Decreased accumulation as a mechanism of resistance to cis-diamminedichloroplatinum(II) in cervix carcinoma HeLa cells: relation to DNA repair.

We previously described a cisplatin-resistant HeLa variant cell line that also exhibited cross-resistance to UV radiation and an enhancement in repair of UV-DNA adducts. In this study, excision repair of cisplatin-DNA adducts in the resistant cell line was investigated by different methods. Using a monoclonal antibody specific for cisplatin-DNA adducts, we found a 2-3-fold decrease in the accumulation of cisplatin-DNA adducts in the resistant cells. This was supported by the direct measurement of the number of cisplatin molecules in cells by atomic absorption spectrophotometry. The repair kinetic curves were composed of two phases, i.e., a rapid phase within the first 4 hr of repair incubation, followed by a slow phase for the cisplatin-DNA adducts. There was a 2.6-fold enhancement in the rapid repair rate in the resistant cells. Dose-response curves from these direct measurements indicated a 2.3-fold reduction in adduct accumulation in the resistant cells. In addition, repair-associated DNA strand breaks, measured using alkaline elution, showed a 1.6-fold increase in the resistant cells. Indirect detection of DNA excision repair, using host cell reactivation of transfected plasmid DNA carrying cisplatin damage, also showed 2.4-fold enhancement in the resistant cells. A phenotypic revertant of the cisplatin-resistant cells displayed reduced DNA repair, compared with the resistant cells. Furthermore, immediately after cisplatin treatment the resistant cells accumulated only 50-60% of the cisplatin-DNA adducts of the parental cells. The results suggest multifactorial mechanisms in cisplatin resistance, including reduced adduct formation and improved excision repair. The findings are also consistent with the notion that the early stage of DNA excision repair is a rate-limiting step in drug resistance.