Dai Xiaofang, Tao Dan, Wu Hongge, Cheng Jing
Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Huazhong Univ Sci Technolog Med Sci. 2009 Feb;29(1):101-6. doi: 10.1007/s11596-009-0122-4. Epub 2009 Feb 18.
The low dose hyper-radiosensitivity (HRS) in human lung cancer cell line A549 was investigated, the changes of ATM kinase, cell cycle and apoptosis of cells at different doses of radiation were observed, and the possible mechanisms were discussed. A549 cells in logarithmic growth phase were irradiated with (60)Co gamma-rays at doses of 0-2 Gy. Together with flow cytometry for precise cell sorting, cell survival fraction was measured by means of conventional colony-formation assay. The expression of ATM1981Ser-P protein was examined by Western blot 1 h after radiation. Apoptosis was detected by Hoechst 33258 fluorescent staining, and Annexin V-FITC/PI staining flow cytometry 24 h after radiation. Cell cycle distribution was observed by flow cytometry 6, 12 and 24 h after radiation. The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy, followed by an increase at >0.2 Gy, and reached the peak at 0.5 Gy, with little further increase as the dose exceeded 0.5 Gy. Twenty-four h after radiation, partial cells presented the characteristic morphological changes of apoptosis, and the cell apoptosis curve was coincident with the survival curve. As compared with control group, the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation (P>0.05). After exposure to 0.3, 0.4 and 0.5 Gy radiation, G(2)/M phase arrest occurred 6 and 12 h after radiation (P<0.05), and the ratio of G(2)/M phase cells was decreased 24 h after radiation (P<0.05). It was concluded that A549 cells displayed the phenomenon of HRS/IRR. The mode of cell death was mainly apoptosis. The activity of ATM and cell cycle change may take an important role in HRS/IRR.
研究了人肺癌细胞系A549的低剂量超放射敏感性（HRS），观察不同辐射剂量下细胞中ATM激酶、细胞周期及凋亡的变化，并探讨其可能机制。对数生长期的A549细胞用0 - 2 Gy剂量的（60）Coγ射线照射。结合流式细胞术进行精确细胞分选，采用传统集落形成试验测定细胞存活分数。辐射1 h后通过蛋白质免疫印迹法检测ATM1981Ser - P蛋白的表达。辐射24 h后，用Hoechst 33258荧光染色检测凋亡，并用膜联蛋白V - FITC/PI染色流式细胞术检测。辐射后6、12和24 h通过流式细胞术观察细胞周期分布。结果显示，在0.2 Gy时观察到ATM1981Ser - P蛋白表达，>0.2 Gy时表达增加，在0.5 Gy时达到峰值，剂量超过0.5 Gy时几乎不再进一步增加。辐射24 h后，部分细胞呈现凋亡的特征性形态变化，细胞凋亡曲线与存活曲线一致。与对照组相比，照射0.1和0.2 Gy辐射后细胞周期几乎无变化（P>0.05）。照射0.3、0.4和0.5 Gy辐射后，辐射后6和12 h出现G(2)/M期阻滞（P<0.05），辐射24 h后G(2)/M期细胞比例降低（P<0.05）。结论是A549细胞表现出HRS/IRR现象。细胞死亡方式主要为凋亡。ATM活性和细胞周期变化可能在HRS/IRR中起重要作用。
