Enhanced repair of DNA interstrand crosslinking in ovarian cancer cells from patients following treatment with platinum-based chemotherapy

Abstract

Despite high tumour response rates to platinum-based chemotherapy in ovarian cancer survival is poor due to the emergence of drug resistance. Mechanistic studies in clinical material have been hampered by the unavailability of sensitive methods to detect the critical drug-induced effects in individual cells. A modification of the single cell gel electrophoresis (comet) assay allows the sensitive detection of DNA interstrand crosslinking in both tumour and normal cells derived directly from clinical material. Tumour cells isolated from 50 ovarian cancer patients were treated ex vivo with 100 microM cisplatin for 1 h and crosslink formation and repair (unhooking) measured. No significant difference in the peak level of crosslinking in tumour cells was observed between patients who were either newly diagnosed or previously treated with platinum-based therapy, or between tumour and mesothelial cells from an individual patient. This indicates no difference in cellular mechanisms such as drug transport or detoxification. In contrast, the percentage repair (unhooking) of DNA interstrand crosslinks was much greater in the group of treated patients. At 24 h in the 36 newly diagnosed patient tumour samples, only one gave >50% repair and 23 gave <10% repair; however, 19 out of 22 treated patient samples gave >10% repair and 14 showed >50% repair. The estimated median difference (newly diagnosed minus treated) was -52 (95% CI -67 to -28), and the P-value from a Mann-Whitney test was <0.001. In eight patients, it was possible to obtain tumour samples prior to any chemotherapy, and also on relapse or at interval debulking surgery following platinum-based chemotherapy. In these patients, the mean % repair prior to therapy was 2.85 rising to 71.23 following treatment. These data demonstrate increased repair of DNA interstrand crosslinks in ovarian tumour cells following platinum therapy which may contribute to clinical acquired resistance.

Figure 1

Time course of DNA interstrand crosslink formation and repair in human ovarian cancer cells from patient 1 (A) and patient 8 (B) as determined by the single cell gel electrophoresis (comet) assay. Cells were treated ex vivo with cisplatin for 1 h at 100 μM. The data points are the values from two independent experiments and the lines are plotted through the mean.

Figure 2

Level of DNA interstrand crosslinking at 9 h following treatment with 100 μM cisplatin in tumour cells from 50 patients as determined by the comet assay. Scatter plot of the percentage decrease in tail moment in newly diagnosed patients, and those previously treated with platinum-based chemotherapy. The horizontal lines indicate the mean value in each group.

Figure 3

DNA interstrand crosslinking at 9 h following treatment with 100 μM cisplatin in tumour cells and mesothelial cells isolated from the same patient. Scatter plot shows the difference in the percentage decrease in tail moment in newly diagnosed and previously treated patients. The horizontal lines indicate the mean value in each group.

Figure 4

Repair (unhooking) of cisplatin-induced DNA interstrand crosslinking in the same tumour samples as Figure 2. Data are expressed as the % repair at 24 h compared to the peak level at 9 h. A negative % repair indicates that the level of crosslinking was higher at the 24 h time point than at the 9 h point. Scatter plot of the percentage repair at 24 h in newly diagnosed and previously treated patients. The distributions are skewed, so the horizontal lines indicate the median value in each group.

Figure 5

Repair of cisplatin-induced DNA interstrand crosslinking in tumour from eight patients where samples were taken both at initial diagnosis and following platinum-based chemotherapy. In (A) the second samples were taken at interval debulking surgery and in (B) the samples were taken at relapse following a treatment-free interval <6 months.