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Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes


Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions1. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence2. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3,4,5), from which proliferative cells emerge to undergo further population doublings (20–70), before entering a second growth plateau (previously termed senescence or M1; refs 4,5,6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

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Figure 1: HMF and HMEC growth curves and cell morphologies in vitro.
Figure 2: Spontaneous genomic instability in human mammary epithelial cells.
Figure 3: Post-selection HMECs continue to shorten telomeres beyond the length detected in senescent HMFs and HMECs at the first growth plateau.


  1. 1

    Shay, J. W., Wright, W. E. & Werbin, H. Toward a molecular understanding of human breast cancer: a hypothesis. Breast Cancer Res. Treatment. 25 83–94 (1993).

    CAS  Article  Google Scholar 

  2. 2

    Hayflick, L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37, 614–636 (1965).

    CAS  Article  Google Scholar 

  3. 3

    Hammond, S. L., Ham, R. G. & Stampfer, M. R. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended passage with pituitary extract. Proc. Natl Acad. Sci. USA 81, 5435–5439 (1984).

    ADS  CAS  Article  Google Scholar 

  4. 4

    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 

  5. 5

    Huschtscha, L. I. et al. Loss of p16INK4 expression by methylation is associated with lifespan extension of human mammary epithelial cells. Cancer Res. 58, 3508–3512 (1998).

    CAS  PubMed  Google Scholar 

  6. 6

    Kiyono, T. et al. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396, 84–88 (1998).

    ADS  CAS  Article  Google Scholar 

  7. 7

    Meyer, K. M., Hess, S. M., Tlsty, T. D. & Leadon, S. A. Human mammary epithelial cells exhibit a differential p53-mediated response following exposure to ionizing or UV light. Oncogene 18, 5792–57805 (1999).

    Article  Google Scholar 

  8. 8

    Walen, K. H. & Stampfer, M. R. Chromosome analyses of human mammary epithelial cells at stages of chemical-induced transformation progression to immortality. Cancer Genet. Cytogenet. 37, 249–261 (1989).

    CAS  Article  Google Scholar 

  9. 9

    Brenner, A. J., Stampfer, M. R. & Aldaz, C. M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene 17, 199–205 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995).

    ADS  CAS  Article  Google Scholar 

  11. 11

    Pignolo, R. J., Rotenberg, M. O. & Cristofalo, V. J. Alterations in contact and density-dependent arrest state in senescent WI-38 cells. In Vitro Cell. Dev. Biol. Anim. 30A, 471–476 (1994).

    CAS  Article  Google Scholar 

  12. 12

    Shay, J. W. & Wright, W. E. Quantitation of the frequency of immortalization of normal human diploid fibroblasts by SV40 large T-antigen. Exp. Cell Res. 184, 109–118 (1989).

    CAS  Article  Google Scholar 

  13. 13

    Taylor-Papadimitriou, J. et al. Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J. Cell Sci. 94, 403–413 (1989).

    PubMed  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

    Lansdorp, P. M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 (1996).

    CAS  Article  Google Scholar 

  16. 16

    van Steensel, B., Smogorzewska, A. & de Lange, T. TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Stampfer, M. R. et al. Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells. Mol. Biol. Cell 8, 2391–2405 (1997).

    CAS  Article  Google Scholar 

  18. 18

    Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & de Lange, T. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283, 1321–1325 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Artandi, S. E. et al. Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice. Nature 406, 641–645 (2000).

    ADS  CAS  Article  Google Scholar 

  20. 20

    Chin, L. et al. p53 Deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell 97, 527–538 (1999).

    CAS  Article  Google Scholar 

  21. 21

    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 92, 13742–13747 (1996).

    ADS  Article  Google Scholar 

  22. 22

    Hara, E. et al. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol. Cell. Biol. 16, 859–867 (1996).

    CAS  Article  Google Scholar 

  23. 23

    Burbano, R. R. et al. Cytogenetics of epithelial hyperplasias of the human breast. Cancer Genet. Cytogenet. 119, 62–66 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Pandis, N. et al. Chromosome abnormalities in bilateral breast carcinomas. Cytogenetic evaluation of the clonal origin of multiple primary tumors. Cancer 76, 250–258 (1995).

    CAS  Article  Google Scholar 

  25. 25

    Berg, J. W. & Hutter, R. V. Breast cancer. Cancer 75, 257–269 (1995).

    CAS  Article  Google Scholar 

  26. 26

    Stoeber, K. et al. Cdc6 protein causes premature entry into S phase in a mammalian cell-free system. EMBO J. 17, 7219–7229 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Wei, W. & Sedivy, J. M. Differentiation between senescence (M1) and crisis (M2) in human fibroblasts cultures. Exp. Cell Res. 253, 519–522 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Tlsty, T. D. et al. Potentiation of genomic instability in normal human mammary epithelial cells by an epigenetic event. J. Mammary Gland Biol. Neoplasia (in the press).

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We thank E. H. Blackburn, I. Herskowitz and J. Li for comments, criticism and reading the manuscript. Stimulating discussion and thoughtful critique were provided by Y. Crawford, G. Whitworth, M. Heiman, M. Springer, D. Crawford, P. Hein and J. Anderson. We thank S. Gilbert for assistance with the figures; P. Ortiz for library support; and G. Williams for MCM2 antibodies. This work was supported by NIH and NIH/NASA grants to T.D.T. and a DOE and NIH grant to M.R.S. C.R.H. is supported by a Howard Hughes Pre-doctoral Fellowship.

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Correspondence to Thea D. Tlsty.

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Romanov, S., Kozakiewicz, B., Holst, C. et al. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409, 633–637 (2001).

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