Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells.
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Abstract:
O(6)-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC(50) values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.Keywords:
K562 cells
O-6-methylguanine-DNA methyltransferase
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Objective: To investigate whether the expression of exogenous tRNAser(CGA) can alter the translation efficiency of the HPV 6bE7 gene in mammalian Cos-1 cell. Methods: The recombinant eukaryotic expressing plasmids encoding tRNAser (CGA) (pSV-tRNAser ) and wild-type (pcDNA3-WtE7) or codon modified (pcDNA3-HuE7) HPV 6b E7 DNA were extracted and identificated by gel analysis of digested products. Cos-1 cells were co-transfected with pSV tRNAser (CGA) and pcDNA3-WtE7 or pcDNA3-HuE7. The expression of the E7 protein were analyzed by immunoflurecence at 48h after transfection. Results: Cos-1 cells co-transfected with pSV-tRNAser and pcDNA3-WtE7 expressed higher levels of E7 protein than similar cells transfected with pcDNA3-WtE7 only, and the cells transfected only by wild-type E7 plasmids were occasionally positive. The expression of codon modified E7 gene were much higher than wild-type E7 gene in transfected Cos-1 cells, but no difference observed between tranfected and co-transfeced groups. The specific tRNAser(CGA) DNA fragment was obtained from the transfected cell by Southern blot analysis. Conclusion: The data indicate that supplement of exogenous tRNAser(CGA)can partly enhance the expression of HPV 6b wild-type E7 gene in mammalian Cos-1 cells.
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Objective To investigate the effect of ribosomal protein L6 (RPL6) gene on the resistance of leukemia cell to antitumor drugs.Methods RPL6 cDNA was obtained by RT-PCR ,Both sense and antisense cDNA recombinants of RPL6-encoding gene were constructed with pcDNA3.1(+) expression vector ,then the sense RPL6 cDNA recombinant was transfected into K562 cells while the antisense one into K562/A02 cells by liposomal reagent.The chemosensitivity of cells was detected by MTT assay.Results Internal control RT-PCR showed higher expression of RPL6 in K562/A02 than in K562;Sense-transfected K562 cells were 3.25 times more resistant to adriamycin than control cells did,whereas resistant to adriamycin of antisense-transfected K562/A02 cells decreased by 62% than that of control cells;Conclusion RPL6 gene plays an important role in the development of drug resistance in K562/A02 cells.
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Objective:To establish the gene expression map of normal peripheral blood mononuclear cells and to study the mechanism and the gene therapy of intracranial aneurysm(IA).Methods:The total RNA was collected from 60 normal people and patients with IA.The gene chip technology was used to analyze the genetic expression of intracranial arterial blood.Results:The gene chip was used to detect 14 112 genes.There were 7 749 genes expressed in the normal peripheral blood.The gene expression levels were more in gene/protein expression categories,metabolism category and the structure/ movement genome.Of which,L41(RPL41),S3A(RPS3A) and L23A(RPL23A) were the first third of the gene expression.Com-pared to IA patients,there were 85 differently expressed genes,the KPNA4 gene expression was up-regulated,which was relat-ed to signal conduction and protein transmission.The SPG3A gene expression was down-regulated,which was related to nu-cleotide conjunctin.Conclusion:The gene expression map of the normal peripheral blood was established.KPNA4 and SPG3A may be related to the development of IA.
Pair-rule gene
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Objective To construct a eukaryotic expression vector for JTV1 gene,stably transfect to human leukemia cellline K562,determine the expression levels of JTV1 mRNA and protein and investigate the effect of expressed product on proliferationof K562 cells. Methods JTV1 gene was cloned from human peripheral blood mononuclear cells(PBMCs)and inserted into expression vector pcDNA3. 1. The constructed plasmid pcDNA3. 1-JTV1 was transfected to K562 cells in mediation of liposome. The ex-pression levels of JTV1 mRNA and protein were determined by RT-PCR and Western blot respectively. The effect of stable expressionof JTV1 on proliferation of K562 cells was determined by MTT method. Results Restriction analysis and sequencing proved that target gene was inserted into recombinant plasmid pcDNA3. 1-JTV1. JTV1 was stably expressed in K562 cells,which inhibited the pro-liferation of K562 cells. Conclusion The eukaryotic expression vector for JTV1 gene was successfully constructed,and the K562cell clones stably expressing JTV1 gene was obtained,which provided a cell model for further study on the function of human JTV1gene as well as its relationship to the proliferation and apoptosis of leukemia cells.
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Objective To investigate the role of SHP1 gene in inducing apoptosis and erythroid differentiation in human erythromyeloblastoid leukemia cell line K562.Methods The full length cDNA of SHP1 gene was cloned by RT-PCR and was subcloned into mammalian expression vector pcDNA3.0.The cDNA sequence of the cloned gene was validated by enzyme digestion and DNA sequencing.Then the recombinant plasmid was used to transfect K562 cells via lipofectin.The apoptosis of K562 cells was examined by Hoechst 33258 staining assay and Annexin Ⅴ/PI double-labeled assay;the differentiation of K562 cells was observed by benzidine staining and expression of glycophorin A (GPA).Results RT-PCR and Western blotting analysis showed expression of SHP1 in K562 cells after transfection with pcDAN3-SHP1 plasmid.Apoptotic cells were detected in the K562 cells 48 h after treatment with pcDNA3-SHP1,with the apoptosis rate being 16.84%,which was significantly higher than that in cells transfected with pcDNA3.0 (6.23%,P=0.000).The positive rate of benzidine staining was 14.67% and the positive rate of GPA expression was 19.38% in cells treated with pcNDA3-SHP1,both were significantly different from those in the cells transfected with pcDNA3.0 (P=0.005).Conclusion Over-expression of SHP1 can effectively induce apoptosis and erythroid differentiation in K562 cells.
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The present study was undertaken to determine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultured cells. Following in situ hybridization with 32P-labeled v-src and v-Ha-ras DNA probes, src and Ha-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-Ha-ras transfected cell lines, the number of silver grains over individual cells were significantly higher (p less than 0.001, t-test) than in a non-transfected, non-tumorigenic, rat esophageal epithelial cell line. There was a highly variable number of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-Ha-ras DNA or that were pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times more oncogene related mRNAs than non-transfected cells as judged by the numbers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirmed that the expression of src and Ha-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of human cells and to human tumor cells in culture.
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Objective To establish leukemia cell mode transfected with anti-VEGF hairpin ribozyme gene and explore the possibility of inhibiting expression of VEGF in leukemia cells. Methods The recombinant eukaryotic expression plasmid(pcDNA3-RZ) including anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation and positive clones were screened by G418. Ribozyme gene in K562 cells was conformed by PCR. Fluorescent real time RT-PCR and western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. The growth rate of endothelium cells was detected by MTT assay after leukemia cells serum was added. Results The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418 for 2 weeks. Stable expression of the ribozyme gene in K562 cells was conformed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC(K562 cell transfected with empty vector) cells. The growth of endothelium cells which was added serum of K562-RZ cells was lower than that of control groups. Conclusion The leukemia cell mode transfected with anti-VEGF hairpin ribozyme gene has been established and ribozyme gene can decrease VEGF expression in K562 cells.
K562 cells
Lipofectamine
Hairpin ribozyme
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Objective To construct the siRNA expression vector for JTV1 gene,transfect to K562 cells and identify its interference effect.Methods The siRNA interfering sequence targeting to JTV1 gene was synthesized and cloned into vector pGeneSil-1.The constructed recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA was transfected to human K562 cells.The positive clones were screened with G418,based on which the effect of recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA on transcription and translation of JTV1 gene in K562 cells was investigated by RT-PCR and Western blot,and that of JTV1 gene expression on proliferation of K562 cells by MTT method.Results Sequencing proved that recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA was constructed correctly.Both the transcription and translation levels of JTV1 gene in K562 cells transfected with the recombinant plasmid decreased significantly.The inhibition of JTV1 expression promoted the proliferation of K562 cells.Conclusion Recombinant plasmid pGeneSil-1-JTV1-1.1 siRNA was successfully constructed,and stable K562 cell clones in which the expression of JTV1 gene was inhibited were obtained,which laid a foundation of further study on function of JTV1 gene and its correlation to tumor.
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Research goal:To do research on the effect of the genes transfected with wild-type p53 on the growth of human Leukemia cell lines.Research method:Transfect human Leukemia cell lines U937,K562 and HL60 with the plasmid cloned with wild-type p53 through Lipofectin and then observe their growth using 4-methy 1 law.Research result: It is apparently observed that the growth of human Leukemia Cell Lines U937,K562 and HL60 are inhibited by the genes transfected with wild-type p53 with the growth-inhibited rates varying from 28.6%,44.9% to 49.0% on the fourth day. Research conclusion:Genes transfected with wild-type p53 can inhibit the growth of human Leukemia cell lines U937, K562 and HL60 effectively.
HL60
K562 cells
U937 cell
Wild type
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