PKB/Akt promotes DSB repair in cancer cells through upregulating Mre11 expression following ionizing radiation
PKB/Akt promotes DSB repair in cancer cells through upregulating Mre11 expression following ionizing radiation
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PKB/Akt 通过电离辐射后上调 Mre11 表达促进癌细胞中的 DSB 修复
DOI：
10.1038/onc.2010.467
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发表时间：
2011-02-01
期刊：
影响因子：
8
通讯作者：
Zhu, X-F
中科院分区：
文献类型：
--
作者：
Deng, R.;Tang, J.;Zhu, X-F
癌细胞中DNA修复能力的提高会导致对辐射的抵抗，并严重限制放射治疗的效果。Akt的激活与放射治疗的抵抗密切相关，而Mre11蛋白在DNA双链断裂的修复过程中起着重要的作用。在本报告中，我们的结果表明，Akt活性的抑制损害了CNE2细胞中DSB的修复，而激活的Akt则促进了HeLa细胞中DSB的修复。Mre11基因的敲除也损害了这两种细胞系的DSB修复过程。更重要的是，我们发现Akt可以调节Mre11的表达。小干扰RNA或LY294002抑制Akt活性可有效下调CNE2细胞中Mre11的表达，而MYR-Akt真核表达载体在HeLa细胞中可上调Mre11的表达。此外，荧光素酶报告基因分析显示，Mre11报告基因的活性在MYR-Akt1载体转染后增强，而这种MYR-Akt1诱导的转录活性在LY294002的存在下被阻断。进一步研究表明，GSK3β/β-连环蛋白/LeF-1通路参与了这一调控过程。β-catenin或Lef-1基因敲除导致mre11表达下调，而β-catenin过表达导致mre11表达上调。染色质免疫沉淀实验表明，β-连环蛋白/LeF-1异源二聚体在体内能直接与mre11启动子结合。PGL3-mre11和pGL3-Lef共转染β-catenin后，其荧光素酶活性增强，但当mre11启动子与Lef-1结合的保守序列发生突变时，荧光素酶活性被抑制。这些结果共同支持Akt可以通过GSK3DSB-连环蛋白/Lef通路上调mre11的表达，从而提高癌细胞的β/β修复能力。
An elevated DNA-repair capacity in cancer cells leads to radiation resistance and severely limits the efficacy of radiation therapy. Activation of Akt is tightly associated with resistance to radiotherapy, and Mre11 protein has important role during the repair of DNA double-strand breaks (DSBs). In this report, our results showed that inhibition of Akt activity impaired the repair of DSBs in CNE2 cells, whereas activated Akt promoted the repair of DSBs in HeLa cells. Knockdown of Mre11 also impaired the process of DSB repair in both these two cell lines. More importantly, we found that Akt could regulate Mre11 expression. Inhibition of Akt activity by small interfering RNA or LY294002 efficiently downregulated the Mre11 expression in CNE2 cells, and transfection with myr-Akt plasmid in HeLa cells upregulated the Mre11 expression. In addition, luciferase reporter analysis revealed that Mre11 reporter activity increased after transfection with myr-Akt1 plasmids, and this myr-Akt1-induced transcriptional activity was blocked in the presence of LY294002. Further study showed GSK3β/β-catenin/LEF-1 pathway was involved in this regulation. Knockdown of β-catenin or LEF-1 led to the downregulation of Mre11, whereas overexpression of β-catenin led to upregulation of Mre11. The chromatin immunoprecipitation assay assay showed β-catenin/LEF-1 heterodimer could directly bind to the promoter of Mre11 in vivo. And the luciferase activity of the pGL3-Mre11 and pGL3-Lef increased in HeLa cells following β-catenin plasmid co-transfected, but was abolished when the LEF-1-binding conserved sequences of Mre11 promoter were mutated. These results together support Akt can upregulate the expression of Mre11 through GSK3β/β-catenin/LEF pathway to elevate DSB-repair capacity in cancer cells.