Ishiwata Nobuo, Inase Naohiko, Fujie Toshihide, Tamaoka Meiyo, Yoshizawa Yasuyuki
Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan.
Anticancer Res. 2003 Mar-Apr;23(2B):1405-9.
To achieve the specific expression of a transfected suicide gene in malignant mesothelioma cells, we applied the enhancer-promoter fusion sequence of the keratin 19 (K19) gene. Northern blot analysis of three mesothelioma cell lines demonstrated that K19 mRNA was expressed most abundantly in the H2052 mesothelioma cell line. Subsequently, in a luciferase reporting assay, K19 promoter (260 bp) exhibited higher promoter activity in H2052 cells than in the other two cell lines. In addition, ligation of a 3' enhancer (80 bp) of the K19 gene to upstream of the K19 promoter sufficiently enhanced the promoter activity. After transfecting an expression vector containing the K19 enhancer-promoter bound thymidine kinase gene (pK19-TK) into the H2052 cells, the pK19-TK transfected cells became more sensitive to GCV than non-transfected cells in vitro and in vivo. In a mouse peritoneal model of malignant mesothelioma, in vivo transfection of pK19-TK by cationic liposome and systemic administration of GCV inhibited the growth of peritoneal tumors. The K19 enhancer-promoter sequence seemed to be specific and efficient enough for the expression of the transfected suicide gene in malignant mesothelioma cells.
为了在恶性间皮瘤细胞中实现转染自杀基因的特异性表达，我们应用了角蛋白19（K19）基因的增强子-启动子融合序列。对三种间皮瘤细胞系进行的Northern印迹分析表明，K19 mRNA在H2052间皮瘤细胞系中表达最为丰富。随后，在荧光素酶报告基因检测中，K19启动子（260 bp）在H2052细胞中表现出比其他两种细胞系更高的启动子活性。此外，将K19基因的3'增强子（80 bp）连接到K19启动子上游可充分增强启动子活性。将含有K19增强子-启动子结合胸苷激酶基因（pK19-TK）的表达载体转染到H2052细胞中后，在体外和体内，pK19-TK转染的细胞比未转染的细胞对GCV更敏感。在恶性间皮瘤的小鼠腹腔模型中，通过阳离子脂质体对pK19-TK进行体内转染并全身给予GCV可抑制腹腔肿瘤的生长。K19增强子-启动子序列似乎对恶性间皮瘤细胞中转染自杀基因的表达具有足够的特异性和有效性。
