| Objective:To abserve the formation of γH2AX foci in cell cycle G2/M phase, and to discussthe characteristics of H2AX phosphorylation related with mitotic progression, in orderto analyze the rationality of γH2AX as the molecular marker of DNA DSB damageand repair. On the other hand, to establish the stable cell lines of Chk2overexpressionand silence, and then to study the effects of them on the mitotic progression, G2/Marrest induced by ionizing radiation, apoptosis and sensitivity of cancer treatment drug,in order to provide ideas and experimental basis for finding the new targets of tumorradiotherapy and chemotherapy.Methods:HeLa cells were synchronized at prometaphase by thymidine double-blockingfollowed by nocodazole treatment. Cell cycle and protein expression level wererespectively analyzed by flow cytometry and western blotting. γH2AX foci weredetected by fluorescent microscopic observation of immunostainning. The exogenousgenes were inserted into eukaryotic expression vectors by PCR. The stable cell lines ofChk2overexpression and silence were obtained by the method of lipofectamin2000transfection and resistance selection by G418or Hygromycin, and then the obtainedcell lines were identified by quantitative Real-Time PCR and western blotting. Thecell cycle of the cell lines were analyzed by thymine double-blocking and releasecombined with flow cytometry. The effect of growth rate and the rate of apoptosisinduced by ionizing radiation of Chk2silent HeLa cells were respectively analyzed bythe methods of cell growth curve and Annexin V-FITC/PI, then the expression levelof p53and pig3protein were analyzed by western blotting. The sensitivity for the antitumor drug Paclitaxel of Chk2silent HeLa cells was also analyzed by MTT. Still,the mitotic progression of the DNA-PKcs silent or activity defective HeLa cells wereobserved by time lapse.Results:1. The expression level of γH2AX was remarkably increased when the cellsentered G2/M and mitotic process. A certain portion of mitotic cells was with a greatnumber of γH2AX foci even without any DNA DSB. As cells exited from M phase,γH2AX expression level was decreased. This G2/M phases-related γH2AX expressionpattern was similar to oscilation of G2/M regulators PLK1and Cyclin B1expression.2. With4Gy of large dose γ-ray irradiation, the cells were arrested in G2/Mphases at8~12h after irradiation. At the8h time point, γH2AX foci number alsoreached the second peak after the first peak formed at1h post-irradiation. However,with1Gy of low dose irradiation, G2/M arrest was very weak, and the number ofγH2AX foci peaked at0.5h after the irradiation, and then declined with the time. Thetiming of γH2AX foci formation and disappearance is consistent with the repairkinetics of ionizing radiation-induced DNA DSB.3. The results of western blotting and qRT-PCR showed that the stable cell linesof Chk2overexpression and silence were established successfully; and the results offlow cytometry showed that the overexpression HeLa cell lines with wild type (Chk2-WT) and continuous activation type (Chk2-T68D) lead the G2/M phase to lengthen,while the continuous inhibition type (Chk2-T68A) had no obvious influence;moreover, the HeLa cell lines of Chk2overexpression enhanced G2/M arrest inducedby ionizing radiation.4. The cell cycle progression and cell proliferation were accelerated; G2/M arrestand the rate of apoptosis induced by ionizing radiation were decreased, the expressionlevel of p53and pig3protein were also reduced; Moreover, the sensitivity foranticancer drug Paclitaxel was reduced, which were in the HeLa cell lines of Chk2silence.5. In M phase, the expression level of Chk2pT68and γH2AX protein werehigher, however, the expression level of these two protein would been reduced when the activity DNA-PKcs was inhibited; also, the expression level of γH2AX proteinwas reduced in the Chk2silent HeLa cells.6. The ratio of abnormal mitosis was increased in the DNA-PKcs silent oractivity defective HeLa cells, and the phenomena of abnormal mitosis includingmisalign chromosome in metaphase, lagging chromosome in anaphase and the mitoticcatastrophe.Conclusion:1. The cell cycle change should be taken into account when γH2AX isconsidered as DSB biomarker for judging the DNA DSB occurrence and repair.However, γH2AX foci can be taken as an indicator of DSB when cells are irradiatedwith a dose of1Gy or lower.2. The stable HeLa cell lines of Chk2overexpression (wild type or mutationaltype) and silence were constructed successfully. Chk2influenced the cell cycle G2/Mprogression which relied on the phosphorylation of Thr68site; Chk2regulated thep53-mediated cell apoptosis, and Chk2silence lead to the reduction of cell apoptosisrate which was induced by ionizing radiation; Chk2silence lead to the sensitivityreduction of HeLa cells for the anticancer drug Paclitaxel which was targeted to thecell cycle.3. DNA-PKcs was the main upstream of Chk2in regulating mitotic progression.Phosphorylation of H2AX in M phase was main regulated by DNA-PKcs-Chk2signaling pathway. |
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