ABSTRACT
Using isogenic mouse embryonic fibroblasts and human cancer cell lines, we show that cells defective in base excision repair (BER) display a cisplatin-specific resistant phenotype. This was accompanied by enhanced repair of cisplatin interstrand cross-links (ICLs) and ICL-induced DNA double strand breaks, but not intrastrand adducts. Cisplatin induces abasic sites with a reduced accumulation in uracil DNA glycosylase (UNG) null cells. We show that cytosines that flank the cisplatin ICLs undergo preferential oxidative deamination in vitro, and AP endonuclease 1 (APE1) can cleave the resulting ICL DNA substrate following removal of the flanking uracil. We also show that DNA polymerase ß has low fidelity at the cisplatin ICL site after APE1 incision. Down-regulating ERCC1-XPF in BER-deficient cells restored cisplatin sensitivity. Based on our results, we propose a novel model in which BER plays a positive role in maintaining cisplatin cytotoxicity by competing with the productive cisplatin ICL DNA repair pathways.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , DNA Repair/drug effects , Animals , Antineoplastic Agents/chemistry , Binding Sites , Cell Line, Tumor , Cisplatin/chemistry , Cross-Linking Reagents/chemistry , DNA Adducts , DNA Damage , DNA-Directed DNA Polymerase/chemistry , Drug Resistance , Humans , Kinetics , Mice , Uracil-DNA Glycosidase/chemistryABSTRACT
Temozolomide (TMZ) is the preferred chemotherapeutic agent in the treatment of glioma following surgical resection and/or radiation. Resistance to TMZ is attributed to efficient repair and/or tolerance of TMZ-induced DNA lesions. The majority of the TMZ-induced DNA base adducts are repaired by the base excision repair (BER) pathway and therefore modulation of this pathway can enhance drug sensitivity. N-methylpurine DNA glycosylase (MPG) initiates BER by removing TMZ-induced N3-methyladenine and N7-methylguanine base lesions, leaving abasic sites (AP sites) in DNA for further processing by BER. Using the human glioma cell lines LN428 and T98G, we report here that potentiation of TMZ via BER inhibition [methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG] is greatly enhanced by over-expression of the BER initiating enzyme MPG. We also show that methoxyamine-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme DNA polymerase ß (Polß), suggesting that cells proficient for BER readily repair AP sites in the presence of MX. Further, depletion of Polß increases PARP inhibitor-induced potentiation in the MPG over-expressing glioma cells, suggesting that expression of Polß modulates the cytotoxic effect of combining increased repair initiation and BER inhibition. This study demonstrates that MPG overexpression, together with inhibition of BER, sensitizes glioma cells to the alkylating agent TMZ in a Polß-dependent manner, suggesting that the expression level of both MPG and Polß might be used to predict the effectiveness of MX and PARP-mediated potentiation of TMZ in cancer treatment.