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Inactivation of the p53 pathway in retinoblastoma

Abstract

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

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Acknowledgements

We thank L. Harris, G. Zambetti and M. Baron for discussions; S. Pounds for statistical analysis; M. Roussel for MDMX and MDM2 retroviruses; B. Schulman and D. Bashford for assistance with MDMX–nutlin-3 modelling; A. McArthur for editorial assistance; J. Gray for assistance with real-time RT–PCR and genomic DNA preparations; and F. Carlotti and M. Rabeling for advice on lentiviral experiments and production of lentivirus stocks. This work was supported by grants (to M.A.D.) from the National Eye Institute, Cancer Center Support from the National Cancer Institute, the American Cancer Society, Research to Prevent Blindness, the Pearle Vision Foundation, the International Retinal Research Foundation and the American Lebanese Syrian Associated Charities (ALSAC). M.A.D. is a Pew Scholar. This work was also supported by funding from the Association for International Cancer Research (A.G.J.) and EC FP6 (A.G.J. and J.-C.M.), the Dutch Cancer Society (Y.R.), the Belgian Foundation against Cancer (J.-C.M.) and Télévie (S.F.). This publication was supported in part by a grant from the National Cancer Institute.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Correspondence to Michael A. Dyer.

Supplementary information

Supplementary Notes

This file contains Supplementary Tables 1–8, Supplementary Figures 1–10 and detailed Supplementary Methods. The Supplementary Tables present data from dissociated cell scoring, FISH analysis and clone size analysis of MDM2Lox/Lox MEFs. The Supplementary Figures provide additional data on modulation of the p53 pathway by MDM2 and MDMX in normal and transformed cells. The figures also provide additional data on the mechanism of topotecan and nutlin-3 mediated killing of retinoblastoma cells and cell-based assays demonstrating that nutlin-3 can kill Mdm2-deficient MEFs by binding to MdmX. The Supplementary Methods provide details about antibodies, cDNAs, siRNAs, microscopy, retinoblastoma tissue analysis, statistical analysis, protein purification and nutlin-3 binding studies and MEF experiments. (PDF 5104 kb)

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Further reading

Figure 1: MDMX amplification correlates with decreased activity of the p53 pathway.
Figure 2: Retinoblastoma cells show a p53 response to DNA damage.
Figure 3: MDMX promotes retinal tumorigenesis in mice.
Figure 4: MDMX rescues cell death in RB1-deficient human retinoblasts.
Figure 5: Nutlin-3 inhibits MDMX activity in retinoblastoma.

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