Cao Limin, Si Jin, Wang Weiyu, Zhao Xiaorong, Yuan Xiaomei, Zhu Huifen, Wu Xiaolong, Zhu Jianzhong, Shen Guanxin
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Mol Cells. 2006 Feb 28;21(1):104-11.
Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex vi-rus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory by-stander effect; (b) short-lived expression of the pro-tein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nu-cleus. The transduced HepG2 cells are killed by exo-genously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the in-troduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nu-clear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activ-ity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.
使用单纯疱疹病毒1型胸苷激酶（HSV1-TK）/更昔洛韦（GCV）自杀基因/前药激活系统的非病毒载体进行基因治疗，由于两个主要因素，在杀死恶性肿瘤细胞方面效率低下：（a）旁观者效应不令人满意；（b）蛋白质的表达寿命短。为了研究HIV-1 TAT蛋白的蛋白质转导结构域（PTD）增强HSV1-TK/GCV癌症基因治疗的能力，我们构建了三种融合蛋白TAT-TK、TK-TAT和TK。TAT-TK保留了与TK一样多的酶活性，而TK-TAT的酶活性则低得多。TAT-TK可以进入HepG2细胞，并且其中大部分被转运到细胞核。转导的HepG2细胞被外源添加的GCV杀死，并且对未转导的HepG2细胞具有旁观者效应。最重要的是，引入的重组蛋白是稳定的，并且至少在几天内保持功能，这可能是因为核定位保护它免受细胞质降解机制的影响，并提供了进入核转录机制的途径。我们的结果表明，TAT融合蛋白在细胞间运输，具有增强的稳定性和前药细胞杀伤活性。我们得出结论，TAT在增强肝癌的酶前药治疗方面具有潜力。
