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J Harbour, L Worley, J Wu; Disruption of the Rb and p53 Tumor Suppressor Checkpoints Occurs Early in the Transformation of Uveal Melanocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1831.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A large number of genetic alterations have been described in uveal melanoma, including cytogenetic changes on chromosomes 3, 6, 8, 13 and 17, and molecular abnormalities of Myc, cyclin D, p16, Rb, p53, HDM2, Bcl-2 and other oncoproteins. However, it has been difficult to distinguish the mutations that are important in the pathogenesis of uveal melanoma from those that represent late secondary changes. To study the early mutational events in the development of uveal melanoma, we examined molecular changes in uveal melanocytes undergoing transformation in culture. Methods: Cultured normal uveal melanocytes at passage 8 underwent crisis, with 99% of melanocytes undergoing cell death, followed by repopulation of the colony with a rapidly growing clone of immortalized melanocytes that were established in nude mice as growing tumors. Pre-crisis and transformed melanocytes were compared by Affymetrix microarray analysis (mRNA expression) and western blot analysis (protein expression) for Rb, p16, cyclin D1, p53, ARF, HDM2, and Myc. Induction of apoptosis by an anti-HDM2 transducible peptide was measured by MTS viability assay. Results: Transformed uveal melanocytes demonstrated an in vitro growth rate 5-fold higher than pre-crisis cells, and they formed tumors in nude mice with a doubling rate of 10 days. Transformed melanocytes demonstrated increased mRNA expression of cyclin D1 (3.4-fold) and HDM2 (2.4-fold), increased protein expression of HDM2, and hyperphosphorylation of Rb. No changes in protein expression levels were detected for Rb, p16, p53, ARF, or Myc. An anti-HDM2 peptide preferentially induced apoptosis in transformed melanocytes compared to pre-crisis cells. Conclusion: Overexpression of HDM2 and cyclin D1 may be two of the earliest mutations in the transformation of uveal melanocytes. These alterations, which occur frequently in primary uveal melanomas, may cooperate in malignant transformation. Cyclin D1 disrupts the Rb cell cycle checkpoint by hyperphosphorylating and inactivating Rb, whereas HDM2 may allow deregulated melanocytes to evade the p53 apoptotic checkpoint by inhibiting p53. These changes, which appear to occur earlier than those reported in Myc, p16, and other oncoproteins, could provide a rational basis for molecular therapy in this eye cancer.
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