Repair of DNA interstrand crosslinks as a mechanism of clinical resistance to platinum drugs in ovarian cancer

Despite improvements in tumour response and survival following platinum based therapy in ovarian cancer, both intrinsic and acquired drug resistance remain a significant problem. Mechanisms of acquired drug resistance have been studied extensively in ovarian cancer cells in culture following repeated drug exposure however the relevance of these mechanisms to the clinical situation still needs to be clearly defined. Studies in clinical material have been hampered by the unavailability of sensitive methods to detect the critical drug-induced effects in individual cells. Recently, a modification of the single cell gel electrophoresis (comet) assay has been developed which allows for the first time the sensitive detection of DNA interstrand crosslinks in both tumour and normal cells derived directly from clinical material. In this study the role of DNA crosslink repair as a potential mechanism of clinical resistance to platinum drugs has been examined. Methods for isolating ovarian tumour cells and mesothelial (normal) cells from ascitic fluid, and to prepare a single cell suspension from primary ovarian tumour tissue from surgery have been established. Cells treated ex vivo with cisplatin were examined for crosslink formation and repair (unhooking) using the comet assay. No significant difference in the peak level of crosslinking was observed between tumour and mesothelial cells from an individual patient, or between patients either untreated or previously treated with platinum-based therapy. In the majority of tumours from nine untreated patients little or no repair of crosslinks was evident at 24 hours (mean of 13.6% repair) with seven patients showing less than 10% repair. In contrast, in the majority of ten treated patients a high level of repair was observed in tumour cells (mean 46.5%), with greater than 70% repair in four at 24 hours. Increased interstrand crosslink repair was also observed in a cisplatin acquired resistant human ovarian tumour cell line. Differences in gene expression pattern between the sensitive and resistant cell lines were compared using microarray analysis. The expression of a number of genes, including ERCC1, were consistently elevated in the resistant cell line, which was confirmed using RT-PCR