ABSTRACT
Objective To study the effect of DNA-PKcs blocking on the expression of autophagic proteins,and proliferation of esophageal squamous cell carcinoma cells(EC109)after irradiation(X-Ray).Methods NU7441 was used to inhibit DNA-PKcs and X-Ray radiation treatment were used to treat EC109 cells.Th experiment was divided into 4 groups,including control group, NU7441 group,X-Ray group,X-Ray+ NU7441 group.The expressions of autophagy protein Beclin-1 and LC3B were detected by Western blot.Apoptosis was detected by flow cytometry.MTT assay was used to detect cell proliferation.Results The expression of p-DNA-PKcs in EC109 cells was decreased after NU7441 treatment and increased after X-ray irradiation.Compared with untreat-ed cells(control group),the expressions of both Beclin-1 and LC3B in X-Ray+NU7441 group were increased.Compared with the X-Ray group,the expression of Beclin-1 in the X-Ray+NU7441 group was increased.Compared with the control group,the apoptotic rate of EC109 cells in the X-Ray group and X-Ray+NU7441 group was significantly increased,the difference was statistically significant(P<0.05).Compared with the X-Ray group the apoptosis rate in the X-Ray-NU7441 group was significantly increased.The MTT results showed that compared with the control group,the proliferation in the X-Ray group and X-Ray+NU7441 group was significantly inhibited, the difference was statistically significant(P<0.05).Conclusion NU7441 inhibits the expression of DNA-PKcs protein in EC109 cells, which could promote the expressions of autophagy protein Beclin-1 and LC3B,promotes apoptosis and inhibits cell proliferation.
ABSTRACT
Objective To quantitatively compare the γ-H2AX foci formation between DNA-PKcs+/+and DNA-PKcs-/-mouse embryonic fibroblast(MEF)cells,and to investigate the dynamic changes in DNA double-strand breaks(DSBs)in human nasopharyngeal carcinoma SUNE-1 cells exposed to X-ray radiation. Methods The expression of DNA-PKcs was determined by Western blot. The γ-H2AX foci formation induced by 5 Gy X-ray radiation was detected by cell immunofluorescence. The ImageJ software was used to quantitatively analyze the γ-H2AX foci formation. Results The expression of DNA-PKcs was silenced in DNA-PKcs-/-MEF cells and normal in DNA-PKcs+/+MEF cells. According to the dynamic analyses of the numbers of γ-H2AX foci/cell and γ-H2AX foci/mm2, a similar tendency was observed in DSB formation in DNA-PKcs+/+MEF cells, DNA-PKcs-/-MEF cells,and SUNE-1 cells exposed to X-ray radiation. A large number of γ-H2AX foci formed at 0.5-1.0 h after radiation. DSBs were repaired at 6 h after radiation in DNA-PKcs+/+MEF cells and 24 h after radiation in DNA-PKcs-/-MEF cells and SUNE-1 cells. The peak values of γ-H2AX foci/cell and γ-H2AX foci/mm2were observed at 1.0 and 0.5 h after radiation, respectively. Compared with DNA-PKcs+/+MEF cells, DNA-PKcs-/-MEF cells had different numbers of γ-H2AX foci/cell at 0.5, 1.0, 3.0, 6.0, and 12.0 h after radiation, as well as different numbers of γ-H2AX foci/mm2at 3.0, 6.0, and 12.0 h after radiation. Conclusions Quantitative measurement of the number of γ-H2AX foci/cell or γ-H2AX foci/mm2by cell immunofluorescence provides new insights into the quantitative and dynamic study of DSB damage and repair.
ABSTRACT
Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim expression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also examined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stability.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.