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Transformation Assays for HPV Oncoproteins

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Human Papillomaviruses

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 119))

Summary

A cornerstone of human papillomavirus (HPV) research was the demonstration that those HPV types associated with the development of cervical cancer encode two potent oncoproteins, while those HPV types associated with only benign lesions do not. Thus both HPV-16 E6 and E7 will transform established rodent cells and will efficiently cooperate with other activated oncogenes in the transformation of primary rodent cells. In addition, the virus also encodes for the E5 oncoprotein, which also possesses a weaker transforming activity in established rodent cells. In this chapter we describe how the transforming activities of the HPV oncoproteins can be assessed.

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References

  1. Land, H., Parada, L. F., and Weinberg, R. A. (1983) Cellular oncogenes and multistep carcinogenesis. Science 222, 771–778.

    Article  PubMed  CAS  Google Scholar 

  2. van der Eb, A. J. and Bernards, R. (1984) Transformation and oncogenicity by adenoviruses. Curr. Top. Microbiol. Immunol. 110, 23–51.

    PubMed  Google Scholar 

  3. Heilmann, V. and Kreienberg, R. (2002) Molecular biology of cervical cancer and its precursors. Curr. Womens Health Rep. 2, 27–33.

    PubMed  Google Scholar 

  4. Sunokawa, Y., Takebe, N., Kasamatsu, T., Terada, M., and Sugimura, T. (1986) Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer. Proc. Natl. Acad. Sci. USA 83, 2200–2203.

    Article  Google Scholar 

  5. Yasumoto, S., Burkhardt, A. L., Doniger, J., and DiPaolo, J. A. (1986) Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells. J. Virol. 57, 572–577.

    PubMed  CAS  Google Scholar 

  6. Matlashewski, G. J., Osborn, K., Murray, A., Banks, L., and Crawford, L. V. (1987) Transformation of mouse fibroblasts with HPV type 16 DNA using a heterologous promoter. In Cancer Cells, Papillomaviruses vol. 5 (Steinberg, B.M., Brandsma, J.L., and Taichman, L. B., eds.), New York, Cold Spring Harbor, pp. 195–199.

    Google Scholar 

  7. Bedell, M. A., Jones, K. H., Grossman, S. R., and Laimins, L. A. (1989) Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells. J. Virol. 63, 1247–1255.

    PubMed  CAS  Google Scholar 

  8. Sedman, S. A., Barbosa, M. S., Vass, W. C., et al. (1991) The full-length E6 protein of human papillomavirus type 16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture. J. Virol. 65, 4860–4866.

    PubMed  CAS  Google Scholar 

  9. Pim, D., Collins, M., and Banks, L. (1992) Human papillomavirus type 16 E5 gene stimulates the transforming activity of the epidermal growth factor receptor. Oncogene 7, 27–32.

    PubMed  CAS  Google Scholar 

  10. Leechanachai, P., Banks, L., Moreau, F., and Matlashewski, G. (1992) The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Oncogene 7, 19–25.

    PubMed  CAS  Google Scholar 

  11. Matlashewski, G., Schneider, J., Banks, L., Jones, N., Murray, A., and Crawford, L. (1987) Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO J. 6, 1741–1746.

    PubMed  CAS  Google Scholar 

  12. Crook, T., Morgenstein, J. P., Crawford, L. V., and Banks, L. (1989) Continued expression of HPV 16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV16 plus EJ-ras. EMBO J. 8, 513–519.

    PubMed  CAS  Google Scholar 

  13. Phelps, W. C., Yee, C. L., Munger, K., and Howley, P. M. (1988) The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A. Cell 53, 539–547.

    Article  PubMed  CAS  Google Scholar 

  14. Storey, A., Pim, D., Murray, A., Osborn, K., Banks, L., and Crawford, L. (1988) Comparison of the in vitro transforming activities of human papillomavirus types. EMBO J. 7, 1815–1820.

    PubMed  CAS  Google Scholar 

  15. Kanda, T., Watanabe, S., and Yoshiike, K. (1988) Immortalisation of primary rat cells by human papillomavirus type 16 subgenomic DNA fragments controlled by the SV40 promoter. Virology 165, 321–325.

    Article  PubMed  CAS  Google Scholar 

  16. Vousden, K. H., Doniger, J., DiPaolo, J. A., and Lowy, D. R. (1988) The E7 open reading frame of human papillomavirus type 16 encodes a transforming gene. Oncogene Res. 3, 167–175.

    PubMed  CAS  Google Scholar 

  17. Storey, A. and Banks, L. (1993) Human papillomavirus type 16 E6 gene cooperates with EJ-ras to immortalize primary mouse cells. Oncogene 8, 919–924.

    PubMed  CAS  Google Scholar 

  18. Pim, D., Storey, A., Thomas, M., Massimi, P., and Banks, L. (1994) Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, abolition of p53 transactivation in vivo and immortalisation of primary BMK cells. Oncogene 9, 1869–1876.

    PubMed  CAS  Google Scholar 

  19. Durst, M., Dzarlieva-Petrusevska, R. T., Boukamp, P., Fusenig, N. E., and Gissmann, L. (1987) Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1, 251–256.

    PubMed  CAS  Google Scholar 

  20. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J., and DiPaolo, J. A. (1987) Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J. Virol. 61, 1061–1066.

    PubMed  CAS  Google Scholar 

  21. Kaur, P. and McDougall, J. K. (1988) Characterization of primary human keratinocytes transformed by human papillomavirus type 18. J. Virol. 62, 1917–1924.

    PubMed  CAS  Google Scholar 

  22. Schlegel, R., Phelps, W. C., Zhang, Y. L., and Barbosa, M. (1988) Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J. 7, 3181–3187.

    PubMed  CAS  Google Scholar 

  23. McCance, D. J., Kopan, R., Fuchs, E., and Laimins, L. A. (1988) Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proc. Natl. Acad. Sci. USA 85, 7169–7173.

    Article  PubMed  CAS  Google Scholar 

  24. Pecoraro, G., Morgan, D., and Defendi, V. (1989) Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells. Proc. Natl. Acad. Sci. USA 86, 563–567.

    Article  PubMed  CAS  Google Scholar 

  25. Woodworth, C. D., Doniger, J., and DiPaolo, J. A. (1989) Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma. J. Virol. 63, 159–164.

    PubMed  CAS  Google Scholar 

  26. Park, N. H., Min, B. M., Li, S. L., Huang, M. Z., Cherick, H. M., and Doniger, J. (1991) Immortalization of normal human oral keratinocytes with type 16 human papillomavirus. Carcinogenesis 12, 1627–1631.

    Article  PubMed  CAS  Google Scholar 

  27. Hudson, J. B., Bedell, M. A., McCance, D. J., and Laiminis, L. A. (1990) Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J. Virol. 64, 519–526.

    PubMed  CAS  Google Scholar 

  28. Halbert, C. L., Demers, G. W., and Galloway, D. A. (1991) The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65, 473–478.

    PubMed  CAS  Google Scholar 

  29. Straight, S. W., Hinkle, P. M., Jewers, R. J., and McCance, D. J. (1993) The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes. J. Virol. 67, 4521–4532.

    PubMed  CAS  Google Scholar 

  30. Venuti, A., Salani, D., Poggiali, F., Manni, V., and Bagnato, A. (1998) The E5 oncoprotein of human papillomavirus type 16 enhances endothelin-1-induced keratinocyte growth. Virology 248, 1–5.

    Article  PubMed  CAS  Google Scholar 

  31. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

    Google Scholar 

  32. Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523.

    Article  PubMed  CAS  Google Scholar 

  33. Southern, P. J. and Berg, P. (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1, 327–341.

    PubMed  CAS  Google Scholar 

  34. Ruley, H. E. (1983) Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304, 602–606.

    Article  PubMed  CAS  Google Scholar 

  35. Rassoulzadegan, M., Naghashfar, Z., Cowie, A., et al. (1983) Expression of the large T protein of polyoma virus promotes the establishment in culture of “normal” rodent fibroblast cell lines. Proc. Natl. Acad. Sci. USA 80, 4354–4358.

    Article  PubMed  CAS  Google Scholar 

  36. Noda, T., Yajima, H., and Ito, Y. (1988) Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function. J. Virol. 62, 313–324.

    PubMed  CAS  Google Scholar 

  37. Miyasaka, M., Takami, Y., Inoue, H., and Hakura, A. (1991) Rat primary embryo fibroblast cells suppress transformation by the E6 and E7 genes of human papillomavirus type 16 in somatic hybrid cells. J. Virol. 65, 479–482.

    PubMed  CAS  Google Scholar 

  38. Hurlin, P. J., Kaur, P., Smith, P. P., Perez-Reyes, N., Blanton, R. A., and McDougall, J. K. (1991) Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Proc. Natl. Acad. Sci. USA 88, 570–574.

    Article  PubMed  CAS  Google Scholar 

  39. Leechanachai, P., Banks, L., Moreau, F., and Matlashewski, G. (1992) The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Oncogene 7, 19–25.

    PubMed  CAS  Google Scholar 

  40. Macpherson, I. and Montagnier, L. (1964) Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291–294.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

    Paola Massimi PhD

  2. Tumour Virology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

    Lawrence Banks PhD

Authors
  1. Paola Massimi PhD
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  2. Lawrence Banks PhD
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Editor information

Editors and Affiliations

  1. Division of Virology, The National Institute for Medical Research, Mill Hill, London, UK

    John Doorbar PhD

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© 2005 Humana Press Inc.

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Massimi, P., Banks, L. (2005). Transformation Assays for HPV Oncoproteins. In: Davy, C., Doorbar, J. (eds) Human Papillomaviruses. Methods in Molecular Medicine, vol 119. Humana Press. https://doi.org/10.1385/1-59259-982-6:381

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  • DOI: https://doi.org/10.1385/1-59259-982-6:381

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-373-2

  • Online ISBN: 978-1-59259-982-0

  • eBook Packages: Springer Protocols

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