title: [Transcriptional targeting gene therapy of double suicide gene driven by hTERT promoter in hepatic carcinoma].
abstract: OBJECTIVE
  To examine the selective killing effects of pEGFP-C1-mediated double suicide gene system driven by the hTERT promoter (hTERT-CDglyTK) on hepatic carcinoma cells.
  
  
  METHODS
  The hTERT promoter and gene fragments SV40, yCD and TKgly were amplified by PCR (polymerase chain reaction) and then inserted into pEGFP-C1. And the constructs of pEGFP-hTERT-CD, pEGFP-hTERT-TK and pEGFP-hTERT-CDglyTK were transfected to SMMC 7721 or HL7702 respectively. The transfection effects were observed and the cellular expressions of suicide genes detected by RT-PCR (reverse transcription-polymerase chain reaction), QPCR (quantitative polymerase chain reaction) and Western blot. The transfected cells were treated with 5-fluorocytosine and ganciclovir at different concentrations and the cell-killing and bystander effects evaluated by the method of MTT (3-(4,5)-dimethyl thiadiazole (-z-y1)-3,5-di-phenytetrazoliumromide). The activity of cell telomerase was detected by the method of TRAP-argentation and the apoptotic rates analyzed by flow cytometry. All results of double and single gene systems were analyzed.
  
  
  RESULTS
  The fragments of enzyme digestion corresponded to the expectations. RT-PCR, QPCR and Western blot demonstrated the expressions of CD, TK and CDglyTK. pEGFP-hTERT-CD, pEGFP-hTERT-TK and pEGFP-hTERT-CDglyTK showed the similar transfection efficiencies in SMMC7721 (74.5%, 76.3%, 76.9%). More sensitive to the prodrugs (P = 0.020, P = 0.015), higher apoptotic rates (P = 0.023, P = 0.017) and bystander effects (P = 0.012, P = 0.001)and lower telomerase activities (P = 0.045, P = 0.038) were observed in double gene system versus those in single gene system. However, the transfection and growth of HL7702 cell could not be infected by this double suicide gene.
  
  
  CONCLUSIONS
  The plasmid of CDglyTK fusion gene system driven by hTERT promoter has been successfully constructed. It has demonstrated highly specific killing effects on hepatic carcinoma cells.
authors.affiliation: Department of Interventional Radiology, Sun Yat-sen University, Guangzhou, China.
authors.forename: Wen-zhe
authors.initials: WZ
authors.lastname: Fan
authors.forename: Jian-yong
authors.initials: JY
authors.lastname: Yang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Yong-hui
authors.initials: YH
authors.lastname: Huang
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Ying-qiang
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Jia-ping
authors.initials: JP
authors.lastname: Li
mesh_terms: D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000628:therapy; D045744:Cell Line, Tumor; D002470:Cell Survival; D018390:Gene Targeting; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy; D010957:Plasmids; D011401:Promoter Regions, Genetic; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D019098:Telomerase