Direct Intratumoral Gene Transfer of the Herpes Simplex Virus Thymidine Kinase Gene with DNA‐liposome Complexes: Growth Inhibition of Tumors and Lack of Localization in Normal Tissues-猫眼课题宝

单纯疱疹病毒胸苷激酶基因与 DNA-脂质体复合物的直接瘤内基因转移：肿瘤生长抑制和正常组织中缺乏定位

DOI：

10.1111/j.1349-7006.1997.tb00362.x

发表时间：

1997

期刊：

Japanese Journal of Cancer Research : Gann

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

K. Takakuwa;K. Fujita;A. Kikuchi;S. Sugaya;T. Yahata;H. Aida;T. Kurabayashi;I. Hasegawa;Kenichi Tanaka

为了构建肿瘤细胞中单纯疱疹病毒胸苷激酶（HSV-TK）基因的位点特异性表达，我们使用荧光素酶表达报告质粒评估了猿猴病毒40（SV 40）启动子和c-erbB-2基因5 '侧翼区的启动子功能。转染c-erbB-2基因启动子区指导的荧光素酶质粒后，在表达c-erbB-2的细胞（Colo 201、MCF-7和HEC 1-A）中观察到大量荧光素酶活性，而在不表达c-erbB-2蛋白的细胞（HRA和KF细胞）中未检测到荧光素酶活性。另一方面，当用SV 40启动子进行转染时，在所有测试的肿瘤细胞系中检测到高水平的荧光素酶活性。在100 μg/ml阿昔洛韦中体外培养5天，重复转染由SV 40启动子或c-erbB-2基因启动子区调控的脂质体偶联HSV-TK基因，导致所有4种检测细胞系或仅c-erbB-2表达细胞（分别存在SV 40启动子或c-erbB-2启动子）的生长抑制。最后，当使用SV 40启动子时，在存在50 mg/kg阿昔洛韦的情况下将DNA-脂质体复合物直接注射到已建立的肿瘤中，导致所有三种测试肿瘤的肿瘤体积显著减小。然而，仅在c-erbB-2-阳性细胞（Colo 201细胞）中，在瘤内注射由c-erbB-2启动子调控的HSV-TK基因后观察到这种抗肿瘤作用。在肿瘤内基因转移的情况下，通过注射后7天进行的聚合酶链反应（PCR）分析，仅在7只小鼠中的1只中检测到外源DNA。当在注射后14或21天进行PCR分析时，根本没有发现DNA信号。然而，在静脉注射的情况下，在所有三次测试中，在几个正常组织中检测到DNA。正常组织的组织学检查或DNA脂质体递送后的血清生化参数未见异常。这些结果表明，由肿瘤特异性转录单位调控的HSV-TK基因的直接基因转移可能是临床上最有前途的抗肿瘤选择性遗传策略之一。

To constitute the site‐specific expression of the herpes simplex virus thymidine‐kinase (HSV‐TK) gene in tumor cells, we have assessed the promoter function of the simian virus 40 (SV40) promoter and the 5’flanking region of c‐erbB‐2 gene using a luciferase‐expressing reporter plasmid. After the transfec‐tion of the luciferase plasmid directed by the promoter region of c‐erbB‐2 gene, a large amount of luciferase activity was observed in c‐erbB‐2‐expressing cells (Colo201, MCF‐7, and HEC1‐A), while none was detected in cells with no expression of c‐erbB‐2 protein (HRA and KF cells). On the other hand, a high level of luciferase activity was detected in all tumor cell lines tested, when the transfection was performed with SV40 promoter. The repeated transfection of the liposome‐conjugated HSV‐TK gene regulated by the SV40 promoter or by the promoter region of c‐erbB‐2 gene with cultivation in 100 μg/ml of aciclovir for 5 days in vitro resulted in growth inhibition for all four cell lines examined or for only c‐erbB‐2‐expressing cells in the presence of SV40 promoter or c‐erbB‐2 promoter, respectively. Finally, direct injection of the DNA‐liposome complex into established tumors in the presence of 50 mg/kg of aciclovir led to significant tumor volume reduction in all three tumors tested when SV40 promoter was employed. However, this anti‐tumor effect was noted only in c‐erbB‐2‐positive cells (Colo201 cells) upon intratumoral injection of HSV‐TK gene regulated by c‐erbB‐2 promoter. In the case of intratumoral gene transfer, foreign DNA was detected in only one of seven mice by polymerase chain reaction (PCR) analysis performed 7 days following injection. When PCR analysis was carried out at 14 or 21 days following injection, no DNA signal was found at all. However, DNA was detected in several normal tissues at all three times tested in the case of intravenous injection. No abnormalities were seen in histologic examinations of normal tissues or in serum biochemical parameters following DNA liposome delivery. These results suggest that the direct gene transfer of HSV‐TK gene regulated by tumor‐specific transcriptional units may be one of the most clinically promising of the selective genetic strategies against cancer.