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Double suicide genes driven by kinase domain insert containing receptor promoter selectively kill human lung cancer cells.

ABSTRACT: BACKGROUND:To investigate the selective killing efficacy of the double suicide genes driven by KDR promoter. MATERIALS AND METHODS:A double suicide gene system with the KDR promoter, pcDNA3-KDRp-CDglyTK, was constructed and transfected into lung cancer cell lines L9981 and NL9980, and human hepatocellular carcinoma cell line HepG2. The efficiency and specificity of the double suicide gene system were assayed by in vitro cellular proliferation and apoptosis, as well as in vivo xenograft studies. RESULTS:The transgenic CD and TK genes were only expressed in L9981 and NL9980 but not in HepG2 cells. Pre-treating transfected cells with 5-Fc and GCV significantly reduced proliferation, enhanced apoptosis in L9981 and NL9980 but not in HepG2 cells. The tumor formed by L9981 and NL9980 cells with the double suicide gene system was much smaller in vivo. CONCLUSION:Tumor targeted expression of CDglyTK gene driven by KDR promotor represents a novel strategy for effective gene therapy of tumor with intrinsic KDR.

SUBMITTER: Ma J

PROVIDER: S-EPMC3068930 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Double suicide genes driven by kinase domain insert containing receptor promoter selectively kill human lung cancer cells.

Ma Junrong J Li Mi M Mei Longyong L Zhou Qinghua Q Liu Lunxu L Yu Xijie X Che Guowei G

Genetic vaccines and therapy 20110322

<h4>Background</h4>To investigate the selective killing efficacy of the double suicide genes driven by KDR promoter.<h4>Materials and methods</h4>A double suicide gene system with the KDR promoter, pcDNA3-KDRp-CDglyTK, was constructed and transfected into lung cancer cell lines L9981 and NL9980, and human hepatocellular carcinoma cell line HepG2. The efficiency and specificity of the double suicide gene system were assayed by in vitro cellular proliferation and apoptosis, as well as in vivo xeno ...[more]

PMID: 21418659

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Project description: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.

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Double suicide genes driven by kinase domain insert containing receptor promoter selectively kill human lung cancer cells.

Publications

Double suicide genes driven by kinase domain insert containing receptor promoter selectively kill human lung cancer cells.

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