Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively), Both of t he cell lines were previously selected by continuous exposure to 9-nitro-CP T. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives in cluding SN-38 and topotecan, but are not cross-resistant to the non-top1 in hibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parenta l cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrat ed top1 catalytic activity and resistance to CPT, in in vitro assays. Rever se transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1, No wild-type top1 RNA or genomic DNA was detected in t he resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the publi shed top1 crystal structure, the R364H mutation is close to the catalytic t yrosine and other well-known mutations leading to CPT resistance.