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.

  • Article
  • Published:

Temporal dissection of p53 function in vitro and in vivo

Nature Genetics volume 37, pages 718–726 (2005)Cite this article

Abstract

To investigate the functions of the p53 tumor suppressor, we created a new knock-in gene replacement mouse model in which the endogenous Trp53 gene is substituted by one encoding p53ERTAM, a p53 fusion protein whose function is completely dependent on ectopic provision of 4-hydroxytamoxifen. We show here that both tissues in vivo and cells in vitro derived from such mice can be rapidly toggled between wild-type and p53 knockout states. Using this rapid perturbation model, we define the kinetics, dependence, persistence and reversibility of p53-mediated responses to DNA damage in tissues in vivo and to activation of the Ras oncoprotein and stress in vitro. This is the first example to our knowledge of a new class of genetic model that allows the specific, rapid and reversible perturbation of the function of a single endogenous gene in vivo.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Generation of the Trp53KI allele and genotype analysis of Trp53KI/KI mice.
Figure 2: 4-Hydroxytamoxifen (4-OHT) restores the p53-mediated damage response in Trp53KI/KI mouse tissues in vivo and cells in vitro.
Figure 3: 4-Hydroxytamoxifen (4-OHT) restores the p53-mediated transcriptional regulation in Trp53KI/KI mouse tissues in vivo and cells in vitro.
Figure 4: The Trp53KI/KI system is competent for deactivation of p53ERTAM.
Figure 5: Persistence of DNA damage–induced p53 activating signal.
Figure 6: Response to Ras deregulation is dependent on 4-hydroxytamoxifen (4-OHT) and reversible.
Figure 7: 4-Hydroxytamoxifen (4-OHT) determines the immortalization, senescence and ploidy states of primary p53ERTAM cells.
Figure 8: Late-passage, but not early-passage, cultured primary fibroblasts activate p53 in the absence of any overt DNA damage or stress.

Similar content being viewed by others

References

  1. Sherr, C.J. Principles of tumor suppression. Cell 116, 235–246 (2004).

    Article  CAS  Google Scholar 

  2. Donehower, L.A. et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221 (1992).

    Article  CAS  Google Scholar 

  3. Vater, C., Bartle, L., Dionne, C., Littlewood, T. & Goldmacher, V. Induction of apoptosis by tamoxifen-activation of a p53-estrogen receptor fusion protein expressed in E1A and T24 H-ras transformed p53-/- mouse embryo fibroblasts. Oncogene 13, 739–748 (1996).

    CAS  PubMed  Google Scholar 

  4. Harvey, M., McArthur, M.J., Montgomery, C.A. Jr., Bradley, A. & Donehower, L.A. Genetic background alters the spectrum of tumors that develop in p53-deficient mice. Faseb J. 7, 938–943 (1993).

    Article  CAS  Google Scholar 

  5. Harvey, M. et al. Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice. Nat. Genet. 5, 225–229 (1993).

    Article  CAS  Google Scholar 

  6. Cui, Y.F. et al. Apoptosis in bone marrow cells of mice with different p53 genotypes after γ-rays irradiation in vitro . J. Environ. Pathol. Toxicol. Oncol. 14, 159–163 (1995).

    CAS  PubMed  Google Scholar 

  7. Botchkarev, V.A. et al. p53 is essential for chemotherapy-induced hair loss. Cancer Res. 60, 5002–5006 (2000).

    CAS  PubMed  Google Scholar 

  8. Fei, P., Bernhard, E.J. & El-Deiry, W.S. Tissue-specific induction of p53 targets in vivo . Cancer Res. 62, 7316–7327 (2002).

    CAS  PubMed  Google Scholar 

  9. Kemp, C.J., Sun, S. & Gurley, K.E. p53 induction and apoptosis in response to radio- and chemotherapy in vivo is tumor-type-dependent. Cancer Res. 61, 327–332 (2001).

    CAS  PubMed  Google Scholar 

  10. Komarova, E.A., Christov, K., Faerman, A.I. & Gudkov, A.V. Different impact of p53 and p21 on the radiation response of mouse tissues. Oncogene 19, 3791–3798 (2000).

    Article  CAS  Google Scholar 

  11. Potten, C.S., Merritt, A., Hickman, J., Hall, P. & Faranda, A. Characterization of radiation-induced apoptosis in the small intestine and its biological implications. Int. J. Radiat. Biol. 65, 71–78 (1994).

    Article  CAS  Google Scholar 

  12. Song, S. & Lambert, P.F. Different responses of epidermal and hair follicular cells to radiation correlate with distinct patterns of p53 and p21 induction. Am. J. Pathol. 155, 1121–1127 (1999).

    Article  CAS  Google Scholar 

  13. Lowe, S.W., Schmitt, E.M., Smith, S.W., Osborne, B.A. & Jacks, T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, 847–849 (1993).

    Article  CAS  Google Scholar 

  14. Attardi, L.D., de Vries, A. & Jacks, T. Activation of the p53-dependent G1 checkpoint response in mouse embryo fibroblasts depends on the specific DNA damage inducer. Oncogene 23, 973–980 (2004).

    Article  CAS  Google Scholar 

  15. Fei, P. & El-Deiry, W.S. p53 and radiation responses. Oncogene 22, 5774–5783 (2003).

    Article  CAS  Google Scholar 

  16. Midgley, C.A. et al. Coupling between γ irradiation, p53 induction and the apoptotic response depends upon cell type in vivo . J. Cell Sci. 108 (Pt 5), 1843–1848 (1995).

    CAS  PubMed  Google Scholar 

  17. Ferbeyre, G. et al. Oncogenic ras and p53 cooperate to induce cellular senescence. Mol. Cell. Biol. 22, 3497–3508 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Harvey, M. et al. In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 8, 2457–2467 (1993).

    CAS  PubMed  Google Scholar 

  21. Fukasawa, K., Wiener, F., Vande Woude, G.F. & Mai, S. Genomic instability and apoptosis are frequent in p53 deficient young mice. Oncogene 15, 1295–1302 (1997).

    Article  CAS  Google Scholar 

  22. Hanawalt, P.C., Ford, J.M. & Lloyd, D.R. Functional characterization of global genomic DNA repair and its implications for cancer. Mutat. Res. 544, 107–114 (2003).

    Article  CAS  Google Scholar 

  23. Adimoolam, S. & Ford, J.M. p53 and regulation of DNA damage recognition during nucleotide excision repair. DNA Repair (Amst) 2, 947–954 (2003).

    Article  CAS  Google Scholar 

  24. Hayflick, L. Living forever and dying in the attempt. Exp. Gerontol. 38, 1231–1241 (2003).

    Article  Google Scholar 

  25. Tarapore, P. & Fukasawa, K. Loss of p53 and centrosome hyperamplification. Oncogene 21, 6234–6240 (2002).

    Article  CAS  Google Scholar 

  26. Bienz, B., Zakut-Houri, R., Givol, D. & Oren, M. Analysis of the gene coding for the murine cellular tumour antigen p53. EMBO J. 3, 2179–2183 (1984).

    Article  CAS  Google Scholar 

  27. Littlewood, T.D., Hancock, D.C., Danielian, P.S., Parker, M.G. & Evan, G.I. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 23, 1686–1690 (1995).

    Article  CAS  Google Scholar 

  28. White, I.N. Tamoxifen: is it safe? Comparison of activation and detoxication mechanisms in rodents and in humans. Curr. Drug Metab. 4, 223–239 (2003).

    Article  CAS  Google Scholar 

  29. Ginzinger, D.G. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512 (2002).

    Article  CAS  Google Scholar 

  30. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Heath for help with interpretation of the pathology of the Trp53KI/KI tumors; P. Rodriquez-Viciana for the H-ras V-12 retroviral vector; T. Littlewood for the p53minER vector; I. Rosewell for electroporation of ES cells; F. Rostker for assistance with animals; D. Ginzinger for Taqman analyses; A. Finch for assistance with histology and imaging; and F. McCormick, K. Shannon, D. Green, T. Tlsty, K. Vousden, R. Treisman, C. O'Shea, D. Iacovides and the members of the laboratory of G.I.E. for discussions, advice and criticism. This work was supported by grants from the US National Institutes of Health and by the University of California San Francisco Comprehensive Cancer Center Cell Cycle and Signaling Disregulation Program. D.M.-Z. was supported by grants from the Spanish Ministry of Science and Education. We dedicate our work to the memory of Stanley Korsmeyer.

Author information

Author notes

  1. Elizabeth R Lawlor

    Present address: University of Southern California Keck School of Medicine, Division of Hematology/Oncology, Children's Hospital of Los Angeles, 4650 Sunset Blvd, Los Angeles, California, 90027, USA

  2. Emmy W Verschuren

    Present address: Pathology Department, Stanford University, 300 Pasteur Drive, MC 5324, Stanford, California, 94305, USA

  3. Maria A Christophorou and Dionisio Martin-Zanca: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Research Institute, University of California San Francisco Comprehensive Cancer Center, San Francisco, 94143-0875, California, USA

    Maria A Christophorou, Dionisio Martin-Zanca, Laura Soucek, Elizabeth R Lawlor, Lamorna Brown-Swigart, Emmy W Verschuren & Gerard I Evan

  2. Instituto de Microbiologia Bioquimica CSIC/Universidad de Salamanca, Campus “Miguel de Unamuno,”, Salamanca, 37007, Spain

    Dionisio Martin-Zanca

Authors
  1. Maria A Christophorou
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Dionisio Martin-Zanca
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Laura Soucek
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Elizabeth R Lawlor
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Lamorna Brown-Swigart
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Emmy W Verschuren
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Gerard I Evan
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to Gerard I Evan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Christophorou, M., Martin-Zanca, D., Soucek, L. et al. Temporal dissection of p53 function in vitro and in vivo. Nat Genet 37, 718–726 (2005). https://doi.org/10.1038/ng1572

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng1572

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