Abstract 2571: Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations

Abstract

Abstract The faithful repair of DNA double strand breaks (DSBs) is crucial for genome integrity, and aberrant repair can result in chromosomal translocations, including those associated with infant leukemia. Evidence from our lab and others suggests that environmental and dietary compounds such as bioflavonoids have the potential to promote chromosomal translocations in utero. DSBs can be repaired by either classical non-homologous end joining (C-NHEJ) that is mostly accurate or by microhomology-mediated alternative end joining (Alt-EJ) that is more error prone and can cause chromosomal translocations. Ku70/80, Ligase 4 (LIG4), X-Ray Repair Cross-Complementing factor 4 (XRCC4), and XRCC4 like factor (XLF) are central to C-NHEJ. Poly ADP Ribose 1 (PARP 1) and C terminal binding protein interacting protein (CtIP) participate in Alt-EJ. CtIP favors Alt-EJ pathway by promoting the resection of DNA ends to uncover microhomologies supporting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in the C-NHEJ repair pathway may promote the use of the other more error-prone pathway such as Alt-EJ. The purpose of the study is to understand the relationship between bioflavonoid exposure, induction of DNA repair proteins, and repair pathway choice to understand the underlying mechanisms of bioflavonoid-induced infant leukemias. Embryonic stem (ES) cells were exposed to genistein 1 hr at doses 0μM-100μM based on determined LD50 values. Following exposure, cells were recovered 4hr, 24hr and 48 hr, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 were measured by densitometry and analyzed using one way ANOVA. CRISPR-Cas9 was used to downregulate C-NHEJ DNA repair protein XRCC4. XRCC4-/- cells will subsequently be exposed to genistein, recovered, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 will measured by densitometry. Protein levels before and after gene editing of XRCC4 will be compared and analyzed using two way ANOVA. We have downregulated the XRCC4 gene in ES cells and anticipate that following exposure to genistein in the absence of XRCC4, we will detect elevated chromosomal translocation frequency in those stem cells by stimulating the Alt-EJ pathway. This research will be highly impactful in understanding how bioflavonoid-induced DNA damage is repaired by cells and whether these compounds promote genome instability analogous to that observed in infant leukemia. Citation Format: Anindita Ghosh, Christine A. Richardson. Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2571.