title: Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma.
abstract: BACKGROUND
  Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate, which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases. The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication, hence leading to cell apoptosis. At present, HSVtk gene usually acts as suicide gene to kill tumor cells. The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the a-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.
  
  
  METHODS
  pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed. HL-7702 liver cells, HUH-7 HCC, and HepG2 HCC were transfected with the recombinant plasmids. HSVtK gene expression was detected using Western blotting analysis. HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent. The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.
  
  
  RESULTS
  Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully. HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells. After G418 selection, a HepG2 cells line stably expressing HSVtk gene was acquired. With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P < 0.05).
  
  
  CONCLUSIONS
  The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.
authors.affiliation: Shanxi Medical University, Taiyuan, Shanxi 030001, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Lai
authors.affiliation: Department of Surgery, Shanxi Provincial People's Hospital, Taiyuan, Shanxi 030012, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Qin
authors.affiliation: Department of Biochemistry and Molecular Biology of Shanxi Medical University, Taiyuan, Shanxi 030001, China.
authors.forename: Baofeng
authors.initials: B
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Molecular Biology of Shanxi Medical University, Taiyuan, Shanxi 030001, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Xie
authors.affiliation: Department of Biochemistry and Molecular Biology of Shanxi Medical University, Taiyuan, Shanxi 030001, China.
authors.forename: Ranpeng
authors.initials: R
authors.lastname: Gao
authors.affiliation: Shanxi Medical University, Taiyuan, Shanxi 030001, China.
authors.forename: Tiantian
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology of Shanxi Medical University, Taiyuan, Shanxi 030001, China.
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Surgery, Shanxi Provincial People's Hospital, Taiyuan, Shanxi 030012, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Niu
authors.affiliation: Department of Surgery, Shanxi Provincial People's Hospital, Taiyuan, Shanxi 030012, China. Email: junxuty@163.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D005326:Fetal Proteins / Q000235:genetics; D015774:Ganciclovir / Q000031:analogs & derivatives* / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014162:Transfection
chemical_list: D005326:Fetal Proteins; C092309:ganciclovir triphosphate; D015774:Ganciclovir