[CANCER RESEARCH 64, 1669–1674, March 1, 2004] Tbx2 Directly Represses the Expression of the p21WAF1 Cyclin-Dependent Kinase Inhibitor Sharon Prince,1,2 Suzanne Carreira,1 Keith W. Vance,1 Amaal Abrahams,2 and Colin R. Goding1 1Signalling and Development Laboratory, Marie Curie Research Institute, Oxted, Surrey, United Kingdom, and 2Division of Medical Biochemistry, Institute for Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa ABSTRACT cycle arrest, apoptosis, and senescence, and deregulation of compo- nents of these key cell cycle regulators can contribute to cancer. The T-box factors play a crucial role in the development of many tissues, p21WAF1/CIP1/SDI1 cdk inhibitor (referred to as p21) is induced in and mutations in T-box factor genes have been implicated in multiple differentiating cells and in response to a wide variety of cellular human disorders. Some T-box factors have been implicated in cancer; for example, Tbx2 and Tbx3 can suppress replicative senescence, whereas stresses including DNA damage, and contributes to stress-induced Tbx3 can cooperate with Myc and Ras in cellular transformation. The growth arrest (24, 25). The promoter of the p21 gene can be induced p21WAF1 cyclin-dependent kinase inhibitor plays a key role in senescence via p53 (26), and p21 expression is necessary for p53-mediated and in cell cycle arrest after DNA damage. Here, using a combination of growth arrest (27–30). In contrast to the p16 cdk inhibitor, mutation of in vitro DNA-binding, transfection, and chromatin immunoprecipitation the p21 gene in human cancers occurs infrequently, and mice lacking assays, we show that Tbx2 can bind and repress the p21 promoter in vitro p21 appear to undergo normal development, with no increase in the and in vivo. Moreover, small interfering RNA-mediated down-regulation frequency of spontaneous tumor formation (27, 28). Nevertheless, of Tbx2 expression results in a robust activation of p21 expression. Taken some studies on the role of p21 in Ras transformation have suggested together, these results implicate Tbx2 as a novel direct regulator of p21 that p21 status is important. For example, in a mouse breast cancer expression and have implications for our understanding of the role of model, the onset of Ras-induced tumors was accelerated in mice T-box factors in the regulation of senescence and oncogenesis, as well as in development. deficient in p21 (31). Any effects on the onset of tumor formation may reflect a role for p21 in replicative senescence. In senescent human fibroblasts, p21 levels are strongly elevated (32–34) and although INTRODUCTION mouse fibroblasts lacking p21 undergo normal senescence and are Recent work has contributed to an increasing body of evidence resistant to transformation by moderate levels of expression of onco- implicating the T-box transcription factor family in the maintenance genic Ras (35), the cell cycle arrest associated with a high-intensity of cell identity during development (reviewed in Refs. 1–3). For Ras/Raf signal is bypassed in p21-deficient fibroblasts (36). Addition- example, Tbx1 mutations have been implicated in DiGeorge syndrome ally, a clone of human diploid fibroblasts in which the p21 gene had (4–6), Holt-Oram syndrome correlates with mutations in Tbx5 (7), been disrupted exhibited a senescence bypass phenotype (37), Tbx4 and Tbx5 play crucial roles in limb bud outgrowth and specifi- whereas p21 expression can induce senescence in a p53-independent cation of limb identity (8–11), and T-Pit determines cell fate in the fashion (38, 39). Given the complex role of p21, identifying the pituitary (12). controls regulating p21 expression is essential for understanding fully In addition to their key function in development, evidence suggest- the balance between proliferation, differentiation, and senescence. ing a role for T-box factors in breast cancer is accumulating. Tbx3 is Here, we used a combination of in vitro DNA binding, transfection, required for normal breast development (13), with mutations in the and chromatin immunoprecipitation assays together with small inter- Tbx3 gene linked to ulnar-mammary syndrome (14). Moreover, Tbx3 fering RNA (siRNA)-mediated down-regulation of Tbx2 to identify can cooperate with Ras and Myc to induce cellular transformation and Tbx2 as a novel negative regulator of p21 expression. These results suppress apoptosis (15). Importantly, both Tbx3 and the highly related have implications for our understanding of the role of T-box factors factor Tbx2 are transcriptional repressors (16–19) and can suppress in the regulation of senescence and oncogenesis, as well as in devel- senescence through a mechanism involving repression of the expres- opment. sion of the p19ARF (p14) gene (20–22). Tbx2, like Tbx3, is also expressed in the developing breast and has been implicated in breast MATERIALS AND METHODS cancer where the Tbx2 gene is preferentially amplified in BRCA1 and BRCA2 mutants (23) and Tbx2 is overexpressed in breast cancer cell siRNA. Suppression of Tbx2 cellular expression was achieved using lines (21). These results suggest that T-box factors may contribute to siRNA that specifically targets Tbx2 mRNA. The siRNA oligonucleotide sense cell cycle control and oncogenesis but it is not known whether their sequence with two uracil residues at the 3Јend was 5Ј-GCCGGAGCGU- effect on proliferation extends beyond regulation of p19ARF. GAUGGCGCUUU-3Ј corresponding to amino acids 302–309 of murine Tbx2 Ј Ј Proliferation of mammalian cells is regulated by signals acting to or 5 -GGAGCUGUGGGACCAGUUCUU-3 corresponding to amino acids 102–108 of human Tbx2. B16 cells or MCF-7 cells were transfected with control the activity and expression of components of the cell cycle anti-Tbx2 siRNA or a control (nonsilencing) 21-mer siRNA 5Ј-UUCUC- machinery. A combination of cyclins, cyclin-dependent kinases CGAACGUGUCACGUTT-3Ј (Xeragon-Qiagen, Crawley, United Kingdom) (cdks), and cdk inhibitors act together to control proliferation, cell using Oligofectamine reagent (Life Technologies, Inc./Invitrogen, Paisley, United Kingdom) according to Dharmacon Research instructions. Received 10/20/03; revised 12/17/03; accepted 12/22/03. Immunofluorescence Microscopy. B16 cells grown on glass coverslips 3 Grant support: The Wellcome Trust, the Association for International Cancer Re- days after siRNA transfection were washed three times with PBS and fixed search, The Medical Research Council, The South African Medical Research Council, and with 3% paraformaldehyde for 20 min at room temperature before permeabi- Marie Curie Cancer Care. The costs of publication of this article were defrayed in part by the payment of page lization with 0.1% Triton X-100 for 10 min at room temperature. Slides were charges. This article must therefore be hereby marked advertisement in accordance with incubated for 1 h with mouse Tbx2 monoclonal antibody (62–2) at a dilution 18 U.S.C. Section 1734 solely to indicate this fact. of 1:750 and with the p21 (C-19) rabbit polyclonal antibody (Santa Cruz Note: S. Prince and S. Carreira contributed equally to this work. Biotechnology, Santa Cruz, CA) at a dilution of 1:250, then incubated with the Requests for reprints: Colin R. Goding, Signalling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, United Kingdom. appropriate secondary antibody coupled to FITC or Texas Red (Vector Lab- Phone: 44- (0)-1883-722306; Fax: 44-(0)-1883-714375; E-mail:[email protected]. oratories, Peterborough, United Kingdom) at 1:100 dilution. Cells were 1669 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. Tbx2 AND REPRESSION OF p21 mounted using Vectashield mounting medium and examined by confocal perature were isolated and expanded under selective conditions. Lysates for microscopy. Western blotting were prepared from each individual clone. Western Blot Analysis. B16 whole-cell extracts were prepared 3 days after Site-Directed Mutagenesis of the p21 Promoter. The wild-type p21-CAT siRNA transfection. Proteins were resolved on 9–15% SDS-polyacrylamide plasmid was modified as follows: the T-element (AGGTGTGA) located at the gels as required and then transferred to Hybond C (Amersham, Amersham, initiator region of the wild-type human p21 promoter was mutated to CTCTGA United Kingdom). The membranes were probed with appropriate primary by site-directed mutagenesis using the Stratagene QuikChange system and antibodies and detected using peroxidase-conjugated antimouse or antirabbit the primer pair 5Ј-GCTGCGCCAGCTGAGCTCTGAGCAGCTGCCGAAG- antibodies and visualized by enhanced chemiluminescence (Amersham). The 3Јand 5Ј-CGACGCGGTCGACTCGAGACTCGTCGACGGCTTC-3Ј. primary antibodies used were mouse monoclonal anti-Tbx2 antibody 62–2, Chromatin Immunoprecipitation Assays. Chromatin immunoprecipita- anti-p21(C-19) rabbit polyclonal antibody (Santa Cruz Biotechnology), and tions were performed essentially as described previously (41). Briefly, mouse mouse monoclonal anti-␣-tubulin (Clone B-5–1-2; Sigma). NIH 3T3 cells stably expressing an SV5-tagged Tbx2 cDNA were treated with Electrophoretic Mobility Shift Assays. Binding reactions were performed formaldehyde, and DNA-containing fractions were incubated overnight with with double-stranded 32P-labeled oligonucleotide probes, in vitro transcribed/ different antibodies and collected on protein G beads. Cross-linked products translated protein,