Suicide gene therapy for hepatocellular carcinoma cells by survivin             promoter-driven expression of the herpes simplex virus thymidine kinase gene.
The aim of this study was to investigate the selective killing effect of             the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system             controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro.             Recombinant plasmid vectors driven by the survivin promoter were constructed.             HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant             plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP             expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression             was detected using RT-PCR and western blot analysis. The selective killing effects             after GCV application were evaluated by tetrazolium assay, FCM and western blot             analysis. Statistical analysis was performed by ANOVA. After transfection with             GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in             the HepG2 cells, but not in the L02 cells and TK gene expression was evidently             detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably             transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were             successfully established. Compared with the HepG2/TK/pSURV group, a significant             'bystander effect' was observed in the HepG2/TAT-TK/pSURV group with the incorporation             of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV             and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly             inhibited. This finding was further corroborated by FCM and immunoblot analysis             revealed the repressed expression of proliferating cell nuclear antigen (PCNA).             Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion             protein gene driven by the survivin promoter were successfully constructed and             their specific expression in HepG2 cells provided the basis for the targeted gene             therapy of HCC.
Department of Laboratory Medicine, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, PR China.
Lili
L
Qu
Yanyun
Y
Wang
Lailing
L
Gong
Jin
J
Zhu
Rujun
R
Gong
Jin
J
Si
D024201:Bystander Effect; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D015774:Ganciclovir / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D011401:Promoter Regions, Genetic; D018139:Simplexvirus; D000077022:Survivin; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics*
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D000077022:Survivin; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir