Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Intracellular Localization and Sustained Prodrug Cell Killing Activity of TAT-HSVTK Fusion Protein in Hepatocelullar Carcinoma Cells

  • Cao, Limin (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology) ;
  • Si, Jin (The Key Laboratory of Molecular Biology, Jiangsu Institute of Parasitic Diseases) ;
  • Wang, Weiyu (Department of General Surgery, Tongji Hospital) ;
  • Zhao, Xiaorong (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology) ;
  • Yuan, Xiaomei (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology) ;
  • Zhu, Huifen (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology) ;
  • Wu, Xiaolong (Department of General Surgery, The Third Affiliated Hospital of NanTong University) ;
  • Zhu, Jianzhong (Department of General Surgery, The Third Affiliated Hospital of NanTong University) ;
  • Shen, Guanxin (Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology)
  • Received : 2005.09.14
  • Accepted : 2005.12.14
  • Published : 2006.02.28
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Abstract

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. 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 nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced 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 nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.

Keywords

Acknowledgement

Supported by : Ministry of Health of China, National Key Basic Research and Development Foundation of China

References

  1. Akyurek, L. M., Nallamshetty, S., Aoki, K., San, H., Yang, Z. Y., et al. (2001) Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. Mol. Ther. 3, 779–786
  2. Benedetti, S., Dimeco, F., Pollo, B., Cirenei, N., Colombo, B. M., et al. (1997) Limited efficacy of the HSV-TK/GCV system for gene therapy of malignant gliomas and perspectives for the combined transduction of the interleukin-4 gene. Hum. Gene Ther. 8, 1345-1353 https://doi.org/10.1089/hum.1997.8.11-1345
  3. Bilbao, R., Gerolami, R., Bralet, M. P., Qian, C., Tran, P. L., et al. (2000) Transduction efficacy, antitumoral effect, and toxicity of adenovirus-mediated herpes simplex virus thymidine kinase/ ganciclovir therapy of hepatocellular carcinoma: the woodchuck animal model. Cancer Gene Ther. 7, 657-662 https://doi.org/10.1038/sj.cgt.7700175
  4. Bremner, K. H., Seymour, L. W., and Pouton, C. W. (2001) Harnessing nuclear localization pathways for transgene delivery. Curr. Opin. Mol. Ther. 3, 170–177
  5. Brunner, S., Sauer, T., Carotta, S., Cotton, M., Saltik, M., et al. (2000) Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 7, 401–407 https://doi.org/10.1038/sj.gt.3301102
  6. Chen, S. H., Kosai, K., Xu, B., Pham-Nguyen, K., Contant, C., et al. (1996) Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival. Cancer Res. 56, 3758-3762
  7. Danthinne, X., Aoki, K., Kurachi, A. L., Nabel, G. J., and Nabel, E. G. (1998) Combination gene delivery of the cell cycle inhibitor p27 with thymidine kinase enhances prodrug cytotoxicity. J. Virol. 72, 9201–9207
  8. Estin, D., Li, M., Spray, D., and Wu, J. K. (1999) Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy. Neurosurgery 44, 361–369 https://doi.org/10.1097/00006123-199902000-00068
  9. Gentry, B. G., Im, M., Boucher, P. D., Ruch, R. J., and Shewach, D. S. (2005) GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity. Gene Ther. 12, 1033-1041 https://doi.org/10.1038/sj.gt.3302487
  10. Gerolami, R., Uch, R., Brechot, C., Mannoni, P., and Bagnis, C. (2003) Gene therapy of hepatocarcinoma: a long way from the concept to the therapeutical impact. Cancer Gene Ther. 10, 649-660 https://doi.org/10.1038/sj.cgt.7700610
  11. Gorlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 https://doi.org/10.1146/annurev.cellbio.15.1.607
  12. Goto, T., Nishi, T., Kobayashi, O., Tamura, T., Dev, S. B., et al. (2004) Combination electro-gene therapy using herpes virus thymidine kinase and interleukin-12 expression plasmids is highly efficient against murine carcinomas in vivo. Mol. Ther. 10, 929-937 https://doi.org/10.1016/j.ymthe.2004.07.028
  13. Kamiya, H., Tsuchiya, H., Yamazaki, J., and Harashima, H. (2001) Intracellular trafficking and transgene expression of viral and non-viral gene vectors. Adv. Drug Deliv. Rev. 52, 153-164 https://doi.org/10.1016/S0169-409X(01)00216-2
  14. Kokoris, M. S., Sabo, P., Adman, E. T., and Black, M. E. (1999) Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant. Gene Ther. 6, 1415-1426 https://doi.org/10.1038/sj.gt.3300966
  15. Kokoris, M. S., Sabo, P., and Black, M. E. (2000) In vitro evaluation of mutant HSV-1 thymidine kinases for suicide gene therapy. Anticancer Res. 20, 959–963
  16. Marconi, P., Tamura, M., Moriuchi, S., Krisky, D. M., Niranjan, A., et al. (2000) Connexin 43-enhanced suicide gene therapy using herpesviral vectors. Mol. Ther. 1, 71-81 https://doi.org/10.1006/mthe.1999.0008
  17. McNeish, I. A., Tenev, T., Bell, S., Marani, M., Vassaux, G., et al. (2001) Herpes simplex virus thymidine kinase/ganciclovir- induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells. Cancer Gene Ther. 8, 308-319 https://doi.org/10.1038/sj.cgt.7700305
  18. Merilainen, O., Hakkarainen, T., Wahlfors, T., Pellinen, R., and Wahlfors, J. (2005) HIV-1 TAT protein transduction domain mediates enhancement of enzyme prodrug cancer gene therapy in vitro: a study with TAT-TK-GFP triple fusion construct. Int. J. Oncol. 27, 203-208
  19. Mohr, L., Yeung, A., Aloman, C., Wittrup, D., and Wands, J. R. (2004) Antibody-directed therapy for human hepatocellular carcinoma. Gastroenterology 127, S225-231 https://doi.org/10.1053/j.gastro.2004.09.037
  20. Okuda, K. (2000) Hepatocellular carcinoma. J. Hepatol. 32, 225–237
  21. O'Malley, B. W., Cope, K. A., Chen, S. H., Li, D., Schwarta, M. R., et al. (1996) Combination gene therapy for oral cancer in a murine model. Cancer Res. 56, 1737–1741
  22. Patterson, A. V., Saunders, M. P., and Greco, O. (2003) Prodrugs in genetic chemoradiotherapy. Curr. Pharm. Des. 9, 2131-2154 https://doi.org/10.2174/1381612033454117
  23. Qian, C., Bilbao, R., Bruna, O., and Prieto, J. (1995) Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. Hepatology 22, 118-123
  24. Ren, W., Strube, R., Zhang, X., Chen, S. Y., and Huang, X. F. (2004) Potent tumor-specific immunity induced by an in vivo heat shock protein-suicide gene-based tumor vaccine. Cancer Res. 64, 6645-6651 https://doi.org/10.1158/0008-5472.CAN-04-1084
  25. Rexach, M. and Blobel, G. (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83, 683–692 https://doi.org/10.1016/0092-8674(95)90181-7
  26. Ruiz, J., Mazzolini, G., Sangro, B., Qian, C., and Prieto, J. (2001) Gene therapy of hepatocellular carcinoma. Dig Dis. 19, 324–332 https://doi.org/10.1159/000050699
  27. Sachs, M. S., Selker, E. U., Lin, B., Roberts, C. J., Luo, Z., et al. (1997) Expression of herpes virus thymidine kinase in Neurospora crassa. Nucleic Acids Res. 25, 2389–2395 https://doi.org/10.1093/nar/25.12.2389
  28. Sa Cunha, A., Bonte, E., Dubois, S., Chretien, Y., Eraiser, T., et al. (2002) Inhibition of rat hepatocellular carcinoma tumor growth after multiple infusions of recombinant Ad.AFPtk followed by ganciclovir treatment. J. Hepatol. 37, 222–230 https://doi.org/10.1016/S0168-8278(02)00111-3
  29. Schwarze, S. R. and Dowdy, S. F. (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol. Sci. 21, 45–48
  30. Stefani, A. L., Barzon, L., Castagliuolo, I., Guido, M., Pacenti, M., et al. (2005) Systemic efficacy of combined suicide/cytokine gene therapy in a murine model of hepatocellular carcinoma. J. Hepatol. 42, 728-735 https://doi.org/10.1016/j.jhep.2004.12.037
  31. Su, H., Lu, R., Ding, R., and Kan, Y. W. (2000) Adeno-associated viral-mediated gene transfer to hepatoma: thymidine kinase/ interleukin 2 is more effective in tumor killing in nonganciclovir (GCV)-treated than in GCV-treated animals. Mol. Ther. 1, 509-515 https://doi.org/10.1006/mthe.2000.0073
  32. Tanaka, M., Fraizer, G. C., De La Cerda, J., Cristiano, R. J., Liebert, M., et al. (2001) Connexin 26 enhances the bystander effect in HSVtk/GCV gene therapy for human bladder cancer by adenovirus/PLL/DNA gene delivery. Gene Ther. 8, 139–148 https://doi.org/10.1038/sj.gt.3301367
  33. Tasciotti, E., Zoppe, M., and Giacca, M. (2003) Transcellular transfer of active HSV-1 thymidine kinase mediated by an 11-amino-acid peptide from HIV-1 Tat. Cancer Gene Ther. 10, 64-74 https://doi.org/10.1038/sj.cgt.7700526
  34. Vives, E., Brodin, P., and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-6017 https://doi.org/10.1074/jbc.272.25.16010
  35. Wadia, J. S. and Dowdy, S. F. (2005) Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv. Drug Deliv. Rev. 57, 579-596 https://doi.org/10.1016/j.addr.2004.10.005