Abstract
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 β (Pol β) expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol β or human Pol β with an inactivating mutation specific to the polymerase active site yet functional for 5′-deoxyribose-phosphate (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 β is required to restore resistance to temozolomide. These results imply that Pol β-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.
Footnotes
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This research was supported by research scholar grant RSG-05-246-01-GMC 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 1P50-CA097190-01A1), 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.
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ABBREVIATIONS: BER, base excision repair; MPG, N-methylpurine DNA glycosylase; APE1, AP endonuclease 1; Pol β, polymerase β;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 β (D256A), polymerase defective mutant of human Pol β; 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.
- Received January 9, 2008.
- Accepted May 13, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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