Sequence specific effects on DNA and cell damage with the PARP inhibitor olaparib (AZD2281) and carboplatin.

Abstract

5025 Background: We have found clinical activity of carboplatin (C) with the PARP inhibitor olaparib (O) in BRCA1/2mut or BRCA-like breast and ovarian cancers. Current clinical trials are testing the hypothesis that PARP inhibition will sensitize tumors to platinum agents. We have examined sequence specificity of C and O combinations in cell lines, including two BRCA1mut (HCC1937, UWB1.289) and two BRCA-WT lines (OVCAR8, HeyA8), on the development of DNA damage and cell injury. Methods: Cell injury was measured with XTT assays. DNA damage was examined using Comet assay with tail quantification and γH2AX foci by immunofluorescence. Platinum DNA-adduct formation was measured using atomic absorption spectrometry and normalized to DNA input. Results: Exposure to O prior to C for 24 hours (O>C) reduced the efficiency of double stranded DNA damage compared to C alone or O+C (p<0.05 for O>C vs. C or O+C) measured by γH2AX foci/nucleus in BRCA1mut cell lines, UWB1.289: 11.0 (O>C), 40.9 (C), 46.4 (O+C); HCC1937: 14.7 (O>C), 53.2 (C), 60.6 (O+C). This trend was not observed in BRCA-WT cell lines. Comet assay demonstrated decreased DNA damage as a reduced percentage of DNA in the comet tail in BRCA1mut cells treated with O>C compared to C (p<0.05) but not O+C (p=not significant), HCC1937: 11.3% (C), 3.4% (O>C), 5.1% (O+C); UWB1.289: 7.3% (C), 2.4% (O>C), 5.1% (O+C). Cell cytotoxicity by XTT assay indicated O>C had decreased cytotoxic effects compared to C or O+C (p<0.05 for O>C vs. C or O+C) in BRCA1mut cell lines, UWB1.289: 69% (O>C), 44% (C), 40% (O+C); HCC1937: 88% (O>C), 68% (C), 63% (O+C). Platinum adduct formation was not affected by treatment with O in either BRCA1mut cell lines or BRCA-WT cell lines. Conclusions: The sequencing of O with C yields changes in cytotoxicity and the quantity of DNA damage observed in both comet analysis and γH2AX focus formation while platinum adducts were not altered by O exposure. This suggests pretreatment of BRCA-deficient cell lines with PARP inhibition may up-regulate other DNA repair pathways and lead to paradoxically less DNA damage than seen with C alone or C+O concurrently. Further studies are ongoing to evaluate this question.