Skip to main content
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1992 Jan;140(1):167–177.

Altered expression of proliferation and differentiation markers in human papillomavirus 16 and 18 immortalized epithelial cells grown in organotypic culture.

D T Merrick 1, R A Blanton 1, A M Gown 1, J K McDougall 1
PMCID: PMC1886258  PMID: 1370596

Abstract

The patterns of expression of keratins K1 and K8, filaggrin, and the proliferation-associated protein, proliferating cell nuclear antigen (PCNA), were studied in normal and human papillomavirus (HPV) 16 or 18 immortalized keratinocyte cell lines grown on organotypic raft cultures. Normal keratinocytes produced an epithelial sheet that closely resembled epidermis in vivo, characterized by lack of K8 expression, PCNA expression restricted to the basal layer, and K1 and filaggrin expression in the suprabasal layers. Although morphologically abnormal in many respects, some HPV-immortalized cell lines produced cornified epithelial layers and approximated the normals in their patterns of expression of keratins and filaggrin. Other HPV-immortalized cell lines produced poorly differentiated epithelial layers that were characterized by loss of filaggrin expression, and the single tumorigenic cell line, 18-11, was distinguished by abundant K8 expression. All of the HPV-immortalized cell lines were distinguished from normal keratinocytes by a common pattern of full-thickness PCNA expression in the epithelial layers they produced, suggesting that maintenance of the proliferative state may be an important contribution made by HPV 16 or 18 sequences toward the production of a malignant phenotype.

Full text

PDF
167

Images in this article

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. Auersperg N., Kruk P. A., MacLaren I. A., Watt F. M., Mydral S. E. Heterogeneous expression of keratin, involucrin, and extracellular matrix among subpopulations of a poorly differentiated human cervical carcinoma: possible relationships to patterns of invasion. Cancer Res. 1989 Jun 1;49(11):3007–3014. [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. Ball R. D., Walker G. K., Bernstein I. A. Histidine-rich proteins as molecular markers of epidermal differentiation. J Biol Chem. 1978 Aug 25;253(16):5861–5868. [PubMed] [Google Scholar]
  5. Banks-Schlegel S. P. Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation. J Cell Biol. 1982 Jun;93(3):551–559. doi: 10.1083/jcb.93.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanton R. A., Perez-Reyes N., Merrick D. T., McDougall J. K. Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture. Am J Pathol. 1991 Mar;138(3):673–685. [PMC free article] [PubMed] [Google Scholar]
  7. Bobrow L. G., Makin C. A., Law S., Bodmer W. F. Expression of low molecular weight cytokeratin proteins in cervical neoplasia. J Pathol. 1986 Feb;148(2):135–140. doi: 10.1002/path.1711480203. [DOI] [PubMed] [Google Scholar]
  8. Bravo R., Frank R., Blundell P. A., Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. doi: 10.1038/326515a0. [DOI] [PubMed] [Google Scholar]
  9. Celis J. E., Celis A. Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc Natl Acad Sci U S A. 1985 May;82(10):3262–3266. doi: 10.1073/pnas.82.10.3262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Celis J. E., Fey S. J., Larsen P. M., Celis A. Expression of the transformation-sensitive protein "cyclin" in normal human epidermal basal cells and simian virus 40-transformed keratinocytes. Proc Natl Acad Sci U S A. 1984 May;81(10):3128–3132. doi: 10.1073/pnas.81.10.3128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  12. Cintorino M., Syrjänen S., Leoncini P., Bellizzi De Marco E., Petracca R., Pallini V., Tosi P., Mäntyjärvi R., Syrjänen K. Altered expression of filaggrin in human papillomavirus (HPV) lesions of the uterine cervix. Arch Gynecol Obstet. 1988;241(4):235–247. doi: 10.1007/BF00931354. [DOI] [PubMed] [Google Scholar]
  13. Cooper J. A., Whyte P. RB and the cell cycle: entrance or exit? Cell. 1989 Sep 22;58(6):1009–1011. doi: 10.1016/0092-8674(89)90495-9. [DOI] [PubMed] [Google Scholar]
  14. Dale B. A., Gown A. M., Fleckman P., Kimball J. R., Resing K. A. Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins. J Invest Dermatol. 1987 Mar;88(3):306–313. doi: 10.1111/1523-1747.ep12466185. [DOI] [PubMed] [Google Scholar]
  15. Dale B. A., Holbrook K. A., Steinert P. M. Assembly of stratum corneum basic protein and keratin filaments in macrofibrils. Nature. 1978 Dec 14;276(5689):729–731. doi: 10.1038/276729a0. [DOI] [PubMed] [Google Scholar]
  16. Dale B. A., Resing K. A., Lonsdale-Eccles J. D. Filaggrin: a keratin filament associated protein. Ann N Y Acad Sci. 1985;455:330–342. doi: 10.1111/j.1749-6632.1985.tb50420.x. [DOI] [PubMed] [Google Scholar]
  17. DeCaprio J. A., Ludlow J. W., Lynch D., Furukawa Y., Griffin J., Piwnica-Worms H., Huang C. M., Livingston D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. doi: 10.1016/0092-8674(89)90507-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Dürst M., Bosch F. X., Glitz D., Schneider A., zur Hausen H. Inverse relationship between human papillomavirus (HPV) type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines. J Virol. 1991 Feb;65(2):796–804. doi: 10.1128/jvi.65.2.796-804.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Fuchs E., Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell. 1980 Apr;19(4):1033–1042. doi: 10.1016/0092-8674(80)90094-x. [DOI] [PubMed] [Google Scholar]
  23. Garcia R. L., Coltrera M. D., Gown A. M. Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis. Am J Pathol. 1989 Apr;134(4):733–739. [PMC free article] [PubMed] [Google Scholar]
  24. Gown A. M., Vogel A. M. Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues. Am J Pathol. 1984 Feb;114(2):309–321. [PMC free article] [PubMed] [Google Scholar]
  25. Gown A. M., Vogel A. M. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies. J Cell Biol. 1982 Nov;95(2 Pt 1):414–424. doi: 10.1083/jcb.95.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Howley P. M., Schlegel R. The human papillomaviruses. An overview. Am J Med. 1988 Aug 29;85(2A):155–158. [PubMed] [Google Scholar]
  27. Hronis T. S., Steinberg M. L., Defendi V., Sun T. T. Simple epithelial nature of some simian virus-40-transformed human epidermal keratinocytes. Cancer Res. 1984 Dec;44(12 Pt 1):5797–5804. [PubMed] [Google Scholar]
  28. Huang H. J., Yee J. K., Shew J. Y., Chen P. L., Bookstein R., Friedmann T., Lee E. Y., Lee W. H. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science. 1988 Dec 16;242(4885):1563–1566. doi: 10.1126/science.3201247. [DOI] [PubMed] [Google Scholar]
  29. Hurlin P. J., Kaur P., Smith P. P., Perez-Reyes N., Blanton R. A., McDougall J. K. Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):570–574. doi: 10.1073/pnas.88.2.570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Itoiz M. E., Conti C. J., Lanfranchi H. E., Mamrack M., Klein-Szanto A. J. Immunohistochemical detection of filaggrin in preneoplastic and neoplastic lesions of the human oral mucosa. Am J Pathol. 1985 Jun;119(3):456–461. [PMC free article] [PubMed] [Google Scholar]
  31. Ivanyi D., Groeneveld E., Van Doornewaard G., Mooi W. J., Hageman P. C. Keratin subtypes in carcinomas of the uterine cervix: implications for histogenesis and differential diagnosis. Cancer Res. 1990 Aug 15;50(16):5143–5152. [PubMed] [Google Scholar]
  32. Jablonska S., Orth G. Epidermodysplasia verruciformis. Clin Dermatol. 1985 Oct-Dec;3(4):83–96. doi: 10.1016/0738-081x(85)90052-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. Koss L. G. Pathogenesis of carcinoma of the uterine cervix. Curr Top Pathol. 1981;70:111–142. doi: 10.1007/978-3-642-68185-1_5. [DOI] [PubMed] [Google Scholar]
  38. Lavker R. M., Sun T. T. Epidermal stem cells. J Invest Dermatol. 1983 Jul;81(1 Suppl):121s–127s. doi: 10.1111/1523-1747.ep12540880. [DOI] [PubMed] [Google Scholar]
  39. Levine A. J. The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology. 1990 Aug;177(2):419–426. doi: 10.1016/0042-6822(90)90505-l. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Miettinen M. Keratin subsets in spindle cell sarcomas. Keratins are widespread but synovial sarcoma contains a distinctive keratin polypeptide pattern and desmoplakins. Am J Pathol. 1991 Feb;138(2):505–513. [PMC free article] [PubMed] [Google Scholar]
  42. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  43. Moll R., Krepler R., Franke W. W. Complex cytokeratin polypeptide patterns observed in certain human carcinomas. Differentiation. 1983;23(3):256–269. doi: 10.1111/j.1432-0436.1982.tb01291.x. [DOI] [PubMed] [Google Scholar]
  44. Nelson W. G., Sun T. T. The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J Cell Biol. 1983 Jul;97(1):244–251. doi: 10.1083/jcb.97.1.244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pecoraro G., Lee M., Morgan D., Defendi V. Evolution of in vitro transformation and tumorigenesis of HPV16 and HPV18 immortalized primary cervical epithelial cells. Am J Pathol. 1991 Jan;138(1):1–8. [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. 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]
  49. 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]
  50. Smedts F., Ramaekers F., Robben H., Pruszczynski M., van Muijen G., Lane B., Leigh I., Vooijs P. Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia. Am J Pathol. 1990 Mar;136(3):657–668. [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. Steinert P. M., Cantieri J. S., Teller D. C., Lonsdale-Eccles J. D., Dale B. A. Characterization of a class of cationic proteins that specifically interact with intermediate filaments. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4097–4101. doi: 10.1073/pnas.78.7.4097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Syrjänen K., Mäntyjärvi R., Väyrynen M., Syrjänen S., Parkkinen S., Yliskoski M., Saarikoski S., Castrén O. Human papillomavirus (HPV) infections involved in the neoplastic process of the uterine cervix as established by prospective follow-up of 513 women for two years. Eur J Gynaecol Oncol. 1987;8(1):5–16. [PubMed] [Google Scholar]
  56. Trask D. K., Band V., Zajchowski D. A., Yaswen P., Suh T., Sager R. Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2319–2323. doi: 10.1073/pnas.87.6.2319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weinberg R. A. The retinoblastoma gene and cell growth control. Trends Biochem Sci. 1990 May;15(5):199–202. doi: 10.1016/0968-0004(90)90162-5. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Wilke M. S., Hsu B. M., Wille J. J., Jr, Pittelkow M. R., Scott R. E. Biologic mechanisms for the regulation of normal human keratinocyte proliferation and differentiation. Am J Pathol. 1988 Apr;131(1):171–181. [PMC free article] [PubMed] [Google Scholar]
  60. Woodworth C. D., Waggoner S., Barnes W., Stoler M. H., DiPaolo J. A. Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNAs exhibit dysplastic differentiation in vivo. Cancer Res. 1990 Jun 15;50(12):3709–3715. [PubMed] [Google Scholar]
  61. Wu Y. J., Parker L. M., Binder N. E., Beckett M. A., Sinard J. H., Griffiths C. T., Rheinwald J. G. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell. 1982 Dec;31(3 Pt 2):693–703. doi: 10.1016/0092-8674(82)90324-5. [DOI] [PubMed] [Google Scholar]
  62. 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]

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

RESOURCES