We have examined the relationship between cis-diamminedichloroplatinum(II) (cisplatin) resistance and replicative bypass in the human ovarian carcinoma cell lines 2008, A2780, and their respective cisplatin-resistant derivatives C13* and A2780/DDP. Replicative bypass is defined as the ability of a replication complex to proceed past a DNA adduct known to block or stall the complex during synthesis. Previous studies in our laboratory have shown a 3–4-fold increase in the replicative bypass of platinum-DNA adducts in platinum-resistant murine leukemia cell lines [G. R. Gibbons et al., Carcinogenesis (Lond.), 12: 2253–2257, 1991]. To test for this effect in the human lines, we used a steady-state replication assay which measures the inhibition of DNA chain elongation (based on the incorporation of [3H]thymidine into nascent DNA strands) as a function of the number of platinum-DNA adducts present on the DNA following cisplatin treatment. With this technique we demonstrated a 4.5-fold increase in the replicative bypass ability of the C13* line compared to the 2008 line and a 2.3-fold increase in the bypass ability of the A2780/DDP line compared to the A2780 line. To confirm these results, we performed a pulse-chase replication assay on the 2008 and C13* lines. This assay differs from the first in that DNA chain elongation is measured in a time-dependent manner. With the pulse-chase assay 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 specificity of this enhanced bypass by repeating the steady-state assay with the 2008 and C13* lines using as damaging agents 1,2-diaminocyclohexanedichloroplatinum(II), UV radiation (producing pyrimidine dimers), and benzo(a)pyrene-7,8-diol-9,10-epoxide. In both cell lines, 1,2-diaminocyclohexancedichloroplatinum(II)-DNA adducts caused a greater inhibition of DNA chain elongation than cisplatin-DNA adducts. The level of enhanced bypass of 1,2-diaminocyclohexanedichloroplatinum(II)-DNA adducts in the resistant line was 2.1-fold (approximately 2-fold less than the level of enhanced bypass observed with cisplatin-DNA adducts). There 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. These results indicate that the bypass response in the C13* line has some degree of specificity for cisplatin adducts. The specificity of bypass in these cell lines coincided well with the specificity of resistance to each agent. We conclude from these studies that enhanced replicative bypass of platinum-DNA adducts occurs in these two cisplatin-resistant 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 replicative bypass may be associated with clinically observed incidences of cisplatin resistance.

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Supported by USPHS Grant CA34082.

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