Purpose: To investigate the changes in expression, localization and functional interaction of Ku70 subunit of DNA-PK after irradiation.
Materials and methods: Protein expression by Western blotting, cellular localization by immunofluorescence and functional interactions by immunoprecipitation were investigated after gamma-irradiation in two different cell systems: (1) quiescent human peripheral lymphocytes (0, 2 and 4 Gy) stimulated to proliferate with PHA; and (2) in a proliferating Nalm-19 cell line (0 and 2 Gy). Cell cycle analysis was performed by FACS. Immunofluorescence on extended chromatin fibres was also performed to show close association of Ku70 with the chromatin fibre.
Results: Gamma-irradiation induced dose-dependent expression of Ku70 in both cell systems. Confocal microscopy on immunostained cells and cell cycle analysis showed that Ku70 protein translocates to the cytoplasm after the G1 phase. There was a delay in the cytoplasmic shift of Ku70 in the irradiated group, corresponding with the G1 delay. The cytoplasmic localization was supported by ultracentrifugation studies. Immunofluorescence with Ku70 antibody on an extended chromatin fibre showed that Ku70 is closely associated with the chromatin fibre, and irradiation produced many spots of intense fluorescence on it. Ku70 coprecipitated with c-ABL and p21 after irradiation. The c-ABL was coprecipitated throughout the time course of observation, whereas p21 was transiently (0.2 h) associated with Ku70 and only in the lower dose groups.
Conclusions: These studies have demonstrated new biological characteristics of Ku70 in the cellular response to irradiation.