title: 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.
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.
authors.affiliation: Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4258, USA.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Kumamoto
authors.forename: Elisa A
authors.initials: EA
authors.lastname: Spillare
authors.forename: Kaori
authors.initials: K
authors.lastname: Fujita
authors.forename: Izumi
authors.initials: I
authors.lastname: Horikawa
authors.forename: Taro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Ettore
authors.initials: E
authors.lastname: Appella
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagashima
authors.forename: Seiichi
authors.initials: S
authors.lastname: Takenoshita
authors.forename: Jun
authors.initials: J
authors.lastname: Yokota
authors.forename: Curtis C
authors.initials: CC
authors.lastname: Harris
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects* / Q000235:genetics; D015536:Down-Regulation; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation; D045325:HCT116 Cells; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D035683:MicroRNAs / Q000235:genetics*; D010879:Piperazines / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D015854:Up-Regulation
chemical_list: D018398:Homeodomain Proteins; C444654:ING2 protein, human; D007093:Imidazoles; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D010879:Piperazines; D018160:Receptors, Cytoplasmic and Nuclear; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C482205:nutlin 3