DNA Repair Enzyme, O-Methylguanine DNA Methyltransferase, Modulates Cytotoxicity of Camptothecin- Derived Topoisomerase I Inhibitors

Two camptothecin-resistant cell lines, CPT30 and KB100, were established and characterized previously in our laboratory. Because enhanced sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and decreased expression of O-methylguanine-DNA methyltransferase (MGMT) protein were observed in these lines, we hypothesized that MGMT may be a determinant of cytotoxicity associated with camptothecin-derived DNA topoisomerase I inhibitors (CPTs). We used the Tet-On system to induce expression of MGMT in Chinese hamster ovary (CHO) cells and RNA interference to knock down MGMT expression in human nasopharyngeal carcinoma HONE-1 cells in order to identify any correlations between MGMT expression and CPTs cytotoxicity. CHO-derived Tet-On-inducible cells (S12 ) showed MGMT overexpression and statistically significant more resistance to BCNU, camptothecin, 7-ethyl-10-hydrocamptothecin (SN38), and topotecan than parental CHO cells (p 0.05), but there was less resistance to CPTs than to BCNU. Knockdown of MGMT expression with small interfering RNA in HONE-1 cells conferred increased sensitivity to BCNU and CPTs compared with mock control. Furthermore, alteration of MGMT expression coincides with CPT-induced cell death and poly(ADP-ribose) polymerase cleavage. There were no differences in protein levels and catalytic activity of topoisomerase I between MGMT-proficient and MGMT-deficient cells from the Tet-On-inducible and small interfering RNA (siRNA) systems. Resistance to CPTs coincided with decreased amounts of protein-linked DNA breaks generated by CPTs in MGMT-proficient cells and vice versa in MGMT-deficient cells. Our data indicate that MGMT can modulate cytotoxicity of CPT-derived topoisomerase I inhibitors. Topoisomerase I (Top I) plays an essential role in controlling DNA supercoiling and relieving torsional stress that is generated during replication, transcription, recombination, and chromatin remodeling (Lee et al., 1993; Wang, 1996). Top I introduces transient single-stranded DNA breaks in one of the phosphodiester backbones of the duplex DNA, resulting in a reversible Top I/DNA covalent complex (Champoux, 1976). Under normal conditions, religation of the DNA cleavage/religation equilibrium is favored and only a small fraction of DNA is cleaved at any given time. Top I is the primary intracellular target of camptothecin (CPT), a plant alkaloid that was isolated from Camptotheca accumulata. Water-soluble derivatives including irinotecan (CPT-11) and topotecan (TPT) have been widely used in clinical practice for various solid tumors (Gore et al., 2001; Vanhoefer et al., 2001). The cytotoxic mechanism of CPT-derived Top I inhibitors (CPTs) involves at least two successive steps. First, the drug stabilizes the covalent enzyme-DNA intermediate through inhibition of religation without affecting the cleavage reaction (Hsiang et al., 1985). Second, cleavable complexes are converted to DNA double-stranded breaks by interaction with moving replication forks, and these DNA breaks are responsible for observed CPTs cytotoxicity (Hsiang et al., 1989; Zhang et al., 1990). This work was supported in part by grants from National Health Research Institutes (NHRI), Taipei, Taiwan (NHRI intramural Grant 92A1-CAPP06-1), and the National Science Council, Taipei, Taiwan (NSC 94-2752-B-400-001PAE). These data were presented in abstract at the American Association for Cancer Research 94th Annual Meeting, July 11–14, 2003, in Washington, D.C. (Abstract No. 3706). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.095919. ABBREVIATIONS: Top I, topoisomerase I; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; CHO, Chinese hamster ovary; CPT, camptothecin; CPTs, CPT-derived topoisomerase I inhibitor(s); Dox, doxycycline; MGMT, O-methylguanine-DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; -MEM, -minimal essential medium; PI, propidium iodide; PLDB, protein-linked DNA break(s); S12, a tetracycline-regulatory MGMT expressing clone derived from CHO cells; S12 , S12 cells without doxycycline addition; S12 , S12 cells with 1 g/ml doxycycline addition; SN38, 7-ethyl-10-hydrocamptothecin; siRNA, small interfering RNA; TPT, topotecan; tTs, transcriptional silencer; Vec, vector. 0022-3565/06/3162-946–954$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 95919/3076506 JPET 316:946–954, 2006 Printed in U.S.A. 946 at A PE T Jornals on A ril 4, 2017 jpet.asjournals.org D ow nladed from The efficacy of CPTs as cancer therapy is explained only in part by their ability to damage DNA. Although Top I is the primary target of CPTs, a lack of correlation has been demonstrated among Top I levels, CPT-induced protein-linked DNA breaks (PLDB), and CPT cytotoxicity (Eng et al., 1988; Nitiss and Wang, 1988; Hsiang et al., 1989). Furthermore, several lines of evidence have shown that a variety of DNA alterations originating from endogenous and exogenous sources (abasic sites, uracil misincorporations, nicks, oxidized bases, UV photolesions, and carcinogenic adducts) induce the formation of the Top I/DNA-cleavable complex (Pourquier and Pommier, 2001). The influence of defective pathways downstream from the cleavable complex may play a key role in selectivity of CPTs toward cancer cells (Pommier et al., 1994). We have established two CPT-resistant cell lines, CPT30 and KB100, from human nasopharyngeal carcinoma HONE-1 and cervical carcinoma KB cell lines, respectively (Beidler et al., 1996; Chang et al., 2002). We demonstrated that the single amino-acid mutation in E418K causes the observed qualitative changes in Top I that are responsible for CPT resistance in CPT30 cells (Chang et al., 2002). The mechanism underlying CPT resistance in KB100 cells is independent from Top I and involves steps subsequent to the formation of PLDB; it may be attributable to alteration of the poly(ADP-ribose) polymerase (PARP)-related DNA repair system (Beidler et al., 1996). It is interesting that CPT30 and KB100 cells are 4and 2-fold more sensitive, respectively, to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than their parental cells. The mechanism of enhanced sensitivity to BCNU in both KB100 and CPT30 cells involves down-regulation of the O-methylguanine-DNA methyltransferase (MGMT) gene (unpublished observation). In addition, previous studies have shown that cells resistant to BCNU exhibit cross-resistance to CPT (Yamauchi et al., 2003). These findings imply that MGMT may be involved in determining CPT cytotoxicity. In the current research, we used a Tet-On-inducible system to overexpress MGMT in Chinese hamster ovary (CHO) cells and a RNA interference method to silence MGMT expression in human nasopharyngeal carcinoma HONE-1 cells to determine any correlation between expression of MGMT and CPTs cytotoxicity. Materials and Methods Materials. CPT and BCNU were purchased from Sigma-Aldrich (St. Louis, MO). SN38 was provided by sanofi-aventis (Bridgewater, NJ). TPT was kindly supplied by GlaxoSmithKline (Welwyn Garden City, Hertfordshire, UK). Plasmids of pTet-On, pTet-tTs, and pTRE2hyg, as well as Tet-On system-approved fetal bovine serum, were purchased from Clontech (Mountain View, CA). Plasmid pBR322 was purchased from Invitrogen (Carlsbad, CA). Monoclonal anti-MGMT antibody (clone MT5.1) was purchased from BD PharMingen (San Diego, CA). Monoclonal anti-Top I antibody (clone 21) was kindly provided by Dr. Yung-Chi Cheng (Yale University, New Haven, CT). Monoclonal anti-PARP antibody was purchased from Trevigen (Gaithersburg, MD). Both G418 sulfate and hygromycin B were purchased from Calbiochem (La Jolla, CA). Cell culture reagents were obtained from Invitrogen. N-[H]-Methyl-N-nitrosurea was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other chemicals were from Merck Biosciences (Darmstadt, Germany) or Sigma-Aldrich and were of standard analytic grade or higher. Cell Culture. Human nasopharyngeal carcinoma HONE-1 cells and colorectal carcinoma HT29 cells were maintained in RPMI 1640 medium supplied with 5% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. CHO cells were maintained in -MEM supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Insertion of MGMT cDNA into Tet-On-Responsive Vector. Total RNA was extracted from human colorectal carcinoma HT29 cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RNA was reverse-transcribed using SuperScript II RNase H reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Human MGMT cDNA was amplified using the following primer pairs: forward primer, 5 -AAGGATCCCCGTTTGCGACTTGGTACTT-3 , and reverse primer, 5 -CGACGATATCAAGCGGCCGCCCGATGCAGTGTTACACG-3 . Polymerase chain reaction amplification was performed under the following conditions. Preincubation was performed at 94°C for 2 min followed by 30 cycles of 94 (30 s), 64 (45 s), and 72°C (1 min), with a final extension at 72°C for 7 min. The amplified 704-base pair product of MGMT cDNA was digested with BamH1 and NotI restriction enzymes and cloned into the pTRE2hyg vector at multiple cloning sites. The plasmid clones were checked by BamH1 and NotI cutting to contain the 0.7-kb fragment, and potential clones were confirmed by sequencing with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). After verification that the sequences were correct, the recombined plasmid MGMT-pTRE2hyg was grown in large scale and purified using plasmid DNA purification kit (Macherey-Nagel Inc., Easton, PA). Establishment of the MGMT Tet-On-Inducible Cell Line. To establish MGMT Tet-On-inducible cell lines, CHO cells were initially transfected with regulatory plasmids pTet-On and pTet-tTs using SuperFect reagent (QIAGEN Operon, Alameda, CA) according to the manufacturer’s instructions. The pTet-tTs plasmid contains the tetracycline-dependent transcriptional silencer (tTs), which binds the tetO-inducible promoter in the absence of doxycycline (Dox), and thus it can potentially reduce high-background activity on the Tet-Oninducible system. The day after transfection, cultures were given medium containing 0.8 mg/ml G418 sulfate. After 14 days, neomycinresistant clones were collected and combined to make a heterogeneous stable population. Responsive vector MGMT-pTRE2hyg was transfected next into this population to obtain Tet-On-regulated MGMT-expressing cells. Transfected cells were double-selected in medium containing 0.8 mg/ml G418 sulfate and 0.75 mg/ml hygromycin B for 2 weeks, and then various cell clones were isolated. Individual clones were expanded and then incubated with or without 1 g/ml Dox for 24 h. MGMT-inducible clones were detected by Western blot analysis. After screening, a stable transfectant of CHO cells expressing human MGMT was obtained and named CHOMGMT-S12. As a control, a mock-transfected CHO-Tet-On cell line named CHO-vector (Vec) (hereinafter referred to as vehicle control) was generated, which was stably transfected with pTet-On, pTet-tTs, and empty pTRE2hyg plasmids. These transfectants were maintained in -MEM supplemented with 5% Tet-On system-approved fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 400 g/ml G418 sulfate, and 400 g/ml hygromycin B. Silencing of MGMT by Small Interfering RNA Transfection. The siRNA sequence targeting MGMT corresponded to two separate coding regions: region 1, 5 -AAGCTGGAGCTGTCTGGTTGT-3 (nucleotides 52–71), and region 2, 5 -AAGGTTGTGAAATTCGGAGAA-3 (nucleotides 310–330). The sequences of both regions are unique to MGMT as indicated by a sequence search. The siRNA sequence targeting luciferase corresponded to the coding region 5 GCCATTCTATCCTCTAGAGGATG-3 , and it was used for mock control. Selected annealed RNA was synthesized by Ambion (Austin, TX). HONE-1 cells in exponential growth phase were plated in 6-cm plates at 5 10 cells/plate, grown for 24 h, and then transfected with MGMT siRNA duplex using RNAiFect transfection kit (QIAMGMT Mediates CPT Cytotoxicity 947 at A PE T Jornals on A ril 4, 2017 jpet.asjournals.org D ow nladed from GEN, Valencia, CA), according to the manufacturer’s instructions. The concentrations of siRNA were chosen based on dose-response studies. Silencing was examined approximately 12 to 60 h after transfection. In addition, control cells were transfected with luciferase siRNA duplex (mock). Growth Inhibition Assay. Cells in logarithmic growth phase were cultured at a density of 5000 cells/ml/well in 24-well plates. Cells were exposed to various concentrations of test drugs for three days. The methylene blue dye assay was used to evaluate the effect of test drugs on cell growth as has been described previously (Finlay et al., 1984). Clonogenic Survival Assay. Cells in logarithmic growth phase were cultured in six-well plates (250 cells/well) for 24 h. Cells were treated next with various concentrations of drugs for the indicated times. Cells were then washed with prewarmed phosphate-buffered saline twice and maintained in drug-free complete medium for 10 to 14 days. At the end of the incubation period, cells were fixed and stained with 50% ethanol containing 0.5% methylene blue for 30 min and then washed with water. The number and size of methylene blue-stained colonies were then recorded. The 50% lethal concentration (LC50) was defined as the drug concentration that inhibited colony formation by 50% compared with formation seen with a vehicle-treated control. Western Blot Analysis. Crude cellular extracts were prepared for Western blot analysis as described previously (Kuo et al., 2004). Detection of immunoreactive signals was accomplished with the Western Blot Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, MA). Band-specific intensity was quantitated using an AlphaImager 2000 system (Alpha Innotech, San Leandro, CA). MGMT Activity Assay. MGMT activity was measured by transfer of H-labeled methyl groups from the O-position of guanine in DNA to the MGMT protein as described previously (Myrnes et al., 1984). Cell extracts were prepared by sonication in MGMT assay buffer [50 mM Tris-HCl (pH 8.3), 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor mixture of 1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin, and 50 g/ml leupeptin] followed by centrifugation at 10,000g for 10 min. Extracts (400 g of protein) were supplemented with [H]DNA enriched for O-methylguanine (200 g; 5000 cpm) incubated for 60 min at 37°C. Reactions were quantitated after acid hydrolysis of DNA substrate, collection of protein precipitates, and radioactivity counting. Top I Catalytic Activity Assay. Top I DNA-catalytic activity using whole-cell lysates was measured by relaxation of supercoiled pBR322 plasmid DNA in vitro as described previously (Liu and Miller, 1981). The reaction mixtures were performed at 37°C for 30 min and terminated by adding 1% SDS and separated in a 1% agarose gel. Photographs of the resulting ethidium bromide-stained agarose gels were taken under UV light, and band intensities were quantitated for DNA relaxation activity using a densitometric scan. Measurement of PLDB by Potassium-SDS Coprecipitation Assay. DNA in logarithmically growing cells (2.5 10 cells/ml) was treated with 1 g/ml MGMT-targeted siRNA and labeled by adding [C]thymidine into medium to a final concentration of 0.5 Ci/ml. After a 24-h incubation, cells were trypsinized and resuspended in fresh medium at a density of 5 10 cells/ml. Cells were aliquoted (1 ml each) into a reaction tube and incubated for another 1 h at 37°C with gentle shaking. Cells were then treated with various concentrations of drugs for 30 min and collected and analyzed for PLDB by the potassium-SDS precipitation method, as described previously (Rowe et al., 1986). Annexin-V/PI Binding Assay. Cells were treated with various concentrations of CPT for 24 h, and the Annexin-V-FLUOS staining kit (Roche Diagnostics, Mannheim, Germany) was used according to manufacturer’s instructions to evaluate Annexin V/PI positivity. Control cells stained with Annexin-V or PI alone were used to compensate for flow-cytometric analysis (FACSVantage; BD Biosciences, San Jose, CA). Annexin-Vand PI-double-negative cells were defined as live cells. Annexin-V-positive, PI-negative cells were defined as early apoptotic cells, and Annexin-Vand PI-double-positive cells were defined as late-arising apoptotic/necrotic cells. Statistical Analysis. All assays were carried out in triplicate. Data were expressed as mean S.D. Student’s t test was used to compare the mean of each group with that of the control group. A p value 0.05 was considered statistically significant.