Utilization of variant-type of human α-fetoprotein promoter in gene therapy targeting for hepatocellular carcinoma
Utilization of variant-type of human α-fetoprotein promoter in gene therapy targeting for hepatocellular carcinoma
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DOI：
10.1038/sj.gt.3300870
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发表时间：
1999-04-01
期刊：
影响因子：
5.1
通讯作者：
Eguchi, K
中科院分区：
文献类型：
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作者：
Ishikawa, H;Nakata, K;Eguchi, K
我们之前报道过，在0.3 kb人甲胎蛋白（AFP）启动子控制下表达单纯疱疹胸苷激酶（HSVtk）基因的逆转录病毒载体（LNAFW0.3tk）在高AFP生成（HuH-7）细胞中提供更昔洛韦（GCV）介导的细胞毒性，而不是在低AFP生成（huH-1/cl.2）人肝癌细胞中提供细胞毒性。在本研究中，我们构建了逆转录病毒载体（LNANM0.3TK），其中HSVtk基因表达由0.3 kb人AFP启动子的变体型调节，该启动子在核苷酸-119处有G到A取代，这是一个负责人AFP遗传持久性的点突变，并将该载体应用于三种人肝癌细胞系：HuH-7、huH-1/c1.2和中间AFP产生细胞（PLC/PRF/5）。报告基因转染实验表明，在HuH-7和huH-1/cl.2细胞中，变异型启动子的活性均远高于野生型启动子。与此一致，LNAFM0.3TK感染可以使huH-1/cl.2细胞以及HuH-7和PLC/PRF/5细胞对GCV敏感，但不影响非肝癌细胞(HeLa)的细胞生长。此外，在HuH-7细胞中，LNAFM0.3TK感染比LNAFW0.3TK感染更有效地实现了旁观者效应。这些结果表明，人AFP启动子的变异型确保了基因治疗中的治疗基因表达，特别是对于低AFP产生的肝癌细胞。
We previously reported that the retroviral vector (LNAFW0.3tk) expressing the herpes simplex thymidine kinase (HSVtk) gene under the control of the 0.3 kb human alpha-fetoprotein (AFP) promoter provided the ganciclovir (GCV)-mediated cytotoxicity in the high AFP-producing (HuH-7) hut not in the low AFP-producing (huH-1/cl.2) human hepatoma cells, in the present study, we constructed the retroviral vector (LNANM0.3TK) in which the HSVtk gene expression is regulated by the variant-type of the 0.3 kb human AFP promoter with a G-to-A substitution at nucleotide -119, a point mutation responsible for hereditary persistence of human AFP and the vector was applied to three human hepatoma cell lines, HuH-7, huH-1/c1.2 and intermediate AFP-producing cells (PLC/PRF/5). By the reporter gene transfection assay, the activity of the variant-type of the promoter was much higher than that of the wild-type of the promoter in both HuH-7 and huH-1/cl.2 cells. consistent with this, LNAFM0.3TK infection could sensitize huH-1/cl.2 cells, as well as HuH-7 and PLC/PRF/5 cells to GCV, but did not affect cell growth of nonhepatoma cells (HeLa). In addition, the bystander effect was achieved more efficiently by LNAFM0.3TK infection than LNAFW0.3TK infection in HuH-7 cells. These results suggest that the variant-type of the human AFP promoter ensures the therapeutic gene expression in gene therapy particularly for the low AFP-producing hepatoma cells.