Abstract
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a distinct factor in the non-homologous end-joining (NHEJ) pathway involved in DNA double-strand break (DSB) repair. We examined the crosstalk between key proteins in the DSB NHEJ repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wild-type cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase. Inhibition of DNA-PKcs prolonged IR-induced G2/M phase arrest because of sequential activation of cell cycle checkpoints. DSBs were introduced, and cell cycle checkpoints were recruited after exposure to IR in nasopharyngeal carcinoma SUNE-1 cells. NU7441 radiosensitized MEF cells and SUNE-1 cells by interfering with DSB repair. Together, these results reveal a mechanism in which coupling of DSB repair with the cell cycle radiosensitizes NHEJ repair-deficient cells, justifying further development of DNA-PK inhibitors in cancer therapy.
Highlights
Cell cycle checkpoints are governed primarily by the kinase Ataxia-telangiectasia mutated (ATM) and Ataxiatelangiectasia and Rad3-related (ATR)
We examined the crosstalk between key proteins in the double-strand break (DSB) non-homologous end-joining (NHEJ) repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wildtype cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxiatelangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase
The results indicated that irradiation induced a reduction of cell survival probability in DNA-PKcs−/− and Ku70−/− cells compared with their wild-type MEF cells (Figure 1B)
Summary
Cell cycle checkpoints are governed primarily by the kinase Ataxia-telangiectasia mutated (ATM) and Ataxiatelangiectasia and Rad3-related (ATR). DSB results in activation of ATM, which triggers phosphorylation of checkpoint effector kinase (CHK2), leading to the arrest of G1/S checkpoints, whereas the intra-S phase or G2/M checkpoints are blocked by CHK1 and activated by ATR. DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase (PI3K) family, represents a central component in NHEJ repair. DNA-PKcs is recruited to the DSB site and binds to the Ku70/80 heterodimer to form the DNA repair complex, which activates the kinase activity of DNA-PKcs through phosphorylation of the serine/threonine [12]. Our previous studies have validated that the absence of Ku protein compromises the ability of cells to repair DSB via the NHEJ repair pathway, elevates radiosensitivity, and enhances radiation-induced apoptosis [13, 14]
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