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Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells

Nature volume 396, pages 84–88 (1998)Cite this article

Abstract

Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence1. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases2,3 and telomere shortening4. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts5. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.

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Figure 1: Telomerase activity and telomere length.
Figure 2: Inactivation of the Rb/p16INK4A pathway.
Figure 3: E6 immortalization does not require inactivation of p53.

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References

  1. Hayflick, L. The limited in vitro lifespan of human diploid cell strains. Exp. Cell Res. 37, 614–621 (1965).

    Article  CAS  Google Scholar 

  2. Loughran, O.et al. Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes. Oncogene 14, 1955–1964 (1997).

    Article  CAS  Google Scholar 

  3. Alcorta, D. A.et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl Acad. Sci. USA 93, 13742–13747 (1996).

    Article  ADS  CAS  Google Scholar 

  4. Allsopp, R. C.et al. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl Acad. Sci. USA 89, 10114–10118 (1992).

    Article  ADS  CAS  Google Scholar 

  5. Bodnar, A. G.et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352 (1998).

    Article  ADS  CAS  Google Scholar 

  6. Wright, W. E. & Shay, J. W. The two-stage mechanism controlling cellular senescence and immortalization. Exp. Gerontol. 27, 383–389 (1992).

    Article  CAS  Google Scholar 

  7. Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy, D. R. & Schiller, J. T. HPV 16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8, 3905–3910 (1989).

    Article  CAS  Google Scholar 

  8. Munger, K., Phelps, W. C., Bubb, V., Howley, P. M. & Schlegel, R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63, 4417–4421 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Halbert, C. L., Demers, G. W. & Galloway, D. A. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65, 474–478 (1991).

    Google Scholar 

  10. Foster, S. A. & Galloway, D. A. Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. Oncogene 12, 1773–1779 (1996).

    CAS  PubMed  Google Scholar 

  11. Foster, S. A., Wong, D. J., Barrett, M. T. & Galloway, D. A. Inactivation of p16 in human mammary epithelial cells by CpG island methylation. Mol. Cell. Biol. 18, 1793–1801 (1998).

    Article  CAS  Google Scholar 

  12. Shay, J. W., Wright, W. E., Brasiskyte, D. & van der Haegen, B. A. E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene 8, 1407–1413 (1993).

    CAS  PubMed  Google Scholar 

  13. Band, V., Zajchowski, D., Kulesa, V. & Sager, R. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proc. Natl Acad. Sci. USA 87, 463–467 (1990).

    Article  ADS  CAS  Google Scholar 

  14. Shay, J. W., van der Haegen, B. A., Ying, Y. & Wright, W. E. The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with SV40 large T-antigen. Exp. Cell Res. 209, 45–52 (1993).

    Article  CAS  Google Scholar 

  15. Nakamura, T. M.et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 277, 955–959 (1997).

    Article  CAS  Google Scholar 

  16. Klingelhutz, A. J., Foster, S. A. & McDougall, J. K. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 380, 79–82 (1996).

    Article  ADS  CAS  Google Scholar 

  17. Wang, J., Xie, L. Y., Allan, S., Beach, D. & Hannon, G. J. Myc activates telomerase. Genes Dev. 12, 1769–1774 (1998).

    Article  CAS  Google Scholar 

  18. Klingelhutz, A. J., Barber, S. A., Smith, P. P., Dyer, K. & McDougall, J. K. Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells. Mol. Cell. Biol. 14, 961–969 (1994).

    Article  CAS  Google Scholar 

  19. Stoppler, H., Hartmann, D. P., Sherman, L. & Schlegel, R. The human papillomavirus type 16 E6 and E7 oncoproteins dissociate cellular telomerase activity from the maintenance of telomere length. J. Biol. Chem. 272, 13332–13337 (1997).

    Article  CAS  Google Scholar 

  20. Khleif, S. N.et al. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F mediated induction of cyclin kinase inhibitor activity. Proc. Natl Acad. Sci. USA 93, 4350–4354 (1996).

    Article  ADS  CAS  Google Scholar 

  21. Demers, G. W., Foster, S. A., Halbert, C. L. & Galloway, D. A. Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7. Proc. Natl Acad. Sci. USA 91, 4382–4386 (1994).

    Article  ADS  CAS  Google Scholar 

  22. Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129–1136 (1990).

    Article  CAS  Google Scholar 

  23. Kiyono, T.et al. Binding of high risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proc. Natl Acad. Sci. USA 94, 11612–11616 (1997).

    Article  ADS  CAS  Google Scholar 

  24. Dalal, S., Gao, Q., Androphy, E. J. & Band, V. Mutational analysis of human papillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells. J. Virol. 70, 683–688 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kamijo, T.et al. Tumor suppression at the mouse INK4A locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659 (1997).

    Article  CAS  Google Scholar 

  26. Gross-Mesilaty, S.et al. Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc. Natl Acad. Sci. USA 95, 8058–8063 (1998).

    Article  ADS  CAS  Google Scholar 

  27. Huibregtse, J. M., Scheffner, M. & Howley, P. M. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol. Cell. Biol. 13, 775–784 (1993).

    Article  CAS  Google Scholar 

  28. Kim, N. W.et al. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2015 (1994).

    Article  ADS  CAS  Google Scholar 

  29. Krupp, G.et al. Molecular basis of artifacts in the detection of telomerase activity and a modified primer for a more robust ‘TRAP’ assay. Nucleic Acids Res. 25, 919–921 (1997).

    Article  CAS  Google Scholar 

  30. Wright, W. E., Shay, J. W. & Piatyszek, M. A. Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucleic Acids Res. 23, 3794–3795 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the Geron Corporation for the hTERT cDNA, E. Espling for maintaining stocks of retroviruses, the Image Analysis Laboratory for help with preparing figures, and members of the McDougall and Galloway laboratories for discussion. This work was supported by grants from the NIH to J.K.M. and D.A.G.

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Author notes

  1. Tohru Kiyono: On leave from the Laboratory of Viral Oncology, Research Institute, Aichi Cancer Center, Nagoya 464, Japan.

Authors and Affiliations

  1. Cancer Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Cl-015, PO Box 19024, Seattle, 98109-1024, Washington, USA

    Scott A. Foster, Jenn I. Koop, James K. McDougall, Denise A. Galloway & Aloysius J. Klingelhutz

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  1. Tohru Kiyono
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  2. Scott A. Foster
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  6. Aloysius J. Klingelhutz
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Correspondence to Denise A. Galloway.

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Kiyono, T., Foster, S., Koop, J. et al. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396, 84–88 (1998). https://doi.org/10.1038/23962

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