To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer,A549 cells were subj...To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer,A549 cells were subjected to irradiation at the dosage ranging from 0.05-2 Gy.Clonogenic survival was measured by using fluorescence-activated cell sorting (FACS) plating technique.Percentage of cells in M-phase after low doses of X-irradiation was evaluated by phospho-histone H3-FITC/PI and Western blotting was used to detect protein expression of Chk2 and phospo-Chk2.DNA DSBs repair efficiency was also measured by induction and persistence of γ-H2AX.The results showed that the killing ability of irradiation with A549 cells increased at low conditioning dose below 0.3 Gy.Within the dose of 0.3 to 0.5 Gy,A549 cells showed a certain extent of radiation resistance.And when the dose was more than 0.5 Gy,survival fraction exhibited a negative correlation with the dosage.There was no difference between the 0.1 or 0.2 Gy dosage groups and the un-irradiated group in terms of the percentage of cells in M phase.But in the high dosage group (0.3-1.0 Gy),the percentage of cells in M phase was decreased markedly.In addition,the percentage of cells in M phase began to decrease two hours after irradiation.One hour after irradiation,there was no conspicuous activation of Chk2 kinase in 0.1 or 0.2 Gy group,but when the irradiation dose reached 0.3 Gy or higher,Chk2 kinase started to be activated and the activation level showed no significant difference among high dosage groups (0.4,0.5,1.0 Gy).Within 1 to 6 h,the DNA DSBs repair efficiency was decreased at 0.2 Gy but increased at 0.5 Gy and 1.0 Gy,which was in line with Chk2 activation.We are led to conclude that the mechanism of HRS/IRR in A549 cell line was probably due to early G2/M checkpoint arrest and enhanced DNA DSBs repair.In this regard,Chk2 activation plays a key role in G2/M checkpoint activation.展开更多
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 d...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 BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.展开更多
The purpose of the study was to investigate if the high gradient strength and slew rate used for long MRI-thermometry monitoring could cause DNA double-stranded breaks (DSBs). To this end, an enzyme-linked immunosorbe...The purpose of the study was to investigate if the high gradient strength and slew rate used for long MRI-thermometry monitoring could cause DNA double-stranded breaks (DSBs). To this end, an enzyme-linked immunosorbent assay (ELISA) was used to quantify γH2AX, a molecular marker for DSBs, in the blood of mice after a 6-hour exposure to magnetic resonance imaging (MRI). Fourteen CF-1 female mice were separated into 4 experimental groups: Untreated negative control, MRI-treated, MRI-Control, and exposed to ionizing radiation positive control. Untreated negative control was used as a baseline for ELISA to quantify γH2AX. MRI-treated consisted of a 6-hour continuous magnetic resonance imaging (MRI) echo planar imaging (EPI) sequence with a slew rate of 192 mT/m/s constituting a significantly longer imaging time than routine clinical imaging. MRI-control mice were maintained under the same conditions outside the MRI scanner for 6-hours. Mice in the irradiation group served as a positive control of DSBs and were exposed to either 2 Gy, 5 Gy or 10 Gy of ionizing radiation. DSBs in the blood lymphocytes from the treatment groups were analyzed using the γH2AX ELISA and compared. Total protein concentration in lysates was determined for each blood sample and averaged 1 ± 0.35 mg/mL. Irradiated positive controls were used to test radiation dose-dependency of the γH2AX ELISA assay where a linear dependency on radiation exposure was observed (r<sup>2</sup> = 0.93) between untreated and irradiated samples. Mean and standard error mean of γH2AX formation were calculated and compared between each treatment group. Repeated measures 1-way ANOVA showed statistically significant differences between the means of irradiated controls and both the MRI-control and MRI-treated groups. There was no statistically significant difference between the MRI-treated samples and the MRI-control groups. Our results show that long MRI exposure at a high slew rate did not cause increased levels of γH2AX when compa展开更多
Yeast strain Saccharornyces cerevisiae was irradiated with different doses of 85 MeV/u 20 Ne 10＋ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand b...Yeast strain Saccharornyces cerevisiae was irradiated with different doses of 85 MeV/u 20 Ne 10＋ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks （DSBs） and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T-G and T-C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.展开更多
DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damag...DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks(DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination(HR) and non-homologous end joining(NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair.Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes(DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.展开更多
基金supported by a grant from a Key ScientificResearch Program of Hubei Provincial Authorities (No.JX2A11)
文摘To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer,A549 cells were subjected to irradiation at the dosage ranging from 0.05-2 Gy.Clonogenic survival was measured by using fluorescence-activated cell sorting (FACS) plating technique.Percentage of cells in M-phase after low doses of X-irradiation was evaluated by phospho-histone H3-FITC/PI and Western blotting was used to detect protein expression of Chk2 and phospo-Chk2.DNA DSBs repair efficiency was also measured by induction and persistence of γ-H2AX.The results showed that the killing ability of irradiation with A549 cells increased at low conditioning dose below 0.3 Gy.Within the dose of 0.3 to 0.5 Gy,A549 cells showed a certain extent of radiation resistance.And when the dose was more than 0.5 Gy,survival fraction exhibited a negative correlation with the dosage.There was no difference between the 0.1 or 0.2 Gy dosage groups and the un-irradiated group in terms of the percentage of cells in M phase.But in the high dosage group (0.3-1.0 Gy),the percentage of cells in M phase was decreased markedly.In addition,the percentage of cells in M phase began to decrease two hours after irradiation.One hour after irradiation,there was no conspicuous activation of Chk2 kinase in 0.1 or 0.2 Gy group,but when the irradiation dose reached 0.3 Gy or higher,Chk2 kinase started to be activated and the activation level showed no significant difference among high dosage groups (0.4,0.5,1.0 Gy).Within 1 to 6 h,the DNA DSBs repair efficiency was decreased at 0.2 Gy but increased at 0.5 Gy and 1.0 Gy,which was in line with Chk2 activation.We are led to conclude that the mechanism of HRS/IRR in A549 cell line was probably due to early G2/M checkpoint arrest and enhanced DNA DSBs repair.In this regard,Chk2 activation plays a key role in G2/M checkpoint activation.
基金supported by a grant from the National Key Technology R&D Program (No. 30270727)
文摘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 BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.
文摘The purpose of the study was to investigate if the high gradient strength and slew rate used for long MRI-thermometry monitoring could cause DNA double-stranded breaks (DSBs). To this end, an enzyme-linked immunosorbent assay (ELISA) was used to quantify γH2AX, a molecular marker for DSBs, in the blood of mice after a 6-hour exposure to magnetic resonance imaging (MRI). Fourteen CF-1 female mice were separated into 4 experimental groups: Untreated negative control, MRI-treated, MRI-Control, and exposed to ionizing radiation positive control. Untreated negative control was used as a baseline for ELISA to quantify γH2AX. MRI-treated consisted of a 6-hour continuous magnetic resonance imaging (MRI) echo planar imaging (EPI) sequence with a slew rate of 192 mT/m/s constituting a significantly longer imaging time than routine clinical imaging. MRI-control mice were maintained under the same conditions outside the MRI scanner for 6-hours. Mice in the irradiation group served as a positive control of DSBs and were exposed to either 2 Gy, 5 Gy or 10 Gy of ionizing radiation. DSBs in the blood lymphocytes from the treatment groups were analyzed using the γH2AX ELISA and compared. Total protein concentration in lysates was determined for each blood sample and averaged 1 ± 0.35 mg/mL. Irradiated positive controls were used to test radiation dose-dependency of the γH2AX ELISA assay where a linear dependency on radiation exposure was observed (r<sup>2</sup> = 0.93) between untreated and irradiated samples. Mean and standard error mean of γH2AX formation were calculated and compared between each treatment group. Repeated measures 1-way ANOVA showed statistically significant differences between the means of irradiated controls and both the MRI-control and MRI-treated groups. There was no statistically significant difference between the MRI-treated samples and the MRI-control groups. Our results show that long MRI exposure at a high slew rate did not cause increased levels of γH2AX when compa
基金supported by Natural Science Foundation of Gansu Province (No.096RJZA126),Department of Science And Technology, Gansu Province, China
文摘Yeast strain Saccharornyces cerevisiae was irradiated with different doses of 85 MeV/u 20 Ne 10＋ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks （DSBs） and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T-G and T-C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.
文摘在自然界中,植物会遭受各种环境或内源因素导致的DNA损伤,其中DNA双链断裂(double strand breaks,DSBs)的影响最为严重,如果修复不当,将导致基因组不稳定、基因突变甚至细胞死亡。一方面,植物进化出了强大且有序的损伤修复机制,以确保其存活及正常繁衍;另一方面,基于修复过程的容错性及致突变性,T-DNA插入、基因编辑、物理诱变等技术广泛应用于动植物品种改良。相较于哺乳动物,植物DSBs修复通路及其分子机制报道较为有限。本文综述了植物对DSBs损伤的响应、主要修复途径及关键因子,介绍了通路机制尚未完全解析的替代末端连接(alternative end joining,Alt-EJ)的最新研究进展;此外,探讨了重离子束引起的植物DSBs修复特征和多途径选择,以及基于不同DSBs修复途径的基因编辑技术的研究进展,旨在为深入了解植物DSBs损伤响应及修复的分子机制和研发高效生物育种技术提供参考。
基金supported by the National Natural Science Foundation of China (Nos. 91749115 and 81872298)the Natural Science Foundation of Jiangxi Province (No. 20181BAB205044), China。
文摘DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks(DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination(HR) and non-homologous end joining(NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair.Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes(DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.
