p14(ARF) silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2

The INK4a/ARF locus encodes two distinct tumor suppressors, p16(INK4a) and p14(ARF). Although the contribution of p16(INKa) to human tumorigenesis thr ough point mutation, deletion, and hypermethylation has been widely documen ted, little is known about specific p14(ARF) lesions and their consequences , Recent data indicate that p14(ARF) suffers inactivation by promoter hyper methylation in colorectal cancer cells. Because it is known that p14(ARF) p revents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenua ting MDM2-mediated degradation, we studied the relationship of p14(ARF) epi genetic silencing to the expression and localization of MDM2 and p53, Cance r cell Lines with an unmethylated p14(ARF) promoter showed strong nuclear e xpression of MDM2, whereas in a colorectal cell line with p14(ARF) hypermet hylation-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 app arent changes in retinoblastoma localization were observed. We also studied the profile of p14(ARF) promoter hypermethylation in an extensive collecti on of 559 human primary tumors of different cell types, observing that in c olorectal, gastric, renal, esophageal, and endometrial neoplasms and glioma s, aberrant methylation of p14(ARF) was a relatively common epigenetic even t. MDM2 expression patterns revealed that lack of p14(ARF) promoter hyperme thylation was associated with tumors showing exclusive nuclear MDM2 stainin g, whereas MDM2 cytosolic staining was frequently observed in neoplasms wit h aberrant p14(ARF) methylation. Taken together, these data support that ep igenetic silencing of p14(ARF) by promoter hypermethylation is a key mechan ism in the disturbance of the MDM2 nuclear localization in human cancer.