ENHANCED REPLICATIVE BYPASS OF PLATINUM-DNA ADDUCTS IN CISPLATIN-RESISTANT HUMAN OVARIAN-CARCINOMA CELL-LINES

We have examined the relationship between cis-diamminedichloroplatinum (II) (cisplatin) resistance and replicative bypass in the human ovaria n carcinoma cell lines 2008, A2780, and their respective cisplatin res istant derivatives C13 and A2780/DDP. Replicative bypass is defined a s the ability of a replication complex to proceed past a DNA adduct km own to block or stall the complex during synthesis. Previous studies i n our laboratory have shown a 3-4-fold increase in the replicative byp ass of platinum-DNA adducts in platinum-resistant murine leukemia cell lines [G. R Gibbons et al., Carcinogenesis (Lond.), 12: 2253-2257, 19 91]. To test for this effect in the human lines, we used a steady-stat e replication assay which measures the inhibition of DNA chain elongat ion (based on the incorporation of [H-3]thymidine into nascent DNA str ands) as a function of the number of platinun-DNA adducts present on t he DNA following cisplatin treatment. With this technique we demonstra ted a 45 fold increase in the replicative bypass ability of the C13 l ine compared to the 2008 line and a 23-fold increase in the bypass abi lity of the A2780/DDP Line compared to the A2780 Line. To confirm thes e results, we performed a pulse-chase replication assay on the 2008 an d C13 lines. This assay differs from the first in that DNA chain elon gation is measured in a time-dependent manner. With the pulse-chase as say we observed a 4.8-fold increase in the replicative bypass ability of the C13 line compared to the 2008 line. We then examined the speci ficity of this enhanced bypass by repeating the steady-state assay wit h the 2008 and C13 lines using as damaging agents 1,2-diaminocyclohex anedichloroplatinum(II), UV radiation (producing pyrimidine dimers), a nd benzo(a)pyrene-7,8-diol-9,10-epoxide. In both cell lines, 1,2-diami nocyclohexanedichloroplatinum(II)-DNA adducts caused a greater inhibit ion of DNA chain elongation than cisplatin-DNA adducts. The level of e nhanced bypass of 1,2-diaminocyclohexanedichloroplatinum(II)-DNA adduc ts in the resistant line was 2.1-fold (approximately 2-fold less than the level of enhanced bypass observed with cisplatin-DNA adducts). The re was no evidence of enhanced bypass in the resistant line when cells were treated with UV light or benzo(a)pyrene-7,8-diol-9,10-epoxide. T hese results indicate that the bypass response in the C13 line has so me degree of specificity for cisplatin adducts. The specificity of byp ass in these cell lines coincided well with the specificity of resista nce to each agent. We conclude from these studies that enhanced replic ative bypass of platinum-DNA adducts occurs in these two cisplatin-res istant human ovarian carcinoma cell lines and that such an enhancement contributes to their overall resistance phenotype. The demonstration of this phenomenon in human cells supports the idea that enhanced repl icative bypass may be associated with clinically observed incidences o f cisplatin resistance.