The Effect Of ATM On Anti-oxidative Damage Competence And Cell Cycle Of AT Cells

Ataxia telangiectasia (AT) is extremely rare. Inheritance follows an autosomal recessive modus characterized by progressive cerebellular degeneration, radiation hypersensitivity, immunodeficiency, genomic instability, premature aging and predisposition to cancer.Its radiation hypersensitivity recently become one hot topic in the sphere of radiation biology .The observed carrier frequency is 1:40,000～100,000. AT is caused by functional inactivation of the ATM gene on 11q22-23.The character of AT cells that correlate with the clinical phenotype is super-recombination, the lack of cell cycle checkpoints, the decurtation of telomere and radiation hypersensitivity. It is assumed that ATM is an early key component of a signal transduction network that mediates cellular responses to certain types of DNA damage. When IR(ionization radiation,IR)induce DNA damage, the ATM can receive the signal and mediated phosphorylation of the Chk2 (checkpoint kinase 2,chk2)and cdc25c .the protein cdc25 belong to phosphatase and eliminate the Thr14 ,Tyr15 phosphorylation of cyclin p34cdc2 , then it sustain the activity of cyclin p34cdc2 and repair IR- induced DNA damgeObjective: In this work, we analyzed the faculty of AT5BIVA cells (AT cells) from skin of Ataxia-telangiectasia (A-T) patients to cope with radiation-induced oxidative damage. we studied The effects of ATM on cell cycle after irradiation by comparing with fibroblast cells GM0639 (GM cells) from the skin of healthy adult and AT cells . we investigated mechanisms of ATM (Ataxia telangiectasia mutated)genes mediating phosphorylation of Chk2(checkpoint kinase 2)and its downstream gene Cdc25c(cell division cycle 25C)in signaling pathway of DNA injury repair.Methods: (1)Using SOD (superoxide dismutase) and MDA (malondialdehyde) kit, the activity of SOD and content of MDA in AT cells, GM cells and AT-ATM+ cells (AT cells transfected with ATM genes) irradiated to 0, 1, 2, 3, 4Gy by 60COγ-rays were detected respectively. The dose-response curves of SOD and MDA in AT cells, GM cells and AT-ATM+ cells were established.(2) Using flow cytometry, the change of cell cycle in AT cells, GM cells and AT-ATM+ cells irradiated to 0, 1, 3,5,7Gy by 60COγ-rays were detected respectively(.3)we applied co-immunocoprecipitate and Western blot to observe changes in the expressions of Chk2 and Cdc25c proteins in AT cells, GM cells and AT-ATM+ cells.Results: (1) In the irradiated cells, the SOD activity of AT cells were significantly lower than that of GM cells and AT-ATM+ cells (p<0.05), while the content of MDA of AT cells were significantly higher than that of GM cells and AT-ATM+ cells (p<0.05). In the three cell types, the activity of SOD had a negative correlation with the irradiation dose (Y=a-bX), whereas the content of MDA had a positive correlation with irradiation dose (Y=a+bX). (2) In the irradiated cells, the proportion of cell cycle phase of AT cells stand still with the raise of irradiation dose, while The rise in G1,G2/M phase fraction of GM cells and AT-ATM+ cells is observed.. (3)After 0,5,10 and 15Gy 60Coγ-ray irradiation at dose rate of 1 Gy/min, The irradiated cells were analyzed to study interactive action between ATM and Chk2 protein,Chk2 and Cdc25c protein by immunocoprecipitate and Western blot.No expression bands of Chk2 were found in AT cells,AT-ATM+ cells and GM cells of the contro1.After irradiation,the expression of the Chk2 and Cdc25c protein were both detected in GM cells and AT-ATM+ cells,and Chk2 protein in AT cells was low expressed merely at 15Gy exposure.Conclusion:(1) the faculty of AT cells to cope with radiation-induced oxidative damage is significantly weaker than that of GM cells and AT-ATM+ cells.This is one reason of AT cells's radiation hypersensitivity (2) ATM is functionally activated in the reparation of DNA after irradiation by delaying G1,G2/M phase. The deprivation of the cell cycle checkpoint of AT cells is another reason of its radiation hypersensitivity. (3)After the irradiation the phosphorylation of Chk2 mediated by ATM could further interact with Cdc25c. This is a cascade in signaling pathway for DNA damage repair and genome stabilization...