Intracellular localization and sustained prodrug cell killing activity of TAT-HSVTK fusion protein in hepatocelullar carcinoma cells.
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.
Cao
Limin
L
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Si
Jin
J
Wang
Weiyu
W
Zhao
Xiaorong
X
Yuan
Xiaomei
X
Zhu
Huifen
H
Wu
Xiaolong
X
Zhu
Jianzhong
J
Shen
Guanxin
G
D024201:Bystander Effect; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology*; D016923:Cell Death; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human; D006801:Humans; D011355:Prodrugs / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase