Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

Nature Cell Biology volume 5, pages 741–747 (2003)Cite this article

An Erratum to this article was published on 01 September 2003

Abstract

Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres1. Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20% oxygen. MEFs did not senesce in physiological (3%) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs suggested that DNA damage limits MEF proliferation in 20% oxygen. Indeed, MEFs accumulated more DNA damage in 20% oxygen than 3% oxygen, and more damage than human fibroblasts in 20% oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Senescent MEFs resemble oxidant-treated human fibroblasts.
Figure 2: Low oxygen abolishes replicative senescence of MEFs.
Figure 3: Growth of repair-deficient MEFs in 3% and 20% oxygen.
Figure 4: MEFs accumulate high levels of oxidative DNA damage in 20% oxygen.

References

  1. Wright, W.E. & Shay, J.W. Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology. Nature Med. 6, 849–851 (2000).

    Article  CAS  Google Scholar 

  2. Campisi, J. Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol. 11, 27–31 (2001).

    Article  Google Scholar 

  3. Shelton, D.N., Chang, E., Whittier, P.S., Choi, D. & Funk, W.D. Microarray analysis of replicative senescence. Curr. Biol. 9, 939–945 (1999).

    Article  CAS  Google Scholar 

  4. Serrano, M. & Blasco, M.A. Putting the stress on senescence. Curr. Opin. Cell Biol. 13, 748–753 (2001).

    Article  CAS  Google Scholar 

  5. Todaro, G.J. & Green, H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established cell lines. J. Cell Biol. 17, 299–313 (1963).

    Article  CAS  Google Scholar 

  6. Kodama, S. et al. Culture condition-dependent senescence-like growth arrest and immortalization in rodent embryo cells. Rad. Res. 155, 254–262 (2001).

    Article  CAS  Google Scholar 

  7. 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 

  8. Sherr, C.J. & DePinho, R.A. Cellular senescence: Mitotic clock or culture shock? Cell 102, 407–410 (2000).

    Article  CAS  Google Scholar 

  9. Meek, R.L., Bowman, P.D. & Daniel, C.W. Establishment of mouse embryo cells in vitro. Relationship of DNA synthesis, senescence and malignant transformation. Exp. Cell Res. 107, 277–284 (1977).

    Article  CAS  Google Scholar 

  10. Seshadri, T. & Campisi, J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science 247, 205–209 (1990).

    Article  CAS  Google Scholar 

  11. Chen, Q.M., Prowse, K.R., Tu, V.C., Purdom, S. & Linskens, M.H. Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts. Exp. Cell Res. 265, 294–303 (2001).

    Article  CAS  Google Scholar 

  12. Robles, S.J. & Adami, G.R. Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts. Oncogene 16, 1113–1123 (1998).

    Article  CAS  Google Scholar 

  13. Dimri, G.P., Itahana, K., Acosta, M. & Campisi, J. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14/ARF tumor suppressor. Mol. Cell. Biol. 20, 273–285 (2000).

    Article  CAS  Google Scholar 

  14. Vaupel, P., Kallinowski, F. & Okunieff, P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49, 6449–6465 (1989).

    CAS  PubMed  Google Scholar 

  15. Ossovskaya, V.S. et al. Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains. Proc. Natl Acad. Sci. USA 93, 10309–10314 (1996).

    Article  CAS  Google Scholar 

  16. Palmero, I., Pantoja, C. & Serrano, M. p19ARF links the tumour suppressor p53 to Ras. Nature 395, 125–126 (1998).

    Article  CAS  Google Scholar 

  17. Boerrigter, M.E., Wei, J.Y. & Vijg, J. Induction and repair of benzo[a]pyrene-DNA adducts in C57BL/6 and BALB/c mice: association with aging and longevity. Mech. Ageing Dev. 82, 31–50 (1995).

    Article  CAS  Google Scholar 

  18. Okayasu, R. et al. A deficiency in DNA repair and DNA-PKcs expression in the radiosensitive BALB/c mouse. Cancer Res. 60, 4342–4345 (2000).

    CAS  PubMed  Google Scholar 

  19. Sharpless, N.E. et al. Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413, 86–90 (2001).

    Article  CAS  Google Scholar 

  20. Smith, G.C.M. & Jackson, S.P. The DNA-dependent protein kinase. Genes Dev. 13, 916–934 (1999).

    Article  CAS  Google Scholar 

  21. Goytisolo, F.A., Samper, E., Edmonson, S., Taccioli, G.E. & Blasco, M.A. The absence of the DNA-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang. Mol. Cell. Biol. 21 3642–3651 (2001).

    Article  CAS  Google Scholar 

  22. de Vries, A. et al. Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA. Nature 377, 169–173 (1995).

    Article  CAS  Google Scholar 

  23. Blasco, M.A. et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34 (1997).

    Article  CAS  Google Scholar 

  24. Karanjawala, Z.E., Murphy, N., Hinton, D.R., Hsieh, C.L. & Lieber, M.R. Oxygen metabolism causes chromosome breaks and is associated with the neuronal apoptosis observed in DNA double-strand break repair mutants. Curr. Biol. 12, 397–402 (2002).

    Article  CAS  Google Scholar 

  25. Collins, A.R., Duthie, S.J. & Dobson, V.L. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733–1735 (1993).

    Article  CAS  Google Scholar 

  26. Rubio, M.A., Kim, S.H. & Campisi, J. Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells. J. Biol. Chem. 277, 28609–28617 (2002).

    Article  CAS  Google Scholar 

  27. Hande, M.P., Samper, E., Lansdorp, P. & Blasco, M.A. Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice. J. Cell Biol. 144, 589–601 (1999).

    Article  CAS  Google Scholar 

  28. Zhu, C., Bogue, M.A., Lim, D.S., Hasty, P. & Roth, D.B. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86, 379–389 (1996).

    Article  CAS  Google Scholar 

  29. Kurimasa, A. et al. Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis. Proc. Natl Acad. Sci. USA 96, 1403–1408 (1999).

    Article  CAS  Google Scholar 

  30. Helma, C. & Uhl, M. A public domain image-analysis program for the single-cell gel- electrophoresis (comet) assay. Mutat. Res. 466, 9–15 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Chen and his group for technical advice and DNA-PKcs−/− MEFs, P. Hasty for Ku80−/− MEFs and critical reading of the manuscript, S. Chang for mTR−/− MEFs, J. Hoeijmakers and H. van Steeg for xpa−/− MEFs, and J. Vijg and his group for helpful discussions. This work was supported by research grants from the National Institutes of Health (AG17242 to J.C.; AG18679 to S.M.), and training grants from the National Institutes of Health (AG00266 to J.G.) and Department of Defence Breast Cancer Research Program (8KB-0100 and BC010658 to S.P.), under contract AC03-76SF00098 to the University of California by the Department of Energy.

Author information

Authors and Affiliations

  1. Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, 94720, CA, USA

    Simona Parrinello, Ana Krtolica, Joshua Goldstein & Judith Campisi

  2. Buck Institute for Age Research, 8001 Redwood Blvd., Novato, 94945, CA, USA

    Enrique Samper, Simon Melov & Judith Campisi

Authors
  1. Simona Parrinello
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Enrique Samper
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana Krtolica
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joshua Goldstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Simon Melov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Judith Campisi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Judith Campisi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information, Fig. S1 Efficacy of GSE, levels of G1 cyclin-dependent kinases, and responsiveness to serum deprivation of MEFs cultured in 3% O2.

Supplementary Information, Fig. S2 Senescence of Balb/c and DNA-PKcs -/- MEFs in low oxygen. (PDF 398 kb)

Supplementary Information, Fig. S3 Telomere-FISH and growth of telomerase-deficient MEFs in 3% and 20% O2.

Supplementary Information, Table S1 Chromosomal aberrations in MEF cultures (DOC 31 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Parrinello, S., Samper, E., Krtolica, A. et al. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 5, 741–747 (2003). https://doi.org/10.1038/ncb1024

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1024

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing