Expression of ERCC1 antisense RNA abrogates gemcitabine-mediated cytotoxicsynergism 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 agen t 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 synergi sm with cisplatin, or cis-diamminedicholoroplatinum(II) (CDDP); however, th e mechanism by which the synergism occurs has not been elucidated. We propo sed that the nucleotide excision repair (NER) process, which is responsible for the cellular removal of CDDP-DNA adducts, may be a target for the mech anism of the cytotoxic synergism of dFdC and CDDP, Because the mismatch rep air (MMR) pathway is involved in mediating CDDP cytotoxicity, making determ ination of the role of the NER in the cytotoxic synergism more complicated, and because tumors are often defective in MMR, we selected an NER-proficie nt, MMR deficient, CP2.0 human colon carcinoma cell line as a model for thi s study. By an in vitro repair synthesis assay, we found that dFdC triphosp hate (dFdCTP), the active metabolite of dFdC, inhibited the incorporation o f [(alpha-P-32]dATP as well as the incorporation of [alpha-P-32]dCTP, sugge sting that the repair inhibition by dFdCTP does not result simply from comp etition for the incorporation site but rather is also due to prevention of chain elongation during the DNA resynthesis process, To determine whether t he repair inhibition contributes to the cytotoxic synergism, we examined th e effect of the constitutive expression of ERCC1 antisense RNA on the inter action of dFdC and CDDP, CP2.0 cells were transfected with pERCC1/AS, an ER CC1 antisense expression vector; eight hygromycine-resistant clones express ing various levels of the antisense RNA were selected for quantification of and correlation between the repair activity and cytotoxic synergism, The r esults show that stable expression of ERCC1 antisense RNA down-regulated th e level of mRNA and repair activity; the down-regulation of the repair acti vity significantly correlated with the reduction of the cytotoxic synergism of the two agents. These data provide direct evidence to support the hypot hesis that inhibition of the repair of CDDP-induced DNA lesions plays a cri tical role in dFdC-mediated cytotoxic synergism with CDDP in MMR-deficient tumor cells.