CHK2 and ERCC1 in the DNA Adduct Repair Pathway that Mediates Acquired Cisplatin Resistance

Abstract

Increased DNA-adduct repair is a leading mechanism of acquired cisplatin resistance. Our previous studies show that overexpression of ERCC1, the essential component of nucleotide excision repair, is associated with enhanced repair of cisplatin-induced DNA-adduct and with clinical resistance to platinum chemotherapy. Current investigations provide extensive data on the mechanism of cisplatin resistance via the DNA-adduct repair pathway. In a study of cisplatin-induced molecular signature in human ovarian cancer A2780 cells, activation of ATM, p53, Chk2, P48, and P21 were observed, with Chk2 identified as an upstream regulator of the ERCC1 recognition/repair pathway. Our data demonstrate that Chk2 is activated and regulated by p53 in wild-type p53-replete cells. We also found that activated Chk2 can be dephosphorylated by PP2A. In other words, PP2A negatively regulates Chk2 by dephosphorylating phosphorylated Chk2. Previous findings by our group suggested that ovarian cancer A2780/CP70 cells, in response to cisplatin exposure, showed an increase of ERCC1 mRNA, with increased transcription and prolonged ERCC1 mRNA half-life. Functional analysis of the ERCC1 promoter by CAT assay indicates that the region from -220 to -110 appears essential to constitutive expression of ERCC1 gene and a more forward upstream region is responsible for cisplatininduced ERCC1 overexpression. Identification of a functional cis-element in the drug-responsive region by EMSA revealed that activator AP1 and repressor MZF1 responded to cisplatin stimulation. Overexpression of MZF1 repressed the ERCC1 promoter activity in cisplatin treated cells, indicating that MZF1 is a repressor in regulation of ERCC1 transcription. After cisplatin exposure, the mRNA level of MZF1 decreased and mRNA levels of c-jun and c-fos increased, suggesting that MZF1 and AP1 coordinately mediate cisplatin-invoked gene expression in these cells. Taken together, in response to cisplatin treatment, decreased MZF1 and increased AP1 binding activities within the drug-responsive region of the ERCC1 promoter appear to be the leading mechanism of up-regulation of ERCC1 expression. In conclusion, our investigations reveal two key factors—Chk2 and ERCC1—that participate in the DNA-adduct repair pathway that mediates acquired cisplatin resistance. Down-regulation of these two critical genes may antagonize cisplatin resistance in the treatment of human ovarian cancer.