Sugaya S, Fujita K, Kikuchi A, Ueda H, Takakuwa K, Kodama S, Tanaka K
Department of Obstetrics and Gynecology, Niigata University School of Medicine, Japan.
Hum Gene Ther. 1996 Jan 20;7(2):223-30. doi: 10.1089/hum.1996.7.2-223.
To establish the expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in tumor cells, we analyzed the promoter function of the SV40 promoter and the nucleotide sequence (CACGTG) to which Myc-Max heterodimers (Myc/Max) were capable of binding in four kinds of cell lines: COLO320 DM, A-431, KF, and Nakajima. When luciferase reporter plasmid under the control of SV40 promoter was transfected into tumor cells in vitro, a high level of luciferase activity was observed in all kinds of cell lines. However, by transfection of the luciferase gene promoted by the Myc/Max binding sequence, accelerated luciferase expression was observed in COLO320 DM and A-431 cells with high expression of c-myc, but not in KF and Nakajima cells, which showed low expression of c-myc. The repeated transfection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter and cultivation in 100 micrograms/ml of aciclovir for 5 days in vitro demonstrated growth inhibition for all four kinds of cell lines. However, cell toxicity was observed only in COLO320 DM and A-431 cells when the HSV-TK gene promoted by the Myc/Max binding sequence was introduced. (The survival rate to 100 micrograms/ml of aciclovir concentration in COLO320 DM, A-431, KF, and Nakajima cells was 59%, 53%, 74%, and 79%, respectively.) In vivo direct injection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter into established tumors and aciclovir administration for 10 days into the mice resulted in significant tumor volume reduction in three tested tumor cells. However, injection of the HSV-TK gene promoted by the Myc/Max binding sequence and aciclovir administration into mice could achieve significant tumor regression only in COLO320 DM and A-431 cells. These results suggest that gene therapy using the HSV-TK gene promoted by the Myc/Max binding sequence can be an attractive approach for treatment against tumor cells expressing high levels of c-myc.
为了在肿瘤细胞中建立单纯疱疹病毒胸苷激酶（HSV-TK）基因的表达，我们分析了SV40启动子的启动子功能以及Myc-Max异二聚体（Myc/Max）能够结合的核苷酸序列（CACGTG），所用细胞系有四种：COLO320 DM、A-431、KF和中岛细胞。当将受SV40启动子控制的荧光素酶报告质粒转染到体外培养的肿瘤细胞中时，在所有细胞系中均观察到高水平的荧光素酶活性。然而，通过转染由Myc/Max结合序列促进的荧光素酶基因，在c-myc高表达的COLO320 DM和A-431细胞中观察到荧光素酶表达加速，但在c-myc低表达的KF和中岛细胞中未观察到。将受SV40启动子调控的脂质体偶联HSV-TK基因重复转染，并在100微克/毫升的阿昔洛韦中体外培养5天，结果显示对所有四种细胞系均有生长抑制作用。然而，当导入由Myc/Max结合序列促进的HSV-TK基因时，仅在COLO320 DM和A-431细胞中观察到细胞毒性。（COLO320 DM、A-431、KF和中岛细胞在100微克/毫升阿昔洛韦浓度下的存活率分别为59%、53%、74%和79%。）在体内，将受SV40启动子调控的脂质体偶联HSV-TK基因直接注射到已形成的肿瘤中，并对小鼠给予阿昔洛韦10天，结果显示在三种受试肿瘤细胞中肿瘤体积显著减小。然而，将由Myc/Max结合序列促进的HSV-TK基因注射到小鼠体内并给予阿昔洛韦，仅在COLO320 DM和A-431细胞中能实现显著的肿瘤消退。这些结果表明，使用由Myc/Max结合序列促进的HSV-TK基因进行基因治疗可能是治疗高表达c-myc的肿瘤细胞的一种有吸引力的方法。
