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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

¹ Laboratory of Human Carcinogenesis and ² Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; ³ Department of Surgery, Toho University Sakura Hospital, Sakura, Japan; ⁴ Second Department of Surgery, Fukushima Medical University School of Medicine, Fukushima, Japan; and ⁵ Biology Division, National Cancer Center Research Institute, Tokyo, Japan

Requests for reprints: Curtis C. Harris, Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, 37 Convent Drive, Building 37, Room 3068, Bethesda, MD 20892-4258. Phone: 301-496-2048; E-mail: harrisc{at}mail.nih.gov.

Key Words: nutlin-3 • p53 • senescence • ING2 • microRNA

Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in 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. [Cancer Res 2008;68(9):3193–203]