Defective chromatin recruitment and retention of NHEJ core components in human tumor cells expressing a Cyclin E fragment

Abstract

Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.

The TMPRSS2–ERG Gene Fusion Blocks XRCC4-Mediated Nonhomologous End-Joining Repair and Radiosensitizes Prostate Cancer Cells to PARP Inhibition

“…These results indicate that by interacting with DNA-PKcs TMPRSS2-ERG does indeed inhibit DNA-PKcs Thr2609 and Ser2056 auto-phosphorylation required for its function. Finally, we and others have shown that Ser1778 is a 53BP1 C-terminal phosphorylation target of DNA-PKcs (37, 38) that contributes significantly to DNA repair after IR (39). Ser1778 53BP1 foci were also absent in PC3 cells expressing TMPRSS2-ERG (Fig.…”

PTEN Activation by DNA Damage Induces Protective Autophagy in Response to Cucurbitacin B in Hepatocellular Carcinoma Cells

“…The key regulators in response to DNA damage are ATM and ATR kinases, which activated Chk1 and Chk2 [40]. The phosphorylation of ATM/ATR and Chk1/Chk2 was increased by Cuc B, which were dramatically inhibited by ATM inhibitor, KU55933 [41], and ATM/ATR inhibitor caffeine [42]. Thus, Cuc B-induced DNA damage response was mediated by ATM/ATR pathways.…”

Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity

“…Furthermore, we investigated whether specific DNA-PKcs inhibitor NU7441 can induce detectable changes in DNA-PKcs phosphorylation status. It has previously been shown that NU7741 inhibits formation of S2056 foci (19), while the inhibitor treatment does not affect formation of T2609 foci or significantly affect ATM or ATR activity (20). AG07217 and HCT116 cells were treated with 5 lM NU7441 for 1 h, irradiated, fixated and stained, and cells in asynchronous population were then analyzed ( Fig.…”

“…The ability to discriminate between wild-type and ligase IV hypomorphic cells can be useful in the characterization of SCID patients and in the selection of appropriate cancer therapies. We and others have previously shown that treatment with NU7441 does not prevent formation of pDNA-PKcs foci (19,22,23). Therefore, in this study we used NU7441 to delay the phosphorylation of DNA-PKcs, which was successfully measured using flow cytometry.…”