Abstract
Purpose: DNA double-strand breaks (DSBs) are considered the most critical determinant of the fate of the cells or organisms exposed to radiation. DSBs are mainly repaired through homologous recombination (HR) or non-homologous end-joining (NHEJ). DNA-PKcs and XRCC4 are thought to play central roles in NHEJ. The present study aimed to clarify the interrelationships between XRCC4, DNA-PK and ATM, which shows a structural similarity to DNA-PKcs.
Methods: We used murine leukemia L5178Y-derived, XRCC4-deficient cell line M10 as a host and introduced empty pCMV10 vector (M10-CMV), normal XRCC4 cDNA (M10-XRCC4) and XRCC4 cDNA mutations on the phosphorylation sites by DNA-PK (M10-S2,3A). NU7026, a DNA-PK inhibitor, KU55933, an ATM inhibitor and wortmannin, inhibiting both of DNA-PK and ATM were added to culture media 1hr prior to irradiation at the concentration of 10microM. Nuclear reactor UTR-KINKI or X-ray generator were used as radiation source. Radiosensitivity was evaluated by colony forming ability in media containing 0.16% agarose.
Results: None of the inhibitors altered the radiosensitivity of M10-CMV cells. On the other hand, all the agents enhanced the radiosensitivity of M10-XRCC4 and M1-S2,3A cells. However, the extent of radiosensitization was greater in M10-XRCC4 than M10-S2,3A and wortmamnnin was more effective than NU7026 and KU55933, which showed similar effects.
Conclusions: These results collectively suggested that DNA-PK and ATM might complementarily regulate XRCC4 via phosphorylation in DSB repair. The existence of additional phosphorylation site(s) was also indicated.
