Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jun;86(11):4102–4106. doi: 10.1073/pnas.86.11.4102

Restoration of growth potential in paraclones of human keratinocytes by a viral oncogene.

Y Barrandon 1, J R Morgan 1, R C Mulligan 1, H Green 1
PMCID: PMC287397  PMID: 2471195

Abstract

Human diploid keratinocytes may be divided into three clonal types with differing capacities for proliferation. The paraclone, which has the shortest life span, is limited to 15 divisions, after which all the cells undergo programmed terminal differentiation. By means of a retroviral vector, paraclones which have not completed their life span and which consist of not more than a few hundred cells can be transduced at a high frequency with DNA complementary to the 12S transcript of the adenovirus early region 1A gene. Transformation can be detected within a single cultivation by the formation of progressively growing colonies. The transformants appear to have an unlimited growth potential, and they form a disorganized epidermis when they are grafted as an epithelium onto athymic mice. These experiments clearly show that, in order to be transformed by a viral oncogene, the target cell need not be a stem cell.

Full text

PDF
4102

Images in this article

4103Image
on p.4103
4103Image
on p.4103
4104Image
on p.4104
4104Image
on p.4104
4105Image
on p.4105
4105Image
on p.4105
4105Image
on p.4105
4105Image
on p.4105

Selected References

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

  1. Allen-Hoffmann B. L., Rheinwald J. G. Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7802–7806. doi: 10.1073/pnas.81.24.7802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baden H. P., Kubilus J., Kvedar J. C., Steinberg M. L., Wolman S. R. Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM 1). In Vitro Cell Dev Biol. 1987 Mar;23(3):205–213. doi: 10.1007/BF02623581. [DOI] [PubMed] [Google Scholar]
  3. Banks-Schlegel S. P., Howley P. M. Differentiation of human epidermal cells transformed by SV40. J Cell Biol. 1983 Feb;96(2):330–337. doi: 10.1083/jcb.96.2.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banks-Schlegel S., Green H. Formation of epidermis by serially cultivated human epidermal cells transplanted as an epithelium to athymic mice. Transplantation. 1980 Apr;29(4):308–313. doi: 10.1097/00007890-198004000-00010. [DOI] [PubMed] [Google Scholar]
  5. Barrandon Y., Green H. Cell size as a determinant of the clone-forming ability of human keratinocytes. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5390–5394. doi: 10.1073/pnas.82.16.5390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barrandon Y., Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2302–2306. doi: 10.1073/pnas.84.8.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barrandon Y., Li V., Green H. New techniques for the grafting of cultured human epidermal cells onto athymic animals. J Invest Dermatol. 1988 Oct;91(4):315–318. doi: 10.1111/1523-1747.ep12475646. [DOI] [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. Boyce S. T., Ham R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol. 1983 Jul;81(1 Suppl):33s–40s. doi: 10.1111/1523-1747.ep12540422. [DOI] [PubMed] [Google Scholar]
  10. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  11. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  12. Cone R. D., Grodzicker T., Jaramillo M. A retrovirus expressing the 12S adenoviral E1A gene product can immortalize epithelial cells from a broad range of rat tissues. Mol Cell Biol. 1988 Mar;8(3):1036–1044. doi: 10.1128/mcb.8.3.1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Freeman A. E., Black P. H., Wolford R., Huebner R. J. Adenovirus type 12-rat embryo transformation system. J Virol. 1967 Apr;1(2):362–367. doi: 10.1128/jvi.1.2.362-367.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fusenig N. E., Breitkreutz D., Dzarlieva R. T., Boukamp P., Bohnert A., Tilgen W. Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo. J Invest Dermatol. 1983 Jul;81(1 Suppl):168s–175s. doi: 10.1111/1523-1747.ep12541032. [DOI] [PubMed] [Google Scholar]
  16. Gallico G. G., 3rd, O'Connor N. E., Compton C. C., Kehinde O., Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med. 1984 Aug 16;311(7):448–451. doi: 10.1056/NEJM198408163110706. [DOI] [PubMed] [Google Scholar]
  17. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  18. Graham F. L., van der Eb A. J. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973 Aug;54(2):536–539. doi: 10.1016/0042-6822(73)90163-3. [DOI] [PubMed] [Google Scholar]
  19. Green H., Kehinde O., Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5665–5668. doi: 10.1073/pnas.76.11.5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. HAYFLICK L. THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS. Exp Cell Res. 1965 Mar;37:614–636. doi: 10.1016/0014-4827(65)90211-9. [DOI] [PubMed] [Google Scholar]
  21. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  22. Morgan J. R., Barrandon Y., Green H., Mulligan R. C. Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science. 1987 Sep 18;237(4821):1476–1479. doi: 10.1126/science.3629250. [DOI] [PubMed] [Google Scholar]
  23. Pierce G. B. Differentiation of normal and malignant cells. Fed Proc. 1970 May-Jun;29(3):1248–1254. [PubMed] [Google Scholar]
  24. 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]
  25. Rassoulzadegan M., Naghashfar Z., Cowie A., Carr A., Grisoni M., Kamen R., Cuzin F. Expression of the large T protein of polyoma virus promotes the establishment in culture of "normal" rodent fibroblast cell lines. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4354–4358. doi: 10.1073/pnas.80.14.4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rheinwald J. G., Beckett M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 1981 May;41(5):1657–1663. [PubMed] [Google Scholar]
  27. Rheinwald J. G., Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature. 1977 Feb 3;265(5593):421–424. doi: 10.1038/265421a0. [DOI] [PubMed] [Google Scholar]
  28. Rheinwald J. G., Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975 Nov;6(3):331–343. doi: 10.1016/s0092-8674(75)80001-8. [DOI] [PubMed] [Google Scholar]
  29. Rhim J. S., Jay G., Arnstein P., Price F. M., Sanford K. K., Aaronson S. A. Neoplastic transformation of human epidermal keratinocytes by AD12-SV40 and Kirsten sarcoma viruses. Science. 1985 Mar 8;227(4691):1250–1252. doi: 10.1126/science.2579430. [DOI] [PubMed] [Google Scholar]
  30. Roberts B. E., Miller J. S., Kimelman D., Cepko C. L., Lemischka I. R., Mulligan R. C. Individual adenovirus type 5 early region 1A gene products elicit distinct alterations of cellular morphology and gene expression. J Virol. 1985 Nov;56(2):404–413. doi: 10.1128/jvi.56.2.404-413.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ruley H. E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature. 1983 Aug 18;304(5927):602–606. doi: 10.1038/304602a0. [DOI] [PubMed] [Google Scholar]
  32. Simon M., Green H. Enzymatic cross-linking of involucrin and other proteins by keratinocyte particulates in vitro. Cell. 1985 Mar;40(3):677–683. doi: 10.1016/0092-8674(85)90216-8. [DOI] [PubMed] [Google Scholar]
  33. Simon M., Green H. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell. 1984 Apr;36(4):827–834. doi: 10.1016/0092-8674(84)90032-1. [DOI] [PubMed] [Google Scholar]
  34. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  35. Steinberg M. L., Defendi V. Altered pattern of growth and differentiation in human keratinocytes infected by simian virus 40. Proc Natl Acad Sci U S A. 1979 Feb;76(2):801–805. doi: 10.1073/pnas.76.2.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. TODARO G. J., GREEN H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963 May;17:299–313. doi: 10.1083/jcb.17.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Williams J. F. Oncogenic transformation of hamster embryo cells in vitro by adenovirus type 5. Nature. 1973 May 18;243(5403):162–163. doi: 10.1038/243162a0. [DOI] [PubMed] [Google Scholar]
  39. Worst P. K., Valentine E. A., Fusenig N. E. Formation of epidermis after reimplantation of pure primary epidermal cell cultures from perinatal mouse skin. J Natl Cancer Inst. 1974 Oct;53(4):1061–1064. doi: 10.1093/jnci/53.4.1061. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES