ATM在G2期同步化A549细胞低剂量放射超敏感性／增加放射抗性现象中作用研究

Effect of ATM on low-dose hyper-radiosensitivity in A549 cells synchronized at G2 phase

目的探讨G2期同步化A549细胞低剂量放射超敏感性／增加放射抗性（HRS／IRR）现象及ATM激酶在其中的作用。方法以人肺腺癌A549细胞为研究对象，阿非迪霉素同步化细胞于G2期．特异性ATM激活剂氯喹和抑制剂KU55933调节ATM活性；克隆形成实验计算细胞存活分数；流式细胞技术检测受照G2期同步化A549细胞周期进程；免疫荧光实验观察γ-H2AX荧光焦点动态变化，评估G2期同步化A549细胞DNA修复效率；Western blot检测p-ATM（Ser 1981）和ATM表达水平。结果G2期同步化A549细胞较非同步化细胞HRS现象更加显著，HRS／IRR转变剂量与早期G2＋M检测点剂量响应模式符合，但早期检查点激活提前且维持时间更久，剂量阈值不变。激活ATM激酶可抑制HRS现象且增强DNA损伤修复，抑制ATM激酶则增强HRS现象且阻碍DNA损伤修复。结论ATM激酶介导的早期G2＋M检查点在同步化A549细胞HRS现象中起着分子开关作用。低剂量放射联合G2期同步化和ATM抑制剂可提高低剂量放射敏感性。

Objective To investigate the low-dose hyper-radiosensitivity （HRS）/induced radioresistance （IRR） in A549 cells synchronized at G2 phase and the role of ATM kinase in the process. Methods Human lung adenocarcinoma cell line A549 was synchronized at G2 phase by aphidicolin. The ATM-specific activator and inhibitor, chloroquine and KU55933, were used to regulate the activity of ATM. The colony formation assay was used to evaluate cell survival. Flow cytometry was used to determine the cell cycle of radiation-exposed A549 cells synchronized at G2 phase. Immunofluorescence was used to observe the dynamics of ＂/-H2AX fluorescence and evaluate the efficiency of DNA repair in A549 cells synchronized at G2 phase. Western blot was used to detect the expression of phosphorylated ATM （ Ser1981 ） and ATM. Results A549 cells synchronized at G2 phase had substantially enhanced HRS than non-synchronized cells. The dose-induced transition from HRS to IRR was in accordance with the dose-response pattern of early G2/M checkpoint. However, with the same threshold dose, the activation of early checkpoint occurred earlier and lasted longer than normal. The activation of ATM kinase inhibited HRS and enhanced DNA repair, while the inhibition of ATM kinase enhanced HRS and hindered DNA repair. Conclusions ATM kinase-mediated early G2 ＋M checkpoint is a molecular switch for HRS in synchronized A549 cells. Low-dose irradiation with G2-phase synchronization and ATM inhibitor can enhance the low-dose radiosensitivity.