Arsenite-Induced Alterations in Ku70-Deficient Cells: A Model to Study Genotoxic Effects
Arsenite-Induced Alterations in Ku70-Deficient Cells: A Model to Study Genotoxic Effects
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Ku70 缺陷细胞中亚砷酸盐诱导的改变：研究基因毒性效应的模型
DOI：
10.1080/15287390701290253
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Z. Zhuang
中科院分区：
文献类型：
--
作者：
Qi;Gao;Yun He;Xin;Jianwei Zhou;Z. Zhuang
Ku70蛋白是DNA依赖性蛋白激酶（DNA- pk）的三个亚基之一，在DNA双链断裂（DNA DSB）的修复中起重要作用。为了进一步了解Ku70蛋白的功能和亚砷酸盐诱导的基因毒性效应的机制，研究了Ku70缺乏的影响。将Ku70基因反sense RNA重组质粒和空载体pEGFP-C1转染人胚胎肺成纤维细胞（HLF），建立Ku70缺陷细胞系HLFK和空载体细胞系HLFC。实验采用中性单细胞凝胶电泳法（SCGE）检测DNA DSB损伤，微核试验检测染色体改变，流式细胞术检测细胞周期进展，分别用对照、1、2.5、5或10 μM亚砷酸钠处理HLFC和HLFK细胞2、4和24小时。Western blot分析结果显示，HLFK细胞中Ku70蛋白含量下降至HLFC细胞的38%。亚砷酸钠对HLFC和HLFK细胞24 h的中位致死浓度（LC50）分别为27.38 μM和21.80 μM。中性SCGE实验结果显示，在HLFK和HLFC细胞中，DNA DSB尾部长度、DNA DSB尾部细胞百分比以及DNA DSB损伤的严重程度均呈浓度依赖性增加。HLFK细胞中这些指标的增加明显高于暴露于相似数量金属的HLFC细胞。DNA DSB在HLFK细胞中的修复能力低于HLFC细胞。亚砷酸钠在微核和异常核形成中产生浓度依赖性升高。ku70缺乏增加了亚砷酸钠诱导染色体改变的易感性。低浓度亚砷酸钠诱导G1期细胞阻滞；然而，在高浓度的金属中，这种G1阻滞效应消失。这些结果表明，Ku70蛋白在DNA DSB损伤的修复和基因组稳定性的维持中起着重要作用。
As one of three subunits of DNA-dependent protein kinase (DNA-PK), Ku70 protein plays an important role in repair of DNA double-strand breaks (DNA DSB). To further understand the functions of Ku70 protein and the mechanisms underlying arsenite-induced genotoxic effects, the effects of Ku70 deficiency were examined. The Ku70-deficient cell line HLFK and null vector cell line HLFC were established after recombinant plasmid of Ku70 gene antisense RNA and null pEGFP-C1 vector were transferred into human embryo lung fibroblasts (HLF) cells. Experiments were undertaken to detect DNA DSB damage by neutral single-cell gel electrophoresis assay (SCGE), chromosomal alterations by micronucleus test, and cell cycle progression by flow cytometry in HLFC and HLFK cells treated with control, 1, 2.5, 5, or 10 μM sodium arsenite for 2, 4, or 24 h, respectively. Western blot analysis results showed that Ku70 protein content in HLFK cells decreased to 38% of those in HLFC cells. The median lethal concentrations (LC50) of sodium arsenite to HLFC and HLFK cells for 24 h were 27.38 μM and 21.80 μM, respectively. Results of neutral SCGE assay showed that there were concentration-dependent increases in tail length of DNA DSB, in percent of cells with DNA DSB tails, and in severity of DNA DSB damage in HLFK and HLFC cells. The increases in these indices in HLFK cells were significantly higher than those found in HLFC cells exposed to similar amounts of metal. The ability of DNA DSB to repair in HLFK cells was less than that seen in HLFC cells. Sodium arsenite produced concentration-dependent elevation in micronuclei and abnormal nuclei formation. The Ku70-deficiency enhanced the susceptibility to chromosomal alterations induced by sodium arsenite. Low concentrations of sodium arsenite induced cell arrest at G1; however, at high concentrations of metal this G1 arrest effect disappeared. These results suggested that Ku70 protein plays an important role in repair of DNA DSB damage and for maintainance of genome stability.
DOI：
10.1073/pnas.0306687101
发表时间：
2004-03-30
影响因子：
11.1
作者：
Chou, WC;Jie, CF;Dang, CV
通讯作者：
Dang, CV
DOI：
10.1073/pnas.94.15.8076
发表时间：
1997-07-22
影响因子：
11.1
作者：
Gu, YS;Jin, SF;Alt, FW
通讯作者：
Alt, FW
影响因子：
16
作者：
Li, GC;Ouyang, HH;Cordon-Cardo, C
通讯作者：
Cordon-Cardo, C