Deficiency in the catalytic subunit of DNA-Dependent protein kinase causes down-regulation of ATM
Deficiency in the catalytic subunit of DNA-Dependent protein kinase causes down-regulation of ATM
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DOI：
10.1158/0008-5472.can-04-3451
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发表时间：
2005-03-01
期刊：
影响因子：
11.2
通讯作者：
Bedford, JS
中科院分区：
文献类型：
--
作者：
Peng, YL;Woods, RG;Bedford, JS
以前的报告表明，DNA依赖性蛋白激酶（DNA-PKcs）的催化亚基的水平降低，非同源DNA双链断裂末端连接系统的组成部分，和ATM的减少之间的连接。我们在其他DNA-PKcs缺陷细胞类型中研究了这种可能的联系，在用小干扰RNA敲除DNA-PKcs后，缺乏DNA-PKcs的中国仓鼠卵巢V3细胞ATM和hSMG-1水平降低，但在用PRKDC转染后两者都恢复了。在小鼠scid细胞中Atm水平也降低。在缺乏DNA-PKcs的人脑胶质瘤细胞系中ATM的减少伴随着通过下游底物的缺陷信号传导，照射后。在用两种DNA-PKcs小干扰RNA序列转染后，在正常人成纤维细胞中实现了DNA-PKcs的大幅减少。与此同时，ATM也在减少。这些数据都证实了免疫细胞化学检测。蛋白质在转染后数小时内，观察到PRKDC mRNA的下降，随后在转染后1天开始DNA-PKcs蛋白的更逐渐的下降。转染后2天未观察到ATM mRNA的变化。只有在DNA-PKcs减少发生后，才观察到ATM mRNA的减少，从转染后2天开始。ATM的量开始下降，从治疗后约3天开始，然后下降到与DNA-PKcs相当的水平。这两种蛋白质在稍后的时间恢复到正常水平。这些数据说明了一个潜在的重要的交叉调节之间的非同源末端连接系统重新连接的DNA双链断裂和ATM依赖的损伤反应网络的途径，这两个操作，以保持基因组的完整性。
Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of. the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.