Hypertonic treatment inhibits radiation-induced nuclear translocation of the Ku proteins G22p1 (Ku70) and Xrcc5 (Ku80) in rat fibroblasts

The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on th e subcellular localization of the Ku proteins G22p1 (also known as Kn70) an d Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivit y were examined using confocal laser microscopy and immunoblotting, Althoug h these proteins were observed mainly in the nuclei of human fibroblasts, a pproximately 80% of the intensities of immunofluorescence from both G22p1 a nd Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cel ls were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation, No significant d ifferences were observed between the total intensities of the cellular fluo rescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm t o the nucleus in the rat cells after X irradiation. The nuclear translocati on of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair pr ocess of potentially lethal damage (PLD). When the rat cells were treated w ith 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited, The surviving fraction was approximately 60% of that of irradiat ed cells that were not treated with 0.5 M NaCl. The,surviving fraction incr eased with incubation time in the growth medium before treatment with NaCl, The proportions of the intensities of fluorescence from G22p1 in the nucle i of X-irradiated cells also increased from 20% to 50% with increasing inte rval between X irradiation and treatment with Nad; These results suggest th at nuclear translocation of G22p1 and Xrcc5 is important for the fast repai r process of PLD in rat cells. (C) 2001 by Radiation Research Society.