【摘要】 为阐明毛细血管扩张性共济失调突变基因(Ataxia telangiectasia mutated,ATM)介导的乳癌基因1 (Breast cancer gene 1,Breal)磷酸化及其下游DNA修复相关蛋白(DNA damage repair protein 51,RAD51) 在DNA损伤修复信号传导通路中作用机理,以源于正常人皮肤的成纤维细胞系GM细胞(Originated from human skin fibroblast GM Cell,GM0639)为对照,用免疫共沉淀与Western blot方法,观察60Coγ射线照射后共济失调症(Ataxia telangiectasia,AT)细胞、ATM转染的AT细胞(ATM+-AT)和GM细胞的BRCA1及 RAD51蛋白表达的变化。经0、5、10、20Gy辐照后,AT细胞通过免疫共沉淀及Western Blot法分析其中 ATM和BRCA1蛋白以及BRCA1和RAD51蛋白之间的相互作用以及PI3K抑制剂对ATM磷酸化其下游基因的影响。0Gy照射ATM+-AT和GM细胞未出现BRCA1表达条带;照射后,GM细胞、ATM细胞BRCA1 和RAD51蛋白均有表达,而AT细胞无表达;PI3 K抑制剂Wortmannin对经电离辐射照射后的AT、ATM+-AT 和GM细胞中BRCA1蛋白表达具有抑制作用;照射后ATM+-AT和GM细胞中BRCA1和RAD51蛋白均有表达。因此,电离辐射照射后,BRCA1由ATM介导磷酸化后可进一步与RAD51相互作用,这是信号通路传导过程中的—个级联反映,从而修复损伤的DNA,保持基因组的稳定性。更多还原

【Abstract】 In order to investigate mechanisms of ATM (Ataxia telangiectasia mutated) genes mediating phos-phorylation of BRCA1(breast cancer gene 1) and its downstream gene RAD51 (DNA damage repair protein 51) in signaling pathway of DNA injury repair, we applied immunocoprecipitate and Western blot to observe changes in the expressions of BRCA1 and RAD51 proteins in AT cells, ATM+-AT cells (AT cells transfected with ATM genes) and GM cells (originated from human skin fibroblast, GM 0639) and used as control, after 5, 10 and 20 Gy 60Co γ-ray irradiation at dose rate of 1.0Gy/min. The irradiated AT cells were analyzed to study interactive action between ATM and BRCA1 protein, BRCA1 and RAD51 protein, and the effect of PI3K inhibitor on ATM phosphorylating, its downstream gene, by immunocoprecipitate and Western blot. No expression bands of BRCA1 were found in ATM+-AT and GM cells of the control. After the irradiation, the BRCA1 and RAD51 were both expressed in GM and ATM cells. The PI3K inhibitor Wortmannin could inhibit the expression of BRCA1 in the AT, ATM+-AT and GM cells. The BRCA1 and RAD proteins were both expressed in ATM+-AT cells and GM cells. Therefore, after the irradiation the phosphorylation of BRCA1 mediated by ATM could further interact with RAD51. This is a cascade in signaling pathway for DNA damage repair and genome stabilization.更多还原