Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice


CDKN2A (INK4a/ARF) is frequently disrupted in various types of human cancer, and germline mutations of this locus can confer susceptibility to melanoma and other tumours1. However, because CDKN2A encodes two distinct cell cycle inhibitory proteins, p16INK4a and p14ARF (p19Arf in mice)2, the mechanism of tumour suppression by CDKN2A has remained controversial. Genetic disruption of Cdkn2a(p19Arf) (hereafter Arf) alone predisposes mice to tumorigenesis3, demonstrating that Arf is a tumour-suppressor gene in mice. We mutated mice specifically in Cdkn2a(p16Ink4a) (hereafter Ink4a). Here we demonstrate that these mice, designated Ink4a*/*, do not show a significant predisposition to spontaneous tumour formation within 17 months. Embryo fibroblasts derived from them proliferate normally, are mortal, and are not transformed by oncogenic HRAS. The very mild phenotype of the Ink4a*/* mice implies that the very strong phenotypes of the original Ink4a/ArfΔ2,3 mice were primarily or solely due to loss of Arf. However, Ink4a*/Δ2,3 mice that are deficient for Ink4a and heterozygous for Arf spontaneously develop a wide spectrum of tumours, including melanoma. Treatment of these mice with the carcinogen 7,12-dimethylbenzanthracene (DMBA) results in an increased incidence of melanoma, with frequent metastases. Our results show that, in the mouse, Ink4a is a tumour-suppressor gene that, when lost, can recapitulate the tumour predisposition seen in humans.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Generation of Ink4a mutant mice.
Figure 2: Growth characteristics of Ink4a*/* MEFs.
Figure 3: Ink4a is a tumour-suppressor gene.
Figure 4: Histology of melanocytic tumours in Ink4a*/Δ2,3 mice.


  1. 1

    Ruas, M. & Peters, G. The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim. Biophys. Acta 1378, F115–F177 (1998).

    CAS  PubMed  Google Scholar 

  2. 2

    Sherr, C. J. Tumor surveillance via the ARF–p53 pathway. Genes Dev. 12, 2984–2991 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Kamijo, T. et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659 (1997).

    CAS  Article  Google Scholar 

  4. 4

    Sherr, C. J. & Roberts, J. M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501–1512 (1999).

    CAS  Article  Google Scholar 

  5. 5

    Sharpless, N. E. & DePinho, R. A. The INK4A/ARF locus and its two gene products. Curr. Opin. Genet. Dev. 9, 22–30 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Arap, W., Knudsen, E. S., Wang, J. Y., Cavenee, W. K. & Huang, H. J. Point mutations can inactivate in vitro and in vivo activities of p16(INK4a)/CDKN2A in human glioma. Oncogene 14, 603–609 (1997).

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  PubMed  Google Scholar 

  8. 8

    Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Carnero, A., Hudson, J. D., Price, C. M. & Beach, D. H. p16INK4A and p19ARF act in overlapping pathways in cellular immortalization. Nature Cell Biol. 2, 148–155 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Sage, J. et al. Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. Genes Dev. 14, 3037–3050 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Dannenberg, J. H., van Rossum, A., Schuijff, L. & te Riele, H. 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 (2000).

    CAS  Article  Google Scholar 

  12. 12

    Wright, W. E. & Shay, J. W. Cellular senescence as a tumor-protection mechanism: the essential role of counting. Curr. Opin. Genet. Dev. 11, 98–103 (2001).

    CAS  Article  Google Scholar 

  13. 13

    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 

  14. 14

    Zhu, J., Woods, D., McMahon, M. & Bishop, J. M. Senescence of human fibroblasts induced by oncogenic Raf. Genes Dev. 12, 2997–3007 (1998).

    CAS  Article  Google Scholar 

  15. 15

    Peeper, D. S., Dannenberg, J. H., Douma, S., te Riele, H. & Bernards, R. Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nature Cell Biol. 3, 198–203 (2001).

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    Schmitt, C. A., McCurrach, M. E., de Stanchina, E., Wallace-Brodeur, R. R. & Lowe, S. W. INK4a/ARF mutations accelerate lymphomagenesis and promote chemoresistance by disabling p53. Genes Dev. 13, 2670–2677 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Serrano, M. et al. Role of the INK4a locus in tumor suppression and cell mortality. Cell 85, 27–37 (1996).

    CAS  Article  Google Scholar 

  19. 19

    Kamijo, T., Bodner, S., van de Kamp, E., Randle, D. H. & Sherr, C. J. Tumor spectrum in ARF-deficient mice. Cancer Res. 59, 2217–2222 (1999).

    CAS  PubMed  Google Scholar 

  20. 20

    Eischen, C. M., Weber, J. D., Roussel, M. F., Sherr, C. J. & Cleveland, J. L. Disruption of the ARF–Mdm2–p53 tumor suppressor pathway in Myc-induced lymphomagenesis. Genes Dev. 13, 2658–2669 (1999).

    CAS  Article  Google Scholar 

  21. 21

    Jacobs, J. J. et al. Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF. Genes Dev. 13, 2678–2690 (1999).

    CAS  Article  Google Scholar 

  22. 22

    Rizos, H., Becker, T. M., Holland, E. A., Kefford, R. F. & Mann, G. J. Differential expression of p16INK4a and p16β transcripts in B-lymphoblastoid cells from members of hereditary melanoma families without CDKN2A exon mutations. Oncogene 15, 515–523 (1997).

    CAS  Article  Google Scholar 

  23. 23

    Randerson-Moor, J. A. et al. A germline deletion of p14(ARF) but not CDKN2A in a melanoma-neural system tumour syndrome family. Hum. Mol. Genet. 10, 55–62 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Burri, N. et al. Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer. Lab. Invest. 81, 217–229 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Robanus-Maandag, E. et al. p107 is a suppressor of retinoblastoma development in pRb-deficient mice. Genes Dev. 12, 1599–1609 (1998).

    CAS  Article  Google Scholar 

  26. 26

    Schwenk, F., Baron, U. & Rajewsky, K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23, 5080–5081 (1995).

    CAS  Article  Google Scholar 

  27. 27

    Morgenstern, J. P. & Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).

    CAS  Article  Google Scholar 

Download references


We thank D. Peeper and M. van Lohuizen for advice and retroviral vectors, M. van der Valk for histological analysis, K. Rajewsky for the Deleter Cre mice, C. Sherr for Arf-/- MEFs, G. Nolan for ΦNX-E retroviral producer cells, and J. Vink and K. van Veen for technical assistance, and the Netherlands Cancer Institute animal facility for providing animal care. Part of this work was supported by the Dutch Cancer Society.

Author information



Corresponding author

Correspondence to Anton Berns.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Krimpenfort, P., Quon, K., Mooi, W. et al. Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 413, 83–86 (2001). https://doi.org/10.1038/35092584

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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