[Killing effect of PNP/MeP-dR suicide gene system driven by an AFP promoter AF0.3 on AFP-positive hepatoma cells].
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.


CONCLUSIONS
PNP/MeP-dR system, driven by AF0.3 promoter, has powerful killing effect on AFP-positive hepatoma HepG2 cells.
Cai
Xiao-Kun
XK
Institute for Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.
Zhou
Jun-Li
JL
Zhou
He-Jun
HJ
Zhang
Ling
L
Wu
Jian-Hong
JH
Lin
Ju-Sheng
JS
D017209:Apoptosis; D024201:Bystander Effect; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D011401:Promoter Regions, Genetic; D011684:Purine Nucleosides / Q000378:metabolism / Q000494:pharmacology; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D000509:alpha-Fetoproteins / Q000235:genetics* / Q000378:metabolism
D011684:Purine Nucleosides; D000509:alpha-Fetoproteins; C032885:6-methylpurine 2'-deoxyriboside; D011683:Purine-Nucleoside Phosphorylase