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Objective:To construct a three-dimensional time-dose-response model for dose estimation and validate its feasibility.Methods:Based on a random number table, mice were divided into 0, 2, 4, 6, and 8 Gy groups for whole-body X-ray irradiation, with each group consisting of three mice. Hair follicle cells of whiskers were sampled at 1, 6, and 24 h after the irradiation. After immunofluorescence staining, the numbers of γ-H2AX foci at different time points from 1 to 24 h post-irradiation were observed using a confocal laser scanning microscope. The average numbers of γ-H2AX foci observed were corrected using the Dolphin’s model, followed by the fitting of dose-response curves. Using the R software, the equations and surfaces of the three-dimensional model for partial-body irradiation were established using the irradiation doses, post-irradiation time, and the corrected average numbers of γ-H2AX foci.Results:The average number of γ-H2AX foci increased with dose at fixed time points 1, 6, and 24 h but decreased with irradiation time at fixed doses 2, 4, 6, and 8 Gy. The dose-response curve equations of partial-body irradiation were fitted as follows: YF = 2.853+ 3.775 D, R2= 0.928, at 1 h after the irradiation; YF = 0.144+ 2.775 D, R2= 0.903, at 6 h after the irradiation; YF = 0.066+ 2.472 D, R2= 0.85, at 24 h after the irradiation. The three-dimensional model equation fitted was YF = 6.837 t-1.728+ 3.113 t-0.071D, R2=0.897. Substituting different post-irradiation time points into the three-dimensional surface model appeared as a two-dimensional linear model. By substituting the number of γ-H2AX foci and irradiation time into the linear and the three-dimensional models, both models yielded relative deviations between the estimated and actual radiation doses of 30% or less. Conclusions:The three-dimensional time-dose-response model, established by using the number of γ-H2AX foci to estimate partial-body irradiation doses, can be preliminarily applied for dose estimation at all time points 1-24 h after irradiation.
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Objective To investigate the inhibitory effect and mechanism of Metformin (Met) combined with irradiation in CT26WT cell lines or mouse models with transplanted tumors.Methods CT26WT cell line was treated with 0.5 μmol/L,1.0 μmol/L,5.0 μmol/L and 10.0 μmol/L Met,and CellTiter Glo kit was used to detect the inhibitory effect of Met at different concentrations on the viability of CT26WT cells.CT26WT cell line was treated with the control,Met (10 pmol/L),15 Gy irradiation and 15 Gy irradiation+Met (10 μmol/L).Clone formation assay was employed to detect the cell proliferation activity.Bablc mouse models of transplanted tumors (tumor size> 150 mm3) were established and randomly divided into the control,15 Gy irradiation,Met and 15Gy irradiation+Met groups.Mice were given with 750 mg/kg Met at 24 h before irradiation.Transplanted tumor volume was measured regularly to delineate the growth curve of transplanted tumors and survival curve.The expression levels of P-H2AX and Sting proteins in CT26WT cells and transplanted tumors were detected by Western blot.The infiltration of CD8a (+) T cells in transplanted tumor tissues was detected by immunohistochemistry.Results The relative cell survival rate was 100%,87.9%,87.8%,87.3% and 76.5% in the 0,0.5,1.0,5.0 and 10.0μmol/L Met groups,respectively (all P<0.05).The inhibitory effect of 10.0 μmol/L was significantly stronger than that of 5.0 μmol/L (P<0.001).The colone formation rate 34.0%,24.0%,22.3% and 14.0% in the control,Met,15 Gy irradiation,Met+ 15Gy irradiation groups,respectively (all P<0.001).Western blot showed that compared with the control group,the expression of Sting protein was increased by 2.99-fold after Met treatment (P<0.001),and increased by 1.37-fold and 4.41-fold in the 15 Gy irradiation and 15Gy irradiation+Met groups (both P<0.01).Compared with the 15 Gy irradiation group,the expression of P-H2AX protein was significantly increased by 1.43 times after treatment with 15Gy+Met (P<0.001).The transplanted tumor growth curve showed that the transplanted tumor growth in the 15 Gy+Met group was slower than that in the control group[(1007.0± 388.5) mm3 vs.(2639.0± 242.9) mm3,P< 0.05)].The overall survival time in the 15 Gy irradiation+Met group was 48 d,significantly longer than 32 d in the control group (P<0.001).Compared with the control group,the expression of P-H2AX and Sting proteins in the 15 Gy+ Met group was increased by 8.8-fold and 1.6-fold (both P<0.001).Immunohistochemical staining showed that the infiltration of CD8a (+) T cells in the 15 Gy irradiation+Met group was significantly higher than that in the control group (P<0.01).Conclusions Met combined with radiotherapy can inhibit the proliferation and clone formation of colon cancer cells,probably by aggravating DNA damage and activating the Sting signaling pathway,eventually leading to the increase of CD8a (+) T cells in tumor tissues and enhancing the killing effect upon transplanted tumor cells.
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OBJECTIVE: To investigate the sensitive target cell population of spermatocyte injury induced by TL in mice. METHODS: Seven to eight-week-old healthy male C57BL/6 mice were orally administered triptolid(TL) of 0.125, 0.25 and 0.5 mg•kg-1 per day, and dissected on days 3, 7, 11 and 15 respectively. The extracted testes were fixed with formaldehyde. Paraffin sections and hematoxylin-eosin (HE) staining were performed to determine the optimal time point and dose level to be applied for sensitive target cell population analysis of spermatocyte injury induced by TL. The primary spermatocytes in different stages were clearly distinguished and counted based on the characteristic distribution profile of γ-H2AX in spermatocytes under immunohistochemical staining. The sensitive target cell populations of spermatocyte injury were determined according to the decreased percentage of spermatocytes in different stages. RESULTS: HE staining showed that the best dose-effect relationship of spermatogenic cell injury in the testes was present on day 11 after TL administration (The severity of the lesion ranged from a minimal degree in the 0.125 mg•kg-1 group to a mild to moderate injury in the 0.25 mg•kg-1 group, and finally to a marked injury in the 0.5 mg•kg-1 group). The degree of injury in the 0.125 and 0.25 mg•kg-1 groups was appropriate and suitable for determination of sensitive target cell populations. γ-H2AX immunohistochemical staining indicated that the γ-H2AX showed different distribution characteristics in nucleus in different stages of spermatocyte differentiation: scattered throughout the nucleus in a few discrete foci to fill the whole nuclear in leptotene; assembled in the chromatin regions in zygotene; located on the edge of the nucleus in a single foci in the pachytene; located in the nucleus in a single foci in the diplotene. The counting results showed that the absolute number of primary spermatocytes in all differentiating stages decreased slightly without statistical significance (P>0.05) in the 0.125 mg•kg-1 dose group; the absolute number of primary spermatocytes decreased significantly with statistical significance (P<0.01 or 0.001) in the 0.25 mg•kg-1 dose group. There was higher decreased percentage of the leptotene primary spermatocyte among the differentiating stages before pachytene stage and with statistical significance (P<0.05) at 0.25 mg•kg-1 when compared with the pachytene primary spermatocyte. CONCLUSION: γ-H2AX immunohistochemical staining can clearly distinguish primary spermatocytes at different stages. The leptotene primary spermatocytes are the most likely sensitive target cells in the testicular spermatocyte-injury induced by TL administration.
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The melamine and cyanuric acid (CA) complex has been suggested to cause the toxic effects observed in melamine-contaminated food or milk. However, the cytotoxic and genotoxic effects of co-exposure to melamine and CA are not fully clear. Therefore, the cytotoxic effects of melamine and CA were first examined by co‐exposure in human kidney 293 cells using the MTT assay. During a 24-h period for the three concentrations tested (0.5, 1, and 5 mg/mL), neither melamine nor CA alone showed significant toxic effects on 293 cells at 0.5 mg/mL, while higher concentrations led to decreased in cell viability. However, co-exposure to several combinations of melamine and CA [100:1, 10:1, 1:10, and 1:100 (v:v), at a final concentration of 0.5 mg/mL] did cause cytotoxicity with higher levels of CA leading to higher cytotoxicity. By contrast, while neither melamine nor CA alone induced phosphorylated-H2AX (γH2AX) foci formation, melamine and CA at a 100:1 ratio induced γH2AX foci 24 h post-treatment. The alkaline comet assay also revealed the presence of DNA damage following melamine and CA co-exposure. In vivo assay also revealed the presence of melamine-CA complex in the kidney. These data indicated that the cytotoxic and genotoxic effects of melamine and CA co-exposure differ from those of melamine or CA alone.
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Humans , Animals , Rats , Triazines/toxicity , DNA Damage/drug effects , Cell Survival/drug effects , Kidney/drug effects , Time Factors , Kidney/embryology , Mutagenicity TestsABSTRACT
Aims:Non-small cell lung cancer (NSCLC) accounts for high lung cancer death that is mostly associated with advanced disease stage at diagnosis and resistance to chemotherapy. In the present study, we investigated whether xanthohumol, a prenylated flavonoid of hop plant, induces metastatic lung cancer H1299 cell death, and whether in combination with cisplatin there are additive effects. Methodology:H1299 cells were grown and treated with xanthohumol (6.25, 12.5, or 25 μM), cisplatin (12.5, 25, or 50 μM) and the combination of cisplatin and xanthohumol for 24 h. Cell viability, cell morphology, chromatin condensation, ɣH2AX, cPARP-1, capsase-3, p21WAF1/CIP1and p14ARFgenes were analyzed Results:Xanthohumol, cisplatin, and the combination of cisplatin and xanthohumol inhibited H1299 cells viability. Cisplatin growth inhibitory effects were potentiated by xanthohumol. Xanthohumol induced chromatin condensation and apoptosis and potentiated cisplatin’s effect vs cisplatin alone. Further investigation of growth inhibitory effects, xanthohumol alone induced γH2AX foci formation and the combination potentiated γH2AX foci formation. Cisplatin, xanthohumol at 25 μM, and the combination of cisplatin and xanthohumol at 6.25 and 12.5 μM increased cPARP-1 level. Active caspase-3 was increased by cisplatin, 12.5 μM of xanthohumol, and the combination of xanthohumol and cisplatin. Xanthohumol at 6.25 or 12.5 μM potentiated cisplatin effect on active caspase-3 and cPARP-1, respectively. Xanthohumol at 25 μM significtly induced the expression cell cycle control genes p21WAF1/CIP1and p14ARF. These results indicate that xanthohumol inhibits proliferation of H1299 cells and induces cell death through cleavage of PARP-1 and activation of caspase-3. The combination of cisplatin and xanthohumol potentiated cytotoxic effects of each other compound.Conclusion:The present study suggests that xanthohumol poses apoptotic effects and potentiates cisplatin’s growth inhibitory effects on metastatic lung cancer cells
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Recent research has shown that cell-free chromatin (cfCh) particles that are released from the billions of cells that die in thebody everyday can enter into healthy cells, integrate into their genomes and induce dsDNA breaks and apoptotic responses.Genomic integration of cfCh activates NFjB suggesting a novel mechanism of induction of systemic inflammation. SinceDNA damage and inflammation are underlying pathologies in multiple devastating acute and chronic disease conditions,the discovery of agents that can inactivate cfCh may provide therapeutic possibilities.
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Objective To investigate the expression of bone marrow γ-H2AX in the patients with multiple myeloma(MM) and its correlation with the prognosis.Methods The patients with newly diagnosed MM in this hospital were selected as the case group,and the patients with non-hemopoietic system tumor without obvious morphological abnormalities by bone marrow smear and biopsy served as the control group.The immunohistochemistry was adopted to detect the expression level of bone marrow γ-H2AX in the cases group and control group,the image-Pro Plus(IPP) semiquantitative analysis was performed.The expression differences were compared between the two groups,moreover the case group was re-divided into the strong expression group and weak expression group according to γ-H2AX expression level.Then the relation ship between γ-H2AX expression level and the prognosis in the patients with MM.Results The bone marrow γ-H2AX expression level in the case group was significantly higher than that in the control group (P<0.05);the level of γ-H2AX expression in the strong expression group was significantly stronger than that in the weak expression group (P<0.05).Conclusion The level of γ-H2AX expression was higher among MM patients,and the over expression of γ-H2AX predicts the shorter survival time.
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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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AIM To investigate the effects of Wuzi Yanzong Prescription (Lycii Fructus,Cuscutae Semen,Rubi Fructus,Plantaginis Semen and Schisandrae chinensis Fructus) on DNA damage of testis cells in adult male mice induced by cyclophosphamide (CTX).METHODS Forty out of fifty adult male Balb/C mice were injected intraperitoneally with CTX and then were randomly and equally divided into model control group (normal saline),Wuzi Yanzong Prescription low-,medium-and high-dose groups (100,200 and 400 mg/kg),and the other ten mice served as normal control group (normal saline).All mice were anesthetized by inhalation of ether,and then were sacrificed by cervical dislocation.The sperm count,sperm motility and malformation rate of sperm were tested.The content of 8-hydroxy-deoxyguanosine (8-OHdG) in serum was measured using ELISA;the DNA damage degree of cells in testis was detected by single cell gel electrophoresis;the protein expressions of p-P53 and γ-H2AX in testis were examined by Western blot.RESULTS Compared with the model control group,Wuzi Yanzong Prescription groups showed significant increase in the sperm count,sperm motility and significantly decreased malformation rate of sperm,the level of 8-OHdG in serum,and the protein expressions of p-P53 and γ-H2AX in testis were also significantly decreased.CONCLUSION Wuzi Yanzong Prescription can significantly alleviate the DNA damage of testis cells in mice induced by CTX through down-regulating protein expressions of p-P53 and γ-H2AX.
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Radiation biodosimetry is an important part of radiation medicine. Some new discoveries and progresses have been made in radiation biodosimetry studies in recent years. Cytogenetic method represented by chromosome aberration analysis as the golden standard of radiation biodosimeter is being transformed to automate analysis, and a number of international, regional and national laboratory networks of radiation biodosimetry are being established. As a widely acceptable molecular marker of DNA damage,γ-H2AX has made rapid progress in radiation dose estimation. Based on the expressions of protein and genes, further advancements have been made in the studies of metabolites and miRNAs. At the same time, with the development of proteomics technology, there are some breakthroughs in the study of using molecular expression profiling to evaluate radiation dose. The research progresses of radiation biodosimetry is reviewed in this paper.
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Radiation biodosimetry is an important part of radiation medicine. Some new discoveries and progresses have been made in radiation biodosimetry studies in recent years. Cytogenetic method represented by chromosome aberration analysis as the golden standard of radiation biodosimeter is being transformed to automate analysis, and a number of international, regional and national laboratory networks of radiation biodosimetry are being established. As a widely acceptable molecular marker of DNA damage,γ-H2AX has made rapid progress in radiation dose estimation. Based on the expressions of protein and genes, further advancements have been made in the studies of metabolites and miRNAs. At the same time, with the development of proteomics technology, there are some breakthroughs in the study of using molecular expression profiling to evaluate radiation dose. The research progresses of radiation biodosimetry is reviewed in this paper.
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Objective To evaluate the effect of iodine contrast agent on the biological responses of CT examination. Methods A total of sixty patients with suspected urinary tract disease who underwent computed tomography urography ( CTU ) examination were randomly divided into control group and experimental group. The control group was treated with routine CTU, where only CT scan was performed on the first day. CTU was added after 3 days. The test group was treated with fractional injection CTU and injected with enhanced scanning agent on the first day. Before and after CT examination, the patients′peripheral blood was collected and the number of γ-H2AX foci in lymphocytes ( mononuclear cells) was measured by immunofluorescence, and the differences of DNA damage in these two groups were observed. Results Before and after CT examination, the number ofγ-H2AX foci was 0. 06 ± 0. 02 and 1. 06 ± 0. 27 in the lymphocytes of control group,0. 06 ± 0. 03 and 1. 42 ± 0. 50 in the test group, respectively. Hence, the number ofγ-H2AX foci in the test group was increased by 38. 14%. Moreover, the change ofγ-H2AX foci in these two groups was not influenced by gender, but correlated with ages( between≤50 years old and>50 years old) in control group (t= -4. 76, P<0. 05) and in test group(t= -8. 16, P <0. 05). Conclusions The iodine contrast agent can increase DNA damage of CT examination, and therefore the use of iodine contrast agent in CT should be reduced as much as possible in clinical work.
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Objective To synthesize novel gold nanoparticles of GAL-PEG-GNPs,study its radiation effect on hepatocellular carcinoma cells HepG2 cells in vitro,and investigate the underlying mechanisms.Methods GAL-PEG-GNPs were synthesized and characterized successfully.HepG2 cells were divided into three groups of control,GNPs and GAL-PEG-GNPs.The cytotoxicities of these compounds were tested by the CCK-8 assay and their IC50 values of HepG2 cells were calculated.Cell uptake of nanoparticles was detected by TEM and ICP-MS.The radiosensitization effect of nanoparticles was tested by the colony formation assay.Cell cycle distribution was detected by FCM.The expressions of CAT,SOD,and total GSH were detected with a microplate reader,and the expressions of apoptosis-related proteins were tested by Western blot.Results The GNPs and GAL-PEG-GNPs had absorption peaks at 520 and 530 nm,respectively,and their diameters were (22.6-±2.12) and (32.0 ± 1.41) nm detected by ICP-MS.The GAL-PEG-GNPs and GNPs had similar cytotoxicity profiles (P > 0.05),while GAL-PEG-GNPs could be more effectively uptaken by HepG2 cells than GNPs.The sensitive enhancement ratio (SER) of GNPs and GAL-PEG-GNPs to HepG2 cells were 1.46 和 1.95,respectively.The percentage of cells at phase of G2/M in HepG2 population treated with GNP was higher than that of untreated cells (t =14.20,P <0.05).The protein expressions of Cytochrome C,Bax,Caspase-3,and Caspase-9 were upregulated while Bcl-2 expression was down-regulated in the cells treated with GNPs/radiation or GAL-PEGGNPs/radiation.The expressions of CAT,SOD and total GSH in the GNP treated groups were significantly decreased compared with the control group(t =12.34,29.39,12.85,P < 0.05).Conclusions GALPEG-GNPs has obvious radiosensitization effect on HepG2 cells,which is related to the induction of cell apoptosis and the generation of free radicals.
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OBJECTIVE: To explore the effect of~(56)Fe~(17+)heavy ion on the expression of phosphorylated histone H2AX( γH2AX) of human lymphccytes. METHODS: The Epstein-Barr virus transformed human B lymphocyte cell lines( PengEBV) were selected and exposed to~(56)Fe~(17+)heavy ion at irradiation dose of 0. 0( control group),0. 1,0. 3,0. 5,0. 7,1. 0 and 2. 0 Gy,respectively,with the dosing rate of 0. 23-0. 55 Gy / min. Flow-cytometry was used to detect the changes of expression of γH2AX at time points of 0,2,4,8,48 and 72 hours after irradiation. RESULTS: The expression of γH2AX showed interaction existed between radiation dose and the treatment time after radiation( P < 0. 01). Compared with the control at the same time points,the expression of γH2AX increased at the dose of 0. 3-2. 0 Gy and the time points of 2-72hours( P < 0. 05). The expression of γH2AX at the dose of 0. 3-2. 0 Gy and time points of 8-72 hours was lower than those at the same dose and time points of 2 and 4 hours( P < 0. 05). When the dose was at 0. 5,1. 0 or 2. 0 Gy,the expression of γH2AX decreased with the increasing time of exposure in 72 hours( P < 0. 05). At the dose of 0. 0-1. 0 Gy and the time points of 2-4 hours,the expression of γH2AX increased with the increasing dose of irradiation( P < 0. 01). CONCLUSION: The expression of γH2AX in Peng-EBV cells shows a dose-response relationship within 2-4 hours after 0. 0-1. 0 Gy irradiation of~(56)Fe~(17+).
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In eukaryotic cells, DNA is packaged inside the nucleus together with histones to form nucleosomes. Each histone molecule contains two of each core histone subunits H2A, H2B, H3 and H4. Among core histones, the H2A family is of great interest due to the high diversity of specialized variants. These variants have shown important role in critical cellular processes. Epigenetic mechanism in oomycetes is barely known. Phytophthora infestans is a severe pathogen and a model species in oomycetes. In this study, we studied the sequence and expression pattern of H2A variants of P. infestans through genome search, sequences alignment, phylogenetic analysis and realtime qPCR detection. P. infestans contains conserved genes encoding histone variants H2A.X.1, H2A.X.2 and H2A.Z, and these genes have specific expression patterns during development and infection stages. Our datasets provide useful inputs to help explore the epigenetic mechanisms of oomycetes.
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Objective To evaluate the potential feasibility of γ-H2AX foci as a biodosimetry after exposure to ionizing radiation by comparing DNA double-strand break repair kinetics in rat blood lymphocytes with that in human lymphocytes.Methods Peripheral blood lymphocytes separated from Sprague-Dawley(SD) male rats and healthy adults were exposed to γ-rays,and some rats were also subjected to total body irradiation.The inductions of DNA repair-related foci of γ-H2AX,pATM (S1981) and pDNA-PKcs (T2609) were detected with immunofluorescence staining technique at different time points post-irradiation,and the status of their co-localization was analyzed.Results The induction kinetics of γ-H2AX foci in rat lymphocytes was similar to that observed in human lymphocytes.The frequencies of γ-H2AX foci peaked at 30 min after γ-ray irradiation (trst =62.64,th =28.52,P < 0.05),then decreased rapidly after 6 h post-irradiation (trat =45.96,th =14.80,P <0.05),and the residual foci number remained only about 3%-8% of its maximal value at 24 h post-irradiation.At 30 min after γ-ray irradiation,the frequencies of pATM (S1981) and pDNA-PKcs (T2609) foci in rat and human lymphocytes significantly higher than those of nonirradiated control (trat =21.05,25.80,th =11.07,29.52,P < 0.05),and the frequencies of co-localization of pATM (S1981) or pDNA-PKcs (T2609) foci with γ-H2AX foci also markedly increased by 26%-32% in irradiated lymphocytes of rat and human (trat =5.34,9.14,thuman =18.32,51.28,P <0.05).Moreover,γ-H2AX foci incidence in rat lymphocytes in vitro was consistent with that induced by total body irradiation of rat.The number of γ-H2AX foci in irradiated rat lymphocytes increased with irradiation dose in a linear dose-dependent manner,its slope was similar to that of irradiated human lymphocytes reported by other laboratory.Conclusions Rat is a useful animal model to evaluate radiation biodosimetry with γ-H2AX foci in lymphocytes.The co-activation of ATM and DNA-PK plays an important role in DSB repair in the irradiated lymphocytes of rat and human.
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AIM:To investigate the effect of MK-2206, an inhibitor of protein kinase B (Akt), on the DNA damage of SGC-7901 cells.METHODS: SGC-7901 cells were treated with different concentrations of MK-2206, and phosphorylated histone H2AX (γ-H2AX) foci formation was detected by immunofluorescence staining .Western blot analy-sis was used to exam the levels of DNA damage-related protein.The expression of LC3-Ⅱ was determined to evaluate the change of autophagy .RESULTS:MK-2206 treatment increased the formation of γ-H2AX foci and histone H2AX phospho-rylation in the SGC-7901 cells.The levels of DNA damage response protein were also increased .In addition, MK-2206-treated SGC-7901 cells increased the expression of LC 3-II, a hallmark of autophagy .Inhibition of autophagy significantly enhanced MK-2206-mediated histone H2AX phosphorylation.CONCLUSION:MK-2206 induces DNA damage and auto-phagy in SGC-7901 cells.Blocking autophagy potentiates the response of MK-2206-induced DNA damage .
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Objective To investigate whether γH2AX could be a useful biomarker for evaluating the DNA double‐stranded . Methods Semem samples in case group were from 27 infertile males who were diagnosed in Andriatrics department or reproductive centre in the First Affiliated Hospital of Guangxi Medical University .The other semen samples were from 23 healthy donors with fertility as comparison .The levels of γH2AX were detected by flow cytometry .Single cell gel electropherosis(SCGE)was applied to assess the level of DSBs of sperm .Density gradient centrifugation(DGC) was applied to optimized spermatozoa .Results TheγH2AX levels and the DSBs of the sperm of the infertile subjects were significantly higher than those of healthy males(P<0 .01) , and the levels of γH2AX and the DSBs of sperm significantly decreased in two groups by DGC(P<0 .01) .Conclusion The level of spermatozoaγH2AX is higher in male infertility patients than in healthy donors with fertility ,which might be a useful biomarker for evaluating DSBs of sperm .
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Aim To investigate the effect of BMS- 345541 on the repair of DNA DSBs induced by VP-16 in AML cells and its possible mechanism. Methods The effects of BMS-345541 on the sensitivity of AML cells to VP-16 were determined by MTT. Flow cytome-try ( FCM) was applied to test the level of DNA dam-age, cell cycle progression and apoptosis in AML cells. High content analysis ( HCA) was used to verify the amount ofγ-H2AX,p-ATM,RAD51 in AML cells. Results BMS-345541 could significantly inhibit the proliferation of AML cells induced by VP-16 . BMS- <br> 345541 increased the amount of RAD51 foci and p-ATM foci in AML cells treated with VP-16 after 6 hours , which led to increased numbers of cells in the G2/M phases of the cell cycle,then induced apoptotic cell death. Conclusion BMS-345541 sensitizes AML cells to VP-16 via selective inhibition of homologous recombinational repair of DNA double-strand breaks.
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Objective To investigate the influence of MgSO4 with different concentrations on cell survival and γ-H2AX expression in HUVEC irradiated with X-rays.Methods Cell proliferation rate was assayed by CCK-8,γ-H2AX foci formationwas observed with a laser confocal microscope,and γ-H2AX protein expression was detected by flow cytometer and Western blot assay.Results MgSO4 with a concentration of 1.25 mg/ml could improve the survival rate of IHUVEC treated with X-rays (t =-6.34,P < 0.05).After irradiation,γ-H2AX loci approached to the highest level from 30 min to 1 h after radiation and then decreased.MgSO4 significantly reduced the foci formation at 0.5,1,2,6 and 12 h postirradiation (t =12.62,6.36,11.93,5.75,9.43,P < 0.05).Both flow cytometry,and Western blot assays showed that MgSO4 inhibited γ-H2AX protein expression at 0.5,1 and 2 h post-irradiation (t =6.07,5.32,11.85,P < 0.05).Conclusions MgSO4 could improve the survival rate and reduced γ-H2AX expression in HUVEC irradiated with X-ray.