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Objective: To silence α-thalassemia/mental retardation syndrome XTinked gene (ATRX) in the cervical cancer HeLa cells, to detect the effect of ionizing radiation on the protein expressions of ATRX, γH2AX, Rad51 and γH2AX, Rad51 foci, and to explore the role of ATRX in DNA damage repair of the HeLa cells after irradiation. Methods: Three ATRX-shRNA and negative Control-shRNA lentiviral vectors were transfected into the 293T cells, and the Antiviruses were collected to infect the HeLa cells; puromycin was used to obtain the HeLa cells stably silencing ATRX named shAi-HeLa, shA2-HeLa, shA3-HeLa, and shCon-HeLa; the silencing efficiency was detected by Western blotting method. After ionizing radiation, the expressions of ATRX, γH2AX, and Rad51 proteins were measured by Western blotting method, and the numbers of γH2AX and Rad51 foci in shCon-HeLa and shAi-HeLa groups were observed and counted by immunofluorescence technique. Results: The ATRX protein expressed in shCon-HeLa cells, but did not express in shA1-HeLa, shA2-HeLa, and shA3-HeLa cells; it indicated that the silencing efficiency was higher. At 1, 6, and 24 h after 2 and 8 Gy irradiation, the ATRX protein expression levels in shCon-HeLa group were increased gradually; it was most at 24 h, and the ATRX was highly expressed at 1, 6, and 24 h after 8 Gy irradiation. Compared with shCon-HeLa group, at 0-6 h after 4 Gy irradiation, the number of γH2AX foci in shA1-HeLa group was significantly increased at 1 h (P<0. 05), then was gradually decreased, but the number of γH2AX foci in shA1-HeLa group was still higher at 6 h (P<0. 01). The number of Rad51 foci was consistent with the changes of γH2AX focus number. Compared with shCon-HeLa group, the number of Rad51 foci was significantly increased at 1 h (P<0. 05), and the number in shA1-HeLa group was still higher at 6 h (P<0.01). At 0-16 h after 4 Gy irradiation, compared with shCon-HeLa, the expression amounts of γH2AX and Rad51 proteins in shAi-HeLa group were increased. Conclusion: The HeLa cell models silencing ATRX are successfully obtained; ionizing radiation can cause the increase of ATRX expression level; the focus number and the protein expression amounts of γH2AX and Rad51 in HeLa cells silencing ATRX are higher than those in control group, which indicates that ATRX involves in the repair of radiation-induced DNA damage.
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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.
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Background Changes in the concentration of oxygen induced retinal neovascularization and DNA damage repair response.Overexpression of angiogenic factors is the main reason of angiogenesis.Whether the DNA damage related to retinal neovascularization is unclear.Objective This study was to study the role of DNA damage marker γH2AX in the process of retinal neovascularization of mouse.Metbods Seventy-two 17-day-old C57BL/6J mice were randomly classified into normal control group,oxygen induced retinopathy (OIR) model group,OIR positive control group and OIR negative control group,18 for each group.The retinal tissue were obtained from the 4 groups,the retinal patch immunofluorescence was used to observe and compare the area of retinal neovascularization and non-perfusion region and γH2AX expression of the four groups.The human umbilical vein endothelial cells (HUVECs) were classified into normal control group,hypoxia model control group,positive interference group and negative interference group.The cells from the 4 groups were obtained 12 hours after treatment,the expression of the γH2AX from different HUVECs groups were compared by immunofluorescence.Western blot was performed to detect the expressions of the γH2AX from different HUVECs groups.Results The retinal patch immunofluorescence showed that the OIR model was successfully established.The area of retinal neovascularization and the area of non-perfusion region among the 4 groups had statistical significances (F=437.62,93.05,both at P＜ 0.01).The area of retinal neovascularization and non-perfusion region in OIR model group and OIR negative control group was larger than that in the normal control group.The non-perfusion region was smaller in the OIR positive control group than that in the OIR model group and OIR negative control group (both at P＜0.01).The appearance of the retinal γH2AX focus congestion was consistent with the area of neovascularization and non-perfusion in the 17-day-old mouse.The difference of γH2AX positive percentage in the four groups of HUVECs was statistically significant (F=64.97,P＜0.01).The percentages of γH2AX positive cells in the hypoxia model control group and negative interference group were significantly higher than that in the normal control group (both at P＜0.01).The percentage of γH2AX positive cells in the positive interference group was lower than that in the hypoxia model control group and negative interference group (both at P＜0.01).Conclusions γH2AX is abundant in OIR neovascularization.Inhibiting the formation of γH2AX may reduce the OIR neovascularization.
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Since γH2 AX was firstly found in 1998 , it has been one of the most important scientific topics and research tools in the related scientific fields. At present, a series of advanced testing methods and analytical technologies have been developed, which exhibited a quite attractive application prospect in the area of life science and medical science. This paper reviewed the latest progress about γH2AX in terms of molecular mechanism of phosphorylation/dephosphorylation, development of testing technologies, and the related applications.
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Objective To observe the relationship between expression changes of γH2AX and the radiosensitivity of lung cancer cells in vitro. Methods The radiosensitivity of lung cancer cell lines A549 and SBC-3 was measured by clone forming assay. The DSBs damage of lung cancer cell lines A549 and SBC-3 was determined by Western blot assay. Re-sults The clone forming rates of lung cancer cell lines A549 and SBC-3 were gradually decreased with the increased radia-tion dose.γH2AX expression was related to the cell radiosensitivity 1 hour and 6 hours after radiated. Conclusion The phosphorylated histoneγH2AX is a powerful tool to monitor DNA DSBs and to predict the radiosensitivity in lung cancer ra-diotherapy.
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Objective To establish an experimental model for the study of α-particle-induced bystander effect of DNA damage and investigate the characteristics of bystander DNA double-strand break (DSB).Methods The red fluorescence fusion protein of HsBrkl-RFP was used to mark the cytoplasm of one cell line to distinguish the irradiated target cells (HFS-RFP) and the non-irradiated bystander cells (HFS) in the co-culture cellular model.After α-particle irradiation,cellular DSB and its repair kinetics were analyzed by the immunofluorescence staining of γH2AX and laser confocal microscope observation.Results A bystander studying model was established by co-culturing human HFS-RFP cells with its partner HSF cells.After 0.1 Gy or 0.2 Gy α-particle irradiation,the similar kinetics of γH2AX foci production and abatement were observed in both irradiated HFS-RFP cells and non-irradiated bystander HFS cells,in which the highest level of γH2AX foci was detected at 1 h post-irradiation.The second peak of γH2AX foci formation appeared at 8 h post-irradiation,which possibly indicates the occurrence of secondary DSB.However,the production of secondary DSB in the bystander cells was weaker than that in the irradiated cells.Conclusions The cell co-culture model can be used for bystander effect investigation.Bystander DSB can be effectively induce by irradiation and the secondary breakage of DNA DSB in the bystander cells may relative to the consequential biochemical processing of clustered DNA damage.
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Objective To investigate the expression and significance of γH2AX in cervical squamous carcinoma.Methods Firstly,DNA were extracted from 74 cervical squamous carcinoma samples and PCR were tested for HPV infection.Secondly,formalin-fixed paraffin-embedded tissue sections (4 μm)were stained with H&E method to detect cervical lesions grading.Thirdly,HPV16 DNA were examined by in situ hybridization(ISH) and γH2AX,p16 were examined by immunohistochemical (IHC) staining.Then,30 cases typical tissue sections in which including the normal cervical tissue,cervical intraepithelial neoplasia and cervical carcinoma in situ were selected for comparing the HPV DNA loading,and the γH2AX and,pl6 expression.Finally,the feasibility of γH2AX serving as a biomarks in HPV infection-related cervical carcinogenesis were analyzed.Results In this study,HPV infection ratio is 98.65％,and HPV16 is the most common type with 74.32％ infection.In situ hybridization showed no HPV16 DNA exist in normal cervical tissues and CINI.In CIN Ⅱ HPV DNA exist mainly as episomal DNA.With the increasing of cervical lesions grade,HPV DNA was integrated into chromosome steadily.The expression of γH2AX and pl6 were positively associated with grading of cervical lesions.HPV DNA and γH2AX protein co-exist primarily in the prickle cell layer and the granular cell layer.The HPV DNA and p16 protein exist in different cell layer.Conclusion γH2AX may be employed as a biomarker for HPV positive cervical carcinogenesis.
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Objective To study the radiosensitization of histone deaeetylases inhibitor(HDACI) panobinistat in prostate cancer cells in vitro,as well as the possible mechanisms.Methods IC20 of two prostate cancer cell lines(LNCaP and PC-3)was determined using MTI assay.Cells received a single dose irradiation of 0,2,4,6,or 8 Gy using 6 MV X-ray for radiosensitivity experiment,but only 2 Gy for western blot and flow cytometry.Radiosensitization of panobinostat was investigated with clonogenic assay,and sensitizing enhancement ratio(SER)was calculated with single-hit multi-target model.Western blot was used to compare γH2AX expression.Flowcytomctry was used to detect the cell cycle distribution.Results IC20 of LNCaP and PC-3 was 2.5 and 10.0 μmol/L,respectively.SER of panobinostat at IC20 was 1.37(D0 ratio)and 1.11(Dq ratio)for LNCaP cells,and 1.78(D0 ratio)and 1.17(Dq ratio)for PC-3 cells.Expression of γH2AX gradually decreased in the 2 Gy irradiation-alone cells standing for the DSB repair,while γH2AX expression was persistent in the combination group.Irradiation triggered a G2/M arrest 6-12 hours after irradiation in LNCaP and PC-3 cells.G2/M arrest was observed when cells were treated with panobinostat for 24 hours,however,no significant change concerning cell cycle distribution was showed when cells received further irradiation.Conclusions Panobinostat Call radiosensitize prostate cancer cells,which may be related with increased DNA DSB,inhibition of DSB repair and attenuation of cell cycle modulation after irradiation.
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DNA damage response (DDR) in different cell cycle starus of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated.The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA).The apoptotic ratio and the phosphorylation H2AX (S139)were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray.The expressions of γH2AX,Bcl-2,caspase-3 and caspase-9 were detected by Western blotting.DDR in 293T cells was detected after H2AX was silenced by RNAi method.Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment.The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P＜0.05).The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased.No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls.It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates.γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.
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DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy.Histone H2AX phosphorylation (ser139) (γH2AX) occurs rapidly at the onset of DNA double strand break (DSB) and is critical to the regulation of DSB repair.If DNA repair is successful,cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs.In this study,we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy.K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin (ADR) and γH2AX formed.Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR.In order to test the potential of γH2AX to reverse drug resistance,K562/A02 cells were treated with PI3K inhibitor LY294002.It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR.Additionally,the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCAl.These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.
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This review describes the observations that constitutive ATM activation (CAA) and H2AX phosphoryla-tion (CHP) caused by endogenous oxidants in normal cells as well in tumor cell lines.This review also reported the findings on differences in CAA and CHP on the effects of several agents and growth conditions.
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We have reported that heat as well as X-rays induced <i>p53</i>-centred signal transduction. The p53 molecule determines the fate of cells, especially apoptosis. Wild-type (wt) <i>p53 </i>cells are resistant to heat as compared with the mutated-type (m) <i>p53 </i>cells. Apoptosis is efficiently induced in the wt<i>p53</i> cells by heat through the activation of Bax and Caspase-3, not but m<i>p53</i> cells. Therefore, we proposed that wt<i>p53</i> patients would be more suitable for hyperthermic therapy than m<i>p53</i> patients. To enhance apoptosis in m<i>p53</i> cells, however, we succeeded the establishing new cancer therapies against m<i>p53</i> cells using chemical chaperon therapy with glycerol and peptide therapy with p53 C-terminal peptide. In addition, we applied siRNA or gene therapy with <i>p53</i>-targeted genes to m<i>p53</i> and <i>p53</i>-deficient cells. It is our hope to show that these new therapies prove more effective as cancer therapies as soon as possible.<br>
