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  • Title: [Killing effect of PNP/MeP-dR suicide gene system driven by an AFP promoter AF0.3 on AFP-positive hepatoma cells].
    Author: Cai XK, Zhou JL, Zhou HJ, Zhang L, Wu JH, Lin JS.
    Journal: Ai Zheng; 2006 Nov; 25(11):1334-9. PubMed ID: 17094897.
    Abstract:
    BACKGROUND & OBJECTIVE: Alpha-fetoprotein (AFP) promoter-driven target gene could be specifically expressed in AFP-positive hepatoma. Escherichia coli purine nucleoside phosphorylase/6-methylpurine-2-deoxyriboside (PNP/MeP-dR) suicide gene system has powerful killing effects on tumor cells. This study was to investigate the specific killing effect of PNP/MeP-dR suicide gene system driven by an AFP promoter, AF0.3, on AFP-positive hepatoma cells. METHODS: Inserting PNP gene into pAF0.3, a eukaryotic expression vector containing PNP gene, pAF0.3/PNP, was constructed. Then it was transfected into AFP-positive HepG2 and AFP-negative SMMC7721 hepatoma cell lines, respectively. Two cell lines HepG2/AF0.3-PNP and SMMC7721/AF0.3-PNP, stably transfected with PNP gene, were obtained with G418 selection. The expression of PNP gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). The proliferation of HepG2/AF0.3-PNP and SMMC7721/AF0.3-PNP cells was determined with trypan blue exclusion. The sensitivity of the cells to MeP-dR and the bystander effects were assessed with MTT assay and flow cytometry (FCM). The enzymatic activities of PNP gene products were determined with high performance liquid chromatography (HPLC). RESULTS: Whether hypoxia or normoxia, HepG2/AF0.3-PNP cells were sensitive to MeP-dR, whereas SMMC7721/AF0.3-PNP cells were not. Under both conditions, obvious cytotoxic effects on HepG2 cells were observed when the proportion of HepG2/AF0.3-PNP cells in the mixture reached 25%. But there were no similar effects on SMMC7721 cells under the same conditions. HPLC assay showed that the product of PNP gene driven by AF0.3 promoter could convert a spot of MeP-dR into 6-MP in HepG2 cells, but not in SMMC7721 cells. CONCLUSION: PNP/MeP-dR system, driven by AF0.3 promoter, has powerful killing effect on AFP-positive hepatoma HepG2 cells.
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