Skip to main content
PMC home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1991 Mar;138(3):673–685.

Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture.

R A Blanton 1, N Perez-Reyes 1, D T Merrick 1, J K McDougall 1
PMCID: PMC1886282  PMID: 1848042

Abstract

Three HPV-16--and four HPV-18--immortalized human foreskin keratinocyte cell lines were analyzed on organotypic epidermal raft cultures at various passage levels. This culture system allowed normal cultured keratinocytes to stratify and differentiate in a manner similar to normal epidermis. All seven HPV-immortalized cell lines displayed epidermal morphologies on organotypic cultures, which were clearly abnormal and resembled premalignant lesions in vivo. Features of premalignant lesions that were shared by all of the HPV-immortalized cell lines included disorganized tissue architecture, mitotic cells present throughout the living layers of the epidermal sheet, abnormal mitoses, enlarged nuclei, and variable cell size and shape. Most HPV-immortalized cell lines were stable in terms of epidermal morphology with long-term passage in culture. Two of the HPV-18--immortalized cell lines, however, lost all morphologically apparent terminal squamous differentiation potential after long-term passage in monolayer culture. These results strongly support the idea that immortalization of squamous epithelial cells in culture by HPV-transforming genes generates a morphologically premalignant cell.

Full text

PDF
673

Images in this article

675Figure 1
on p.675
676Figure 2
on p.676
677Figure 3
on p.677
678Figure 4
on p.678
679Figure 5
on p.679
680Figure 6
on p.680
681Figure 7
on p.681
682Figure 8
on p.682

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Asselineau D., Bernard B. A., Bailly C., Darmon M., Pruniéras M. Human epidermis reconstructed by culture: is it "normal"? J Invest Dermatol. 1986 Feb;86(2):181–186. doi: 10.1111/1523-1747.ep12284237. [DOI] [PubMed] [Google Scholar]
  2. Asselineau D., Bernard B. A., Bailly C., Darmon M. Retinoic acid improves epidermal morphogenesis. Dev Biol. 1989 Jun;133(2):322–335. doi: 10.1016/0012-1606(89)90037-7. [DOI] [PubMed] [Google Scholar]
  3. Baker C. C., Phelps W. C., Lindgren V., Braun M. J., Gonda M. A., Howley P. M. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol. 1987 Apr;61(4):962–971. doi: 10.1128/jvi.61.4.962-971.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banks L., Crawford L. Analysis of human papillomavirus type 16 polypeptides in transformed primary cells. Virology. 1988 Jul;165(1):326–328. doi: 10.1016/0042-6822(88)90695-2. [DOI] [PubMed] [Google Scholar]
  5. Barbosa M. S., Schlegel R. The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes. Oncogene. 1989 Dec;4(12):1529–1532. [PubMed] [Google Scholar]
  6. Bedell M. A., Jones K. H., Grossman S. R., Laimins L. A. Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells. J Virol. 1989 Mar;63(3):1247–1255. doi: 10.1128/jvi.63.3.1247-1255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boshart M., Gissmann L., Ikenberg H., Kleinheinz A., Scheurlen W., zur Hausen H. A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 1984 May;3(5):1151–1157. doi: 10.1002/j.1460-2075.1984.tb01944.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988 Mar;106(3):761–771. doi: 10.1083/jcb.106.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boukamp P., Rupniak H. T., Fusenig N. E. Environmental modulation of the expression of differentiation and malignancy in six human squamous cell carcinoma cell lines. Cancer Res. 1985 Nov;45(11 Pt 2):5582–5592. [PubMed] [Google Scholar]
  10. Cole S. T., Danos O. Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products. J Mol Biol. 1987 Feb 20;193(4):599–608. doi: 10.1016/0022-2836(87)90343-3. [DOI] [PubMed] [Google Scholar]
  11. DiPaolo J. A., Woodworth C. D., Popescu N. C., Notario V., Doniger J. Induction of human cervical squamous cell carcinoma by sequential transfection with human papillomavirus 16 DNA and viral Harvey ras. Oncogene. 1989 Apr;4(4):395–399. [PubMed] [Google Scholar]
  12. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  13. Dürst M., Dzarlieva-Petrusevska R. T., Boukamp P., Fusenig N. E., Gissmann L. Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene. 1987;1(3):251–256. [PubMed] [Google Scholar]
  14. Galloway D. A., McDougall J. K. Human papillomaviruses and carcinomas. Adv Virus Res. 1989;37:125–171. doi: 10.1016/s0065-3527(08)60834-9. [DOI] [PubMed] [Google Scholar]
  15. Hawley-Nelson P., Vousden K. H., Hubbert N. L., Lowy D. R., Schiller J. T. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 1989 Dec 1;8(12):3905–3910. doi: 10.1002/j.1460-2075.1989.tb08570.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hornung J., Bohnert A., Phan-Than L., Krieg T., Fusenig N. E. Basement membrane formation by malignant mouse keratinocyte cell lines in organotypic culture and transplants: correlation with degree of morphologic differentiation. J Cancer Res Clin Oncol. 1987;113(4):325–341. doi: 10.1007/BF00397716. [DOI] [PubMed] [Google Scholar]
  17. Howley P. M., Schlegel R. The human papillomaviruses. An overview. Am J Med. 1988 Aug 29;85(2A):155–158. [PubMed] [Google Scholar]
  18. Hudson J. B., Bedell M. A., McCance D. J., Laiminis L. A. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J Virol. 1990 Feb;64(2):519–526. doi: 10.1128/jvi.64.2.519-526.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kanda T., Furuno A., Yoshiike K. Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells. J Virol. 1988 Feb;62(2):610–613. doi: 10.1128/jvi.62.2.610-613.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaur P., McDougall J. K. Characterization of primary human keratinocytes transformed by human papillomavirus type 18. J Virol. 1988 Jun;62(6):1917–1924. doi: 10.1128/jvi.62.6.1917-1924.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaur P., McDougall J. K., Cone R. Immortalization of primary human epithelial cells by cloned cervical carcinoma DNA containing human papillomavirus type 16 E6/E7 open reading frames. J Gen Virol. 1989 May;70(Pt 5):1261–1266. doi: 10.1099/0022-1317-70-5-1261. [DOI] [PubMed] [Google Scholar]
  22. Kaur P., McDougall J. K. HPV-18 immortalization of human keratinocytes. Virology. 1989 Nov;173(1):302–310. doi: 10.1016/0042-6822(89)90247-x. [DOI] [PubMed] [Google Scholar]
  23. Kopan R., Traska G., Fuchs E. Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization. J Cell Biol. 1987 Jul;105(1):427–440. doi: 10.1083/jcb.105.1.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matlashewski G., Osborn K., Banks L., Stanley M., Crawford L. Transformation of primary human fibroblast cells with human papillomavirus type 16 DNA and EJ-ras. Int J Cancer. 1988 Aug 15;42(2):232–238. doi: 10.1002/ijc.2910420215. [DOI] [PubMed] [Google Scholar]
  25. McCance D. J., Kopan R., Fuchs E., Laimins L. A. Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7169–7173. doi: 10.1073/pnas.85.19.7169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Münger K., Werness B. A., Dyson N., Phelps W. C., Harlow E., Howley P. M. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 1989 Dec 20;8(13):4099–4105. doi: 10.1002/j.1460-2075.1989.tb08594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pecoraro G., Morgan D., Defendi V. Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):563–567. doi: 10.1073/pnas.86.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pirisi L., Creek K. E., Doniger J., DiPaolo J. A. Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA. Carcinogenesis. 1988 Sep;9(9):1573–1579. doi: 10.1093/carcin/9.9.1573. [DOI] [PubMed] [Google Scholar]
  29. Pirisi L., Yasumoto S., Feller M., Doniger J., DiPaolo J. A. Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J Virol. 1987 Apr;61(4):1061–1066. doi: 10.1128/jvi.61.4.1061-1066.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Regnier M., Desbas C., Bailly C., Darmon M. Differentiation of normal and tumoral human keratinocytes cultured on dermis: reconstruction of either normal or tumoral architecture. In Vitro Cell Dev Biol. 1988 Jul;24(7):625–632. doi: 10.1007/BF02623599. [DOI] [PubMed] [Google Scholar]
  31. Schlegel R., Phelps W. C., Zhang Y. L., Barbosa M. Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J. 1988 Oct;7(10):3181–3187. doi: 10.1002/j.1460-2075.1988.tb03185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schneider-Gädicke A., Schwarz E. Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes. EMBO J. 1986 Sep;5(9):2285–2292. doi: 10.1002/j.1460-2075.1986.tb04496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schwarz E., Freese U. K., Gissmann L., Mayer W., Roggenbuck B., Stremlau A., zur Hausen H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature. 1985 Mar 7;314(6006):111–114. doi: 10.1038/314111a0. [DOI] [PubMed] [Google Scholar]
  34. Smith P. P., Bryant E. M., Kaur P., McDougall J. K. Cytogenetic analysis of eight human papillomavirus immortalized human keratinocyte cell lines. Int J Cancer. 1989 Dec 15;44(6):1124–1131. doi: 10.1002/ijc.2910440631. [DOI] [PubMed] [Google Scholar]
  35. Smotkin D., Wettstein F. O. The major human papillomavirus protein in cervical cancers is a cytoplasmic phosphoprotein. J Virol. 1987 May;61(5):1686–1689. doi: 10.1128/jvi.61.5.1686-1689.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smotkin D., Wettstein F. O. Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4680–4684. doi: 10.1073/pnas.83.13.4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Spence R. P., Murray A., Banks L., Kelland L. R., Crawford L. Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers. Cancer Res. 1988 Jan 15;48(2):324–328. [PubMed] [Google Scholar]
  38. Stanley M. A., Browne H. M., Appleby M., Minson A. C. Properties of a non-tumorigenic human cervical keratinocyte cell line. Int J Cancer. 1989 Apr 15;43(4):672–676. doi: 10.1002/ijc.2910430422. [DOI] [PubMed] [Google Scholar]
  39. Stoler A., Kopan R., Duvic M., Fuchs E. Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J Cell Biol. 1988 Aug;107(2):427–446. doi: 10.1083/jcb.107.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsunokawa Y., Takebe N., Kasamatsu T., Terada M., Sugimura T. Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2200–2203. doi: 10.1073/pnas.83.7.2200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Watanabe S., Kanda T., Yoshiike K. Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. J Virol. 1989 Feb;63(2):965–969. doi: 10.1128/jvi.63.2.965-969.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Werness B. A., Levine A. J., Howley P. M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76–79. doi: 10.1126/science.2157286. [DOI] [PubMed] [Google Scholar]
  43. Woodworth C. D., Bowden P. E., Doniger J., Pirisi L., Barnes W., Lancaster W. D., DiPaolo J. A. Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA. Cancer Res. 1988 Aug 15;48(16):4620–4628. [PubMed] [Google Scholar]
  44. Woodworth C. D., Doniger J., DiPaolo J. A. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma. J Virol. 1989 Jan;63(1):159–164. doi: 10.1128/jvi.63.1.159-164.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yee C., Krishnan-Hewlett I., Baker C. C., Schlegel R., Howley P. M. Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am J Pathol. 1985 Jun;119(3):361–366. [PMC free article] [PubMed] [Google Scholar]
  46. de Villiers E. M. Heterogeneity of the human papillomavirus group. J Virol. 1989 Nov;63(11):4898–4903. doi: 10.1128/jvi.63.11.4898-4903.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. de Villiers E. M., Wagner D., Schneider A., Wesch H., Miklaw H., Wahrendorf J., Papendick U., zur Hausen H. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet. 1987 Sep 26;2(8561):703–706. doi: 10.1016/s0140-6736(87)91072-5. [DOI] [PubMed] [Google Scholar]

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES