Poly(Adp)Ribose Inhibition Forces The Repair Of Topoisomerase I Cleavage Complexes Through Homologous Recombination And Non-Homologous End Joining

Abstract Poly(ADP-Ribose) polymerase (PARP) inhibitors represent a promising class of novel anticancer agents. In the present study, we explored the strategy of combining the classical topoisomerase I (Top1) inhibitor camptothecin (CPT) and the novel PARP inhibitor ABT-888 in human cancer cells. ABT-888 potentiated cell killing by CPT. By colony forming assay, the survival ratio of CPT-treated colon cancer HT-29 cells was 52%, while the survival fraction were decreased to 35% when HT-29 cells were co-treated with CPT and ABT-888. By measuring γH2AX, a DNA double-strand break marker, we found that ABT-888 enhanced the γH2AX level induced by CPT. The enhanced γH2AX response in the presence of ABT-888 affected both replication-related γH2AX but also transcription-related γH2AX. In osteosarcoma U2OS cells, ABT-888 also enhanced γH2AX in both EdU (5-ethynyl-2′- deoxyuridine) positive and negative cells. Even in non-replicating human primary lymphocytes, ABT-888 enhanced CPT-induced γH2AX foci (1.3 ± 1.2 vs. 5.1 ± 3.1 γH2AX foci/per cell in the absence and presence of co-treatment with ABT-888, respectively). Proteasome inhibition with MG-132 blocked the γH2AX induction by CPT, and PARP inhibition could not overcome the block, which demonstrates that PARP acts downstream from the proteasome. Neutral Comet assays showed that the enhanced γH2AX response was related to DNA breaks. Alkaline elution assays revealed that ABT-888 increased CPT-induced DNA single-strand breaks (SSBs) but without a corresponding increase in DNA protein crosslinks indicating that ABT-888 was inducing DNA breaks in addition to the Top1-DNA cleavage complexes trapped by CPT. These additional DNA breaks were dependent on XPF/ERCC1 endonuclease. Furthermore, after removing CPT, reversal of γH2AX in the absence or presence of ABT-888 showed the same reversal rate, although there was more γH2AX in the presence of ABT-888, suggesting that PARP inhibition did not affect the repair of double-strand breaks. Moreover, PARP inhibition enhanced DNA-PK and ATM activation with increased formation of Rad51 foci. Therefore, we propose that PARP inhibition forces the cells to activate alternative endonucleolytic and recombination pathways in response to Top1-mediated DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2973.