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Mutant P53 Protects Cells from 12-O-Tetradecanoylphorbol-13- Acetate–Induced Death by Attenuatingactivating Transcription Factor 3 Induction

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Mutant P53 Protects Cells from 12-O-Tetradecanoylphorbol-13- Acetate–Induced Death by Attenuatingactivating Transcription Factor 3 Induction Research Article Mutant p53 Protects Cells from 12-O-Tetradecanoylphorbol-13- Acetate–Induced Death by AttenuatingActivating Transcription Factor 3 Induction Yosef Buganim,1 Eyal Kalo,1 Ran Brosh,1 Hila Besserglick,1 Ido Nachmany,3 Yoach Rais,2 Perry Stambolsky,1 Xiaohu Tang,1 Michael Milyavsky,1 Igor Shats,1 Marina Kalis,1 Naomi Goldfinger,1 and Varda Rotter1 1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; 2Department of Life Science, Bar-Ilan University, Ramat Gan, Israel; and 3Department of General Surgery B, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Abstract mutated. Notably, the predominant mode of p53 inactivation is by Mutations in p53 are ubiquitous in human tumors. Some p53 point mutation rather than by deletion or truncation. These data mutations not only result in loss of wild-type (WT) activity but coupled with the observation that mutant p53 is generally highly also grant additional functions, termed ‘‘gain of function.’’ overexpressed in tumors have led to the hypothesis that mutant In this study, we explore how the status of p53 affects the p53 possesses gain-of-function activities. This hypothesis is immediate response gene activating transcription factor 3 supported by the results of in vivo and in vitro studies. For (ATF3) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)- example, mice harboring mutant p53 display allele-specific tumor protein kinase C (PKC) pathway. We show that high doses of spectra, higher metastatic frequency, enhanced cell proliferation, TPA induce ATF3 in a WT p53-independent manner correlat- and higher transformation potential compared with their p53-null ingwith PKCs depletion and cell death. We show that cells counterparts (8, 9). In addition, endogenous mutant p53 or harboringmutant p53 have attenuated ATF3 induction and reconstitution of mutant p53 expression in p53-null cells augments are less sensitive to TPA-induced death compared with their the transformed phenotype (10–13). Although the question of how p53-null counterparts. Mutagenesis analysis of the ATF3 pro- mutant p53 contributes to tumor initiation and progression has moter identified the regulatory motifs cyclic AMP-responsive been addressed intensively, the molecular mechanism that under- element bindingprotein/ATF and MEF2 as beingresponsible lies the role of mutant p53 in malignant transformation remains for the TPA-induced activation of ATF3. Moreover, we show unclear. One putative mechanism is that mutant p53 alters the that mutant p53 attenuates ATF3 expression by two comple- expression of specific genes and thus interferes with the onset of the apoptotic process. This is supported by recent data showing mentary mechanisms. It interacts with the ATF3 promoter and j influences its activity via the MEF2 site, and additionally, that the EGR1 and NF B2 genes can be activated and the CD95 and it attenuates transcriptional expression of the ATF3 activator MSP-1 genes repressed by mutant p53 (14–17). These findings MEF2D. These data provide important insights into the prompted the present study to investigate whether mutant p53 molecular mechanisms that underlie mutant p53 gain of influences cell fate by altering gene expression. Due to the fact that function. (Cancer Res 2006; 66(22): 10750-9) activation of the protein kinase C (PKC) pathway by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) has been associated with neoplastic transformation, carcinogenesis, and tumor cell Introduction invasion, we decided to pursue the role of mutant p53 in this pathway. At low doses, TPA is a potent PKC activator and its major Multiple stress responses are regulated by the p53 tumor suppressor gene (1). p53 is a transcription factor that activates biological effects are exerted via the PKCs (18), which in turn can induce either cell survival or cell death (19, 20). In contrast, high specific target genes through direct binding to a p53 consensus WAF1 doses of TPA lead to a down-regulation of PKC activity (21–23). sequence (2). p21 and GADD45 are activated transcriptionally by p53 in response to DNA damage, and this activation is involved Notably, in certain cell types, attenuation of PKC activity, either due in the control of cell cycle checkpoints (3, 4). Alternatively, p53 can to exposure to high doses of TPA or due to PKC inhibitors, results in cell death (24, 25). transactivate genes, such as BAX, PUMA, and Noxa, leading to The cyclic AMP-responsive element binding protein (CREB)/ induction of apoptosis (5–7). Wild-type (WT) p53 plays a pivotal role in preventing tumor activating transcription factor (ATF) and activator protein-1 (AP-1) transcription factor families were shown to be induced in cells development. Indeed, in >50% of human primary tumors p53 is treated with low doses of TPA (26, 27). ATF3 is a member of the CREB/ATF subfamily of bZIP transcription factors (28). It has been shown to stabilize WT p53 by blocking its ubiquitination (29) and is Note: Supplementary data for this article are available at Cancer Research Online also a p53 downstream target gene, creating a regulatory feedback (http://cancerres.aacrjournals.org/). This publication reflects the authors’ views and not necessarily those of the loop (30). Evidence supporting interactions between p53 and ATF3 European Community (EC). The EC is not liable for any use that may be made of the proteins has led to the proposal that ATF3 can inhibit p53 information contained herein. V. Rotter is the incumbent of the Norman and Helen Asher Professorial Chair Cancer Research at the Weizmann Institute. transactivation capacity (31). ATF3 is induced in response to stress Requests for reprints: Varda Rotter, Department of Molecular Cell Biology, agents, such as UV and ionizing radiation (IR), not only via p53 but Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-9344070; Fax: 972- also by p53-independent pathways (32, 33). Similar to p53, ATF3 8-9465265; E-mail: [email protected]. I2006 American Association for Cancer Research. expression is associated with cell cycle arrest and apoptosis. doi:10.1158/0008-5472.CAN-06-0916 Overexpression of ATF3 in HeLa and HT-1080 cells induces G1 Cancer Res 2006; 66: (22). November 15, 2006 10750 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Novel Mutant p53 Gain of Function arrest on IR treatment (31, 33), whereas in HeLa-S3 cells treated Fluorescence-activated cell sortinganalysis. Cells were plated in with etoposide ATF3 enhances apoptosis (34). 10-cm dishes and treated with 10 Ag/mLTPA for 72 hours. Cells were We report here that expression of mutant p53 results in reduced subsequently trypsinized and fixed in 70% ethanol/30% HBSS for 24 hours. sensitivity to cell death induced by high doses of TPA. We show Cells were then rehydrated for at least 30 minutes in PBS, washed, resuspended in PBS containing 50 Ag/mLpropidium iodide and 10 Ag/mL that this protection is mediated by attenuation of p53-independent RNase A, and subjected to fluorescence-activated cell sorting (FACS)-based ATF3 induction. Furthermore, we show that mutant p53 attenuates cell cycle analysis. ATF3 expression by two complementary mechanisms involving Cell proliferation assay. Cells were seeded in 24-well culture dishes at the MEF2 motif on the ATF3 promoter and expression of the 60% confluency. Cells were incubated with 10 Ag/mLTPA or 0.2 Ag/mL MEF2D gene. This study provides novel insights into the molecular doxorubicin for 72 hours. Cell proliferation was determined by using a mechanisms by which mutant p53 exerts gain-of-function activity. colorimetric assay with WST-1 reagent (Roche, Mannheim, Germany) following the manufacturer’s instructions. Western blot and retroviral infections. Western blot analysis and Materials and Methods retroviral infection were done as described in ref. 35. The following primary Chemicals and reagents. TPA, doxorubicin, and DMSO were from antibodies were used: anti-p53 (DO-1; kindly provided by Dr. D. Lane, Sigma (St. Louis, MO). Go6976 and rottlerin were from Calbiochem- Ninewells Hospital and Medical School, Dundee, Scotland), anti-PKCa Novabiochem (Bad Soden, Germany). Tumor necrosis factor a (TNFa) was and PKCy (kindly provided by Y. Dicken, Bar-Ilan University), anti-p21, from Biological Industries (Beit Haemek, Israel). Transforming growth anti-ATF3, anti-MEF2 (C-19; Santa Cruz Biotechnology, Santa Cruz, CA), factor h (TGFh) was from R&D Systems (Minneapolis, MN). anti-poly(ADP-ribose) polymerase-1 (PARP-1; C-2-10; Biomol, Plymouth Cell culture and treatments. The amphotropic and ecotropic Phoenix Meeting, PA), anti-vinculin (Sigma), anti-glyceraldehyde-3-phosphate dehy- retrovirus-producing cells were from the American Type Culture Collection drogenase (GAPDH; MAB374; Chemicon, Temecula, CA), and anti-tubulin (Manassas, VA). The immortalized primary human embryonic lung (T7816; Sigma). fibroblasts (WI-38) were created and described previously by our laboratory Chromatin immunoprecipitation analysis. Chromatin immunopre- (35). The ovarian cancer SKOV3 cell line stably expressing either an empty cipitation (ChIP) was conducted as described in ref. 14. To detect binding of vector, p53R175H, or p53R248W was a gift from Prof. P.M. Chumakov p53R175H or MEF2 to the ATF3 promoter, quantitative RT-PCR (QRT-PCR) (University of California, San Diego, CA). The fibrosarcoma HT-1080 cell line primers amplifying the ATF3 promoter were used. To detect binding of was kindly provided by Dr. M. Brandeis (Hebrew University, Jerusalem, p53R175H or MEF2 to the MEF2D promoter, QRT-PCR primers amplifying Israel). WI-38 cells were grown in MEM supplemented with 10% FCS, the MEF2D promoter were used (see Supplementary Materials). 1 mmol/Lsodium pyruvate, 2 mmol/L L-glutamine, and antibiotics. Phoenix, Real-time RT-PCR analysis. Total RNA was extracted using the HT-1080, and SKOV3 cells were grown in DMEM supplemented with 10% Versagene RNA cell kit (Gentra Systems, Inc., Minneapolis, MN). An aliquot FCS and antibiotics.
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