EXPRESSION, LOCALIZATION AND FUNCTIONAL INTERACTIONS OF KU70 SUBUNIT OF DNA-PK IN PERIPHERAL LYMPHOCYTES AND NALM-19 CELLS AFTER IRRADIATION

Purpose: To investigate the changes in expression, localization and fu nctional interaction of Ku70 subunit of DNA-PK after irradiation. Mate rials and methods: Protein expression by Western blotting, cellular lo calization by immunofluorescence and functional interactions by immuno precipitation were investigated after gamma-irradiattion in two differ ent cell systems: (1) quiescent human peripheral lymphocytes (0, 2 and 4Gy) stimulated to proliferate with PHA; and (2) in a proliferating N alm-19 cell line (0 and 2 Gy). Cell cycle analysis was performed by FA CS. 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 ce ll 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 th e irradiated group, corresponding with the G1 delay. The cytoplasmic l ocalization was supported by ultracentrifugation studies. Immuno fluor escence with Ku70 antibody on an extended chromatin fibre showed that Ku70 is closely associated with the chromatin fibre, and irradiation p roduced many spots of intense fluorescence on ir. Ku70 coprecipitated with c-ABL, and p21 after irradiation. The c-ABL was coprecipitated th roughout the time course of observation, whereas p21 was transiently ( 0-2 h) associated with Ku70 and only in the lower dose groups. Conclus ions: These studies have demonstrated new biological characteristics o f Ku70 in the cellular response to irradiation.