Severe hypoxia increases expression of ATM and DNA-PKcs and it increases their activities through Src and AMPK signaling pathways
Severe hypoxia increases expression of ATM and DNA-PKcs and it increases their activities through Src and AMPK signaling pathways
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DOI：
10.1016/j.bbrc.2018.09.068
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发表时间：
2018-10-20
影响因子：
3.1
通讯作者：
Hosoi, Yoshio
中科院分区：
文献类型：
--
作者：
Hashimoto, Takuma;Murata, Yasuhiko;Hosoi, Yoshio
背景：实体瘤通常含有缺氧区域，因为异常和低效的肿瘤血管系统无法提供足够的氧气。组织缺氧通常定义为低于2%的低氧浓度。众所周知，在氧浓度低于0.1%的严重缺氧条件下，肿瘤细胞表现出辐射抗性。据报道，在严重缺氧下的细胞显示出与在氧浓度为0.5- 2.0%的轻度缺氧下的细胞不同的反应。在本研究中，我们研究了严重缺氧对ATM和DNA依赖性蛋白激酶催化亚基（DNA-PKcs）的表达和活性的影响，这两个亚基都调节DNA双链断裂（DSB）修复和辐射敏感性。方法：以SV 40转化的人成纤维细胞株LM 217和LM 205及正常人皮肤成纤维细胞（NHDF）为研究对象，观察缺氧条件下ATM和DNA-PKcs表达和活性的变化。将细胞在低于0.05%的氧浓度下培养12或24小时。结果：重度缺氧可使ATM、DNA-PKcs、Src、Caveolin-1、EGFR、HIF-1 α、PDK 1、Akt、AMPK α和mTOR的表达和活性增加，而mTOR的表达和活性降低。作为一种特异性Src抑制剂，PP 2在严重缺氧条件下抑制ATM、DNA-PKcs、Caveolin-1、EGFR和Akt的活化。结论：缺氧可通过Src和AMPK信号通路增加ATM和DNA-PKcs的活性，缺氧时AMPK的激活可导致ATM表达增加。由于ATM和DNA-PKcs在电离辐射诱导的DSB修复中发挥重要作用，这些数据为严重缺氧条件下细胞辐射抗性的分子机制提供了新的见解。(C)2018爱思唯尔公司All rights reserved.
Background: Solid tumors often contain hypoxic regions because an abnormal and inefficient tumor vasculature is unable to supply sufficient oxygen. Tissue hypoxia is generally defined as a low oxygen concentration of less than 2%. It is well known that tumor cells under severe hypoxia, where oxygen concentration is less than 0.1%, show radioresistance. It has been reported that cells under severe hypoxia show different responses from those under mild hypoxia, where oxygen concentration is 0.5-2.0%. In the present study, we investigated the effects of severe hypoxia on expression and activities of ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), both of which regulate DNA double-strand breaks (DSBs) repair and radiation sensitivity. Signaling pathways for increasing expression and activities of ATM and DNA-PKcs under severe hypoxia were also investigated.Methods: SV40-transformed human fibroblast cell lines, LM217 and LM205, and normal human dermal fibroblasts (NHDF) were used. Cells were cultured at an oxygen concentration of less than 0.05% for 12 or 24 h. Activities and/or expression of ATM, DNA-PKcs, Src, Caveolin-1, EGFR, HIF-1 alpha, PDK1, Akt, AMPK alpha, and mTOR were estimated by Western blot analyses.Results: Severe hypoxia increased expression and activities of ATM, DNA-PKcs, Src, Caveolin-1, EGFR, PDK1, Akt, and AMPK alpha, and decreased expression and activity of mTOR. A specific Src inhibitor, PP2 suppressed activation of ATM, DNA-PKcs, Caveolin-1, EGFR, and Akt under severe hypoxia. Treatment with siRNA for AMPK alpha suppressed activation of ATM and DNA-PKcs and increase of ATM expression under severe hypoxia.Conclusion: Our data show that severe hypoxia increases activities of ATM and DNA-PKcs through Src and AMPK signaling pathways, and that activation of AMPK under hypoxia causes increase of ATM expression. Since ATM and DNA-PKcs play important roles in DSBs repair induced by ionizing radiation, those data provide novel insights on the molecular mechanism of the cellular radioresistance under severe hypoxia. (C) 2018 Elsevier Inc. All rights reserved.