共济失调毛细血管扩张突变基因对氢醌诱导人外周血淋巴细胞DNA损伤及S期阻滞的影响

Effect of ataxia telangiectasia-mutated gene on DNA damage and S-phase arrest in human peripheral blood lymphocytes induced by hydroquinone

目的 探讨共济失调毛细血管扩张突变基因(ataxia telangiectasia-mutated gene, ATM)对氢醌(hydroquinone, HQ)诱导人外周血淋巴细胞(JHP)DNA损伤及细胞周期S期阻滞的影响。方法 以不同浓度的HQ(0、1、5、10μmol/L)处理JHP 12 h后,采用流式细胞术检测细胞周期分布情况,活细胞成像技术检测活性氧(reactive oxygen species, ROS)释放水平,Western blot法检测DNA损伤标志蛋白γ-H2AX、抗氧化相关蛋白Nrf2、HO-1、NQO1以及细胞周期相关蛋白CyclinA、CDK2、CyclinE的表达;利用ATM化学抑制剂KU-60019预处理JHP 1 h,以10μmol/L HQ处理12 h后观察上述指标的变化。结果(1)与0μmol/L HQ组相比,随着HQ浓度的增加,ROS释放水平逐渐升高,ATM、γ-H2AX及抗氧化相关蛋白Nrf2、HO-1、NQO1表达水平呈剂量依赖性增加,CDK2蛋白表达呈剂量依赖性降低(P<0.05);10μmol/L HQ组S期细胞比例显著增加,CyclinA表达水平显著降低,与0μmol/L HQ组相比差异有统计学意义(P<0.05)。(2)与HQ组相比,HQ+KU-60019组ROS的释放水平显著升高,S期阻滞减轻(P<0.05);HQ+KU-60019组γ-H2AX、Nrf2、HO-1、NQO1和CDK2蛋白的表达均显著升高(P<0.05)。结论 HQ可诱导JHP的ROS释放,促进ATM蛋白、DNA损伤标志蛋白γ-H2AX以及抗氧化相关蛋白表达升高,诱导细胞发生S期阻滞;ATM表达抑制后,ROS释放增多,γ-H2AX以及抗氧化相关蛋白表达进一步升高,S期阻滞减轻。

Objective To investigate the effects of ataxia telangiectasia-mutated gene(ATM) on DNA damage and cell cycle S-phase arrest of human peripheral blood lymphocytes(JHP) induced by hydroquinone(HQ).Methods JHP cells were treated with different concentrations of HQ(0,1,5,10 μmol/L) for 12 hours.Then, detect the distribution of cell cycle was detected by flow cytometry, the release level of reactive oxygen species(ROS) by live cell imaging, and the expression levels of DNA damage marker protein(γ-H2AX),antioxidant related proteins(Nrf2,HO-1,NQO1) and cell cycle related proteins(CyclinA,CDK2,CyclinE) by Western blot after one hour and 12 h pretreatments with KU-60019,an ATM chemical inhibitor, and 10 μmol/L of HQ,respectively.Results(1) With the increase of HQ concentration, ROS released level gradually increased, the expression levels of ATM,γ-H2AX and antioxidant related proteins such as Nrf2,HO-1 and NQO1 were all increased in dose-dependent manner, while the expression levels of CDK2 decreased compared with 0 μmol/L HQ group(P<0.05). Additionally, the proportion of S-phase cells in 10 μmol/L HQ group was significantly increased, and the expression level of CyclinA was significantly decreased compared with 0 μmol/L HQ group(P<0.05).(2) Compared with HQ group, the released level of ROS was significantly increased(P<0.05),the S-phase arrest was reduced(P<0.05),and the western blot detection also showed that the protein expressions of γ-H2AX,Nrf2,HO-1,NQO1 and CDK2 were significantly increased as well(P<0.05).Conclusion The results suggested that HQ can induce the release of ROS,promote the expressions of ATM protein, DNA damage marker protein and antioxidant related proteins, and induce cell S-phase arrest in JHP cells;after inhibiting the expression of ATM,the release of ROS increased, the expressions of γ-H2AX and antioxidant related protein further increased, and S-phase arrest relieved too.