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.

  • Original Article
  • Published:

Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression

Oncogene volume 26, pages 7665–7674 (2007)Cite this article

Abstract

Cell-cycle inhibitors of the Cip/Kip and INK4 families are involved in cellular senescence and tumor suppression. Some of these proteins, p21Cip1, p16INK4a and p15INK4b, are coexpressed in response to antiproliferative signals such as cellular senescence resulting in cell-cycle arrest. To understand the roles of these inhibitors and their synergistic effect, we have characterized the growth properties and senescent behavior of primary cells deficient in p21Cip1 and expressing an endogenous Cdk4R24C (cyclin-dependent kinase) mutant (Cdk4R24C knock-in cells) insensitive to INK4 proteins. Inactivation of both p21Cip1 and INK4 pathways strongly cooperate in suppressing cellular senescence in vitro. These double mutant cells behavior as immortal cultures and display high sensitivity to cellular transformation by oncogenes. Moreover, mice double mutant in the INK4 and p21Cip1 pathways (Cdk4R24C; p21Cip1-null mice) display an increased incidence of specific sarcomas, suggesting a significant cooperation between these two families of cell-cycle inhibitors in senescence responses and tumor suppression in vivo.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  • Adnane J, Jackson RJ, Nicosia SV, Cantor AB, Pledger WJ, Sebti SM . (2000). Loss of p21WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/knockout mammary cancer model. Oncogene 19: 5338–5347.

    Article  CAS  Google Scholar 

  • Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC . (1996). Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci USA 93: 13742–13747.

    Article  CAS  Google Scholar 

  • Attwooll C, Lazzerini Denchi E, Helin K . (2004). The E2F family: specific functions and overlapping interests. EMBO J 23: 4709–4716.

    Article  CAS  Google Scholar 

  • Bagui TK, Mohapatra S, Haura E, Pledger WJ . (2003). P27Kip1 and p21Cip1 are not required for the formation of active D cyclin-cdk4 complexes. Mol Cell Biol 23: 7285–7290.

    Article  CAS  Google Scholar 

  • Bearss DJ, Lee RJ, Troyer DA, Pestell RG, Windle JJ . (2002). Differential effects of p21(WAF1/CIP1) deficiency on MMTV-ras and MMTV-myc mammary tumor properties. Cancer Res 62: 2077–2084.

    CAS  Google Scholar 

  • Braig M, Schmitt CA . (2006). Oncogene-induced senescence: putting the brakes on tumor development. Cancer Res 66: 2881–2884.

    Article  CAS  Google Scholar 

  • Brown JP, Wei W, Sedivy JM . (1997). Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277: 831–834.

    Article  CAS  Google Scholar 

  • Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ . (1995). Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377: 552–557.

    Article  CAS  Google Scholar 

  • Carnero A, Hudson JD, Hannon GJ, Beach DH . (2000). Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model. Nucleic Acids Res 28: 2234–2241.

    Article  CAS  Google Scholar 

  • Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM et al. (1999). The p21(Cip1) and p27(Kip1) CDK ‘inhibitors’ are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 18: 1571–1583.

    Article  CAS  Google Scholar 

  • Coqueret O . (2002). Linking cyclins to transcriptional control. Gene 299: 35–55.

    Article  CAS  Google Scholar 

  • Coqueret O . (2003). New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 13: 65–70.

    Article  CAS  Google Scholar 

  • Dannenberg JH, van Rossum A, Schuijff L, te Riele H . (2000). Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions. Genes Dev 14: 3051–3064.

    Article  CAS  Google Scholar 

  • De la Cueva E, Garcia-Cao I, Herranz M, Lopez P, Garcia-Palencia P, Flores JM et al. (2006). Tumorigenic activity of p21Waf1/Cip1 in thymic lymphoma. Oncogene 25: 4128–4132.

    Article  CAS  Google Scholar 

  • Duan Z, Zarebski A, Montoya-Durango D, Grimes HL, Horwitz M . (2005). Gfi1 coordinates epigenetic repression of p21Cip/WAF1 by recruitment of histone lysine methyltransferase G9a and histone deacetylase 1. Mol Cell Biol 25: 10338–10351.

    Article  CAS  Google Scholar 

  • Franklin DS, Godfrey VL, Lee H, Kovalev GI, Schoonhoven R, Chen-Kiang S et al. (1998). CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev 12: 2899–2911.

    Article  CAS  Google Scholar 

  • Franklin DS, Godfrey VL, O'Brien DA, Deng C, Xiong Y . (2000). Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity. Mol Cell Biol 20: 6147–6158.

    Article  CAS  Google Scholar 

  • Gregory DJ, Garcia-Wilson E, Poole JC, Snowden AW, Roninson IB, Perkins ND . (2002). Induction of transcription through the p300 CRD1 motif by p21WAF1/CIP1 is core promoter specific and cyclin dependent kinase independent. Cell Cycle 1: 343–350.

    Article  CAS  Google Scholar 

  • Hannon GJ, Sun P, Carnero A, Xie LY, Maestro R, Conklin DS et al. (1999). MaRX: an approach to genetics in mammalian cells. Science 283: 1129–1130.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM et al. (2006). Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 443: 421–426.

    Article  CAS  Google Scholar 

  • Korenjak M, Brehm A . (2005). E2F-Rb complexes regulating transcription of genes important for differentiation and development. Curr Opin Genet Dev 15: 520–527.

    Article  CAS  Google Scholar 

  • Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A . (2001). Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 413: 83–86.

    Article  CAS  Google Scholar 

  • Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S et al. (2006). p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 443: 453–457.

    Article  CAS  Google Scholar 

  • Latres E, Malumbres M, Sotillo R, Martin J, Ortega S, Martin-Caballero J et al. (2000). Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis. EMBO J 19: 3496–3506.

    Article  CAS  Google Scholar 

  • Macaluso M, Montanari M, Giordano A . (2006). Rb family proteins as modulators of gene expression and new aspects regarding the interaction with chromatin remodeling enzymes. Oncogene 25: 5263–5267.

    Article  CAS  Google Scholar 

  • Malumbres M, Barbacid M . (2001). To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer 1: 222–231.

    Article  CAS  Google Scholar 

  • Malumbres M, Barbacid M . (2005). Mammalian cyclin-dependent kinases. Trends Biochem Sci 30: 630–641.

    Article  CAS  Google Scholar 

  • Malumbres M, Carnero A . (2003). Cell cycle deregulation: a common motif in cancer. Prog Cell Cycle Res 5: 5–18.

    Google Scholar 

  • Malumbres M, Perez De Castro I, Hernandez MI, Jimenez M, Corral T, Pellicer A . (2000). Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b). Mol Cell Biol 20: 2915–2925.

    Article  CAS  Google Scholar 

  • Malumbres M, Sotillo R, Santamaria D, Galan J, Cerezo A, Ortega S et al. (2004). Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell 118: 493–504.

    Article  CAS  Google Scholar 

  • Martin-Caballero J, Flores JM, Garcia-Palencia P, Collado M, Serrano M . (2004). Different cooperating effect of p21 or p27 deficiency in combination with INK4a/ARF deletion in mice. Oncogene 23: 8231–8237.

    Article  CAS  Google Scholar 

  • Martin A, Odajima J, Hunt SL, Dubus P, Ortega S, Malumbres M et al. (2005). Cdk2 is dispensable for cell cycle inhibition and tumor suppression mediated by p27(Kip1) and p21(Cip1). Cancer Cell 7: 591–598.

    Article  CAS  Google Scholar 

  • Martin-Caballero J, Flores JM, Garcia-Palencia P, Serrano M . (2001). Tumor susceptibility of p21(Waf1/Cip1)-deficient mice. Cancer Res 61: 6234–6238.

    CAS  Google Scholar 

  • Martins CP, Berns A . (2002). Loss of p27(Kip1) but not p21(Cip1) decreases survival and synergizes with MYC in murine lymphomagenesis. EMBO J 21: 3739–3748.

    Article  CAS  Google Scholar 

  • McConnell BB, Starborg M, Brookes S, Peters G . (1998). Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr Biol 8: 351–354.

    Article  CAS  Google Scholar 

  • Medcalf AS, Klein-Szanto AJ, Cristofalo VJ . (1996). Expression of p21 is not required for senescence of human fibroblasts. Cancer Res 56: 4582–4585.

    CAS  Google Scholar 

  • Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J et al. (2006). Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature 443: 448–452.

    Article  CAS  Google Scholar 

  • Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR . (1994). Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 211: 90–98.

    Article  CAS  Google Scholar 

  • Palmero I, McConnell B, Parry D, Brookes S, Hara E, Bates S et al. (1997). Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status. Oncogene 15: 495–503.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Pantoja C, Serrano M . (1999). Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras. Oncogene 18: 4974–4982.

    Article  CAS  Google Scholar 

  • Philipp J, Vo K, Gurley KE, Seidel K, Kemp CJ . (1999). Tumor suppression by p27Kip1 and p21Cip1 during chemically induced skin carcinogenesis. Oncogene 18: 4689–4698.

    Article  CAS  Google Scholar 

  • Rane SG, Cosenza SC, Mettus RV, Reddy EP . (2002). Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence. Mol Cell Biol 22: 644–656.

    Article  CAS  Google Scholar 

  • Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP et al. (1999). Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia. Nat Genet 22: 44–52.

    Article  CAS  Google Scholar 

  • Sage J, Mulligan GJ, Attardi LD, Miller A, Chen S, Williams B et al. (2000). Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. Genes Dev 14: 3037–3050.

    Article  CAS  Google Scholar 

  • Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW . (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88: 593–602.

    Article  CAS  Google Scholar 

  • Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, Aguirre AJ et al. (2001). Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413: 86–91.

    Article  CAS  Google Scholar 

  • Sherr CJ . (2000). The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 60: 3689–3695.

    CAS  Google Scholar 

  • Sherr CJ, Roberts JM . (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13: 1501–1512.

    Article  CAS  Google Scholar 

  • Sotillo R, Dubus P, Martin J, de la Cueva E, Ortega S, Malumbres M et al. (2001a). Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors. EMBO J 20: 6637–6647.

    Article  CAS  Google Scholar 

  • Sotillo R, Garcia JF, Ortega S, Martin J, Dubus P, Barbacid M et al. (2001b). Invasive melanoma in Cdk4-targeted mice. Proc Natl Acad Sci USA 98: 13312–13317.

    Article  CAS  Google Scholar 

  • Stein GH, Drullinger LF, Soulard A, Dulic V . (1999). Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 19: 2109–2117.

    Article  CAS  Google Scholar 

  • Topley GI, Okuyama R, Gonzales JG, Conti C, Dotto GP . (1999). p21(WAF1/Cip1) functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential. Proc Natl Acad Sci USA 96: 9089–9094.

    Article  CAS  Google Scholar 

  • Vogt M, Haggblom C, Yeargin J, Christiansen-Weber T, Haas M . (1998). Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts. Cell Growth Differ 9: 139–146.

    CAS  Google Scholar 

  • Wang YA, Elson A, Leder P . (1997). Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice. Proc Natl Acad Sci USA 94: 14590–14595.

    Article  CAS  Google Scholar 

  • Weinberg WC, Fernandez-Salas E, Morgan DL, Shalizi A, Mirosh E, Stanulis E et al. (1999). Genetic deletion of p21WAF1 enhances papilloma formation but not malignant conversion in experimental mouse skin carcinogenesis. Cancer Res 59: 2050–2054.

    CAS  Google Scholar 

  • Wolfel T, Hauer M, Schneider J, Serrano M, Wolfel C, Klehmann-Hieb E et al. (1995). A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269: 1281–1284.

    Article  CAS  Google Scholar 

  • Yang WC, Mathew J, Velcich A, Edelmann W, Kucherlapati R, Lipkin M et al. (2001). Targeted inactivation of the p21(WAF1/cip1) gene enhances Apc-initiated tumor formation and the tumor-promoting activity of a Western-style high-risk diet by altering cell maturation in the intestinal mucosal. Cancer Res 61: 565–569.

    CAS  Google Scholar 

  • Zindy F, van Deursen J, Grosveld G, Sherr CJ, Roussel MF . (2000). INK4d-deficient mice are fertile despite testicular atrophy. Mol Cell Biol 20: 372–378.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Mariano Barbacid for helpful discussions, and Sheila Rueda and Blanca Velasco for their valuable help in the management of the mouse colony. This work was supported by grants from the INSERM, and Association pour la Recherche contre le Cancer and the Région Aquitaine (to PD); Ministerio de Sanidad (FIS-02/0126), Fundación Mutua Madrileña and the Ministerio de Educación y Ciencia (SAF2005-00944) (to AC); and from the Ministerio de Educación y Ciencia (SAF2006-05186), Fundación Científica de la Asociación Española contra el Cáncer, Fundación Ramón Areces and Fundación Médica Mutua Madrileña Automovilística (to MM). The Cell Division and Cancer Group of the CNIO is supported by the OncoCycle program from the Comunidad de Madrid (S-BIO-0283-2006).

Author information

Authors and Affiliations

  1. Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain

    V Quereda, J Martinalbo & M Malumbres

  2. EA2406, Histologie et Pathologie Moleculaire, University of Bordeaux 2, Bordeaux, France

    P Dubus

  3. Assays Development Group, CNIO, Madrid, Spain

    A Carnero

Authors
  1. V Quereda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Martinalbo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P Dubus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A Carnero
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M Malumbres
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to A Carnero or M Malumbres.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Quereda, V., Martinalbo, J., Dubus, P. et al. Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression. Oncogene 26, 7665–7674 (2007). https://doi.org/10.1038/sj.onc.1210578

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1210578

Keywords

This article is cited by

Search

Advanced search

Quick links