Cai Xiao-Kun, Zhou Jun-Li, Zhou He-Jun, Zhang Ling, Wu Jian-Hong, Lin Ju-Sheng
Institute for Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.
Ai Zheng. 2006 Nov;25(11):1334-9.
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.
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).
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.
PNP/MeP-dR system, driven by AF0.3 promoter, has powerful killing effect on AFP-positive hepatoma HepG2 cells.
甲胎蛋白（AFP）启动子驱动的靶基因可在AFP阳性肝癌中特异性表达。大肠杆菌嘌呤核苷磷酸化酶/6-甲基嘌呤-2-脱氧核糖苷（PNP/MeP-dR）自杀基因系统对肿瘤细胞具有强大的杀伤作用。本研究旨在探讨由AF0.3（一种AFP启动子）驱动的PNP/MeP-dR自杀基因系统对AFP阳性肝癌细胞的特异性杀伤作用。
将PNP基因插入到含有PNP基因的真核表达载体pAF0.3中，构建pAF0.3/PNP。然后分别将其转染到AFP阳性的HepG2和AFP阴性的SMMC7721肝癌细胞系中。通过G418筛选获得稳定转染PNP基因的两种细胞系HepG2/AF0.3-PNP和SMMC7721/AF0.3-PNP。采用逆转录-聚合酶链反应（RT-PCR）检测PNP基因的表达。用台盼蓝拒染法测定HepG2/AF0.3-PNP和SMMC7721/AF0.3-PNP细胞的增殖情况。用MTT法和流式细胞术（FCM）评估细胞对MeP-dR的敏感性及旁观者效应。用高效液相色谱法（HPLC）测定PNP基因产物的酶活性。
无论缺氧还是常氧条件下，HepG2/AF0.3-PNP细胞对MeP-dR敏感，而SMMC7721/AF0.3-PNP细胞不敏感。在两种条件下，当混合物中HepG2/AF0.3-PNP细胞比例达到25%时，对HepG2细胞观察到明显的细胞毒性作用。但在相同条件下对SMMC7721细胞没有类似作用。HPLC检测显示，AF0.3启动子驱动的PNP基因产物能在HepG2细胞中将少量MeP-dR转化为6-巯基嘌呤，但在SMMC7721细胞中不能。
由AF0.3启动子驱动的PNP/MeP-dR系统对AFP阳性肝癌HepG2细胞具有强大的杀伤作用。
