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Objective:To obtain the relative biological effectiveness (RBE) of DNA double strand breaks (DSB) clusters by tracing the mechanism of radiated DNA damage, and explore the relationship among the biological effectiveness of DNA damage, chromosomal aberrations and germ cell death.Methods:Taking low-energy electrons, protons, and α particles as the research objects, this study simulated the process that cell nuclei were exposed to particle radiation using a radiation-related physicochemical model. On the ground of the DSB density-based spatial clustering of applications with noise (DBSCAN) algorithm, the DSB cluster classification method was improved to weaken the connection between the DSBs and the random distribution assumptions of energy depositions during the simulation. In this manner, the DSB clusters can be much closer to a non-random distribution. Furthermore, this study obtained the yields of DSB clusters and proposed a method to calculate the RBE values of DSB clusters.Results:The calculated RBE value (12.29) of DSB clusters of 2 MeV α particles was similar to the experimental RBE values of chromosomal fragments (15.3±5.9) and cell survival (14.7±5.1).Conclusions:After high-LET ionizing radiation, unlike the single DSB, the RBE of DSB clusters was similar to that of chromosomal aberration and cell survival.
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Objective:To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods:TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy, the TAB182 knockdown MCF-7 as the TAB182 knockdown group, and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group. RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression. Western blot was used to detect protein expression. Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3′ ssDNA formation by the end resection. The cell cycle arrest and apoptosis were measured by flow cytometry. Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results:Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2( t=17.97, P<0.05). Compared with the TAB182 negative control group, the protein level of RPA2, the number of RAD51 foci, and the 3′ ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced. At 4, 8, and 12 h after actinomycin D treatment, the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated ( t=5.37, 3.79, 3.69, P<0.05). Compared with the TAB182 negative control group, the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased ( t=3.48, 11.05, P<0.05), and at 24 h after irradiation, the cell cycle block time was prolonged ( t=8.40, P<0.01). Conclusions:TAB182 plays a role in maintaining RPA2 mRNA stability, thereby promoting HR repair. TAB182 knockdown cells are highly sensitive to ionizing radiation.
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Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining (NHEJ)-mediated DSB repair pathway that rejoins DSB ends. New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. This review focuses on the up- and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair, which in turn promotes the sensitivity of poly(ADP-ribose) polymerase inhibitor (PARPi) in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.
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Objective:To construct a random forest classification model of DNA double strand breaks (DSB) induced by ionizing radiation and investigate the genome-wide distribution of DSB.Methods:The GRCh38 reference genome was divided into 50 kilobase fragments. Then these genomic fragments were separated into low-level or high-level regions of ionizing radiation-induced DSB according to the sequencing data of MCF-7 cells. The data of eight epigenetic features were used as input. Two thirds of the data were randomly assigned to the training set, and the rest of the data was assigned to the test set. A random forest classification model with 100 decision trees was constructed. The importance of epigenetic features in the classification model was analyzed and displayed.Results:The accuracy score of the random forest classification model on the test set was 99.4%, the precision score was 98.9% and the recall score was 99.9%. The area under the receiver operating characteristic curve was 0.994. Among the eight epigenetic features, H3K36me3 and DNase markers were the most important variables. The enrichments of the two markers in DSB high-level regions were much higher than those in DSB low-level regions.Conclusions:The random forest classification model could precisely predict the genome-wide levels of DSB induced by ionizing radiation in the 50 kilobase window based on epigenetic features. Analysis revealed that these DSB might primarily distribute in the actively transcribed sites in the genome.
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@#Objective To explore the effects and molecular mechanisms of histone methylase G9a inhibitor BIX-01294 on apoptosis in esophageal squamous cell carcinoma (ESCC). Methods MTT assay and Colony-forming Units were adopted to determine the effects of BIX-01294 on the growth and proliferation of ESCC cell lines EC109 and KYSE150. Flow cytometry was used to analyze the apoptosis status of ESCC cells after the treatment of BIX-01294. The effects of BIX-01294 treatment on the expressions of G9a catalytic product H3K9me2, DNA double-strand break (DSB) markers, and apoptosis-related proteins were detected by Western blotting. Results BIX-01294 inhibited the growth of EC109 and KYSE150 cells in a dose-dependent manner (P<0.05), and BIX-01294 with the inhibitory concentration 50%(IC50) significantly inhibited the formation of colony (P<0.05). After 24 hours treatment of BIX-01294 (IC50), the apoptosis rate of EC109 cells increased from 11.5%±2.1% to 42.5%±5.4%, and KYSE150 cells from 7.5%±0.9% to 49.2%±5.2%(P<0.05). The expression level of the G9a catalytic product, H3K9me2, significantly decreased (P<0.05); while the expression of the DSB marker γH2AX was dramatically enhanced (P<0.05). We also found that the mitochondrial apoptosis pathway was activated and the expression levels of cleaved caspase3 and cleaved PARP were significantly elevated (P<0.05). Conclusion BIX-01294, the inhibitor of methyltransferase G9a, prompted apoptosis in ESCC cells by inducing DSB damage and activating mitochondrial apoptosis pathway.
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Meiosis is an essential step in gametogenesis which is the key process in sexually reproducing organisms as meiotic aberrations may result in infertility. In meiosis, programmed DNA double-strand break (DSB) formation is one of the fundamental processes that are essential for maintaining homolog interactions and correcting segregation of chromosomes. Although the number and distribution of meiotic DSBs are tightly regulated, still abnormalities in DSB formation are known to cause meiotic arrest and infertility. This review is a detailed account of molecular bases of meiotic DSB formation, its evolutionary conservation, and variations in different species. We further reviewed the mutations of DSB formation genes in association with human infertility and also proposed the future directions and strategies about the study of meiotic DSB formation.
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Humans , DNA Breaks, Double-Stranded , DNA Repair/genetics , Infertility/genetics , Meiosis/physiologyABSTRACT
Objective To investigate the expressions of phosphorylated H2AX (γH2AX) and p53-binding protein 1 (53BP1) in DNA oxidative damage of human bronchial epithelial (HBE) cells.Methods The HBE cells were treated with 0,25,50,100,200,400 μmol/L of hydrogen peroxide (H2O2) for 1 hour,respectively,and their DNA oxidative damages displaying as double-strand breaks (DSBs) were induced.The viability and apoptosis of HBE cells were measured by the CCK-8 method and flow cytometry,respectively.The expression status of γH2AX and 53BP1 in nucleus of HBE cells was observed by a fluorescence microscope.The expression levels of γH2AX,53BP1 and BRCA1 were determined by western blot.Results Compared with the control (0 μmol/L of H2O2),the via bility of HBE cells treated with 25 μmol/L of H2O2 (1.07 ±0.01) increased,while those with 50,100,200,400 μmol/L of H2O2 (0.97 ± 0.01,0.96 ± 0.01,0.95 ± 0.01,0.94 ± 0.01) decreased significantly (F =50.35,P ＜ 0.01).The apoptosis rates of HBE cells treated with 50,100,200,400 μ mol/L of H2O2 ([7.54 ± 0.57] ％,[7.84 ± 0.68] ％,[8.40 ± 0.50] ％ and [14.03 ± 1.03] ％) were significantly higher than that with 0 μmol/L of H2O2 ([4.65 ± 0.32] ％,F =35.879,P ＜ 0.01).Compared with the control (0 μmol/L of H2O2),the average fluorescence intensity of γH2AX in nucleus of HBE cells treated with 25,50,100,200,400 μmol/L of H2O2 increased significantly (F =223.97,P ＜ 0.01),while those of 53BP1 in nucleus of HBE cells treated with 50,100,200,400 μmol/L of H2O2 decreased significantly (F =117.78,P ＜ 0.01).The results of western blot showed that the expres sion levels of γH2AX increased with the increase of H2O2 concentration,while that of 53BP1 and BRCA1 was on the contrary (F =96.20,21.92 and 11.55,respectively,P ＜0.01).Conclusion In the oxidative damage of HBE cells induced by H2O2,γH2AX may be used as a marker of DNA oxidative damage,while the decreased expression of 53BP1 suggests that other mechanisms to repair the DNA damage sites may exist.
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Objective To study the effect of neuroepithelial cell transforming gene 1 (Net1) on the cellular radiosensitivity and underlying mechanism.Methods Real-time quantitative PCR was used to measure the variations in Net1 expression level upon irradiation.Radiosensitivity was analyzed by colonyforming assay after Net1-siRNAs.Net1-associated proteins were identified by co-immunoprecipitation.Results The Net1 mRNA level in the cells was increased significantly (t =-10.52,P ＜ 0.05) after irradiation.Compared to the control group,siRNA-mediated silencing of Net1 enhanced cell radiosensitivity (t =15.31,11.65,P ＜0.05).Net1 was found to interact with Ku70,Ku80 and DNA-PKcs under either normal conditions or after irradiation.Conclusions Net1 could protect cells from irradiation by interaction with DNA repair proteins in non-homologous end joining pathway.
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Objective To investigate the characteristics of repair of DNA double strand breaks (DSB) induced by high-LET α-particle irradiation and their relationship with chromatin structure in the G0 lymphocytes of human peripheral blood,in order to provide the experimental basis for the judgement and dose evaluation of internal α-particle radiation.Methods Peripheral whole blood were collected from four healthy adults and lymphocytes were separated.A monocellular layer of human lymphocytes attached in Mylar film were irradiated with 0 and 0.5 Gy of α-particles and the lymphocytes suspensions were irradiated with 0 and 0.5 Gy of γ-rays.The formations of γH2AX foci as a surrogate marker of DSB and Rad51 foci as a marker of homologous recombination (HR) repair and their spatial localization in chromatin structure were measured by immunofluorescence staining technique at 10 min-48 h post-irradiation.Results Linear-γH2AX foci tracks were observe at 10 min-2 h post-irradiation in lymphocytes exposed to α-particle irradiation(t =11.12,14.40,16.56,P ＜ 0.05),and almost completely disppeared at 6 h postirradiation.The frequencies of γH2AX foci peaked at 30 min after α-particle irradiation (t =51.72,P ＜0.05) and then decreased rapidly during 6 h post-irradiation (t =29.83,P ＜ 0.05).The average number of foci remained only about 16％ at 24-48 h post-irradiation.Moreover,27％ of γH2AX foci located at DAPI-bright heterochromatin region at 10 min after α-particle radiation,suggesting that the efficacy of DSB repair may be decreased.In contrast,at 10 min-48 h after γ-ray irradiation,no linear γH2AX foci track was observed and the γH2AX foci diffused randomly in nucleus and predominantly located in DAPI-weak euchromatin region.The numbers of formative and residual γH2AX foci after γ-ray irradiation were significantly less than those after α-particle radiation.During 30 min-2 h after α-particle and γ-ray irradiation,the frequencies of Rad51 foci slightly but not significantly increased in comparison with background level,and the frequencies of co-localization of Rad51 foci and γH2AX foci were only 3％-8％.Conclusions The formation of linear γH2AX foci tracks induced by high-LET α-particle irradiation in Go human lymphocyte could be used as biological indicator to estimate whether a person has been exposed to internal α-particle radiation.Prolonged persistence of residual γH2AX foci may be applicable for biological dosimetry.
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Objective To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism.Methods siRNA and anacardic acid (AA,an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity,respectively.Radiosensitivity was analyzed by colony-forming ability assay.γ-H2AX foci were detected to analyze the DNA double-strand break (DSB).Immunoprecipitation was used to determine the interaction of proteins.Results siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1,2 Gy after γ-ray irradiation,but had no significant effect at 4 Gy post-irradiation ( t =3.364,3.979,P ＜ 0.05 ).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA doublestrand break repair at 1,4 and 8 h after irradiation( t =3.875,3.183 and 3.175,respectively,P ＜ 0.05 ).The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation.Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation.Conclusions Tip60plays a role in the cellular response to ionizing radiation-induced DNA damage through,at least in part,interacting with DNA-PKcs and regulating its phosphorylation.
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Objective To study the association between DNA double-strand break repair gene NBS1(nijmegen breakage syndrome gene)polymorphisms and the susceptibility to lung cancer.Methods A case-control study design was applied.PCR-RFLP was used to identify NBS1 polymorphisms among 575 lung cancer cases and 575 controls.Results The frequencies of C/C,C/G and G/G genotypes at NBS1 rs 1805794 site were 25.9%,51.8%,22.3% among controls compared to 20.5%,52.3%,27.1% among cases.There was significant difference between controls and cases(χ~2=6.38,P=0.04).Individuals carrying C/G + G/G genotypes had an increased risk for lung cancer (OR=1.46,95%CI:1.09-1.97)compared to the C/C genotype.The frequencies of G/G,G/C and C/C genotypes at NBS1 rs2735383 site were 37.9%,47.0%,15.1% among controls compared to 35.5%,48.5%,16.0% among cases,with no significant difference between the two groups(χ~2=0.75,P=0.69).Individuals earning Hap4-GC haplotype(OR=1.70,95%CI:1.24-2.31)and Hap4/Hap2 dihaplotype(OR=1.75,95%CI:1.11-2.76)had an increased risk on lung cancer.Joint associations of smoking and the NBS1 polymorphism with the risk of lung cancer were observed(P＜0.05).Conclusion The G/G genotype at NBS1 rs1805794 site and the Hap4-GC haplotype and Hap4/Hap2 dihaplotype from rs1805794 and rs2735383 were both associated with lung cancer.
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DNA double strand breaks are potentially lethal DNA lesions induced by ionizing radiation, which can be repaired by homologous recombination and non- homologous end joining. Inhibition of the expression and function of genes in double strand breaks repair pathways using RNA interference and anti- sense techniques could improve the radiosensitivity of cancer cells.
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Objective To explore the inhibitory effects of Tanshinone Ⅱ A on the radiationinduced microglia activation and the possible mechanism.Methods Microglia cells BV-2 were irradiated with 2,4,8,16,and 32 Gy doses or sham-irradiated in presence or absence of 1.0 μg/ml Tanshinone Ⅱ A for 12 h,respectively.The effects of Tanshinone Ⅱ A on radiation-induced pro-inflammatory cytokines were evaluated using real-time PCR.The expression level of NF-κB p65 in cytoplasm and nucleus was measured by using Western blot.Immunofluorescence staining and confocal microscopy analysis were applied to detect the expression of γ-H2AX and p65 post-irradiation.Results The microglia cells were activated at 16,32 Gy post-irradiation.Radiation-induced release of the pro-inflammatory cytokines in BV-2 cells was detectable after irradiation.Tanshinone Ⅱ A decreased radiation-induced release of proinflammatory cytokines(t=5.56,P ＜ 0.05).Furthermore,western blotting showed that Tanshinone Ⅱ A could attenuate the nuclear translocation of NF-κB p65 submit post-irradiation.Immunofluorescence staining showed that γ-H2AX foci formation while p65 translocation into nucleus post-irradiation.Conclusions Tanshinone Ⅱ A exerts anti-inflammatory properties by suppressing the transcription of proinflammatory cytokine genes that might be associated with NF-κB signaling pathway.It is postulated that irradiation causes immediate cellular reaction and DSB triggers the molecular response which leads to NFκB pathway activation.
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BACKGROUND/AIMS: DNA double strand break (DSB) is one of the critical types of DNA damage. When unrepaired DSB is accumulated in the nucleus of the cells having mutations in such genes as p53, it will lead to chromosomal instability and further more to mutation of tumor-activating genes resulting in tumorogenesis. Some of malignant cancers and its premalignant lesions were proven to have DSB in their nuclei. The aim of this study was to define the differences in expression of 53BP1 and gamma-H2AX, the markers of DSB, among normal, gastric adenoma, and gastric adenocarcinoma tissues. METHODS: Tissue microarray was made with the tissues taken from 121 patients who underwent gastrectomy for gastric adenocarcinoma, and 51 patients who underwent endoscopic mucosal resection for gastric adenoma. Immunochemical stain was performed for the marker of DSB, 53BP1 and gamma-H2AX in the tissue microarray. The normal tissues were collected from histologically confirmed tissues with no cellular atypia obtained from the patients with gastric adenocarcinoma. RESULTS: In gastric carcinoma cells, 53BP1 and gamma-H2AX were highly expressed as compared to normal epithelial cells and gastric adenoma (p<0.01). There were no differences in the expression of 53BP1 and gamma-H2AX between normal epithelium and gastric adenoma. The expression of 53BP1 in the adenoma with grade II and III atypism was more elevated than in those with grade I atypism. The expression of 53BP1 and gamma-H2AX were not significantly different according to the clinicopathologic parameters in the patients with gastric adenocarcinoma. CONCLUSIONS: The DSB in DNA seems to be associated with the development of gastric adenocarcinoma, but does not affect the premalignant adenoma cells.
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Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Adenocarcinoma/genetics , Adenoma/genetics , Chromosomal Instability , DNA Breaks, Double-Stranded , Histones/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasm Staging , Stomach Neoplasms/geneticsABSTRACT
Objective To investigate the expression and clinical significance of Ku70 in nasopha ryngeal oarcinoma(NPC).Methods The expression of Ku70 protein in 223 specimens of nasopharyngeal carcinoma was examined by immunohistochemistry.Based on the levels of ku70 immunoreactivities,the 223 specimens were divided into high Ku70 expression group and low Ku70 expression group.The correlation of Ku70 expression with clinicopathologic features and prognosis of NPC was analyzed according to the clinical data. Results The rate of high Ku70 expression was 63.7%.Univariate survival analysis suggested that the overall survival rate was significantly lower in high KuTO expression group than in low Ku70 expression group (X2 = 7.88,P = 0.005).Cox multivariate analysis indicated that T stage,N stage and M stage were the independent prognostic predictors (X2 = 8.02,7.22,36.86;P =0.005,0.007,0.000),but not with gender, age or pathological type(X2 = 0.08 ,1.04,2.34;P = 0.780,0.308,0.126),the influence of Ku70 on the o verall survival rate was close to critical value(P = 0.085).Conclusions Ku70 is positively expressed in the majority of NPC.KuT0 expression has significant correlations with T stage and N stage.The results of our study suggest that Ku70 is a valuable prognostic factor of NPC.
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Objective To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients,and to explore the predictor of radiotherapy responses of cancers. Methods DNA double-strand breaks (DSBs) were induced by 60Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results A wide variation of radiosensitivity, e.g. The survival parameter of D0 varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radioseusitivity (D0 or SF2). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy.