Oncolysis of canine tumor cells by myxoma virus lacking the serp2 gene

Ashlee S. Urbasic Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Stacy Hynes Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Amy Somrak Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Stacey Contakos Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Masmudur M. Rahman Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32611.

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Jia Liu Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32611.

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Amy L. MacNeill Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Abstract

Objective—To determine the oncolytic efficacy of an attenuated form of myxoma virus lacking the serp2 gene in canine tumor cells.

Sample—Primary cells were isolated from tumors that were surgically removed from dogs and from connective tissue obtained from the cadaver of a dog. Cells of various established cell lines from tumors and nontumorous tissues were obtained.

Procedures—Experiments were performed with cells in monolayer culture. Cell cultures were inoculated with wild-type myxoma viruses or myxoma viruses lacking the serp2 gene, and measures of cytopathic effects, viral growth kinetics, and cell death and apoptosis were determined.

Results—Myxoma viruses replicated in cells of many of the primary and established canine tumor cell lines. Canine tumor cells in which expression of activated protein kinase B was upregulated were more permissive to myxoma virus infection than were cells in which expression of activated protein kinase B was not upregulated. Myxoma viruses lacking the serp2 gene caused more cytopathic effects in canine tumor cells because of apoptosis than did wild-type myxoma viruses.

Conclusions and Clinical Relevance—Results of the present study indicated myxoma viruses lacking the serp2 gene may be useful for treatment of cancer in dogs.

Impact for Human Medicine—Results of the present study may be useful for development of novel oncolytic treatments for tumors in humans.

Abstract

Objective—To determine the oncolytic efficacy of an attenuated form of myxoma virus lacking the serp2 gene in canine tumor cells.

Sample—Primary cells were isolated from tumors that were surgically removed from dogs and from connective tissue obtained from the cadaver of a dog. Cells of various established cell lines from tumors and nontumorous tissues were obtained.

Procedures—Experiments were performed with cells in monolayer culture. Cell cultures were inoculated with wild-type myxoma viruses or myxoma viruses lacking the serp2 gene, and measures of cytopathic effects, viral growth kinetics, and cell death and apoptosis were determined.

Results—Myxoma viruses replicated in cells of many of the primary and established canine tumor cell lines. Canine tumor cells in which expression of activated protein kinase B was upregulated were more permissive to myxoma virus infection than were cells in which expression of activated protein kinase B was not upregulated. Myxoma viruses lacking the serp2 gene caused more cytopathic effects in canine tumor cells because of apoptosis than did wild-type myxoma viruses.

Conclusions and Clinical Relevance—Results of the present study indicated myxoma viruses lacking the serp2 gene may be useful for treatment of cancer in dogs.

Impact for Human Medicine—Results of the present study may be useful for development of novel oncolytic treatments for tumors in humans.

Contributor Notes

Supported by University of Illinois College of Veterinary Medicine Department of Pathology, the University of Illinois College of Veterinary Medicine Companion Animal Grants Memorial Fund, the Wayne D. and Josephine Spangler Fund, and the National Center for Research Resources (National Institutes of Health, T35 RR020292).

Presented as an oral presentation at the American College of Veterinary Pathologists–American Society for Veterinary Clinical Pathology Annual Meeting, Monterey, Calif, June 2010.

The authors thank Drs. Gail Scherba and Joanna Shisler for assistance with reporting of data and Karen Kelly for performance of transmission electron microscopy.

Address correspondence to Dr. MacNeill (almac@illinois.edu).
  • 1 Stojdl DF, Lichty B, Knowles S, et al. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 2000; 6: 821825.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2 Cahill DP, Kinzler KW, Vogelstein B, et al. Genetic instability and darwinian selection in tumours. Trends Cell Biol 1999; 9: M57M60.

  • 3 Vaha-Koskela MJ, Heikkila JE, Hinkkanen AE. Oncolytic viruses in cancer therapy. Cancer Lett 2007; 254: 178216.

  • 4 Jiang BH, Liu LZ. PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv Cancer Res 2009; 102: 1965.

  • 5 Soares JA, Leite FG, Andrade LG, et al. Activation of the PI3K/Akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication. J Virol 2009; 83: 68836899.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6 Ricketts CR. Molecular permeability of the burned area. Proc Soc Exp Biol Med 1966; 121: 770772.

  • 7 McCart JA, Ward JM, Lee J, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res 2001; 61: 87518757.

    • Search Google Scholar
    • Export Citation
  • 8 Thorne SH, Bartlett DL, Kirn DH. The use of oncolytic vaccinia viruses in the treatment of cancer: a new role for an old ally? Curr Gene Ther 2005; 5: 429443.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9 Merchlinsky M, Eckert D, Smith E, et al. Construction and characterization of vaccinia direct ligation vectors. Virology 1997; 238: 444451.

  • 10 Karupiah G, Blanden RV. Anti-asialo-GM1 inhibits vaccinia virus infection of murine ovaries: asialo-GM1 as an additional virus receptor? Immunol Cell Biol 1990; 68: 343346.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11 Selin LK, Santolucito PA, Pinto AK, et al. Innate immunity to viruses: control of vaccinia virus infection by gamma delta T cells. J Immunol 2001; 166: 67846794.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12 Xu R, Johnson AJ, Liggitt D, et al. Cellular and humoral immunity against vaccinia virus infection of mice. J Immunol 2004; 172: 62656271.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13 Chaudhri G, Panchanathan V, Bluethmann H, et al. Obligatory requirement for antibody in recovery from a primary poxvirus infection. J Virol 2006; 80: 63396344.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14 Fenner F, Woodroffe GM. The pathogenesis of infectious myxomatosis: the mechanism of infection and the immunological response in the European rabbit (Oryctolagus cuniculus). Br J Exp Pathol 1953; 34: 400411.

    • Search Google Scholar
    • Export Citation
  • 15 Andrewes CH, Harisijades S. Propagation of myxoma virus in one-day old mice. Br J Exp Pathol 1955; 36: 1821.

  • 16 Jackson EW, Dorn CR, Saito JK, et al. Absence of serological evidence of myxoma virus infection in humans exposed during an outbreak of myxomatosis. Nature 1966; 211: 313314.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17 Gorski J, Mizak B, Chrobocinska M. Control of rabbit myxomatosis in Poland. Rev Sci Tech 1994; 13: 869879.

  • 18 Fenner F. Adventures with poxviruses of vertebrates. FEMS Microbiol Rev 2000; 24: 123133.

  • 19 McCabe VJ, Tarpey I, Spibey N. Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein. Vaccine 2002; 20: 24542462.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20 McCabe VJ, Spibey N. Potential for broad-spectrum protection against feline calicivirus using an attenuated myxoma virus expressing a chimeric FCV capsid protein. Vaccine 2005; 23: 53805388.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21 Pignolet B, Boullier S, Gelfi J, et al. Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants. J Gen Virol 2008; 89: 13711379.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22 Sypula J, Wang F, Ma Y, et al. Myxoma virus tropism in human tumor cells. Gene Ther Mol Biol 2004; 8: 103114.

  • 23 Lun X, Yang W, Alain T, et al. Myxoma virus is a novel oncolytic virus with significant antitumor activity against experimental human gliomas. Cancer Res 2005; 65: 99829990.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24 Lun XQ, Zhou H, Alain T, et al. Targeting human medulloblastoma: oncolytic virotherapy with myxoma virus is enhanced by rapamycin. Cancer Res 2007;67: 88188827.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25 Stanford MM, Shaban M, Barrett JW, et al. Myxoma virus oncolysis of primary and metastatic B16F10 mouse tumors in vivo. Mol Ther 2008; 16: 5259.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26 Lun X, Alain T, Zemp FJ, et al. Myxoma virus virotherapy for glioma in immunocompetent animal models: optimizing administration routes and synergy with rapamycin. Cancer Res 2010; 70: 598608.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27 Guo ZS, Naik A, O'Malley ME, et al. The enhanced tumor selectivity of an oncolytic vaccinia lacking the host range and antiapoptosis genes SPI-1 and SPI-2. Cancer Res 2005; 65: 99919998.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28 Messud-Petit F, Gelfi J, Delverdier M, et al. Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus. J Virol 1998; 72: 78307839.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29 MacNeill AL, Turner PC, Moyer RW. Mutation of the Myxoma virus SERP2 P1-site to prevent proteinase inhibition causes apoptosis in cultured RK-13 cells and attenuates disease in rabbits, but mutation to alter specificity causes apoptosis without reducing virulence. Virology 2006; 356: 1222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30 Nathaniel R, MacNeill AL, Wang YX, et al. Cowpox virus crmA, myxoma virus serp2 and baculovirus P35 are not functionally interchangeable caspase inhibitors in poxvirus infections. J Gen Virol 2004; 85: 12671278.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31 Johnston JB, Barrett JW, Chang W, et al. Role of the serinethreonine kinase PAK-1 in myxoma virus replication. J Virol 2003; 77: 58775888.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32 Liu J, Wennier S, Reinhard M, et al. Myxoma virus expressing interleukin-15 fails to cause lethal myxomatosis in European rabbits. J Virol 2009; 83: 59335938.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33 Duteyrat JL, Gelfi J, Bertagnoli S. Ultrastructural study of myxoma virus morphogenesis. Arch Virol 2006; 151: 21612180.

  • 34 Barrett JW, Alston LR, Wang F, et al. Identification of host range mutants of myxoma virus with altered oncolytic potential in human glioma cells. J Neurovirol 2007; 13: 549560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35 Wang G, Barrett JW, Stanford M, et al. Infection of human cancer cells with myxoma virus requires Akt activation via interaction with a viral ankyrin-repeat host range factor. Proc Natl Acad Sci U S A 2006; 103: 46404645.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36 Kylarova D, Prochazkova J, Mad'arova J, et al. Comparison of the TUNEL, lamin B and annexin V methods for the detection of apoptosis by flow cytometry. Acta Histochem 2002; 104: 367370.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37 Porrello A, Cardelli P, Spugnini EP. Oncology of companion animals as a model for humans. an overview of tumor histotypes 2. J Exp Clin Cancer Res 2006; 25: 97105.

    • Search Google Scholar
    • Export Citation
  • 38 Gentschev I, Stritzker J, Hofmann E, et al. Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent. Cancer Gene Ther 2009; 16: 320328.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39 Gentschev I, Ehrig K, Donat U, et al. Significant growth inhibition of canine mammary carcinoma xenografts following treatment with oncolytic vaccinia virus GLV-1h68. J Oncol 2010; 2010: 736907.

    • Search Google Scholar
    • Export Citation
  • 40 Gentschev I, Adelfinger M, Josupeit R, et al. Preclinical evaluation of oncolytic vaccinia virus for therapy of canine soft tissue sarcoma. PLoS One [serial online] 2012; 7:e37239. Available at: www.plosone.org/article/fetchObjectAttachment;jsessionid=EB9DA36616175442A5E5F9C7E105C6FD?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0037239&representation=PDF. Accessed May 15, 2012.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41 Kennedy RB, Ovsyannikova IG, Jacobson RM, et al. The immunology of smallpox vaccines. Curr Opin Immunol 2009; 21: 314320.

  • 42 Liu TC, Hwang T, Park BH, et al. The targeted oncolytic poxvirus JX-594 demonstrates antitumoral, antivascular, and anti-HBV activities in patients with hepatocellular carcinoma. Mol Ther 2008; 16: 16371642.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43 Amato RJ, Shingler W, Goonewardena M, et al. Vaccination of renal cell cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) alone or administered in combination with interferon-alpha (IFN-alpha): a phase 2 trial. J Immunother 2009; 32: 765772.

    • Crossref
    • Search Google Scholar
    • Export Citation

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