Abstract 3924: A proteolytic fragment of cyclin E enhances apoptosis by increasing the DNA damage response and inhibiting DNA repair

Abstract

Abstract Exposure to genotoxic agents, such as ionizing radiation (IR) can initiate double strand breaks (DSBs) that in mammalian cells are predominantly repaired by nonhomologous end-joining (NHJE). NHEJ requires the Ku70-Ku80 heterodimer, DNA-PK catalytic subunit (DNA-PKcs), XRCC4, Ligase IV and accessory factors, such as XLF/Cernunnos. Impairment of DNA repair may lead to either cell death or genomic instability. Previously we have identified Ku70 as a novel interacting partner of p18CycE, a proteolytic fragment of Cyclin E (CycE) that is produced during IR-induced apoptosis in all hematopoietic tumor cells that we have examined. To investigate the role of p18CycE in NHEJ, we have generated HEK 293T cells that stably express p18CycE at a non-toxic level. These low levels of p18CycE sensitized these cells to genotoxic stress agents, such as IR and etoposide. Neutral comet assays that measure residual DSBs, indicative of an ineffective NHEJ, showed significant difference in both tail length and tail moment in the presence of p18CycE following IR and etoposide treatment. Moreover, gamma-H2AX foci formation, another indicator of DSBs, showed a marked difference in p18CycE-expressing cells as compared to parental cells following IR. There were 2.5 foci/1 Gy at maximal DNA damage detected in the parental cells following IR while 4 foci were obtained in p18CycE-expressing cells. Moreover, the timing of DNA damage foci resolution revealed a slower DNA repair kinetics in p18CycE-expressing cells, with the number of foci in parental cells being decreased to 50% after 30 min while it took the p18CycE-expressing cells > 1 hour. Surprisingly, DNA pull-down assays demonstrated that the assembly of Ku70-Ku80 and DNA-PKcs on DSBs was not affected by the presence of p18CycE. However, the recruitment of XRCC4, Ligase IV, and the recently identified accessory factor XLF/Cernunnos was significantly impaired by p18CycE. Similarly, the number of DNA-PKcs foci observed was affected as an indicator of the DNA-PK activity. Moreover, a plasmid reactivation assay indicated that p18CycE reduced bacterial colony formation known to be associated with NHEJ activity to ∼10% as compared to ∼60% for cell lysates containing wild-type CycE. Gel electrophoretic analyses indicated an impairment of end-ligation dependent on the expression levels of p18CycE. These data indicate a profound effect of p18CycE on NHEJ that is most likely dependent on its interaction with Ku70 and probably caused by interference with the recruitment of the XRCC4-Ligase IV heterocomplex to the sites of DSBs. Interaction of p18CycE with nuclear Ku70 interferes with the NHEJ process and makes the p18CycE-expressing cells more sensitive towards genotoxic agents such as IR. Therefore, these studies provide mechanistic insights into the choice between cell death and DNA repair depending on the cell type and nature of the genotoxic insult. 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 3924.