Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells
Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells
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DOI：
10.2307/3579783
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发表时间：
1998-05-01
影响因子：
3.4
通讯作者：
Ullrich, RL
中科院分区：
文献类型：
--
作者：
Okayasu, R;Suetomi, K;Ullrich, RL
Wortmannin是PI-3激酶的特异性抑制剂，最近被发现在各种人和小鼠细胞系中是有效的放射增敏剂。另一项研究表明，使用中性洗脱试验，渥曼青霉素抑制了受辐射的中国仓鼠卵巢细胞中DNA双链断裂（DSB）的修复。为了进一步阐明渥曼青霉素放射增敏的机制，我们用脉冲场凝胶电泳研究了γ射线照射后正常人成纤维细胞的DSB修复。当向细胞中加入20 μ M或更多的渥曼青霉素时，受照射细胞中DSB的再连接被显著抑制。在用渥曼青霉素处理的培养物中的殖民地形成试验表明，放射增敏以依赖于药物浓度的方式发生。然而，仅在渥曼青霉素浓度为20 μ M或更高时观察到显著的致敏作用，这反映了DSB再加入研究的结果。对于单独用渥曼青霉素处理的细胞，没有观察到平板接种效率的显著降低。对DNA依赖性蛋白激酶（DNA-PK）表达水平的研究表明，当Ku蛋白的表达没有显著变化时，使用针对DNA-PK催化亚基（DNA-PKcs）C-末端区域的抗体用渥曼青霉素处理的培养物中DNA-PK催化亚基（DNA-PKcs）的表达显著降低。此外，使用识别这种大蛋白质的中间区域的抗体，在用渥曼青霉素处理的细胞中没有观察到DNA-PKcs表达水平的降低。这些结果与相关研究的结果一起表明，渥曼青霉素通过抑制DSB修复而使正常人细胞放射增敏，并且这种抑制是激酶活性失活和/或由渥曼青霉素与DNA-PKcs的C-末端区域结合引起的结构变化的结果。(C)1998年，辐射研究学会。
Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 mu M or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 mu M or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs. (C) 1998 by Radiation Research Soeiety.