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Transcellular transfer of active HSV-1 thymidine kinase mediated by an 11-amino-acid peptide from HIV-1 Tat

Abstract

Suicide gene therapy using herpes simplex virus type-1 (HSV-1) thymidine kinase (TK) is a widely exploited approach for gene therapy of cancer and other hyperproliferative disorders. Despite its popularity, clinical success has been so far hampered mostly by the relative inefficiency of TK gene transfer and its limited bystander effect. Here we report that fusion of TK to an 11-amino-acid peptide from the basic domain of the HIV-1 Tat protein (Tat11) imparts cell membrane translocating ability to the enzyme and significantly increases its cytotoxic efficacy. In cells expressing Tat11-TK, this protein is found extracellularly, associated with cell surface heparan sulfate proteoglycans, and is released into the cell culture medium. Based on its interaction with HSPGs, the protein is then internalized by neighboring, nonexpressing cells, which become susceptible to cell death when treated with the nucleoside analogue acyclovir. As a consequence, co-cultures of wild-type cells with cells expressing Tat11-TK show increased sensitivity to ACV through a mechanism involving apoptosis. Modification of TK by fusion with Tat11 might constitute an important step for the optimization of TK suicide gene strategy for gene therapy of cellular proliferation.

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References

  1. Aghi M, Hochberg F & Breakefield XO . Prodrug activation enzymes in cancer gene therapy. J Gene Med. 2000; 2: 148–164.

    CAS  Article  PubMed  Google Scholar 

  2. Miller WH & Miller RL . Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase. J Biol Chem. 1980; 255: 7204–7207.

    CAS  PubMed  Google Scholar 

  3. Rainov NG . A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther. 2000; 11: 2389–2401.

    CAS  Article  PubMed  Google Scholar 

  4. Culver KW, Ram Z & Wallbridge S, et al. In vivo gene transfer with retroviral vector–producer cells for treatment of experimental brain tumors. Science. 1992; 256: 1550–1552.

    CAS  Article  PubMed  Google Scholar 

  5. Freeman SM, Abboud CN & Whartenby KA, et al. The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res. 1993; 53: 5274–5283.

    CAS  PubMed  Google Scholar 

  6. Ishii-Morita H, Agbaria R & Mullen CA, et al. Mechanism of ‘bystander effect’ killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment. Gene Ther. 1997; 4: 244–251.

    CAS  Article  PubMed  Google Scholar 

  7. Trask TW, Trask RP & Aguilar-Cordova E, et al. Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther. 2000; 1: 195–203.

    CAS  Article  PubMed  Google Scholar 

  8. Niranjan A, Moriuchi S & Lunsford LD, et al. Effective treatment of experimental glioblastoma by HSV vector–mediated TNF alpha and HSV-tk gene transfer in combination with radiosurgery and ganciclovir administration. Mol Ther. 2000; 2: 114–120.

    CAS  Article  PubMed  Google Scholar 

  9. Palu G, Cavaggioni A & Calvi P, et al. Gene therapy of glioblastoma multiforme via combined expression of suicide and cytokine genes: a pilot study in humans. Gene Ther. 1999; 6: 330–337.

    CAS  Article  PubMed  Google Scholar 

  10. Shand N, Weber F & Mariani L, et al. A phase 1–2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. Hum Gene Ther. 1999; 10: 2325–2335.

    CAS  Article  PubMed  Google Scholar 

  11. Klatzmann D, Valery CA & Bensimon G, et al. A phase I/II study of herpes simplex virus type 1 thymidine kinase “suicide” gene therapy for recurrent glioblastoma. Study Group on Gene Therapy for Glioblastoma. Hum Gene Ther. 1998; 9: 2595–2604.

    CAS  PubMed  Google Scholar 

  12. Ram Z, Culver KW & Oshiro EM, et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med. 1997; 3: 1354–1361.

    CAS  Article  PubMed  Google Scholar 

  13. Frankel AD & Pabo CO . Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 1988; 55: 1189–1193.

    CAS  Article  PubMed  Google Scholar 

  14. Green M & Loewenstein PM . Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell. 1988; 55: 1179–1188.

    CAS  Article  PubMed  Google Scholar 

  15. Elliott G & O'Hare P . Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell. 1997; 88: 223–233.

    CAS  Article  PubMed  Google Scholar 

  16. Oess S & Hildt E . Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens. Gene Ther. 2000; 7: 750–758.

    CAS  Article  PubMed  Google Scholar 

  17. Sosnowski BA, Gonzalez AM & Chandler LA, et al. Targeting DNA to cells with basic fibroblast growth factor (FGF2). J Biol Chem. 1996; 271: 33647–33653.

    CAS  Article  PubMed  Google Scholar 

  18. Rubartelli A, Cozzolino F & Talio M, et al. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J. 1990; 9: 1503–1510.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Derossi D, Joliot AH & Chassaing G, et al. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem. 1994; 269: 10444–10450.

    CAS  PubMed  Google Scholar 

  20. Rojas M, Yao S & Lin YZ . Controlling epidermal growth factor (EGF)–stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor. J Biol Chem. 1996; 271: 27456–27461.

    CAS  Article  PubMed  Google Scholar 

  21. Nagahara H, Vocero-Akbani AM & Snyder EL, et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med. 1998; 4: 1449–1452.

    CAS  Article  PubMed  Google Scholar 

  22. Fawell S, Seery J & Daikh Y, et al. Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci USA. 1994; 91: 664–668.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Schwarze SR, Ho A & Vocero-Akbani A, et al. In vivo protein transduction: delivery of a biologically active protein into the mouse [see comments]. Science. 1999; 285: 1569–1572.

    CAS  Article  PubMed  Google Scholar 

  24. Demarchi F, d'Adda di Fagagna F & Falaschi A, et al. Activation of transcription factor NF-κB by the Tat protein of human immunodeficiency virus-1. J Virol. 1996; 70: 4427–4437.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Marzio G, Tyagi M & Gutierrez MI, et al. HIV-1 tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter. Proc Natl Acad Sci USA. 1998; 95: 13519–13524.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Tyagi M, Rusnati M & Presta M, et al. Internalization of HIV-1: Tat requires cell surface heparan sulfate proteoglycans. J Biol Chem. 2001; 276: 3254–3261.

    CAS  Article  PubMed  Google Scholar 

  27. Derossi D, Calvet S & Trembleau A, et al. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem. 1996; 271: 18188–18193.

    CAS  Article  PubMed  Google Scholar 

  28. Johnson DE & Williams LT . Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res. 1993; 60: 1–41.

    CAS  PubMed  Google Scholar 

  29. Fidler IJ . Selection of successive tumour lines for metastasis. Nat New Biol. 1973; 242: 148–149.

    CAS  Article  PubMed  Google Scholar 

  30. Fabrizi C, Colasanti M & Persichini T, et al. Interferon gamma up-regulates alpha 2 macroglobulin expression in human astrocytoma cells. J Neuroimmunol. 1994; 53: 31–37.

    CAS  Article  PubMed  Google Scholar 

  31. Rusnati M, Tulipano G & Spillmann D, et al. Multiple interactions of HIV-1 Tat protein with size-defined heparin oligosaccharides. J Biol Chem. 1999; 274: 28198–28205.

    CAS  Article  PubMed  Google Scholar 

  32. Rusnati M, Tulipano G & Urbinati C, et al. The basic domain in HIV-1 Tat protein as a target for polysulfated heparin-mimicking extra-cellular Tat antagonists. J Biol Chem. 1998; 273: 16027–16037.

    CAS  Article  PubMed  Google Scholar 

  33. Rusnati M, Coltrini D & Oreste P, et al. Interaction of HIV-1 Tat protein with heparin. J Biol Chem. 1997; 272: 11313–11320.

    CAS  Article  PubMed  Google Scholar 

  34. Wallace H, Clarke AR & Harrison DJ, et al. Ganciclovir-induced ablation non-proliferating thyrocytes expressing herpesvirus thymidine kinase occurs by p53-independent apoptosis. Oncogene. 1996; 13: 55–61.

    CAS  PubMed  Google Scholar 

  35. Hamel W, Magnelli L & Chiarugi VP, et al. Herpes simplex virus thymidine kinase/ganciclovir–mediated apoptotic death of bystander cells. Cancer Res. 1996; 56: 2697–2702.

    CAS  PubMed  Google Scholar 

  36. Samejima Y & Meruelo D . “Bystander killing” induces apoptosis and is inhibited by forskolin. Gene Ther. 1995; 2: 50–58.

    CAS  PubMed  Google Scholar 

  37. Spencer DM . Developments in suicide genes for preclinical and clinical applications. Curr Opin Mol Ther. 2000; 2: 433–440.

    CAS  PubMed  Google Scholar 

  38. Guzman RJ, Hirschowitz EA & Brody SL, et al. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA. 1994; 91: 10732–10736.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Chang MW, Ohno T & Gordon D, et al. Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery. Mol Med. 1995; 1: 172–181.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Steg PG, Tahlil O & Aubailly N, et al. Reduction of restenosis after angioplasty in an atheromatous rabbit model by suicide gene therapy. Circulation. 1997; 96: 408–411.

    CAS  Article  PubMed  Google Scholar 

  41. Tiberghien P, Cahn JY & Brion A, et al. Use of donor T-lymphocytes expressing herpes-simplex thymidine kinase in allogeneic bone marrow transplantation: a phase I–II study. Hum Gene Ther. 1997; 8: 615–624.

    CAS  Article  PubMed  Google Scholar 

  42. Helene M, Lake-Bullock V & Bryson JS, et al. Inhibition of graft-versus-host disease. Use of a T cell–controlled suicide gene. J Immunol. 1997; 158: 5079–5082.

    CAS  PubMed  Google Scholar 

  43. Bonini C, Ferrari G & Verzeletti S, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997; 276: 1719–1724.

    CAS  Article  PubMed  Google Scholar 

  44. Shinoura N, Chen L & Wani MA, et al. Protein and messenger RNA expression of connexin43 in astrocytomas: implications in brain tumor gene therapy. J Neurosurg. 1996; 84: 839–845.

    CAS  Article  PubMed  Google Scholar 

  45. Estin D, Li M & Spray D, et al. Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy. Neurosurgery. 1999; 44: 361–368.

    CAS  Article  PubMed  Google Scholar 

  46. Marconi P, Tamura M & Moriuchi S, et al. Connexin 43–enhanced suicide gene therapy using herpesviral vectors. Mol Ther. 2000; 1: 71–81.

    CAS  Article  PubMed  Google Scholar 

  47. Tanaka M, Fraizer GC & De La Cerda J, et al. Connexin 26 enhances the bystander effect in HSVtk/GCV gene therapy for human bladder cancer by adenovirus/PLL/DNA gene delivery. Gene Ther. 2001; 8: 139–148.

    CAS  Article  PubMed  Google Scholar 

  48. Kokoris MS, Sabo P & Adman ET, et al. Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant. Gene Ther. 1999; 6: 1415–1426.

    CAS  Article  PubMed  Google Scholar 

  49. Su H, Lu R & Ding R, et al. Adeno-associated viral-mediated gene transfer to hepatoma: thymidine kinase/interleukin 2 is more effective in tumor killing in non-ganciclovir (GCV)–treated than in GCV-treated animals. Mol Ther. 2000; 1: 509–515.

    CAS  Article  PubMed  Google Scholar 

  50. Chen SH, Kosai K & Xu B, et al. Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival. Cancer Res. 1996; 56: 3758–3762.

    CAS  PubMed  Google Scholar 

  51. O'Malley BW, Cope KA & Chen SH, et al. Combination gene therapy for oral cancer in a murine model. Cancer Res. 1996; 56: 1737–1741.

    CAS  PubMed  Google Scholar 

  52. Benedetti S, Dimeco F & Pollo B, et al. 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. 1997; 8: 1345–1353.

    CAS  Article  PubMed  Google Scholar 

  53. Danthinne X, Aoki K & Kurachi AL, et al. Combination gene delivery of the cell cycle inhibitor p27 with thymidine kinase enhances prodrug cytotoxicity. J Virol. 1998; 72: 9201–9207.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Akyurek LM, Nallamshetty S & Aoki K, et al. Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. Mol Ther. 2001; 3: 779–786.

    CAS  Article  PubMed  Google Scholar 

  55. McNeish IA, Tenev T & Bell S, et al. Herpes simplex virus thymidine kinase/ganciclovir–induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells. Cancer Gene Ther. 2001; 8: 308–319.

    CAS  Article  PubMed  Google Scholar 

  56. Liu CS, Kong B & Xia HH, et al. VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the level of ganciclovir needed to cause suicide cell death. J Gene Med. 2001; 3: 145–152.

    CAS  Article  PubMed  Google Scholar 

  57. Dilber MS, Phelan A & Aints A, et al. Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22. Gene Ther. 1999; 6: 12–21.

    CAS  Article  PubMed  Google Scholar 

  58. Chang HC, Samaniego F & Nair BC, et al. HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix–associated heparan sulfate proteoglycans through its basic region. AIDS. 1997; 11: 1421–1431.

    CAS  Article  PubMed  Google Scholar 

  59. Nicoletti I, Migliorati G & Pagliacci MC, et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991; 139: 271–279.

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by Grants from the National Research Program on AIDS of the Istituto Superiore di Sanità (Rome, Italy) and from the Ministero dell'Istruzione, Universitá e Ricerca. We are grateful to M Janicot for the anti–TK antibody, and to B Boziglav and ME Lopez for excellent technical assistance.

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Correspondence to Monica Zoppè.

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Tasciotti, E., Zoppè, M. & Giacca, M. 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 (2003). https://doi.org/10.1038/sj.cgt.7700526

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  • DOI: https://doi.org/10.1038/sj.cgt.7700526

Keywords

  • acyclovir
  • bystander effect
  • cancer gene therapy
  • ganciclovir
  • intercellular translocation
  • protein transduction
  • HIV-1 Tat
  • thymidine kinase

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