Authors: Kumamoto K; Spillare EA; Fujita K; Horikawa I; Yamashita T; Appella E; Nagashima M; Takenoshita S; Yokota J; Harris CC
Abstract: Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in approximately 50% of all cancers, p53 is still functionally active in the other 50%. Consequently, nutlin-3 and similar drugs could be candidates for neoadjuvant therapy in cancers with a functional p53. Cellular senescence is also a phenotype induced by p53 activation and plays a critical role in protecting against tumor development. In this report, we found that nutlin-3a can induce senescence in normal human fibroblasts. Nutlin-3a activated and repressed a large number of p53-dependent genes, including those encoding microRNAs. mir-34a, mir-34b, and mir-34c, which have recently been shown to be downstream effectors of p53-mediated senescence, were up-regulated, and inhibitor of growth 2 (ING2) expression was suppressed by nutlin-3a treatment. Two candidates for a p53-DNA binding consensus sequence were found in the ING2 promoter regulatory region; thus, we performed chromatin immunoprecipitation and electrophoretic mobility shift assays and confirmed p53 binding directly to those sites. In addition, the luciferase activity of a construct containing the ING2 regulatory region was repressed after p53 activation. Antisense knockdown of ING2 induces p53-independent senescence, whereas overexpression of ING2 induces p53-dependent senescence. Taken together, we conclude that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, and p53-mediated mir34a, mir34b, and mir34c up-regulation and ING2 down-regulation may be involved in the senescence pathway.
Keywords: Base Sequence; Cell Aging/*drug effects/genetics; Cells, Cultured; Down-Regulation; Fibroblasts/drug effects/physiology; Gene Expression Regulation; HCT116 Cells; Homeodomain Proteins/*genetics; Humans; Imidazoles/*pharmacology; MicroRNAs/*genetics; Piperazines/*pharmacology; Promoter Regions, Genetic; Protein Binding/drug effects; Receptors, Cytoplasmic and Nuclear/*genetics; Tumor Suppressor Protein p53/genetics/*metabolism/physiology; Tumor Suppressor Proteins/*genetics; Up-Regulation|
Journal: Cancer research Volume: 68 Issue: 9 Pages: 3193-203 Date: May 3, 2008 PMID: 18451145 |
Kumamoto K, Spillare EA, Fujita K, Horikawa I, Yamashita T, Appella E, Nagashima M, Takenoshita S, Yokota J, Harris CC (2008) Nutlin-3a activates p53 to both down-regulate inhibitor of growth 2 and up-regulate mir-34a, mir-34b, and mir-34c expression, and induce senescence. Cancer research 68: 3193-203.
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