1.Mechanism involving blm gene underlies repair of DNA damage of Jurkat cells induced by mitomycin C.
Xue YI ; Hui CHENG ; Ping ZOU ; Ling-Bo LIU ; Ting ZHANG ; Dan YU ; Xiao-Ming ZHU ; Liang ZOU
Journal of Experimental Hematology 2010;18(5):1155-1158
The defect or block of apoptosis is an important factor involved in the drug resistance of tumor cells. Blm gene plays a great role in DNA damage and repair. This study was aimed to explore the relationship of blm gene expression with cell cycle and apoptosis after Jurkat DNA damage. The apoptosis rate and change of cell cycle were detected by flow cytometry, the expression level of blm mRNA in Jurkat cells was determined by semi-quantitative RT-PCR. The results indicated that after induction with 0.4 g/L of mitomycin C (MMC) for 24 hours the apoptosis rate of Jurkat cells were (11.42±0.013)%, and (66.08±1.60)% Jurkat cells were arrested in G2/M phase. After induction for 48 hours, the apoptosis rate of Jurkat cells declined from (11.42±0.013)% to (8.08±0.27)%, and cell count of Jurkat cells arrested in G2/M phase decreased from (66.08±1.60)% to (33.96±1.05)%. When induced with 0.4 g/L of MMC for 24 hours, the apoptosis rate of fibroblasts and the percentage of fibroblasts in G2/M, G0-G1 and S phase all showed no significant change until 48 hours. The range of apoptosis rate and the change of cell percentage in three phases were significantly different between Jurkat cells and fibroblasts (p<0.01). Expression level of blm mRNA in Jurkat cells was remarkably higher than that in normal fibroblasts (p<0.01), at 48 hours expression level of blm mRNA was remarkably higher than that at 24 hours. The 2 groups showed clear difference of blm mRNA expression after treated by MMC (p<0.01). It is concluded that the blm gene may play a significant role in repair of DNA damage of Jurkat cells after MMC induction. Abnormal expression of blm is correlated to the drug resistance of leukemia cells.
Apoptosis
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Cell Cycle
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DNA Damage
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drug effects
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DNA Repair
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drug effects
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Humans
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Jurkat Cells
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Mitomycin
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pharmacology
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RecQ Helicases
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genetics
2.Potent in vitro interference of fleroxacin in DNA-binding, unwinding and ATPase activities of Bloom helicase.
Heng LUO ; Hou Qiang XU ; Xiang CHEN ; Mei DING ; Qi Xin YANG ; Kun LI
Biomedical and Environmental Sciences 2013;26(4):231-242
OBJECTIVETo study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules.
METHODSDNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra.
RESULTSThe DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands.
CONCLUSIONFLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.
DNA ; metabolism ; Fleroxacin ; pharmacology ; Nucleic Acid Synthesis Inhibitors ; pharmacology ; RecQ Helicases ; antagonists & inhibitors ; metabolism ; Spectrometry, Fluorescence ; Spectrophotometry, Ultraviolet
3.Expression of BLM mRNA in six tumor cell strains.
Xue YI ; Ping ZOU ; Ling-Bo LIU ; Lei TIAN ; Fang LIU ; Xian-Qi FENG ; Juan XIAO ; Zhao-Dong ZHONG ; Ping XIONG
Journal of Experimental Hematology 2005;13(5):823-826
The mutations of BLM gene may result in Bloom's syndrome which includes immunodeficiency, predisposition to malignant tumors and so on, and enhances sister chromati exchange (SCE), DNA replication failure, genome instability, and increases cancer susceptibility. This study was aimed to investigate the variability of mRNA expression level and cDNA structure of BLM gene in tumor cell strains so as to look for a new cancerogenic mechanism and to find a new therapeutic target. The expression level of mRNA and the structure of cDNA of BLM gene in six tumor cell strains and the normal human bone marrow mononuclear cells were detected with RT-PCR and DNA sequencing was performed. The results indicated that these tumors cells expressed BLM mRNA higher than the normal human bone marrow mononuclear cells (P < 0.01), but no cDNA sequence abnormality of BLM gene in these tumors cells was observed. It is concluded that the increase of expressing level of BLM mRNA may play an important role in the development of these tumors.
Base Sequence
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Cell Line, Tumor
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DNA Helicases
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genetics
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Gene Expression Regulation, Neoplastic
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HL-60 Cells
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Humans
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Jurkat Cells
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Molecular Sequence Data
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RNA, Messenger
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biosynthesis
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genetics
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RecQ Helicases
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Reverse Transcriptase Polymerase Chain Reaction
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Sequence Analysis, DNA
4.Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo.
Yixu CHEN ; Wen DUI ; Zhongsheng YU ; Changqing LI ; Jun MA ; Renjie JIAO
Protein & Cell 2010;1(5):478-490
RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Animals
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DNA Repair
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genetics
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DNA, Single-Stranded
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genetics
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Drosophila Proteins
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genetics
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metabolism
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Drosophila melanogaster
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genetics
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metabolism
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Loss of Heterozygosity
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genetics
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RecQ Helicases
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genetics
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metabolism
5.Correlation between the methylation of SULF2 and WRN promoter and chemosensitivity to irinotecan in gastric cancer.
Lin WANG ; Jie SHEN ; Li-juan MENG ; Wei-fei FAN ; Jun WANG ; Bao-rui LIU
Chinese Journal of Oncology 2013;35(6):418-422
OBJECTIVETo explore the relationship between SULF2 and WRN promoter methylation and chemosensitivity to irinotecan, and also the clinicopathological features in patients with gastric cancer.
METHODSThe chemosensitivity to irinotecan was tested by MTT assay. The methylation of SULF2 and WRN promoter in the fresh gastric cancer tissues was detected by methylation specific PCR. The differences of chemosensitivity and clinicopathological features of the methylation group were compared with that of the non-methylation group. The tumor growth in nude mice bearing human gastric cancer xenografts treated with CPT-11was also observed.
RESULTSThe methylation rates of SULF2 and WRN were 28.4% (29/102) and 23.5% (24/102), respectively. There were no significant association between promoter methylation and clinicopathological features of patients including age, gender, histologic type, lymphatic invasion, and TNM Stage. In all the 102 cases, there were 30 cases of irrinotecan-sensitive group, and 72 cases of the irrinotecan-resistant group. The SULF2 methylation rate was 46.7% (14/30)in the sensitive group, and 20.8% (15/72) in the resistant group (P = 0.008),The WRN methylation rate was 33.3% (10/30) in the sensitive group, and 19.4% (14/72) in the resistant group (P = 0.214). Gastric cancer tissues were more sensitive to irrinotecan when both the genes were methylated. The nude mice bearing human gastric cancer xenografts with SULF2 methylation were more sensitive to irrinotecan.
CONCLUSIONSThe detection of SULF2 and WRN promoter methylation may provide evidence for screening and targeting the most sensitive gastric cancer subpopulation suitable for personalized irrinotecan chemotherapy.
Antineoplastic Agents, Phytogenic ; pharmacology ; Camptothecin ; analogs & derivatives ; pharmacology ; DNA Methylation ; Exodeoxyribonucleases ; metabolism ; Humans ; Methylation ; Promoter Regions, Genetic ; RecQ Helicases ; metabolism ; Stomach Neoplasms ; metabolism ; Sulfotransferases ; metabolism ; Werner Syndrome Helicase
6.Effects of mercury on the structure and activity of BLM642-1290 recombinant helicase.
Xiang CHEN ; Heng LUO ; Lixia DUAN ; Qinghe XU ; Yong ZHANG ; Houqiang XU
Biomedical and Environmental Sciences 2011;24(1):47-55
OBJECTIVEBloom's syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene (encoding a BLM helicase) may form a structure of the etiology of this disease. As a global pollutant, mercury poses a major threat to human health. The current study was conducted to elucidate the effects of Hg(2+) on the structure and activity of BLM642-1290 recombinant helicase, and to further explore the molecular mechanisms of mercury toxicity to the DNA helicase.
METHODSThe effects of Hg(2+) on biological activity and structure of BLM642-1290 recombinant helicase were determined by fluorescence polarized, ultraviolet spectroscopic, and free-phosphorus assay technologies, respectively.
RESULTSThe helicase activity, the DNA-binding activity, and the ATPase activity of BLM642-1290 recombinant helicase were inhibited by Hg(2+) treatment. The LMCT (ligand-to-metal charge transition) peaks of the helicase were enhanced with the increase of the Hg(2+) level. The LMCT peaks of the same concentration of helicase gradually increased over time.
CONCLUSIONThe biological activity of BLM642-1290 recombinant helicase is inhibited by Hg(2+) treatment. The conformation of the helicase is significantly altered by Hg(2+). There exist two binding sites between Hg(2+) and the helicase, which are located in the amino acid residues 1063-1066 and 940-944 of the helicase, respectively.
Adenosine Triphosphatases ; metabolism ; Base Sequence ; DNA Primers ; Fluorescence Polarization ; Humans ; Mercury ; toxicity ; Protein Conformation ; RecQ Helicases ; chemistry ; drug effects ; metabolism ; Recombinant Proteins ; chemistry ; drug effects ; metabolism ; Spectrophotometry, Ultraviolet ; Structure-Activity Relationship
7.Enhancing alkylating agent resistance through ERCC2 gene transfection in human glioma cell line.
Zhongping CHEN ; Junying ZHANG ; Gérard MOHR
Chinese Medical Journal 2003;116(8):1171-1174
OBJECTIVETo confirm the enhancing effect of excision repair cross complementing rodent repair deficiency gene 2 (ERCC2) on alkylating agents resistance.
METHODSThe authors constructed a pcDNA3-ERCC2 plasmid. The pcDNA3-ERCC2 was transfected into a selected ERCC2 negative human glioma cell line, SKMG-4, using liposome-mediated transfection. After G418 selection, a stable transfected cell line was obtained and tested for cytotoxicity of several alkylating agents.
RESULTSThe stable transfectant was obtained and confirmed by RT-PCR as well as Western blot analysis to be strongly expressing ERCC2 at both mRNA and protein levels. The IC(90) ( micro mol/L) of two alkylating agents, cisplatin and melphalan, increased from 1.0 to 1.75 (75%) and 5.6 to 9.0 (61%), respectively, compared with control cell line.
CONCLUSIONThe present data provided evidences and confirmed the authors' previous results that ERCC2 contributes, at least partially, to alkylating agent resistance in human glioma cell line.
Antineoplastic Agents, Alkylating ; pharmacology ; Cisplatin ; pharmacology ; DNA Helicases ; DNA-Binding Proteins ; Drug Resistance, Neoplasm ; genetics ; Glioma ; Humans ; Melphalan ; pharmacology ; Proteins ; genetics ; Transcription Factors ; Transfection ; Tumor Cells, Cultured ; Xeroderma Pigmentosum Group D Protein
8.Study on the expression of DNA excision repair biomarkers in cispatin-treated lung cancer cell line.
Zhi-yong WANG ; Sheng ZHOU ; Bin LU ; Xia ZHANG ; Qiao-ling HAO ; Yi-kai ZHOU
Chinese Journal of Preventive Medicine 2003;37(2):109-111
OBJECTIVETo study the expression levels of ERCC2, UDG, and PCNA in cisplatin-treated A549 cell line.
METHODComet assay, RT-PCR, and western blot were used to study the mRNA and protein expression levels of ERCC2, UDG, and PCNA.
RESULTSWhen treated with IC(20) cisplatin, the DNA damage level increased as the cisplatin treated time increased within 24 h of cisplatin treatment. The tail state 12 h and 24 h after treatment was 5.02 +/- 0.68 and 7.22 +/- 0.53 respectively, which was significantly higher than those of the controls (2.73 +/- 0.29). The tail state 24 h after treatment was not significantly different from that of the controls. The DNA damage level decreased to normal after cisplatin treatment in 24 h (tail state 3.64 +/- 0.7). The expression levels of ERCC2, UDG, PCNA protein (4.37 +/- 0.57, 5.47 +/- 0.46, 2.21 +/- 0.47 respectively) and mRNA (0.71 +/- 0.08, 0.74 +/- 0.06, 0.82 +/- 0.09) were increased significantly within 24 h exposure and decreased to normal 24 h after cisplatin treatment. The 3 enzymes' mRNA and protein expression increased when treated with cisplatin, but the changes of protein level were slower than those of mRNA levels.
CONCLUSIONSThe DNA repair capability in A549 cells increases after cisplatin treatment. Cisplatin was a positive regulation of ERCC2, UDG, PCNA expression levels, which causes the increase of mRNA, and protein. The positive regulation only works in a short time and returns normal after 24 h of cisplatin treatment.
Antineoplastic Agents ; pharmacology ; Biomarkers, Tumor ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Comet Assay ; DNA Glycosylases ; biosynthesis ; genetics ; DNA Helicases ; DNA Repair ; drug effects ; DNA-Binding Proteins ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Protein Biosynthesis ; Proteins ; genetics ; RNA, Messenger ; biosynthesis ; Transcription Factors ; Xeroderma Pigmentosum Group D Protein
9.Association of genetic polymorphisms in the DNA repair gene XPD with risk of lung and esophageal cancer in a Chinese population in Beijing.
De-yin XING ; Jun QI ; Wen TAN ; Xiao-ping MIAO ; Gang LIANG ; Chun-yuan YU ; Wen-fu LU ; Chuan-nong ZHOU ; Min WU ; Dong-xin LIN
Chinese Journal of Medical Genetics 2003;20(1):35-38
OBJECTIVEXPD polymorphisms at Asp312Asn and Lys751Gln sites have been shown to modulate DNA repair capacity. The authors therefore assessed the relationship between these XPD polymorphisms and susceptibility to lung and esophageal cancer in a Chinese population via a hospital-based, case-control study.
METHODSGenotypes were determined by PCR-restriction fragment length polymorphism approaches in 383 healthy controls, 351 patients with lung cancer, and 325 patients with esophageal squamous cell carcinoma (SCC). The adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using multivariate logistic regression.
RESULTSIndividuals carrying at least one 312Asn variant allele (Asp/Asn and Asn/Asn genotypes) were at an increased risk for lung SCC as compared with those with the Asp/Asp genotype (OR 1.80; 95% CI: 1.10-2.93; adjusted for age, sex and smoking), but this increased risk was not observed among patients with adenocarcinoma of the lung (adjusted OR: 1.07; 95% CI: 0.55-2.08). Furthermore, stratified analysis indicated a multiplicative interaction between tobacco smoking and the variant XPD 312Asn and 751Gln alleles on risk of lung SCC. The ORs of lung SCC for the variant XPD 312Asn and 751Gln alleles with smoking>or=29 pack/year were 12.44 (95% CI: 4.97-31.17) and 10.74 (95% CI: 4.51-25.57), respectively. No significant association between the Asp312Asn or Lys751Gln polymorphism and the risk of esophageal cancer was found.
CONCLUSIONThe above findings indicate that the Asp312Asn and Lys751Gln polymorphisms in the XPD locus are associated with the risk of lung SCC but not lung adenocarcinoma or esophageal SCC in this Chinese population.
Adenocarcinoma ; genetics ; Asparagine ; genetics ; Aspartic Acid ; genetics ; Carcinoma, Squamous Cell ; genetics ; China ; DNA ; genetics ; DNA Helicases ; DNA Repair ; genetics ; DNA-Binding Proteins ; Esophageal Neoplasms ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Logistic Models ; Lung Neoplasms ; genetics ; Male ; Multivariate Analysis ; Odds Ratio ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Proteins ; genetics ; Risk Factors ; Smoking ; Transcription Factors ; Xeroderma Pigmentosum Group D Protein