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p14^ARF Silencing by Promoter Hypermethylation Mediates Abnormal Intracellular Localization of MDM2¹

Department of Oncology, The Johns Hopkins Comprehensive Cancer Center, Baltimore, Maryland 21231 [M. E., P. G. C., D. N. W., S. B. B., J. G. H.]; Cancer Epigenetics Laboratory, Molecular Pathology Program, Centro Nacional de Investigaciones Oncologicas, Majadahonda 28220, Spain [M. E.]; Department of Pathology, Division of Molecular Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York [C. C.-C., K. S. P.]; Department of Medicine, Gastroenterology Division, Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201 [S. J. M.]; Cancer Research Institute, Hospital Duran i Reynals, Barcelona 08907, Spain [G. C., M. A. P.]; and Department of Pathology, Hospital Sant Pau, Barcelona 08025, Spain [X. M-G., J. P.]

The INK4a/ARF locus encodes two distinct tumor suppressors, p16^INK4a and p14^ARF. Although the contribution of p16^INK4a to human tumorigenesis through point mutation, deletion, and hypermethylation has been widely documented, little is known about specific p14^ARF lesions and their consequences. Recent data indicate that p14^ARF suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14^ARF prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14^ARF epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14^ARF promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14^ARF hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2'-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14^ARF promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14^ARF was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14^ARF promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14^ARF methylation. Taken together, these data support that epigenetic silencing of p14^ARF by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.

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