Expression of ERCC1 antisense RNA abrogates gemicitabine-mediated cytotoxic synergism with cisplatin in human colon tumor cells defective in mismatch repair but proficient in nucleotide excision repair.

Gemcitabine, or 2',2'-difluorodeoxycytidine (dFdC) is a new anticancer agent with significant activity against a broad spectrum of tumors either as a single agent or in combination with other active anticancer drugs. Studies in vitro and in vivo have demonstrated that dFdC produces cytotoxic synergism with cisplatin, or cis-diamminedi-choloroplatinum(II) (CDDP); however, the mechanism by which the synergism occurs has not been elucidated. We proposed that the nucleotide excision repair (NER) process, which is responsible for the cellular removal of CDDP-DNA adducts, may be a target for the mechanism of the cytotoxic synergism of dFdC and CDDP. Because the mismatch repair (MMR) pathway is involved in mediating CDDP cytotoxicity, making determination of the role of the NER in the cytotoxic synergism more complicated, and because tumors are often defective in MMR, we selected an NER-proficient, MMR-deficient, CP2.0 human colon carcinoma cell line as a model for this study. By an in vitro repair synthesis assay, we found that dFdC triphosphate (dFdCTP), the active metabolite of dFdC, inhibited the incorporation of [alpha-32P]dATP as well as the incorporation of [alpha-32P]dCTP, suggesting that the repair inhibition by dFdCTP does not result simply from competition for the incorporation site but rather is also due to prevention of chain elongation during the DNA resynthesis process. To determine whether the repair inhibition contributes to the cytotoxic synergism, we examined the effect of the constitutive expression of ERCC1 antisense RNA on the interaction of dFdC and CDDP. CP2.0 cells were transfected with pERCC1/AS, an ERCC1 antisense expression vector; eight hygromycine-resistant clones expressing various levels of the antisense RNA were selected for quantification of and correlation between the repair activity and cytotoxic synergism. The results show that stable expression of ERCC1 antisense RNA down-regulated the level of mRNA and repair activity; the down-regulation of the repair activity significantly correlated with the reduction of the cytotoxic synergism of the two agents. These data provide direct evidence to support the hypothesis that inhibition of the repair of CDDP-induced DNA lesions plays a critical role in dFdC-mediated cytotoxic synergism with CDDP in MMR-deficient tumor cells.