Skip to main content
SpringerLink
Log in
Book cover

Human Papillomaviruses pp 187–202Cite as

Retrovirus-Mediated Gene Transfer to Analyze HPV Gene Regulation and Protein Functions in Organotypic “Raft” Cultures

  • Protocol

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

Summary

The productive phase of human papillomavirus (HPV) infection is dependent on squamous differentiation of epithelial keratinocytes. Organotypic culture systems of primary human keratinocytes (PHKs) or immortalized keratinocytes that contain HPV genomes were developed to recapitulate this permissive environment. A complementary approach to determine the functions of individual HPV genes and to examine the virus-host interactions is to introduce the gene, alone or in combination, into keratinocytes that are then grown in organotypic cultures. The success of the latter approach depends on the methodology of retrovirus-mediated gene transfer, which can transduce the viral gene or genes into an entire population of PHKs. In this chapter, we describe the strategies and methods of retrovirus-mediated gene transfer into keratinocytes grown into organotypic cultures.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Stoler, M. H. and Broker, T. R. (1986) In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma. Human Pathol. 17, 1250–1258.

    Article  CAS  Google Scholar 

  2. Stoler, M. H., Wolinsky, S. M., Whitbeck, A., Broker, T. R., and Chow, L. T. (1989) Differentiation-linked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes. Virology 172, 331–340.

    Article  PubMed  CAS  Google Scholar 

  3. Stoler., M. H., Whitbeck, A., Wolinsky, S. M., et al. (1990) Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice. J. Virol. 64, 3310–3318.

    PubMed  CAS  Google Scholar 

  4. Stoler, M. H, Rhodes, C. R., Whitbeck, A., et al. (1992) Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Human Pathol. 23, 117–128.

    Article  CAS  Google Scholar 

  5. Dollard, S. C., Wilson, J. L., Demeter, L. M., et al. (1992) Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. Genes Dev. 6, 1131–1142.

    Article  PubMed  CAS  Google Scholar 

  6. Wilson, J. L., Dollard, S. C., Chow, L. T., and Broker, T. R. (1992) Epithelialspecific gene expression during differentiation of stratified primary human keratinocyte cultures. Cell Growth Diff. 3, 471–483.

    PubMed  CAS  Google Scholar 

  7. Meyers, C., Frattini, M. G., Hudson, J. B., and Laimins, L. A. (1992) Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Science 257, 971–973.

    Article  PubMed  CAS  Google Scholar 

  8. Chow, L. T. and Broker, T. R. (1997) In vitro experimental systems for HPV: epithelial raft cultures for investigations of viral reproduction and pathogenesis and for genetic analyses of viral proteins and regulatory sequences. Clin. Dermatol. 15, 217–227.

    Article  PubMed  CAS  Google Scholar 

  9. 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 

  10. Blanton, R. A., Perez-Reyes, N., Merrick, D. T., and McDougall J. K. (1991) Epithellial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic raft culture. Am. J. Pathol. 138, 673–685.

    PubMed  CAS  Google Scholar 

  11. Steenbergen, R. D. M., Parker, J. N., Isern, S., et al. (1998) Viral E6-E7 transcription in the basal layer of organotypic cultures without apparent p21cip1 protein precedes immortalization of human papillomavirus type 16-and 18-transfected human keratinocytes. J. Virol. 72, 749–757.

    PubMed  CAS  Google Scholar 

  12. Frattini, M. G., Lim, H. B., and Laimins, L. A. (1996) In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression. Proc. Natl. Acad. Sci. USA 93, 3062–3067.

    Article  PubMed  CAS  Google Scholar 

  13. Flores, E. R., Allen-Hoffmann, B. L., Lee, D., Sattler, C. A., and Lambert, P. F. (1999) Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line. Virology 30, 262, 344–354.

    Google Scholar 

  14. Flores, E. R., Allen-Hoffmann, B. L., Lee, D., and Lambert, P. F. (2000) The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle. J. Virol. 74, 6622–6631.

    Article  PubMed  CAS  Google Scholar 

  15. Blanton, R. A., Coltrera, M. D., Gown, A. M., Halbert, C. L., and McDougall J. K. (1992) Expression of the HPV 16 E7 gene generates proliferation in stratified squamous cell cultures which is independent of endogenous p53 levels. Cell Growth Diff. 3, 791–802.

    PubMed  CAS  Google Scholar 

  16. Halbert, C. L., Demers, G. W., and Galloway D. A. (1992) The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells. J. Virol. 66, 2125–2134.

    PubMed  CAS  Google Scholar 

  17. Cheng, S., Schmidt-Grimminger, D., Murant, T., Broker, T. R., and Chow L. T. (1995) Differentiation dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. Genes Dev. 9, 2335–2349.

    Article  PubMed  CAS  Google Scholar 

  18. Jian, Y., Schmidt-Grimminger, D.-C., Wu, X., Broker, T. R., and Chow, L. T. (1998) Post-transcriptional induction of p21cip1 protein by HPV E7 in differentiated epithelial cells inhibits reactivated unscheduled DNA synthesis. Oncogene 17, 2027–2038.

    Article  PubMed  CAS  Google Scholar 

  19. Jian, Y., Van Tine, B. A., Chien, W.-M., Shaw, G. M., Broker, T. R., and Chow, L. T. (1999) Concordant induction of cyclin E and p21cip1 in differentiated keratinocytes by the HPV E7 protein inhibits cellular and viral DNA synthesis. Cell Growth Diff. 10, 101–111.

    PubMed  CAS  Google Scholar 

  20. Chien, W-M., Parker, J. N., Schmidt-Grimminger, D.-C., Broker T. R., and Chow L. T. (2000) Casein kinase II phosphorylation of the human papillomavirus-18 E7 protein is critical for promoting S-phase entry. Cell Growth Diff. 11, 425–435.

    PubMed  CAS  Google Scholar 

  21. Chien, W-M., Noya, F., Benedict-Hamilton, H. M., Broker, T. R., and Chow, L. T. (2002) Alternative fates of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation. J. Virol. 76, 2964–2972.

    Article  PubMed  CAS  Google Scholar 

  22. Noya, F., Chien, W-M., Broker, T. R., and Chow L. T. (2001) p21cip1 degradation in differentiated keratinocytes is abrogated by costabilization with cyclin E induced by human papillomavirus E7. J. Virol. 75, 6121–6134.

    Article  PubMed  CAS  Google Scholar 

  23. Garner-Hamrick, P. A., Fostel, J. M., Chien, W.-M., et al. (2004) Global effects of human papillomavirus 18 (HPV-18) E6/E7 in an organotypic culture system. J. Virol. 78, 9041–9050.

    Article  PubMed  CAS  Google Scholar 

  24. Boccardo, E., Noya, F., Broker, T. R., Chow, L. T., and Villa, L. L. (2004) Resistance to TNF-α mediated cell proliferation arrest and DNA synthesis inhibition by HPV-18 oncoproteins in organotypic cultures of primary human keratinocytes. Virology 328, 233–244.

    Article  PubMed  CAS  Google Scholar 

  25. Parker, J. N., Zhao, W., Askins, K. J., Broker, T. R., and Chow, L. T. (1997) Mutational analyses of differentiation dependent human papillomavirus type 18 enhancer elements in epithelial raft cultures of neonatal foreskin keratinocytes. Cell Growth Diff. 8, 751–762.

    PubMed  CAS  Google Scholar 

  26. Zhao, W., Broker, T. R., and Chow, L. T. (1997) Transcriptional activities of human papillomavirus type-11 promoter-proximal elements in raft and submerged cultures of foreskin keratinocytes. J. Virol. 71, 8832–8840.

    PubMed  CAS  Google Scholar 

  27. Zhao, W., Chow, L. T., and Broker T. R. (1999) A distal element in the HPV-11 upstream regulatory region contributes to promoter repression in basal keratinocytes in squamous epithelium. Virology 253, 219–29.

    Article  PubMed  CAS  Google Scholar 

  28. Zhao, W., Noya, F., Chen, W. Y., Townes, T. M., Chow, L. T., and Broker, T. R. (1999) Trichostatin A up-regulates human papillomavirus type 11 upstream regulatory region-E6 promoter activity in undifferentiated primary human keratinocytes. J. Virol. 73, 5026–5033.

    PubMed  CAS  Google Scholar 

  29. Korman, A. J., Frantz, F. D., Strominger, J., and Mulligan, R. (1987) Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc. Natl. Acad. Sci. USA 84, 2150–2154.

    Article  PubMed  CAS  Google Scholar 

  30. Miller, A. D. and Rosman, G. J. (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–990.

    PubMed  CAS  Google Scholar 

  31. Morganstern, J. P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucl. Acid Res. 18, 3587–3596.

    Article  Google Scholar 

  32. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl, Acad. Sci. USA 90, 8392–8396.

    Article  CAS  Google Scholar 

  33. Markowitz, D., Goff, S., and Bank, A. (1988) Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167, 400–406.

    PubMed  CAS  Google Scholar 

  34. Noya, F., Chien, W-M., Wu, X., et al. (2002) The promoter of the human proliferating cell nuclear antigen gene is not sufficient for cell cycle-dependent regulation in organotypic raft cultures of keratinocytes. J. Biol. Chem. 277, 17,271–17,280.

    Article  PubMed  CAS  Google Scholar 

  35. Kozak, S. L. and Kabat, D. (1990) Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production. J. Virol. 64, 3500–3508.

    PubMed  CAS  Google Scholar 

  36. Van Tine, B. A, Kappes, J.C., Banerjee, N.S., et al. (2004) Clonal selection for transcriptionally active viral oncogenes during progression to cancer by DNA methylation-mediated silencing. J. Virol. 78, 11,172–11,186.

    Article  PubMed  Google Scholar 

  37. Yu, S. F., von Ruden, T., Kantoff, P. W., et al. (1986) Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc.Natl. Acad. Sci. USA 83, 3194–3198.

    Article  PubMed  CAS  Google Scholar 

  38. Naldini, L., Blömer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996) Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93, 11,382–11,388.

    Article  PubMed  CAS  Google Scholar 

  39. Dull, T., Zufferey, R., Kelly, M., et al. (1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471.

    PubMed  CAS  Google Scholar 

  40. Zufferey, R., Dull, T., Mandel, R.J., et al. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delievery. J. Virol. 72, 9873–9880.

    PubMed  CAS  Google Scholar 

  41. Wu, X., Wakefield, J. K., Liu, H., et al. (2000) Development of a novel trans-lentiviral vector that affords predictable safety. Mol. Ther. 2, 47–55.

    Article  PubMed  CAS  Google Scholar 

  42. Pfeifer, A., Kessler, T., Silletti, S., Cheresh, D. A., and Verma, I. M. (2000) Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2. Proc. Natl. Acad. Sci. USA 97, 12,227–12,232.

    Article  PubMed  CAS  Google Scholar 

  43. Kumar, K., Keller, B., Makalou, N., and Sutton, R. E. (2001) Systematic determination of the packaging limit of lentivral vectors. Human Gene Ther. 12, 1893–1905.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL

    N. Sanjib Banerjee PhD, Louise T. Chow PhD & Thomas R. Broker PhD

Authors
  1. N. Sanjib Banerjee PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Louise T. Chow PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas R. Broker PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

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

    John Doorbar PhD

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Banerjee, N.S., Chow, L.T., Broker, T.R. (2005). Retrovirus-Mediated Gene Transfer to Analyze HPV Gene Regulation and Protein Functions in Organotypic “Raft” Cultures. In: Davy, C., Doorbar, J. (eds) Human Papillomaviruses. Methods in Molecular Medicine, vol 119. Humana Press. https://doi.org/10.1385/1-59259-982-6:187

Download citation

  • DOI: https://doi.org/10.1385/1-59259-982-6:187

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-373-2

  • Online ISBN: 978-1-59259-982-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation