RESUMO
Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase ß (Polß) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polß in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polß in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polß misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.
Assuntos
Cisplatino/farmacologia , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Epistasia Genética , Regulação Neoplásica da Expressão Gênica , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Mapeamento de Interação de Proteínas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Testes de ToxicidadeRESUMO
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.