【作者】 张凤梅；

【导师】 刘秉慈；

【作者基本信息】 中国疾病预防控制中心 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 背景与目的:DNA依赖蛋白激酶(DNA dependent protein kinase,DNA-PK)是DNA损伤感受器(sensors)之一,参与DNA双链断裂(DNA double strand breaks,DSBs)非同源末端连接(nonhomologous end-joining,NHEJ)的修复。DNA-PK由调节亚单位Ku(Ku70和Ku80)和催化亚单位(DNA-PK catalytic subunit,DNA-PKcs)组成。石英粉尘(简称石英)是我国最严重的职业危害因素之一,既可致矽肺,也可致癌。本课题组前期研究表明:石英刺激可致细胞周期S期阻滞;蛋白激酶B(serine/threonine kinase protein kinase B,PKB/Akt)通过激活丝裂素活化蛋白激酶(MAPKs)家族的ERK和JNK调控活化蛋白-1(activator protein-1,AP-1)的活性,对石英诱导的细胞周期蛋白D1(cyclin D1)的表达及细胞周期改变起调节作用。然而,石英所致DSBs修复感受器方面的研究尚未见报道。本研究拟以人胚肺成纤维细胞(human embryo lung fibroblasts,HELF)为实验模型,以DSBs及其修复为切入点,通过使用RNAi、显性失活突变体和化学抑制剂等研究手段,将我室以往的MAPKs通路研究向上溯源至DNA-PKcs,向下追踪到DSBs的修复,研究DNA-PKcs/JNK/p53通路在石英所致DSBs修复中的作用及DNA-PKcs/p53通路对石英诱导的细胞周期改变的影响,并探讨通路中信号分子的上下游关系。方法:1、采用脂质体转染法将DNA-PKcs的小干扰RNA(small interference RNA,siRNA)质粒稳定转染HELF细胞,用免疫印迹(westernblot,WB)技术鉴定。2、采用中性彗星实验和/或γH2AX识别抗体技术检测DSBs的发生情况。并根据中性彗星的实验结果,计算DNA修复能力(DNA repair compentence,DRC),DRC值越大,DSBs修复能力越强。3、采用WB技术测定蛋白表达及其磷酸化水平。免疫荧光技术检测γH2AX焦点在细胞内的定位及其水平。荧光素酶报告基因技术检测AP-1的活性。流式细胞术检测细胞周期。结果:1、成功建立稳定共转染的细胞系。2、200μg/ml石英致HELF细胞DSBs在12h达峰值,24h残留损伤减少,提示石英诱导的DSBs在24h内可被一定程度的修复。3、石英所致H2AX磷酸化是DNA-PKcs依赖性的,DNA-PKcs表达影响石英所致DSBs的识别。提示DNA-PK是石英所致DSBs感受器。4、DNA-PKcs、Akt、JNK、AP-1、p53在石英所致DSBs修复中的作用石英作用阴性转染对照细胞,DRC=59.67%;抑制DNA-PKcs表达后,DRC=22.47%,DSBs修复能力降低。石英作用空载体对照细胞,DRC=67.25%;抑制Akt表达后,DRC=26.13%,DSBs修复能力降低。抑制JNK表达后,在检测的24h点内,Olive尾矩呈现持续增高趋势,表明石英所致DSBs修复能力明显降低。石英作用对照细胞,DRC=77.20%;抑制AP-1活性后,DRC=21.08%,表明石英所致DSBs修复能力明显降低。石英作用p53 siRNA的阴性对照细胞,DRC=57.19%;抑制p53表达后,DRC=87.68%,石英所致的DSBs修复能力增强。以上结果提示:DNA-PKcs、Akt、JNK和AP-1促进石英所致DSBs的修复;p53抑制石英所致DSBs的修复。5、DNA-PKcs、p53在石英致细胞周期改变中作用石英处理可增加S期细胞百分比;显著诱导cyclin D1、E2F1、p21表达及pRb-Ser780水平的增加,且呈现明显的时间反应关系;pRb及CDK4表达无明显改变。分别沉默DNA-PKcs和p53表达后,石英诱导的S期细胞百分比均进一步增加;石英诱导的E2F1表达及pRb-Ser780水平进一步增加,石英诱导的p21表达受抑制,石英诱导的cyclin D1表达无明显改变。提示DNA-PKcs和p53参与调控石英诱导的细胞周期改变。6、DNA-PKcs、Akt、JNK、AP-1、p53在石英致DSBs修复通路研究中的上下游关系石英诱导Akt、JNK、p53和AP-1活性增强。抑制DNA-PKcs表达,可显著抑制石英诱导的Akt、JNK、p53磷酸化水平增加及AP-1转录活性的增强。分别抑制Akt、JNK、AP-1后,石英诱导的p53表达无明显改变。结论:1、DNA-PK是石英所致DSBs的感受器;2、DNA-PKcs/Akt/JNK/AP-1通路可促进石英所致DSBs的修复;而p53表达则抑制石英所致DSBs的修复;3、DNA-PKcs/p53通路通过活化p21,负性调控E2F1表达及pRb-Ser780水平参与调控石英所致的细胞周期改变。总之,本研究初步证明了DNA-PKcs/JNK/p53通路在石英所致DSBs修复中的作用,所得结果加深了对石英致病分子机制的理解。更多还原

【Abstract】 Background and objective:DNA-dependent protein kinase (DNA-PK), composing of a large catalytic subunit, DNA-PKcs, and a regulatory component, the Ku70-Ku80 heterodimer, is a molecular sensor for DNA damage, and is involved in the repair of DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ) pathway.Silica is one of the most serious occupational hazards capable of inducing lung fibrosis and lung cancer after chronic exposure. Our previous studies showed that silica exposure can induce cell cycle alternations, accompanied with the increased percentages of cells in S phase, and the marked activation of serine/threonine kinase protein kinase B (PKB/Akt), activator protein-1 (AP-1) as well as mitogen activated protein kinase (MAPK). Moreover, Akt/ERK, JNK pathway medicates silica-induced activator protein 1 (AP-1) transactivation, the expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) as well as cell cycle alternations. However, the sensor molecules of silica-induced DNA double strand breaks are not clear.Based on above studies, our current studies, which take DNA double strand breaks and the repair as the breakthrough point, further focused on tracing the upstream sensor of our previous MAPKs pathway, the catalytic subunit of DNA-PK, and the biological endpoints of DNA double strand breaks repair effect. In this study, RNAi, dominant negative mutants as well as chemical inhibitors, were used to investigate the role of DNA-PKcs/JNK/p53 pathway in silica-induced DNA double strand breaks repair as well as the potential effect of this pathway on silica-induced cell cycle and cell cycle regulatory proteins alternations in human embryonic lung fibroblast (HELF), and to detect the upstream or downstream relationship of signaling pathway.Methods1. DNA-PKcs siRNA expression vectors together with AP-1 luciferase reporter plasmid were transfected into HELF by lipofectamine. Hygromycin was used to select the transfected cell lines, which were consequently identified by western blot (WB).2. Neutral comet assay and/orγH2AX recognition technology were applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair ability (DNA repair compentence, DRC) was calculated.3. The expression levels and activity of protein in HELF, such as DNA-PKcs, Akt, JNK, p53, p21, cyclin D1, CDK4, E2F1, pRb, were determined by WB. Cell cycle changes were identified by flow cytometry in HELF. The formation ofγH2AX foci in HELF were analyzed by immunofluorescence microscopy. AP-1 luciferase activity was determined by the luciferase reporter gene assay using a luminometer.Results1. Stable transfectants were established successfully.2. Silica induces the expression ofγH2AX in HELF in a dose- and time-dependent manner.After treatment with different doses of silica for 12 h, 25μg/ml silica can dramatically induce the expression ofγH2AX. Along with the concentration increasing, theγH2AX level was increased gradually and reached a peak at 200μg/ml. After treatment with 200μg/ml silica for different times, the levels ofγH2AX increased in a time-dependent manner. The expression ofγH2AX was significantly increased at 1 h, and reaching maximum at 12 h and then decreasing at 24 h.3. Analysis of DNA double strand breaks and repairAfter treatment with different doses of silica for 12 h, Olive tail moment increased in concentration-dependent manner. After treatment with 200μg/ml silica for different times, Olive tail moment increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h.4. DNA-PKcs is silica-induced DNA double strand breaks damage sensor Both western blot and immunofluorescence assay analysis indicated thatsiRNA-mediated silencing of DNA-PKcs strikingly downregulated silica-induced the expression ofγH2AX in HELF. It indicates that H2AX phosphorylation is through DNA-PKcs dependent pathway, and DNA-PKcs is silica-induced DNA double strand breaks damage sensor.5. The role of DNA-PKcs, Akt, INK, AP-1 and p53 in silica induced DNA double strand breaks repair by neutral comet assay.Silencing of DNA-PKcs in HELF cells resulted in a decreased of silica-induced DNA damage repair competence (DRC=22.47 %), compared with the negative control cell induced by silica (DRC=59.67 %). Silica-induced DNA damage repair competence was markedly decreased in dominant negative mutants of Akt or JNK. Inhibition of the activation of AP-1 by curcumin significantly inhibited DNA double strand breaks repair in response to silica treatment. After p53 expression was inhibited, silica-induced DNA damage repair competence was markedly increased. These results indicate that DNA-PKcs, Akt, JNK and AP-1 can promote silica-induced DNA damage repair, however, the expression of p53 inhibit silica-induced DNA damage repair.6. Effect of DNA-PKcs and p53 on cell cycle and cell cycle regulatory proteins.Silica increased the percentage of S phase as compared to the controls. Silica markedly increases in the expression of cyclin D1, E2F1 and p21 as well as the phosphorylation level of pRb-Ser780. There were not marked changes of pRb and CDK4 expression. When DNA-PKcs and p53 expression was inhibited, the number of S phase cells was marked increased, and the overexpression of p21 was inhibited, however, the overexpression of E2F1 and the phosphorylation level of pRb-Ser780 were further increased. Expression of cyclin D1 were not changed after the inhibiton of DNA-PKcs and p53 expression. These results suggest that DNA-PKcs and p53 are involved in silica-induced cell cycle change through positively regulating the expression of p21, negatively regulating the overexpression of E2F1 and the phosphorylation level of pRb-Ser780.7. Relationship among DNA-PKcs, Akt, JNK, AP-1 and p53 in silica-treated HELFExposure of HELF cells to silica markedly increased the phosphorylation of Akt at Ser473 and Thr308, JNK at Thrl83/Tyrl85,and p53 at Ser15 as well as the expression of p53 and transactivation of AP-1. After the expression of DNA-PKcs was inhibited, silica-induced phosphorylation level of Akt-Ser473, JNK and p53 were potently blocked, indicating that DNA-PKcs is upstream kinase of Akt, JNK, AP-1, p53. After the expression of Akt、JNK and AP-1 transactivation were inhibited , silica-induced the phosphorylation of p53 and the expression was not changed, suggesting that the phosphorylation of p53 is through Akt、JNK and AP-1 independent pathway. In our previous study, Akt/JNK/AP-1 signaling pathway has been tested. These results indicate that DNA-PKcs activate two pathways in silica-induced DNA double strand breaks repair, including DNA-PKcs/Akt/JNK/AP-1 and DNA-PKcs/p53 pathway, which is different in the role of silica-induced DNA double strand breaks repair.Conclusion 1. DNA-PK is silica-induced DNA double strand breaks damage sensor.2. DNA-PKcs/Akt/JNK/AP-1 signaling pathway promote silica-induced DNA double strand breaks repair, however, the expression of p53 inhibited silica-induced DNA double strand breaks repair.3. DNA-PKcs/p53 pathway mediates silica-induced cell cycle change through positively regulating the expression of p21, negatively regulating the overexpression of E2F1 and the phosphorylation level of pRb-Ser780.In brief, this research had initially proven the role of the DNA-PKcs/JNK/p53 pathway in silica-induced DNA double strand breaks repair. These findings will help us to understand the signal transduction mechanisms involved in the pathogenesis effects of silica at DNA damage reponse level.更多还原