Human Methyl Purine DNA Glycosylase and DNA Polymerase Expression Collectively Predict Sensitivity to Temozolomide

Overexpression of N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize tumor cells to the cell-killing effects of temozolomide, an imidazotetrazine-class chemotherapeutic alkylating agent. In the present study, we show that both elevated MPG expression and short hairpin RNA-mediated loss of DNA polymerase (Pol ) expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol or human Pol with an inactivating mutation specific to the polymerase active site yet functional for 5 -deoxyribosephosphate (5 dRP) lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in base excision repair (BER), leading to an accumulation of cytotoxic 5 dRP lesions, and that the 5 dRP lyase activity of Pol is required to restore resistance to temozolomide. These results imply that Pol -dependent 5 dRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as N7-methylguanine and N3-methyladenine. Furthermore, we find that 5 dRP-mediated cell death is independent of caspase-3 activation and does not induce the formation of autophagosomes, as measured by green fluorescent protein-light chain 3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the temozolomide-mediated resistance seen in some tumors and that balanced BER protein expression and overall BER capacity may help predict sensitivity to temozolomide. Base excision repair (BER) is the predominant pathway for the repair of base damage mediated by endogenous and exogenous stressors (Lindahl and Wood, 1999; Almeida and Sobol, 2007). The repair of DNA bases damaged by alkylation is initiated in mammalian cells by N-methylpurine DNA glycosylase (MPG), also known as alkyladenine DNA glycosylase (Wood et al., 2001). The majority of repair that is initiated by MPG occurs via short-patch BER, a mechanism whereby only one nucleotide is replaced. Once the modified base is removed by MPG, the resulting abasic site is hydrolyzed by AP endonuclease (APE1) (Wood et al., 2001), catalyzing the incision of the damaged strand, leaving a 3 OH and a 5 -deoxyribose-phosphate moiety (5 dRP) at the margins of the repair site. DNA polymerase (Pol ) subsequently hydrolyzes the 5 dRP moiety and fills the single nucleotide gap, preparing the strand for ligation by either DNA ligase I or a complex of DNA ligase III and XRCC1. As with many DNA repair processes, BER functions via a series of repair complexes that assemble at the site of the DNA lesion. For the repair of DNA damaged by alkylation, MPG, APE1, Pol , and XRCC1 are essential, with little evidence of effective complementary repair capacity (Almeida and Sobol, 2007). This would suggest that inhibition of or a This research was supported by research scholar grant RSG-05-246-01GMC from the American Cancer Society and grants from the Susan G. Komen Breast Cancer Foundation (grant BCTR0403276), National Institutes of Health (1R01-AG24364-01, P20-CA103730, 1P20-CA132385-01, and 1P50CA097190-01A1), the Brain Tumor Society, the UPMC Health System Competitive Medical Research Fund, and the University of Pittsburgh Cancer Institute (to R.W.S.). This project is also funded, in part, by a grant from the Pennsylvania Department of Health. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.045112. ABBREVIATIONS: BER, base excision repair; MPG, N-methylpurine DNA glycosylase; APE1, AP endonuclease 1; Pol , polymerase ; 5 dRP, 5 deoxyribose-phosphate; TMZ, temozolomide; shRNA, small hairpin RNA; RNAi, RNA interference; GFP-LC3, green fluorescent protein-light chain 3; mAb, monoclonal antibody; PCNA, proliferating cell nuclear antigen; FLAG-Pol (D256A), polymerase defective mutant of human Pol ; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; WT, wild type; FIV, feline immunodeficiency virus; KD, knockdown; ETO, etoposide; 3-MA, 3-methyladenine; IP, immunoprecipitation; IB, immunoblot; PAGE, polyacrylamide gel electrophoresis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ORF, open reading frame; UTR, untranslated region. 0026-895X/08/7402-505–516$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 2 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 45112/3363894 Mol Pharmacol 74:505–516, 2008 Printed in U.S.A. 505 at A PE T Jornals on Jauary 2, 2018 m oharm .aspeurnals.org D ow nladed from deficiency in one or more of these essential BER proteins will lead to DNA repair-intermediate induced cell death (e.g., alkylated bases, abasic sites, 5 dRP-containing lesions or DNA single-strand breaks). When cells, or in some cases mice, are deficient in Mpg, Xrcc1, or Pol , they are hypersensitive to alkylating agents (Engelward et al., 1996; Sobol et al., 1996; Elder et al., 1998; Paik et al., 2005; Horton et al., 2008). In addition, decreased APE1 expression can also lead to an increase in sensitivity to alkylating agents (Ono et al., 1994; Walker et al., 1994). We have extended our characterization of the role of Pol in the repair of alkylation damage in mouse cells, demonstrating that Pol provides cellular resistance to the clinical alkylating agent temozolomide (TMZ; an imidazotetrazine-class chemotherapeutic alkylating agent) by repairing lesions that ultimately trigger activation of the DNA damage response checkpoint (Trivedi et al., 2005). Interestingly, both MPG knockdown (Paik et al., 2005) and increased expression of MPG have been found to sensitize cells to alkylators (Rinne et al., 2004; Fishel et al., 2007). To avoid alkylation damage-induced mutations that accumulate in the absence of MPG expression, forced overexpression of MPG has been suggested as a strategic and viable gene therapy approach to sensitize tumor cells to TMZ. To directly evaluate the role of MPG and Pol in human tumor cells with regard to the cellular response to alkylation damage, we developed human Pol -specific shRNA-expressing lentiviruses to completely deplete human tumor cells of Pol , as well as vectors for ectopic expression of MPG and RNAi-resistant human Pol transgenes so as to define the enzymatic activity of Pol (5 dRP lyase or DNA polymerase activity) that confers TMZ resistance. We show that both elevated MPG expression and shRNA-mediated loss of Pol expression increase cellular sensitivity to TMZ in human breast cancer cells. In both cases, resistance to TMZ is restored by complementation of either wild-type human Pol or human Pol with an inactivating mutation specific to the polymerase active site yet functional for 5 dRP lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in BER by saturating the Pol -dependent removal of the cytotoxic 5 dRP lesion. Failure to remove this cytotoxic lesion does not induce caspase-3 activation and does not induce the formation of autophagosomes, as measured by GFP-LC3 localization. These studies support the possibility that the lyase activity of Pol is the rate-limiting step in BER in human cells and suggest that BER is a tightly balanced pathway for the repair of alkylated bases such as N7methylguanine and N3-methyladenine. Materials and Methods Chemicals and Reagents. RPMI 1640 medium and heat-inactivated fetal bovine serum were from Lonza Walkersville (Walkersville, MD) and Invitrogen (Carlsbad, CA). Temozolomide (NSC 362856; IUPAC name, 3-methyl-2-oxo-1,3,4,5,8-pentazabicyclo[4.3.0]nona4,6,8-triene-7-carbo oxamide; CAS no. 85622-93-1) (Sobol, 2008b) was from the National Cancer Institute Developmental Therapeutics Program (Bethesda, MD) and prepared as 100 mM stock in dimethyl sulfoxide. We used the following primary antibodies: anti-Pol (mAb clone 61; Thermo Fisher Scientific, Waltham, MA); anti-human MPG (mAb clone 506-3D; kindly provided by Dr. S. J. Kennel, Oak Ridge National Laboratory, Oak Ridge, TN); anti-APE1 (EMD Biosciences, San Diego, CA); anti-PCNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and anti-FLAG (M2 mAb; Sigma-Aldrich, St. Louis, MO). All electrophoresis reagents were from Bio-Rad Laboratories (Hercules, CA). Neomycin and Dynabeads protein G were purchased from Invitrogen. Puromycin, gentamicin sulfate (10 mg/ml), and 3 FLAG peptide were from Clontech (Mountain View, CA), Irvine Scientific (Santa Ana, CA), and Sigma-Aldrich, respectively. Etoposide (ETO) and 3-methyladenine (3-MA) were obtained from Sigma-Aldrich. Z-VAD-FMK was