1.Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells.
Wan-ling HUANG ; Chang-zheng LI ; Zhen-rui CHEN ; Wei HE ; Ye ZHOU ; Zhi-gang ZHOU ; Shu-wen LIU ; Chen ZHOU
Journal of Southern Medical University 2010;30(1):111-113
OBJECTIVETo study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells.
METHODSThe expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed.
RESULTSThe transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments.
CONCLUSIONIn construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.
Bacteria ; genetics ; DNA Damage ; radiation effects ; DNA Repair ; Genetic Vectors ; radiation effects ; Transformation, Bacterial ; radiation effects ; Ultraviolet Rays
2.Effect of acute exposure to microwave from mobile phone on DNA damage and repair of cultured human lens epithelial cells in vitro.
Li-xia SUN ; Ke YAO ; Ji-liang HE ; De-qiang LU ; Kai-jun WANG ; Hong-wu LI
Chinese Journal of Industrial Hygiene and Occupational Diseases 2006;24(8):465-467
OBJECTIVETo investigate the DNA damage of human lens epithelial cells (LECs) caused by acute exposure to low-power 217 Hz modulated 1.8 GHz microwave radiation and DNA repair.
METHODSCultured LECs were exposed to 217 Hz modulated 1.8 GHz microwave radiation at SAR (specific absorption rate) of 0, 1, 2, 3 and 4 W/kg for 2 hours in an sXc-1800 incubator and irradiate system. The DNA single strand breaks were detected with comet assay in sham-irradiated cells and irradiated cells incubated for varying periods: 0, 30, 60, 120 and 240 min after irradiation. Images of comets were digitized and analyzed using an Imagine-pro plus software, and the indexes used in this study were tail length (TL) and tail moment (TM).
RESULTSThe difference in DNA-breaks between the exposure and sham exposure groups induced by 1 and 2 W/kg irradiation was not significant at every detect time (P > 0.05). As for the dosage of 3 and 4 W/kg there was difference in both group immediately after irradiation (P < 0.01). At the time of 30 min after irradiation the difference went on at both group (P < 0.01). However, the difference disappeared after one hour's incubation in 3 W/kg group (P > 0.05), and existed in 4 W/kg group.
CONCLUSIONNo or repairable DNA damage was observed after 2 hour irradiation of 1.8 GHz microwave on LECs when SAR < or = 3 W/kg. The DNA damages caused by 4 W/kg irradiation were irreversible.
Cell Phone ; Cells, Cultured ; Comet Assay ; DNA Damage ; radiation effects ; DNA Repair ; Dose-Response Relationship, Radiation ; Epithelial Cells ; radiation effects ; Humans ; Lens, Crystalline ; cytology ; radiation effects ; Microwaves
3.Effects of 50 Hz magnetic fields on DNA double-strand breaks in human lens epithelial cells.
Xiao-gang DU ; Shan-shan XU ; Qing CHEN ; De-qiang LU ; Zheng-ping XU ; Qun-li ZENG
Journal of Zhejiang University. Medical sciences 2008;37(1):9-14
OBJECTIVETo investigate the effects of 50 Hz magnetic fields (MF) on DNA double-strand breaks in human lens epithelial cells (hLECs).
METHODSThe cultured human lens epithelial cells were exposed to 0.4 mT 50 Hz MF for 2 h, 6 h, 12 h, 24 h and 48 h. Cells exposed to 4-nitroquinoline-1-oxide, a DNA damage agent, at a final concentration of 0.1 micromol/L for 1 h were used as positive controls.After exposure, cells were fixed with 4 % paraformaldehyde and for H2AX (gamma H2AX) immunofluorescence measurement. gamma H2AX foci were detected at least 200 cells for each sample. Cells were classified as positive when more than three foci per cell were observed. Mean values of foci per cell and percentage of foci positive cells were adopted as indexes of DNA double-strand breaks.
RESULTThe mean value of foci per cell and the percentage of gamma H2AX foci positive cells in 50 Hz MF exposure group for 24 h were (2.93 +/-0.43) and (27.88 +/-2.59)%, respectively, which were significantly higher than those of sham-exposure group [(1.77 +/-0.37) and (19.38+/-2.70)%, P <0.05], and the mean value of foci per cell and the percentage of gamma H2AX foci positive cells in 50 Hz MF exposure group for 48 h were (3.14 +/-0.35) and (31.00 +/-3.44)%, which were significantly higher than those of sham-exposure group (P <0.01). However there was no significant difference between 50 Hz MF exposure groups for 2 h, 6 h, 12 h and sham-exposure group for above two indexes (P >0.05).
CONCLUSION0.4 mT 50 Hz MF exposure for longer duration might induce DNA double-strand breaks in human lens epithelial cells in vitro.
Cells, Cultured ; DNA ; radiation effects ; DNA Breaks, Double-Stranded ; radiation effects ; DNA Damage ; radiation effects ; DNA Repair ; radiation effects ; Electromagnetic Fields ; Epithelial Cells ; metabolism ; radiation effects ; Humans ; Lens, Crystalline ; cytology
4.Detection of DNA injury and repair with single cell gel electrophoresis radiation in biodosimetry.
Qiang LIU ; En-Hai JIANG ; Jin LI ; Wei-Sheng TANG ; Zhi-Quan WANG ; Yong-Cheng ZHAO ; Fei-Yue FAN
Chinese Journal of Industrial Hygiene and Occupational Diseases 2006;24(12):734-738
OBJECTIVETo explore the relationship between DNA repair in vitro and in vivo after irradiation, and to describe the curves of DNA repair which can improve the accuracy of radiation dose estimation.
METHODSThe DNA double-strand break in lymphocytes of human and mouse was detected using neutral single cell gel electrophoresis (SCGE) after radiation and the curves of DNA repair individually were estimated, which were compared later.
RESULTSAlong with the time lapsing, the DNA repair of human peripheral blood and mice increased significantly and the residual damage decreased gradually, which showed significant time-effect relationship. The curve of DNA repair in vitro of human lymphocytes presented the same log model as that of mouse DNA repair in vivo. The curve showed as followed respectively: Mice: Y(TM) = 55.8256 - 10.792 lnX (R(2) = 0.629, P < 0.01) and Y(OTM) = 25.4173 - 4.5273 lnX (R(2) = 0.661, P < 0.01); Human: Y(TM) = 30.242 7 - 7.383 6 lnX (R(2) = 0.686, P < 0.01) and Y(OTM) = 17.9772 - 3.9125 lnX (R(2) = 0.752, P < 0.01).
CONCLUSIONThe curve of DNA repair in vitro of human lymphocytes could be considered in biodosimetry estimation because the process of DNA repair in vitro could display the repair level and speed of DNA double-strand break in vivo.
Animals ; Cell Survival ; Comet Assay ; DNA Damage ; radiation effects ; DNA Repair ; radiation effects ; Dose-Response Relationship, Radiation ; Female ; Humans ; Lymphocytes ; radiation effects ; Male ; Mice ; Mice, Inbred Strains ; Radiation Dosage ; Single-Cell Analysis
5.Radiosensitivity detected by "comet" assay in two human tumor cell lines.
Yuan-hong GAO ; Wei-zhi YANG ; Jie YAN ; Zhi-yong YUAN ; Xin-fan LIU ; Guo-zhen XU
Chinese Journal of Oncology 2004;26(4):217-219
OBJECTIVETo evaluate the value of the "comet" assay in detecting the radiosensitivity in human tumor cell lines.
METHODSThe radiation-induced primary DNA damage and repair were detected by the comet assay in CNE-1 and 973 cell lines. The tail moment was used as the end point, to quantitate the primary DNA damage and subsequent repair ability. The cell-survival curve was plotted by the classical colony assay, to detect the D(0) value and Dq value. The results from the above two assays were compared.
RESULTS1. With the increment of irradiation doses, under the same experimental condition, the radiation-induced primary DNA damage was more severe in CNE-1 cells than in 973 cells (P < 0.01). From the cell-survival curves, the D(0) value was 1.631 and 1.822 in CNE-1 and CNE-1 973 cells respectively, indicating that CNE-1 cells were more sensitive to irradiation than 973 cells. The radiosensitivity detected by comet assay and by colony assay in the two cell lines tended to be consistent. 2. The half-repair time of 973 and CNE-1 cell line was 33 min and 41 min detected by comet assay, which indicats that the ability of DNA damage and repair in CNE-1 cells was weaker than in 973 cells. The Dq value of the cell survival curve was 2.152 for 973 and 0.626 for CNE-1 cell line detected by the colony assay, which indicates that the sublethal damage repair in 973 cells being much faster than in CNE-1 cells. The repair ability reflected by the results in the two cell lines was consistent.
CONCLUSIONThe radiosensitivities reflected by the results of the primary DNA damage and repair detected by both comet assay and colony assay in CNE-1 and 973 cells are consistent. It suggests that comet assay is a good method for detecting the radiosensitivity of tumor cells.
Adenocarcinoma ; pathology ; Carcinoma, Squamous Cell ; pathology ; Cell Line, Tumor ; Cell Survival ; radiation effects ; Comet Assay ; DNA Damage ; radiation effects ; DNA Repair ; drug effects ; Humans ; Lung Neoplasms ; pathology ; Nasopharyngeal Neoplasms ; pathology ; Particle Accelerators ; Radiation Dosage ; Radiation Tolerance
6.DNA-PKcs silencing inhibit the DNA repair induced by low dose radiation on human breast epithelial cells.
Wei ZOU ; Jian CHE ; Chongjie WANG ; Yuying CUI ; Qinming ZHANG
Chinese Journal of Biotechnology 2009;25(5):727-732
DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), plays an important role in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand breaks (DSBs) repair. To investigate the effects of DNA-PKcs down-regulation on cell growth and sensitization to low dose radiation (LDR), we transfected DNA-PKcs siRNA into human mammary epithelia cells MCF10F, then, detected the proliferation curve of the cells and the expression of protiens in DNA repair pathways. The results showed that DNA-PKcs gene silencing, induced by the transfection of DNA-PKcs siRNA could suppress significantly the cell proliferation and inhibit the expression of the DNA repair proteins, such as Ku80, ATM and P53 after 50 cGy 137Cs gamma-irradiation.The results suggested that DNA-PKcs gene silencing could increase the sensitivity of human breast epithelial cells to the LDR, which might be relative with the decrease of the proteins.
Breast
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cytology
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Cell Line
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DNA Repair
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drug effects
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radiation effects
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DNA-Activated Protein Kinase
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genetics
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Dose-Response Relationship, Radiation
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Epithelial Cells
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metabolism
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radiation effects
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Gene Silencing
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Humans
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Nuclear Proteins
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genetics
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RNA, Small Interfering
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genetics
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Transfection
7.Differential gene expression profiles of DNA repair genes in esophageal cancer cells after X-ray irradiation.
Hai ZHANG ; Xian-Shu GAO ; Jing ZHAO ; Wei XIONG ; Min ZHANG ; Hong-Zhen LI ; De-Min ZHOU ; Xin JIN ; Dan-Shen ZHANG
Chinese Journal of Cancer 2010;29(10):865-872
BACKGROUND AND OBJECTIVEVarious factors affect the radioresistance of tumor cells, with unknown molecular mechanism(s). Many genes have been found to associate with the radioresistance of tumor cells, however, the precise mechanism of these genes have not been elucidated. This paper was to analyze the differential expressions of DNA repair genes in esophageal carcinoma cells at different time after X-ray irradiation, and to investigate the role of these DNA repair genes in radiation resistance.
METHODSEsophageal cancer parental cells Seg-1 were treated with continuous 2 Gy of fractionated irradiation until the total dose reached 60 Gy to establish the radioresistant cell line Seg-1R. Total RNA was extracted from each cell line at 0, 8, and 24 h after irradiation. Illumine Human-6 V3 microarray was used to identify differentially expressed genes between parental and radioresistant cells. Ten genes involved in DNA repair were obtained and their expressions at different time points after irradiation were analyzed by Gene Ontology analysis.
RESULTSTen DNA repair associated genes were found to be differentially expressed. Three of these genes, SLK, HMGB1, and PMS1, were not only differentially expressed between parental and radioresistant cell lines, but also expressed differently at different time points after irradiation in the same cell line.
CONCLUSIONSPMS1 may be an important factor involved in the mechanism of radioresistance of esophageal carcinoma cells.
Cell Line, Tumor ; radiation effects ; DNA Repair ; genetics ; DNA, Neoplasm ; genetics ; Esophageal Neoplasms ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; radiation effects ; Humans ; MutL Proteins ; Neoplasm Proteins ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; Radiation Tolerance ; Transcriptome ; X-Rays
8.Detecting DNA repair capacity of human lymphocytes exposed to ultraviolet C with comet assay.
Wei ZHENG ; Ji-liang HE ; Li-fen JIN ; Jian-lin LOU ; Bao-hong WANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2004;22(2):93-95
OBJECTIVETo assess DNA repair capacity of human lymphocytes with comet assay.
METHODSFresh lymphocytes form twelve 26-year old donors (6 males, 6 females) were exposed to ultraviolet C (UVC, 254 nm) at the dose rate of 1.5 J/m(2). The lymphocytes of each donor were divided into three parts: UVC group, UVC + aphidicolin (APC) group, UVC + novobiocin (NOV) group. DNA single strand breaks were detected with comet assay in UVC-irradiated cells and unirradiated cells incubated for 30, 60, 90, 120, 180 and 240 min. DNA repair rate (DRR) was calculated and served as an indicator of DNA repair capacity.
RESULTSThe maximum average comet tail length (MTL) in three groups appeared 90 min after UVC exposure. The DRR range of UVC group was 81.84% (62.84% - 98.71%); There was no significant difference in DRR between males and females (P > 0.05). However, the average DRRs of UVC + NOV group and UVC + APC group (52.98% and 39.57% respectively) were significantly lower than that of UVC group (P < 0.01).
CONCLUSIONComet assay is a rapid and simple screening test to assess DNA repair capacity. DRR, as an indicator, may express the individual DNA repair capacity.
Aphidicolin ; pharmacology ; Comet Assay ; methods ; DNA ; drug effects ; genetics ; radiation effects ; DNA Repair ; Enzyme Inhibitors ; pharmacology ; Female ; Humans ; Lymphocytes ; drug effects ; metabolism ; radiation effects ; Male ; Novobiocin ; pharmacology ; Ultraviolet Rays
9.Differential expression of thymic DNA repair genes in low-dose-rate irradiated AKR/J mice.
Jin Jong BONG ; Yu Mi KANG ; Suk Chul SHIN ; Seung Jin CHOI ; Kyung Mi LEE ; Hee Sun KIM
Journal of Veterinary Science 2013;14(3):271-279
We previously determined that AKR/J mice housed in a low-dose-rate (LDR) (137Cs, 0.7 mGy/h, 2.1 Gy) gamma-irradiation facility developed less spontaneous thymic lymphoma and survived longer than those receiving sham or high-dose-rate (HDR) (137Cs, 0.8 Gy/min, 4.5 Gy) radiation. Interestingly, histopathological analysis showed a mild lymphomagenesis in the thymus of LDR-irradiated mice. Therefore, in this study, we investigated whether LDR irradiation could trigger the expression of thymic genes involved in the DNA repair process of AKR/J mice. The enrichment analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways showed immune response, nucleosome organization, and the peroxisome proliferator-activated receptors signaling pathway in LDR-irradiated mice. Our microarray analysis and quantitative polymerase chain reaction data demonstrated that mRNA levels of Lig4 and RRM2 were specifically elevated in AKR/J mice at 130 days after the start of LDR irradiation. Furthermore, transcriptional levels of H2AX and ATM, proteins known to recruit DNA repair factors, were also shown to be upregulated. These data suggest that LDR irradiation could trigger specific induction of DNA repair-associated genes in an attempt to repair damaged DNA during tumor progression, which in turn contributed to the decreased incidence of lymphoma and increased survival. Overall, we identified specific DNA repair genes in LDR-irradiated AKR/J mice.
Animals
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DNA Repair/*radiation effects
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Dose-Response Relationship, Radiation
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Female
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Gene Expression Regulation/*radiation effects
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Gene Regulatory Networks/radiation effects
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Lymphoma/etiology/*genetics
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Mice
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Mice, Inbred AKR
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Oligonucleotide Array Sequence Analysis
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*Radiation, Ionizing
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Reverse Transcriptase Polymerase Chain Reaction
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Thymus Gland/*radiation effects
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Thymus Neoplasms/etiology/*genetics
10.Time-course analysis of DNA damage response-related genes after in vitro radiation in H460 and H1229 lung cancer cell lines.
Kang Ho KIM ; Hae Yong YOO ; Kyeung Min JOO ; Yong JUNG ; Juyoun JIN ; Yonghyun KIM ; Su Jin YOON ; Seung Ho CHOI ; Ho Jun SEOL ; Woong Yang PARK ; Do Hyun NAM
Experimental & Molecular Medicine 2011;43(7):419-426
Radiation is the most useful treatment modality for cancer patients. It initiates a series of signal cascades such as DNA damage response (DDR) signaling for repairing damaged DNA, arresting the cell cycle, and inducing cell death. Until now, few genes have been found to be regulated by radiation, which explains the molecular mechanisms of cellular responses to radiation. Although the transcriptional changes caused by radiation have been widely investigated, little is known about the direct evidence for the transcriptional control of DDR-related genes. Here, we examined the radiosensitivity of two non-small cell lung cancer cell lines (H460 and H1299), which have different p53 status. We monitored the time-dependent changes of 24 DDR-related gene expressions via microarray analysis. Based on the basal expression levels and temporal patterns, we further classified 24 DDR-related genes into four subgroups. Then, we also addressed the protein levels of several DDR-related genes such as TopBP1, Chk1 and Chk2, confirming the results of microarray analysis. Together, these results indicate that the expression patterns of DDR-related genes are associated with radiosensitivity and with the p53 statuses of H460 and H1299, which adds to the understanding of the complex biological responses to radiation.
Adaptor Proteins, Signal Transducing/genetics
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Cell Cycle Proteins/genetics
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Cell Line, Tumor
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Cell Survival/radiation effects
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DNA Damage/*radiation effects
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DNA Repair Enzymes/genetics
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DNA-Binding Proteins/genetics
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Gene Expression Profiling
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Gene Expression Regulation, Neoplastic/*radiation effects
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Humans
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Lung Neoplasms
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Radiation Tolerance/genetics
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Signal Transduction