Abstract 5205: Poly(ADP-ribose) polymerase inhibitors induce β-radiosensitization through an altered selection of DNA double-strand break repair pathways

Abstract

Abstract Background: Systematically reviewing published studies on a broad range of potential radiosensitizers, we previously reported that poly(ADP-ribose) polymerase inhibitors (PARPi) may have a unique radiosensitizing effect to enhance β-components of the linear-quadratic model. In contrast, many other radiosensitizing agents predominantly modified α-components. The aim of this study is to elucidate underlying mechanisms of “β-radiosensitization”. Methods and Results: We first confirmed that treatments with PARPi PJ34, olaparib, or veliparib two hours prior to and 48 hours after 10 Gy of ionizing radiation (IR) potentiated radiation-induced cell death in human cancer cells HCT116, HT29, H460 and A549 using the colony-forming assay. Tumor regrowth assay with 3-dimensional multicellular spheroids of HCT116 and H460 cells showed that PJ34 significantly delayed tumor regrowth when combined with single dose of 10 Gy but not in tumors treated with 10 Gy in 5 fractions. Similar results were found using HCT116 tumor xenograft model in mice. We next examined what cellular events may underlie the radiosensitization using PARPi PJ34 in HCT116 cells. Kinetics of DNA damage responses were first measured by phosphorylation of histone H2AX (γH2AX). PJ34 did not change an initial increase of γH2AX following 10 Gy IR, but γH2AX levels decreased faster in PJ34-treated cells than control cells. Immunocytochemical analysis of Rad51 showed that PJ34 reduced Rad51 foci formations up to 6 hours following 10 Gy IR. IR-induced chromosomal aberrations were quantitated by the C-banding of metaphase spreads. More dicentric chromosomes were found in PJ34-treated cells than the control. These data suggested that PARPi did not inhibit total DNA double-strand break (dsb) repair but altered the selection in DNA dsb repair pathways with increased end-joining type(s) of (mis)repair compensating for reduced homologous recombination repair. Cell cycle profiles showed sustained 4C DNA cell peaks in cells treated with PJ34 up to 72 hours following 10 Gy IR. Cell cycle markers (cyclin B1 and phosphorylated histone H3) indicated that the 4C DNA peaks of the PJ34- treated cells included substantial amount of G1 tetraploid cells in addition to G2- or M-phase diploid cells. Additionally, senescence-associated β-galactosidase staining showed that PJ34 promoted radiation-induced premature senescence, while smaller IR-induced sub-G1 populations were found in PJ34-treated cells than the control. Conclusion: PARPi increased IR-induced chromosomal aberrations through the altered selection in DNA dsb repair pathways. The increased quadratic misrepair enhanced β-components of IR-induced cell death mediated by the increased formation of G1 tetraploid cells and premature senescence. DNA dsb repair pathway selection may be a novel and effective target to elicit β-radiosensitization. Citation Format: Yuji Seo, Keita Yoshizaki, Keisuke Tamari, Yutaka Takahashi, Keisuke Otani, Masahiko Koizumi, Kazuhiko Ogawa. Poly(ADP-ribose) polymerase inhibitors induce β-radiosensitization through an altered selection of DNA double-strand break repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2017-5205