CHARACTERIZATION OF BINDING-PROTEINS FROM OVARIAN-CARCINOMA AND KIDNEY TUBULE CELLS THAT ARE SPECIFIC FOR CISPLATIN MODIFIED DNA

We have detected proteins in nuclear extracts from ovarian carcinoma cells and kidney tubule cells that bind specifically to platinated DNA. A 123-bp restriction fragment was platinated with cisplatin (DDP) to a formal molar platinum to nucleotide ratio of 0.05 and end-labeled with [P-32]-dCTP. Incubation with nuclear extracts from 2008 human ovarian carcinoma cells caused shifts in the mobility of this probe in non-denaturing polyacrylamide gels. Proteinase K, but not ribonuclease A, destroyed the bands. Comparison of the shifted bands generated by DDP-resistant 2008 and A2780 human ovarian carcinoma cell nuclear extracts with bands from the corresponding sensitive cells showed no differences in protein levels. The affinity of the proteins for the probe was the same in sensitive and resistant 2008 nuclear extracts as determined by competition with platinated salmon sperm DNA. These proteins also bound to a probe damaged with 1,2-diaminocyclohexaneplatinum(II) dichloride but did not bind to a trans-DDP platinated probe. No differences were found in the levels in UV4 or UV5 Chinese hamster ovary cells, which were hypersensitive to DDP compared to wild-type AA8 cells. MDCK and LLC-PK1 kidney tubule cells, which were more resistant to DDP cytotoxicity than 2008 cells, exhibited decreased levels of these proteins. We conclude that, although these proteins that recognize DDP damage in DNA may be involved in excision repair, their levels did not correlate with DDP sensitivity in this panel of cell lines.