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Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model

Cancer Gene Therapy volume 10, pages 626–636 (2003)Cite this article

Abstract

Latent membrane protein 1 (LMP1) is an Epstein–Barr virus (EBV)-encoded oncogene expressed in EBV-associated nasopharyngeal carcinoma (NPC). Previous studies indicate that a strategy combining LMP1-mediated NF-κB activation and the HSV thymidine kinase/Ganciclovir (HSVtk/GCV) prodrug system leads to regression of tumor growth in nude mice. To improve the efficacy of this strategy in immunocompetent hosts, we developed a therapeutic cassette, p6κB-EDL1E-tk, containing six copies of the NF-κB binding motif and an epithelial-specific EBV promoter, ED-L1E. The cassette was tested in a murine CT-26 carcinoma model in syngenic Balb/c mice. Coinjection of an LMP1-expressing vector and p6κB-EDL1E-tk by in vivo electroporation in mouse muscle revealed at least two-fold higher TK enzymatic activity than that of previously tested pLTR-tk. Furthermore, growth was attenuated in a group of mice containing LMP1-positive tumors that were intratumorally injected with the p6κB-EDL1E-tk cassette and GCV via in vivo electroporation, but not in mice treated with p6κB-EDL1E-tk or GCV alone. Similarly, no retardation of tumor growth was observed in mice containing LMP1-negative CT-26 tumors injected with both the p6κB-EDL1E-tk cassette and GCV. We propose that intratumoral injection of therapeutic agents, such as DNA of transcription-regulated cassette and GCV, via in vivo electroporation may be used as an alternative treatment for EBV LMP1-expressing cancers.

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References

  1. Shanmugaratnam K . World Health Organization Series International Histological Classification of Tumors. Histological Typing of Tumors of the Upper Respiratory Tract and Ear, 2nd edn. 1991.

  2. Vokes EE, Liebowitz DN, Weichselbaum RR . Nasopharyngeal carcinoma (review). Lancet. 1997;350:1087–1091.

    Article  CAS  PubMed  Google Scholar 

  3. Fahraeus R, Rymo L, Rhim JS, Klein G . Morphological transformation of human keratinocytes expressing the LMP gene of Epstein–Barr virus. Nature. 1990;345:447–449.

    Article  CAS  PubMed  Google Scholar 

  4. Dawson CW, Rickinson AB, Young LS . Epstein–Barr virus latent membrane protein inhibits human epithelial cell differentiation. Nature. 1990;334:777–780.

    Article  Google Scholar 

  5. Wilson JB, Weinberg W, Johnson R, Yuspa S, Levine AJ . Expression of the BNLF-1 oncogene of Epstein–Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6. Cell. 1990;61:1315–1327.

    Article  CAS  PubMed  Google Scholar 

  6. Kulwichit W, Edwards RH, Davenport EM, Baskar JF, Godfrey V, Raab-Traub N . Expression of the Epstein–Barr virus latent membrane protein 1 induces B lymphoma in transgenic mice. Proc Natl Acad Sci USA. 1998;95:11963–11968.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mann KP, Staunton D, Thorley-Lawson DA . Epstein–Barr virus encoded protein found in plasma membranes of transformed cells. J Virol. 1985;55:710–720.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Mosialos G, Birkenbach M, Yalamanchili R, Van Arsdale T, Ware C, Kieff E . The Epstein–Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell. 1995;80:389–399.

    Article  CAS  PubMed  Google Scholar 

  9. Izumi KM, Mcfarland EC, Ting AT, Riley EA, Seed B, Keiff E . The Epstein–Barr virus oncoprotein latent membrane protein q engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-κB activation. Mol Cell Biol. 1999;19:5759–5767.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Devergne O, Hatzivassiliou E, Izumi KM et al. Association of TRAF1, TRAF2, and TRAF3 with an Epstein–Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-κB activation. Mol Cell Biol. 1996;16:7098–7108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kayes K, Ziegler L, Yu CP, Brownie AC, Gallant S . The resistance of the Wistar/Furth rat strain to steroid hypertension. Endocr Res. 1996;22:681–689.

    Article  CAS  PubMed  Google Scholar 

  12. Eliopoulos AG, Rickinson AB . Epstein–Barr virus: LMP1 masquerades as an active receptor. Curr Biol. 1998;12:R196–R198.

  13. Eliopoulos AG, Gallagher NJ, Blake SMS, Dawson CW, Young LS . Activation of the p38 mitogen-activated protein kinase pathway by Epstein–Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem. 1999;274:16085–16096.

    Article  CAS  PubMed  Google Scholar 

  14. Herrero J A, Mathew P, Paya CV . LMP-1 activates NF-κB by targeting the inhibitory molecule IκBα. J Virol. 1995;69:2168–2174.

    PubMed  PubMed Central  Google Scholar 

  15. Wu CJ, Leu CY, Liu ST, Chow KP, Meng CL, Chang YS . Transcriptional activation of NF-kB activity by Epstein–Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases. Gene Therpay. 1998;5:905–912.

    Article  CAS  Google Scholar 

  16. Chang M-S, Ng C-K, Lin Y-J, et al. Identification of a promoter for the latent membrane protein 1 gene of Epstein–Barr virus that is specifically activated in human epithelial cells. DNA and Cell Biol. 1997;16:829–837.

    Article  CAS  Google Scholar 

  17. Chen ML, Tsai CN, Liang CL, et al. Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein–Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population. Oncogene. 1992;7:2131–2140.

    CAS  PubMed  Google Scholar 

  18. Hammarskjold ML, Simurda MC . Epstein–Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-κB activity. J Virol. 1992;66:6496–6501.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Goto T, Nishi T, Takahiko T, et al. Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA. 2000;97:354–359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Huber BE, Richards CA, Krenitsky TA . Retrovirus-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy. Proc Natl Acad Sci USA. 1991;88:8039–8043.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vile RG, Hart IR . Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanoma following direct intratumoral injection of DNA. Cancer Res. 1993;53:3860–3864.

    CAS  PubMed  Google Scholar 

  22. Osaki T, Tanio Y, Tachibana I, et al. Gene therapy for carcinoembryonic antigen-producing human lung cancer cells by cell type-specific expression of herpes simplex virus thymidine kinase gene. Cancer Res. 1994;54:5258–5261.

    CAS  PubMed  Google Scholar 

  23. Kumagai T, Tanio Y, Osaki T, et al. Eradication of Myc-overexpressing small cell lung cancer cells transfected with herpes simplex virus thymidine kinase gene containing Myc-Max response elements. Cancer Res. 1996;56:354–358.

    CAS  PubMed  Google Scholar 

  24. Gu J, Andreeff M, Roth JA, Fang B . hTERT promoter induces tumor-specific Bax gene expression and cell killing in syngenic mouse tumor model and prevents systemic toxicity. Gene Therapy. 2002;9:30–37.

    Article  CAS  PubMed  Google Scholar 

  25. Koga S, Hirohata S, Kondo Y, et al. A novel telomerase-specific gene therapy: gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter. Human Gene Ther. 2000;11:1397–1406.

    Article  CAS  Google Scholar 

  26. Mir L, Bureau M, Gehl J, et al. High-efficiency gene transfer into skeletal muscle mediated by electric pluses. Proc Natl Acad Sci USA. 1999;96:4262–4267.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Titmitov A, Sukharev S, Kistanova E . In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA vector. Biochem Biophys Acta. 1991;1088:131–134.

    Google Scholar 

  28. Suzukin T, Shin B, Fujikura K, Matsuzaki T, Takata K . Direct gene transfer into rat liver cells by in vivo electroporation. FEBS Lett. 1998;425:436–440.

    Article  Google Scholar 

  29. Shibata MA, Morimoto J, Otsuki Y . Suppression of murine mammary carcinoma growth and metastasis by HSVtk/GCV gene therapy using in vivo electroporation. Cancer Gene Ther. 2002;9:16–27.

    Article  CAS  PubMed  Google Scholar 

  30. Rols MP, Teissie J . Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J. 1998;75:1415–1423.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Heller L, Pottinger C, Jaroszeski MJ, Gilbert R, Heller R . In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity. Melanoma Res. 2000;10:577–583.

    Article  CAS  PubMed  Google Scholar 

  32. Niu G, Heller R, Catlett-Falcone R et al. Gene therapy with dominant-negative Stat3 suppresses growth of the murine melanoma B16 tumor in vivo. Cancer Res. 1999;59:5059–5063.

    CAS  PubMed  Google Scholar 

  33. Lohr F, Huang Q, Hu K, Dewhirst MW, Li CY . Systemic vector leakage and transgene expression by intratumorally injected recombinant adenovirus vectors. Clin Cancer Res. 2001;7:3625–3628.

    CAS  PubMed  Google Scholar 

  34. Jaroszeski MJ, Gilbert R, Nicolau C, Heller RI . In vivo gene delivery by electroporation. Adv Drug Rev. 1999;35:131–137.

    Article  CAS  Google Scholar 

  35. Lucas ML, Heller L, Coppola D, Heller R . IL-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous B16.F10 melanoma. Mol Ther. 2002;5:668–675.

    Article  CAS  PubMed  Google Scholar 

  36. Klatzmann D, Valery C, Bensimon G, et al. A Phase I/II study of herpes simplex virus type I thymidine kinase “suicide” gene therapy for recurrent glioblastoma. Hum Gene Ther. 1998;9:2595–2604.

    CAS  PubMed  Google Scholar 

  37. Sterman D, Treat J, Litzky L, et al. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther. 1998;9:1083–1092.

    Article  CAS  PubMed  Google Scholar 

  38. Shalev M, Kadmon D, The B, et al. Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer. J Urol. 2000;163:1747–1750.

    Article  CAS  PubMed  Google Scholar 

  39. Kwong Y-L, Chen S-H, Kosai K, Finegold M, Woo S . Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer. Caner Gene Ther. 1996;3:339–344.

    CAS  Google Scholar 

  40. Kuriyama S, Kikukawa M, Masui K, et al. Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo. Int J Cancer. 1999;83:374–380.

    Article  CAS  PubMed  Google Scholar 

  41. Rivas C, Chandler P, Melo J, Simpson E, Apperley J . Absence of in vitro or in vivo bystander effects in a thymidine kinase-transduced murine T lymphoma. Cancer Gene Ther. 2000;7:954–962.

    Article  CAS  PubMed  Google Scholar 

  42. Golumbek PT . Herpes simplex virus thymidine kinase gene is unable to completely eliminate live, nonimmunogenic tumor cell vaccines. J Immunother. 1992;12:224–230.

    Article  CAS  PubMed  Google Scholar 

  43. Osaki T, Tanio Y, Tachibana I, et al. Gene therapy for carcinoembryonic antigen-producing human lung cancer cells by cell type-specific expression of herpes simplex virus thymidine kinase gene. Cancer Res. 1994;54:5258–5261.

    CAS  PubMed  Google Scholar 

  44. Mesnil M, Yamasaki H . Bystander effect in herpes simplex virus thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication (review). Cancer Res. 2000;60:3989–3999.

    CAS  PubMed  Google Scholar 

  45. Bi WU, Parysek LM, Warnick KR, Stambrook PJ . In vitro evidence that metabolic cooperation in responsible for the bystander effect observed with HSV-TK retrovial gene therapy. Human Gene Ther. 1993;4:725–731.

    Article  CAS  Google Scholar 

  46. McMasters RA, Saylors RL, Johns KE, Hendrix ME, Moyer MP, Drake RR . Lack of bystander killing in herpes simplex virus thymidine kinase-transduced colon cell lines due to deficient connexin43 gap junction formation. Human Gene Ther. 1998;9:2253–2261.

    Article  CAS  Google Scholar 

  47. Touraine RL, Vahanian N, Ramsey WJ, Blaese RM . Enhancement herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions. Human Gene Ther. 1998;9:2385–2391.

    Article  CAS  Google Scholar 

  48. Princen F, Pierre R, Chantal L, et al. A cell type-specific and gap junction independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir mediated bystander effect. Clincal Cancer Res. 1999;5:3639–3644.

    CAS  Google Scholar 

  49. Hayashi K, Hayashi T, Sun HD, Takeda Y . Potentiation of ganciclovir toxicity in the herpes simplex virus thymidine kinase/ganciclovir administration system by ponicidin. Cancer Gene Ther. 2000;7:45–52.

    Article  CAS  PubMed  Google Scholar 

  50. Hasenburg A, Tong XW, Rojas-Martinez A, et al. Thymidine kinase gene therapy with concomitant topotecan chemotherapy for recurrent ovarian cancer. Cancer Gene Ther. 2000;7:839–852.

    Article  CAS  PubMed  Google Scholar 

  51. Judde JG, Spangler G, Magrath I, Bhatia K . Use of Epstein–Barr virus nuclear antigen-1 in targeted therapy of EBV-associated neoplasis. Hum Gene Ther. 1996;7:647–653.

    Article  CAS  PubMed  Google Scholar 

  52. Roger RP, Ge JQ, Holley-Guthrie E, et al. Killing Epstein–Barr virus-positive B lymphocytes and gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase. Hum Gene Ther. 1996;7: 2235–2245.

    Article  Google Scholar 

  53. Li J-H, Chia M, Shi W, et al. Tumor-targeted gene therapy for nasopharyngeal carcinoma. Cancer Res. 2002;62:171–178.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Ling-Ling Hsieh, Department of Public Health, Chang-Gung University for help with the statistic analysis. This work was supported by National Science Council Grant NSC89-2318B-182-005, MOE Program for Promoting Academic Excellency in Universities (Grant number 89-B-FA04-1-4), and Chang-Gung University and Chang-Gung Memorial Hospital Grant CMRP721.

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Authors and Affiliations

  1. Institute of Microbiology and Immunology, National Yang-Ming University, Shih-pai, Taipei, 112, Republic of China

    Yu-hua Hsieh & Chia-lung Tsai

  2. Institute of Preventive Medicine, National Defense Medical Center, Taipei, 114, Republic of China

    Chang-jer Wu

  3. Graduate Institute of Basic Medical Sciences, Chang-Gung University, Kwei-shan, Taoyuan 333, Taiwan, Republic of China

    Kai-ping Chow & Yu-sun Chang

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Correspondence to Yu-sun Chang.

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Hsieh, Yh., Wu, Cj., Chow, Kp. et al. Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model. Cancer Gene Ther 10, 626–636 (2003). https://doi.org/10.1038/sj.cgt.7700609

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