81.3 1.3 81.5 (E) ND PaTrin-2 17 nmol/L 78.4 2.3 78.3 0.8 oblimersen 17 nmol/L oblimersen 57.1 2.4 57.3 0.9 +10Amol/L PaTrin-2 33 nmol/L oblimersen 66.3 1.5 64.7 1.4 Figure 3. Effect of oblimersen, reverse, and mismatch oligonucleotides 33 nmol/L oblimersen 25.1 1.6 24.5 0.5 on sensitization of A2780 cells to temozolomide (values obtained from +10Amol/L PaTrin-2 a MTT-based assay). A2780 cells were treated for 5 days, prior to temozolomide treatment, with 33 nmol/L oblimersen (n), reverse sequence NOTE: Values were obtained from the MTT-based and clonogenic assays oligonucleotide (4; dotted line), or 2-bp mismatch oligonucleotide (o) and shown in Figs. 3 to 5. Cell survival is expressed as percentage of control. 30 Amol/L jetPEI transfection reagent or the transfection reagent alone (E). The lowest dose of temozolomide in those experimental conditions was Points, percentage cell growth of the group control; bars, 1 SD. 10 Ag/mL. ND, not determined. E, extrapolated values.

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An important caveat to be addressed when considering the use of combined therapies in clinical trials is the potential for additional toxicity in normal tissues. PaTrin-2 depletes MGMT not only in tumors but also in other tissues such as the bone marrow. The cytotoxic effect of temozolomide combined with PaTrin-2 is therefore enhanced in tumors, but myelosuppression is also increased, forcing a modest reduction of the dose of the alkylating agent in phase I studies. Strategies to protect the bone marrow against such combinations are now being considered. They involve the transduction of hematopoietic stem cell with a pseudosubstrate-resistant mutant MGMT gene, so that tumor sensitization, by inactivation of endogeneous MGMT, and bone marrow protection can be achieved simultaneous- Figure 4. Effect of PaTrin-2 and oblimersen on sensitization of A2780 cells to temozolomide (values obtained from a MTT-based assay). A2780 ly even in the presence of a MGMT inactivator (20). cells (n); A2780 cells + 10 Amol/L PaTrin-2 (5); A2780 cells + 17 nmol/L Oblimersen has undergone clinical study as a single oblimersen (.); A2780 cells + 10 Amol/L PaTrin-2 + 17 nmol/L agent. Dose-limiting toxicities were seen (at doses >3 mg/ o oblimersen ( ); A2780 cells + 33 nmol/L oblimersen (E); A2780 cells kg/d) in patients with non-Hodgkin’s lymphoma and +10Amol/L PaTrin-2 + 33 nmol/L oblimersen (4). Points, percentage cell growth of the group control; bars, 1 SD. chronic lymphocytic leukemia that consisted of fever, thrombocytopenia, fatigue, and hypotension. However, these tumor types seem to be the only ones in which this increases the sensitivity of A2780 cells to temozolomide. toxicity is shown (21–23). Toxicities induced by the This increase was not extensive, which may have been a combination of fludarabine, cytarabine, and filgrastim with consequence of the high levels of expression of Bcl-2 in oblimersen in refractory or relapsed myeloid leukemias these cells. We further showed that oblimersen treatment were not dose limiting and were not clearly attributable attenuated Bcl-2 expression in A2780 cells and that this to oblimersen (24). This was also shown in patients with resulted in an enhanced sensitivity of the cells to temozo- chemorefractory small cell lung cancer treated with the lomide. This effect, which was not observed with either of combination of oblimersen and paclitaxel (25). Further- the control oligonucleotides, was even noted at the lowest more, toxic side effects observed in patients with advanced dose of oblimersen used and the sensitivity of cells was malignant melanoma after treatment with the methylating increased further by increasing the oblimersen dose. This agent DTIC and oblimersen have been shown to be may be related to the observation that Bcl-2 staining was comparable with those observed with single-agent DTIC visibly less intense after 33 nmol/L oblimersen compared therapy (26). The lack of increased toxicity when obli- with 17 nmol/L oblimersen, indicating a greater extent of mersen is used as part of a combination therapeutic ablation. Finally, we observed using both growth inhibition regimen is promising and may be a consequence of the and clonogenic survival assays that the combination of relatively low levels of expression of Bcl-2 in normal cells. oblimersen and PaTrin-2 resulted in a further enhancement of the sensitivity of A2780 cells to temozolomide. The analysis of the data for oblimersen and temozolomide has revealed an interaction between the two drugs. This would suggest that O6-meG DNA adducts induced by temozolomide lead the cell to death via the Bcl-2-controlled mitochondria-mediated apoptosis pathway. Oblimersen, by attenuating the expression of Bcl-2, renders the cell more susceptible to O6-meG-triggered apoptosis, thus enhancing the effect of temozolomide. The key role played by Bcl-2 in the regulation of apoptosis induced by O6-meG DNA adducts has been shown in mammalian cells (17). On the other hand, recent evidence suggests that O6-meG DNA adducts trigger apoptosis in human peripheral lymphocytes by activating the receptor-mediated apoptosis pathway independently of Bcl-2 (18). However, because Bcl-2 Figure 5. Effect of PaTrin-2 and oblimersen on sensitization of A2780 and MGMT are frequently overexpressed in human cancers cells to temozolomide (values obtained from a clonogenic assay). A2780 (10, 19) and are associated with tumor cell resistance to cells (n); A2780 cells + 10 Amol/L PaTrin-2 (5); A2780 cells + 17 nmol/L chemotherapeutic agents, it is reasonable to suggest that oblimersen (.); A2780 cells + 10 Amol/L PaTrin-2 + 17 nmol/L o oblimersen and PaTrin-2 may be a useful combination to oblimersen ( ); A2780 cells + 33 nmol/L oblimersen (E); A2780 cells +10Amol/L PaTrin-2 + 33 nmol/L oblimersen (4; dotted lines) extrap- explore in clinical studies involving alkylating agents such olation of curves. Points, percentage cell survival of the group control; as temozolomide or DTIC. bars, 1 SD.

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Downloaded from mct.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. Sensitization of a human ovarian cancer cell line to temozolomide by simultaneous attenuation of the Bcl-2 antiapoptotic protein and DNA repair by O6 -alkylguanine-DNA alkyltransferase

Vincent A. Barvaux, Paul Lorigan, Malcolm Ranson, et al.

Mol Cancer Ther 2004;3:1215-1220.

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