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协同抑制Ku80和DNA-PKcs对HeLa细胞放射生物学功能的影响 被引量：2

The effect of co-inhibition of Ku80 and DNA-PKcs on radiobiological function of HeLa cells
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摘要 目的:研究小干扰RNA(small interfering RNA,siRNA)和LY29400抑制单个或多个DNA双链断裂(DNA double-strand break,DSB)修复蛋白后,HeLa细胞放射敏感性和细胞周期的变化。方法:Ku80受抑细胞HeLa/Ku80-siRNA和对照细胞HeLa/Neg-siRNA分别转染可以靶向抑制DNA依赖蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)表达的siRNA,或用DNA-PKcs活性抑制剂LY294002处理,经6 MV X线照射后,克隆形成实验检测细胞放射敏感性的变化,FCM法检测细胞周期。结果:DNA-PKcs-siRNA转染后的HeLa/Ku80-siRNA细胞,其2 Gy照射后的存活分数(sur-vival fraction,SF2)为0.08±0.01,LY294002作用后HeLa/Ku80-siRNA的SF2达0.03±0.01,均远低于未处理HeLa/Ku80-siRNA细胞的0.20±0.05。各组细胞照射6 Gy后均出现G2/M期阻滞,转染DNA-PKcs-siRNA的HeLa/Neg-siRNA细胞以及经LY294002处理的HeLa/Ku80-siRNA、HeLa/Neg-siRNA细胞G2/M期阻滞逐渐缓慢出现,照射后72 h仍未达到顶点,而其他细胞G2/M期阻滞均于照射后48 h达到顶点。结论:在抑制Ku80已达95%的基础上,DSB修复功能可以由其他DSB修复蛋白如DNA-PKcs和共济失调毛细血管扩张症突变基因(ataxia-telangiectasia mutant,ATM)代偿,协同抑制上述蛋白可以明显增加,如HeLa细胞的辐射敏感性;Ku80、DNA-PKcs和ATM在细胞的G2/M期阻滞过程中发挥的作用不同。 Objective：To study the changes in radiosensitivity and cell cycle distribution of HeLa cells after inhibition of one or several DNA double-strand break （DSB） repair proteins by small interfering RNA （siRNA） and LY294002. Methods：Ku80 silenced cells （HeLa/Ku80-siRNA） and control cells （HeLMNeg-siRNA） were transfected with siRNA targeting DNA-dependent protein kinase catalytic subunit （DNA-PKcs） or pretreated with 50μmol/L LY294002, a chemically specific inhibitor of DNA-PKcs. After the cells received 6MV X-ray irradiation, the radiosensitivity of the cells was detected by clony formation assay and cell cycle distribution was analyzed by flow cytometry. Results：The survival fraction at 2 Gy （SF2） value was 0. 08±0. 01 for HeLa/Ku80-siRNA cells after being transfected with DNA-PKcs siRNA ; the SF2 value reached 0.03 ± 0.01 when HeLa/Ku80-siRNA cells were pretreated with LY294002. Both of them were significantly lower than that of untreated HeLa/Ku80-siRNA cells （0.20 ± 0.05）. The cells in all the groups were arrested in G2/M phase after irradiaton with 6 Gy X-ray. The G2/M arrest occurred slowly in the DNA-PKcs-siRNA-transfected HeLa/ Neg-siRNA cells and LY294002-pretreated HeLa/Ku80-siRNA and HeLa/Neg-siRNA cells, which did not reach the peak at 72 h postirradiation. The G2/M accumulation was maximal at 48 h post-irradiation in other cell lines. Conclusion：Based on 95% inhibition of Ku80 protein, DNA-PKcs or ataxia-telangiectasia mutant （ATM） gene could compensate the DSB repair function. Co-inhibition of these proteins led to increase in radiosensitivity of HeLa cells; Ku80, DNA-PKcs and ATM play different roles in G2/M arrest.
作者 庄亮 于世英 黄晓园 李小兰 熊华 冷彦
机构地区 华中科技大学同济医学院附属同济医院肿瘤中心
出处 《肿瘤》 CAS CSCD 北大核心 2008年第8期640-645,共6页 Tumor
基金 国家自然科学基金资助项目(编号:30672426)
关键词 HELA细胞 RNA 小分子干扰 辐射耐受性 细胞周期 HeLa cells RNA,small interfering Radiation tolerance Cell cycle
分类号 R730.55 [医药卫生—肿瘤]
  • 相关文献

参考文献14

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二级参考文献13

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共引文献19

同被引文献12

  • 1安静,徐勤枝,隋建丽,白贝,周平坤.沉默DNA-PKcs对细胞信号转导相关基因转录的影响[J].中国生物化学与分子生物学报,2005,21(5):649-655. 被引量：2
  • 2程光惠,赵红光,李艳博,郭伟,龚守良.p53蛋白在低剂量电离辐射诱导EL-4细胞适应性反应中的变化[J].肿瘤,2007,27(4):269-271. 被引量：4
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  • 10余子建,徐勤枝,周丽君,隋建丽,张士猛,安静,王豫,周平坤.肝癌组织DNA-PKcs过量表达及其靶向siRNA分子的抗增殖作用[J].世界华人消化杂志,2007,15(36):3815-3821. 被引量：6

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肿瘤
2008年 第8期

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