The Effect And Mechanism Of ATM On Radiation Sensitivity Of AT Cells

Objective: to investigate DNA injury and repair in GM, ATM and AT cells mediated by radiation, to investigate the mechanism for ATM mediated phosphorylation of BRCA1 and downstream gene RAD51 in signaling pathway of DNA injury repair; to investigate the expressions of BRCA1, RAD51 proteins in GM, ATM⁺-AT, AT cells before and after radiation by Coγ.Methods: (1) GM, ATM⁺-AT and AT cells treated with wortmannin (WT), a PI3K inhibitor, or DMSO as control were radiated by y ray in different doses. Alkaline comet assay also named alkaline single gel electrophoresis assay were applied to determine comet rate and comet tail length. (2) applying immunocoprecipitation and Western blotting to analyze the interactive action between ATM and BRCA1 protein, BRCA1 and RAD51 protein (DNA repair protein RAD51) in AT cells after ⁶⁰Coγradiation.(3) the localizations and expressions of BRCA1, RAD51 proteins were detected in GM, ATM⁺-AT, AT cells by immunofluorescence staining and laser scanning confocal microscopy after 0 and 10 Gy⁶⁰Coγ radiation. Quantitative analysis was also processed.Results: (1) Comet tail length significantly increased with the doses of radiation increasing in three kinds of cells (P<0.001) and with the different cells ranking as GM cells, ATM⁺-AT cells and AT cells (P<0.05). The comet tail length was shorter in cells treated with WT than control (P<0.05). The comet tail length in WT treated cells increased in the order of GM cells, ATM⁺-AT cells and AT cells (P<0.05). (2) BRCA1 and RAD51 protein were expressed in GM cells and ATM⁺-AT cells, not in AT cells.(3) Without radiation, BRCA1, RAD51 proteins were little expressed and no colocalization in GM, ATM⁺-AT, AT cells, and lowest expression were found in AT cells. After 10Gy⁶⁰Coγ radiation, the expressions of BRCA1, RAD51 proteins were increased in GM, ATM⁺-AT, AT cells. There were colocalized expressions in GM, ATM⁺-AT cells, while no in AT cells. The expressions of BRCA1, RAD51 proteins in GM, ATM⁺-AT cells significantly differentiated from the expressions in AT cells, respectively (P <0.01).