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

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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. All cells were maintained in a humidified incubator of 2 Ag of total RNA was reverse transcribed using Moloney murine at 37jC and 5% CO2. leukemia virus reverse transcriptase (Promega) and random hexamer TPA and rottlerin were applied in DMSO to a final concentration of primers. QRT-PCR was done using the ABI 7000 machine (Applied 1 Ag/ALand 500 Amol/L, respectively. Treatment of cells with doxorubicin Biosystems, Foster City, CA) with SYBR Green PCR Master Mix (Applied (0.2 Ag/mL), TNFa (10 ng/mL), TGFh (2 ng/mL), TPA (100 ng/mL, 10 Ag/mL), Biosystems). Specific primers were designed to the following genes: ATF3, rottlerin (5 Amol/L), and Go6976 (10 nmol/L) was conducted at 60% p21WAF, MEF2A, MEF2B, MEF2C, and MEF2D. cDNA levels were normalized confluency. Equal amounts of DMSO were added to the control plates. to GAPDH levels and amplified with appropriate primers (see Supplemen- Plasmids. The ATF3 expression plasmid was constructed by cloning the tary Materials for all primers). ATF3 open reading frame, obtained by reverse transcription-PCR (RT-PCR) with specific primers (see Supplementary Materials), into the pGEMT-easy Results vector (Promega, Madison, WI) and then restricted with EcoRI and inserted into the pBabe-puro expressing vector. Four ATF3 promoter-luciferase ATF3 induction followinghigh-dose TPA is attenuated reporters were constructed by cloning the ATF3 promoter, obtained by by mutant p53. Because p53 and ATF3 are cross-talking regu- genomic PCR amplification with specific primers (see Supplementary lators of cell fate decisions, we decided to explore further the Materials), into the pGEMT-easy vector with subsequent subcloning into molecular relationships between these two transcription factors. the pGL3-Basic luciferase plasmid (Promega). Seven motifs along the ATF3 Furthermore, as both p53 and CREB/ATF are involved in the PKC promoter were mutated using the Site-Directed Mutagenesis kit (Stra- pathway that has been implicated in malignant progression, we tagene, La Jolla, CA) following the manufacturer’s instructions (see focused on p53-ATF3 cross-talk following treatment with three Supplementary Materials for primer sequences). pLXSN-GSE56-neo was compounds that modulate PKC activity: TGFh, TNFa, and TPA. obtained by subcloning the GSE56 BamHI-restricted fragment from pBabe- To that end, we established congenic cell lines differing in their GSE56-puro into the pLXSN vector (Ossovskaya et al., 1996). The pLXSN- p53R175H-neo was obtained by subcloning the p53R175H BamHI-restricted p53 status. hTERT-immortalized normal human fibroblasts fragment from pcDNA-p53R175H into the pLXSN vector. The expression (WI-38) and a human fibrosarcoma cell line (HT-1080), both har- plasmids MEF2A and MEF2D were kindly provided by Dr. E. Bengal boring WT p53, were infected stably with a retrovirus contain- (Technion-Israel Institute of Technology, Haifa, Israel). The p53 short hair- ing either an empty vector (neo), a dominant-negative p53 peptide pin RNA (shRNA) vector and its mouse NOXA shRNA control vector were (GSE56), or a tumor-associated p53 mutant R175H (p53R175H). The kindly provided by Dr. D. Ginsburg (Bar-Ilan University, Ramat Gan, Israel). efficiency of GSE56 and p53R175H in blocking WT p53 activity was Transfections and reporter assays. p53-null and p53R175H SKOV3 validated by measuring expression of the p53 target gene p21WAF1 at Â 4 cells were plated at 2 10 per well in a 24-well plate 24 hours before both mRNA and protein levels (Supplementary Fig. S1). transfection. Cells were transfected with jetPEI transfection reagent Whereas 24 hours of treatment with TNFa (10 ng/mL) or TGFh (Polyplus Transfection, Illkirch, France) using 300 ng/well of the relevant (2 ng/mL) did not change the levels of ATF3, low doses of TPA ATF3 promoter-luciferase reporter construct, 600 ng/well Bluescript carrier plasmid, and 100 ng/well of pCMV-h-galactosidase expression vector for (100 ng/mL) down-regulated ATF3 expression (Supplementary A normalization of transfection efficiency. Cells were treated with 10 Ag/mL Fig. S2). In contrast, high doses of TPA (10 g/mL) induced ATF3 TPA 48 hours after transfection. Following 24 hours of treatment, cell expression independently to the p53 status (Fig. 1A, compare lanes extracts were prepared and luciferase and h-galactosidase activities were neo and GSE56). Notably, ATF3 induction was significantly determined using Promega materials and procedures. attenuated in cells expressing the p53R175H mutant in both the www.aacrjournals.org 10751 Cancer Res 2006; 66: (22). November 15, 2006

Figure 1. ATF3 induction following TPA in cells differing in their p53 status. Cell lines differing in their p53 status WI-38, HT-1080 (A), and SKOV3 (B) were treated with TPA at a concentration of 10 Ag/mL for 24 hours. ATF3 mRNA level was measured by QRT-PCR and normalized to the GAPDH housekeeping control gene. C, Western blot analysis of p53 and ATF3 in p53-null, p53R175H, and p53R248W SKOV3 cells following 72 hours of 10 Ag/mL TPA treatment. D, effects of p53 mutants on ATF3 promoter following TPA. SKOV3 p53-null cells were cotransfected with pLucATF3-1287 and expression plasmids encoding for various forms of p53. Fold activation was calculated as the ratio of promoter activity in TPA-treated cells to nontreated cells.

WI-38 and HT-1080 backgrounds. In light of the finding that p53 (pLucATF3-1287) was cotransfected into p53-null SKOV3 cells status influences ATF3 expression specifically under conditions of together with different p53 mutants, and reporter activity high-dose TPA treatment, we focused on p53-ATF3 cross-talk was assayed after treatment with TPA for 24 hours. Notably, under this condition, and therefore, from this point on in this study p53 mutants R175H, H179G, R248W, and D281G reduced signifi- ‘TPA treatment’ refers only to high-dose TPA treatment, unless cantly ATF3 promoter induction following TPA treatment when stated otherwise. compared with the empty vector. In contrast, p53R273H as well It was important to evaluate the response of ATF3 to TPA in cells as WT p53 lacked this repressor activity (Fig. 1D). A same that express mutant p53 in a p53-null background as opposed to phenomenon was observed with the partially truncated promoter the p53 WT background of HT-1080 and WI-38. To that end, ATF3 pLucATF3-293 (Supplementary Fig. S3). Thus, the capacity to gene expression was examined in the genetic background of interfere with TPA induction of ATF3 is not restricted to specific SKOV3, a p53-null ovarian cancer cell line. It was observed that mutant. SKOV3 cells stably expressing mutant p53 R175H (p53R175H) or Mutant p53 protects cells from TPA-induced cell death. R248W (p53R248W) exhibited attenuated ATF3 induction in Having established that mutant p53 interferes with TPA-induced response to TPA, both at the mRNA and protein levels, when ATF3 activation, we wanted to examine the consequences of compared with SKOV3 cells expressing empty vector (p53 null; mutant p53 presence during TPA exposure for cell fate. To that end, Fig. 1B and C). Taken together, these results suggest that mutant p53-null and p53R175H SKOV3 cells were treated with TPA for p53 interferes with p53-independent cellular pathways that 72 hours and assayed for cell proliferation using a colorimetric mediate ATF3 induction on TPA treatment. This represents a assay. Cell proliferation values indicate that already at 48 hours novel gain-of-function activity for mutant p53. p53R175H presence confers cells with a significant proliferation Next, we tested whether this gain-of-function activity is a fea- advantage on TPA treatment (Fig. 2A). To delineate whether this ture of other p53 mutants. To this end, a genomic fragment of was due to inhibition of cell death or of cell cycle arrest, DNA f1.3 kbp corresponding to the ATF3 promoter (28) was cloned content was measured by flow cytometry to reveal changes in cell upstream of a luciferase reporter. This full-length promoter cycle distribution following TPA treatment. Whereas p53-null

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SKOV3 cells exhibited G1 arrest following 48 hours of treatment ATF3 sensitizes cells to TPA-induced cell death. The data and pronounced cell death following 72 hours, p53R175H and thus far indicate that mutant p53 inhibits TPA-induced cell death p53R248W SKOV3 cells displayed prolonged G1 arrest with a low and that this correlates with the ability of mutant p53 to attenuate amount of cell death even after 72 hours (Fig. 2B). To further ATF3 gene expression. This raises the question whether the characterize the cellular death observed, the cleavage of the cell attenuation of ATF3 induction by p53R175H is required for death marker PARP-1 (36) was measured. The level of full-size p53R175H to exert its protective effect. To answer this question, PARP-1 protein was reduced dramatically in p53-null cells we examined the consequences of expressing ectopically ATF3 in following 72 hours of TPA treatment (Fig. 2C). In contrast, in the SKOV3 cell lines during TPA treatment. Both p53-null and p53R175H cells its level remained unchanged during the entire p53R175H SKOV3 cells were infected with either an ATF3 carrying course of treatment. This protection from TPA by p53R175H retrovirus (pBabe-ATF3) or an empty vector (pBabe-puro; Fig. 3A, SKOV3 cells was abolished when a p53 shRNA vector (shp53) was compare lanes 1 and 7 and lanes 4 and 10). Importantly, whereas introduced to the cells as indicated by the PARP-1 analysis at in SKOV3 cells expressing p53R175H the protein level of ATF3 72-hour time point (Fig. 2D). In summary, these data suggest that was transiently up-regulated before returning to basal levels after p53-null cells cease to proliferate sooner than p53R175H cells as 72 hours, in SKOV3 cells expressing both p53R175H and ATF3 indicated by the lower mitochondrial activity observed at 48 hours sustained high levels of ATF3 protein were observed (Fig. 3A, after TPA treatment. More importantly, at 72 hours past TPA compare lanes 6 and 12). treatment, p53-null SKOV3 cells display significantly higher To assess the phenotypic effect of this elevation of ATF3 protein amount of cell death as measured by FACS and PARP-1 cleavage levels, the SKOV3 cell lines with each p53 status, with or without compared with their mutant p53-expressing counterparts. These ATF3 ectopic expression, were exposed to either TPA (10 Ag/mL) or data support the hypothesis that mutant p53 possesses a gain- doxorubicin (0.2 Ag/mL) and cell proliferation was assessed. Both of-function activity whereby it blocks TPA-induced cell death. treatments resulted in pronounced cell death (Fig. 3B). For the

Figure 2. Characterization of cellular responses to TPA. p53-null and p53R175H SKOV3 cells were treated with TPA at 10 Ag/mL for the indicated times. A, cells were analyzed for cellular proliferation by WST-1. B, DNA content of SKOV3 p53-null, p53R175H, and p53R248W was measured by FACS analysis using propidium iodide. C, Western blot analysis of the full-size PARP-1as a marker of cell death. D, shRNA against p53 resensitizes the p53R175H cells to TPA. p53R175H SKOV3 cells were infected either with a retrovirus harboring a p53 shRNA vector (shp53) or with a control vector, mouse NOXA shRNA (shmNOXA). Cells were treated with TPA at 10 Ag/mL for the indicated times. Western blot analysis depicting the level of ATF3, p53, and the full-size PARP-1. www.aacrjournals.org 10753 Cancer Res 2006; 66: (22). November 15, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research doxorubicin-treated cells, ectopic ATF3 expression did not affect p53R175H is actingdownstream to the PKC proteins. It significantly the amount of cell death following 72 hours of was shown that TPA exerts its cellular effects mainly via modu- treatment in either p53-null or p53R175H cells. In contrast, when lation of the PKC proteins (18). Whereas treatment with TPA cells were treated with TPA, ATF3 ectopic expression enhanced cell at low doses activates most PKC isoforms, high doses of TPA result death in p53R175H cells at the 72-hour time point. Moreover, in depletion of PKC isoforms and consequently in inhibition of Western blot analysis of extracts generated from these SKOV3 cell the PKC pathway. To discover whether the gain-of-function acti- lines was conducted to measure the level of the full-size PARP-1. In vity of p53R175H is mediated by PKCs, we monitored the pro- p53-null cells, the full-size PARP-1 protein completely disappeared tein levels of two major PKC isoforms that were shown to influence following 72 hours of TPA treatment both in the presence and in cell survival, PKCa and PKCy, in SKOV3 and WI-38 cell lines the absence of ectopic ATF3 expression (Fig. 3A, compare lanes 3 treated with TPA for 72 hours. Both PKC isoforms were depleted and 9). In contrast, in p53R175H cells the full-size PARP-1 protein following the treatment independently of the p53 status (Fig. 3D). was observed to disappear more completely in the cells expressing These results suggest that p53R175H is acting downstream to ectopic ATF3 than in those without ectopic ATF3 (Fig. 3A, compare the PKC proteins, but upstream to ATF3, in the TPA response lanes 6 and 12). In agreement with that, p53-null SKOV3 cells pathway. Drugs that inhibit PKC activity have the potential to harboring an ATF3 shRNA vector (shATF3) were less sensitive to mimic the phenotype induced by high doses of TPA. We examined TPA compared with their control vector (shmNOXA) counterparts ATF3 gene expression after treatment with isoform-specific as indicated by the PARP-1 analysis at 72-hour time point (Fig. 3C). inhibitors of PKC activity. PKCy is inhibited specifically by rottlerin These results implicate ATF3 as a cell death promoter under at 5 Amol/L(37), whereas the activity of PKC a, as well as that of conditions of TPA treatment. Moreover, these data extend the PKCh1, is inhibited by Go6976 at 10 nmol/L(38). In p53-null correlation between ATF3 expression and the ability of p53R175H SKOV3 cells, treatment with rottlerin induced ATF3 protein to a to influence TPA-induced cell death, lending support to the level similar to that induced by TPA treatment (Fig. 3D). As hypothesis that one mechanism by which p53R175H protects cells expected, cells expressing mutant p53 were less sensitive to from TPA-induced cell death partially involves the attenuation of rottlerin treatment compared with their p53-null counterparts, ATF3 expression. similarly to the results obtained with the TPA treatment

Figure 3. Negative correlation between PKCy activity and ATF3 level and the effect of ATF3 ectopic expression on TPA-induced cell death. p53-null and p53R175H SKOV3 cells were infected either with a retrovirus encoding ATF3 (pBabe-ATF3) or with an empty vector (pBabe-puro). A, cells were treated with TPA at 10 Ag/mL for the indicated times. Western blot analysis depicting the level of ATF3, p53, and the full-size PARP-1. B, cells were treated with either TPA at 10 Ag/mL or doxorubicin at 0.2 Ag/mL for the indicated times. Cellular proliferation was measured by WST-1. C, shRNA against ATF3 partially protects cells from TPA-induced cell death. p53-null SKOV3 cells were infected either with a retrovirus harboring an ATF3 shRNA vector (shATF3) or with a control vector, mouse NOXA shRNA (shmNOXA). Cells were treated with TPA at 10 Ag/mL for the indicated times. Western blot analysis depicting the level of ATF3, p53, and the full-size PARP-1. D, Western blot analysis depicting the protein levels of two PKC isoforms and ATF3 in WI-38 and SKOV3 cell lines treated with TPA and PKC inhibitors. Cells were treated with TPA at 10 Ag/mL for 72 hours or with either rottlerin at 5 Amol/L or Go6976 at 10 nmol/L for 24 hours.

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Figure 4. ATF3 induction following TPA treatment is mediated via the MEF2 and CREB/ATF motifs. A, schematic representation of ATF3 promoter-luciferase reporters. Numbers on the left, position relative to the TSS. Boxes, known and putative regulatory motifs (predicted by MatInspector) and are shaded according to the legend (bottom). B, mapping the region responsible for ATF3 induction on TPA treatment. ATF3 promoter-luciferase reporters of various lengths were transfected into p53-null SKOV3 cells. Cells were treated with TPA at 10 Ag/mL after 48 hours of transfection for a period of 24 hours. C, mutations of the MEF2 and the CREB/ATF abolish the TPA-induced activation of ATF3 promoter. SKOV3 cells were transfected with either the WT construct pLucATF3-293 or its mutated derivatives and treated as described above. The name of each construct corresponds to the mutated regulatory site as depicted in (A). Numbers above columns, fold activation calculated as the ratio of promoter activity in TPA-treated cells to nontreated cells.

(Supplementary Fig. S4). In contrast, treatment with Go6976 did latory motifs [i.e., MEF2, CREB/ATF, and CAAT/enhancer binding not affect ATF3 levels. These results point to a specific role of the protein (C/EBP)]. Directed mutagenesis of either the CREB/ATF or PKCy isoform in ATF3 induction by TPA. the MEF2 sites, but not of the C/EBP site, in the pLucATF3-293 Activation of ATF3 by TPA is mediated via CREB/ATF and reporter resulted in partial loss of the reactivity to TPA (Fig. 4C). MEF2 motifs. To map the region responsible for ATF3 transcrip- Simultaneous mutations of both CREB/ATF and MEF2 motifs tional activation on TPA treatment, a deletion analysis of the completely abolished the induction of pLucATF3-293 reporter ATF3 promoter was carried out. Several 5¶-truncated constructs activity on TPA treatment, suggesting that these two transcription were generated and placed upstream of the luciferase reporter factor binding motifs are required for the induction of ATF3 gene (Fig. 4A). These were each transfected transiently into p53- by TPA. null SKOV3 cells that were then treated with TPA for 24 hours ATF3 induction by TPA is attenuated by p53R175H via the before assessment of luciferase activity. pLucATF3-1287 was MEF2 site. Having established that ATF3 induction by TPA is induced approximately 2- to 3-fold on TPA treatment (Fig. 4B). mediated by the CREB/ATF and MEF2 sites, we wanted to A similar response to TPA was observed with the partially elucidate whether either of these or any other binding sites mediate truncated construct pLucATF3-293. Although further truncation, attenuation of ATF3 induction by p53R175H. Although the majority pLucATF3-104, resulted in a construct with significantly reduced of p53 tumor-derived mutants, including R175H, are impaired in basal activity, the transcriptional activity of this construct was their DNA-binding domain, it has been reported that certain p53 still induced by TPA. In contrast, truncation of another 39 nucleo- mutants can nevertheless bind specific regulatory sites within tides resulted in a construct pLucATF3-65 that had lost its ability promoters by interacting with other transcription factors, such as to respond to TPA but which still exhibited basal activity higher ETS-1 and SP1 (40–42). QRT-PCR-ChIP analysis was conducted on than that of the control empty construct (pLucEMPTY). These data p53R175H-expressing SKOV3 cells following TPA treatment to suggest that the genomic region spanning from À65 to À104 discover whether p53R175H interacts with the ATF3 promoter. relative to the transcription start site (TSS) is responsible for ATF3 Indeed, p53R175H was interacted with the ATF3 promoter in induction on TPA treatment. Using the MatInspector database (39), nontreated cells and this interaction was enhanced following TPA we found that this promoter region harbors three known regu- treatment (compare ap53 with aHA in Fig. 5A). An in silico search www.aacrjournals.org 10755 Cancer Res 2006; 66: (22). November 15, 2006

Figure 5. p53R175H attenuates ATF3 induction via the MEF2 site. A, QRT-PCR-ChIP analysis was carried out on p53R175H SKOV3 cells treated with TPA at 10 Ag/mL for 24 hours. Protein-DNA complexes were immunoprecipitated with antibodies against p53 and MEF2 and with the control antibody against hemagglutinin (HA) tag. The amount of precipitated DNA was measured by QRT-PCR with specific primers directed at a region corresponding to ATF3 promoter. Amplification of nonprecipitated DNA was conducted as well (1% input). B and C, luciferase reporter activities in p53-null and p53R175H SKOV3 cells. Cells were transfected with either the WT construct pLucATF3-293 or its mutated derivatives and treated with TPA for 24 hours. Numbers above columns, fold activation of promoter activity was calculated. D, schematic representation of transcription factor motifs along ATF3 promoter. Numbers, position relative to the TSS. Motif names are depicted within the boxes according to their position along ATF3 promoter. Motif consensus sequence is presented below with core consensus sequence (uppercase). ATF3 reporters that harbor mutations in specific motifs were created. Underlined letters, corresponding mutations. for putative transcription factor binding sites within the ATF3 p53R175H attenuates TPA-induced MEF2D expression. The promoter with known involvement in ATF3 regulation revealed data presented above implicate the involvement of the MEF2 five motifs in addition to MEF2 and CREB/ATF: SP1, ATF4, ETS-1, family of transcription factors both in TPA-induced ATF3 C/EBP, and CDE. To investigate which of these sites mediates expression and in the attenuation of this induction by p53R175H. attenuation of ATF3 induction by p53R175H, seven ATF3 promoter To study in more detail this role of MEF2 proteins, we monitored constructs upstream of the luciferase reporter gene were generated, the mRNA levels of the four MEF2 members in p53-null and each having one of the sites mutated (Fig. 5D). Mutation in the p53R175H SKOV3 cells following 24 hours of TPA treatment using MEF2 motif abolished most effectively the effect of p53R175H the QRT-PCR analysis. Whereas MEF2A, MEF2C, and MEF2D were on the induction of ATF3 by TPA (Fig. 5B and C), for the fold induced following TPA treatment, no significant induction of induction of all ATF3 reporters except for the one harboring a MEF2B was observed (Fig. 6A). MEF2A and MEF2C mRNA levels mutant MEF2 site was lower in p53R175H cells than in p53-null were induced similarly in both p53-null and p53R175H cells. In cells. This suggests that p53R175H attenuates the ATF3 promoter contrast, MEF2D induction was much stronger in the p53-null cells activity primarily via the MEF2 site. compared with their p53R175H counterparts. A differential

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Figure 6. The effect of p53R175H on the TPA-induced activation of MEF2 proteins and MEF2-dependent activation of ATF3. A, analysis of induction of the MEF2 family of transcription factors following TPA treatment in p53-null and p53R175H SKOV3 cells. TPA was applied at 10 Ag/mL for 24 hours, and mRNA levels were measured for all the MEF2 members by QRT-PCR. SKOV3 cells were treated with 10 Ag/mL TPA for the indicated time, and lysates were blotted with an anti-MEF2 antibody specific for MEF2D, MEF2A, and, to a lower extent, MEF2C. B, QRT-PCR-ChIP analysis was carried out on p53R175H SKOV3 cells treated with TPA at 10 Ag/mL for 24 hours. Protein-DNA complexes were immunoprecipitated with antibodies against p53 and MEF2 and with the control antibody against HA. The amount of precipitated DNA was measured by QRT-PCR with specific primers directed at a region corresponding to MEF2D promoter. C, effect of MEF2A and MEF2D on ATF3 promoter activity. SKOV3 p53-null or p53R175H cells were cotransfected with either pLucATF3-293 or pLucATF3-293 + mCREB/ATF and with either an empty vector (pBabe-puro), MEF2A (pBabe-MEF2A), or MEF2D (pBabe-MEF2D). Cells were treated with 10 Ag/mL TPA 48 hours after transfection for 24 hours. Luciferase activity was measured and normalized. D, model for ATF3 transcriptional regulation on TPA treatment. Regulation of ATF3 promoter in both p53-null and p53R175H cells in basal condition and after TPA treatment. Boxes, ATF3 promoter with its two regulatory motifs that are involved in TPA-induced activation. Ovals, various transcription factors that regulate ATF3 expression with the corresponding transcription factors written inside. Lines with arrow tip, transcriptional activation; lines that end with a perpendicular line, repression. www.aacrjournals.org 10757 Cancer Res 2006; 66: (22). November 15, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research induction in response to TPA in p53-null versus p53R175H cells was In agreement with others, we showed here that high doses of also observed for MEF2 protein levels (Fig. 6A). TPA act to inhibit PKCs due to their ability to promote depletion of To examine the possibility that MEF2D mediates TPA-induced the PKC isoforms (21–23). Consistent with these previous studies, ATF3 expression, p53-null and p53R175H SKOV3 cells were we observed that ATF3 induction following high doses of TPA is transiently cotransfected with the reporter pLucATF3-293 and WT p53 independent and is correlated negatively with the levels of either with a control vector (pBabe-puro), MEF2A (pBabe-MEF2A), PKCs. However, we discovered that expression of mutant p53 or MEF2D (pBabe-MEF2D) expression constructs. Notably, ectopic disrupted this correlation and resulted in attenuated ATF3 expression of both MEF2A and MEF2D increased reporter gene induction. Importantly, this attenuation of ATF3 by mutant p53 expression in the absences of treatment, indicating that these was shown to correlate with protection of cells from TPA-induced transcription factors are activators of ATF3 (Fig. 6C). The death. apparently contradictory results that both overexpression of Having shown a novel gain-of-function activity for mutant p53, MEF2 (Fig. 6C) and mutation in the MEF2 motif (Fig. 4C) lead to we investigated the molecular mechanism underlying this activity. up-regulation of ATF3 promoter activity can be resolved by the First, we revealed that both CREB/ATF and MEF2 sites are hypothesis that in the absence of TPA treatment a repressor responsible to TPA-induced ATF3 activation. Moreover, we show protein is bound to the MEF2 site, which is released from the that both mutation in the MEF2 site and expression of the MEF2D promoter by MEF2D on TPA treatment. When the p53-null SKOV3 protein up-regulate the basal activity of the ATF3 promoter, cell expressing the pLucATF3-293 reporter gene and either empty suggesting that a repressor protein binds to the MEF2 site in basal vector, MEF2D, or MEF2A were treated with TPA, only for MEF2D conditions. In contrast, mutations of both MEF2 and CREB/ATF was TPA-induced expression attenuated (Fig. 6C). This result sites do not result in increased promoter activity. These results may indicates that specifically MEF2D is responsible for the transcrip- suggest that a potential repressor bound to MEF2 site that tional induction of ATF3 following TPA treatment. The same result interferes with the binding of an activator to the CREB/ATF. Next, was observed when using the reporter pLucATF3-293 + mCREB/ we found that p53R175H interacts with the ATF3 promoter and ATF, which implies that the combination of mutant CREB/ATF provide evidence that it attenuates ATF3 induction via this site and MEF2D expression can mimic the induction of ATF3 as promoter binding specifically at the MEF2 site. Furthermore, we was observed in the response to TPA. Based on these experiments, uncovered a second mechanism by which p53R175H influences we hypothesize that in p53R175H-expressing SKOV3 cells in ATF3 expression, for p53R175H interacts also with the MEF2D response to TPA treatment the removal of a repressor from the promoter and attenuates the expression of the ATF3 activator MEF2 site by MEF2D is prevented, leading to attenuated induction MEF2D. (Fig. 6D). Increased activation of the TPA-PKC pathway of ATF3. has been associated with malignant transformation in breast, lung, The next question pertains to how p53R175H might exert this and gastric carcinoma cell lines. Conversely, inhibition of the TPA- action, of preventing the removal of a repressor bound to the MEF2 PKC pathway has been shown to inhibit the invasive and motif. The observation that MEF2D induction was much stronger metastasis potential of some malignant cells. Therefore, inhibiting in p53-null cells raised the possibility that p53R175H influences the TPA-PKC pathway by antisense or inhibitors is considered events at the MEF2 site in the ATF3 promoter not only via its likely to result in tumor regression and such inhibitors are used in interacting there but also indirectly through binding to the MEF2D cancer therapy today, such as UCN01, PKC412, bryostatin, and promoter and inhibiting MEF2D induction. QRT-PCR-ChIP analysis ISIS3521 (reviewed in ref. 46). Our discovery that mutant p53 was conducted on p53R175H SKOV3 cells following 24 hours of interferes with TPA-induced cell death at a point in the pathway TPA treatment. Indeed, p53R175H displayed interaction with the below the PKCs raises the worrying possibility that its expression MEF2D promoter (Fig. 6B), although this interaction was weak in human tumors may desensitize them to such therapies. compared with ATF3 promoter binding (Fig. 5A). These results Furthermore, ATF3 was shown to enhance cell death in several support the premise that an additional mechanism by which cell systems (34, 47, 48). Recently, it was shown that ATF3 can p53R175H attenuates ATF3 induction following TPA treatment stabilize the tumor suppressor gene p53 and suppress Ras- might be via direct binding to the MEF2D promoter and inhibition stimulated tumorigenesis (29, 49). These tumor-suppressive of MEF2D expression. activities of ATF3 have led to the hypothesis that down-regulated ATF3 expression may be an important feature of tumors compared with normal tissues. Indeed, Yan and Boyd (50) have Discussion shown recently that ATF3 expression is down-regulated in tumor Expression of several genes was shown to be modulated by versus normal tissues. Thus, in light of the results of the present mutant p53, including MDR1, EGFR, PCDNA, IL-6, BFGF, HSP70, and study, it is tempting to speculate that the clinical aggressive BAG-1 (reviewed in ref. 43). In this study, we explore how mutant features displayed by mutant p53-expressing tumors are at least p53 affects the immediate early response gene ATF3 and exerts its partially exerted via the negative regulation of mutant p53 of the gain of function in the TPA-PKC pathway. ATF3 gene. Kieser et al. (44) showed a link between mutant p53 and the PKC pathways by showing that mutant, but not WT, p53 increases the transcription of the vascular endothelial growth factor (VEGF) gene Acknowledgments following low doses of TPA treatment. They suggested that VEGF induction following PKC activation is mediated by a member of the Received 3/10/2006; revised 7/21/2006; accepted 9/19/2006. Grant support: Flight Attendant Medical Research Institute Center of Excellence, AP-1 family of transcription factors and that mutant p53 might European Commission’s Sixth Framework Programme grant LSHC-CT-2004-503576, enhance AP-1 activity. Indeed, c-fos, a member of the AP-1 family and Yad Abraham Center for Cancer Diagnosis and Therapy. The costs of publication of this article were defrayed in part by the payment of page of transcription factors, was shown to be induced in mutant p53 charges. This article must therefore be hereby marked advertisement in accordance cells (45). with 18 U.S.C. Section 1734 solely to indicate this fact.

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MEETING OF THE RADIATION RESEARCH SOCIETY

The annual meeting of the Radiation Research Socie S. Harris. (2) On Monday night, June 22, a lecture by ty will be held at the State University of Iowa, Iowa Dr. L. W. Alvarez on meson physics has been tentative City, on June 22â€”24,1953. The Society will be the guest ly scheduled. On Tuesday night, June 23, Dr. L. H. of the University, and all meetings will be held on the Gray of the Hammersmith Hospital, London, will speak campus. The program will consist of: (1) Two symposia, on a topic to be announced. Dr. Gray's lecture is spon one on â€œTheEffects of Rwliation on Aqueous Solu sored by the Iowa Branch of the American Cancer Soci tions,â€• which includes the following speakers: E. S. G. ety. Those desiring to report original research in radia Barren, Edwin J. Hart, Warren Garrison, J. L. Magee, tion effects, or interested in attending or desiring addi and A. 0. Allen. The second is â€œPhysicalMeasurements tional information, please contact the Secretary of the for Radiobiologyâ€•and companion talks by Ugo Fano, Society, Dr. A. Edelmann, Biology Department, Brook Burton J. Moyer, G. Failla, L. D. Marinelli, and Payne haven National Laboratory, Upton, L.I., New York.

ERRATUM The following correction should be made in the arti by the glucose utilized by 16 per cent in CLL. If the as cle by Beck and Valentine, â€œTheAerobic Carbohydrate sumption is made that, in this respect, the myeloid and Metabolism of Leukocytes in Health and Leukemia. I. lymphoid celLsof leukemia are similar to those of nor Glycolysis and Respiration,â€• November, 1952, page 821; ma! blood, it may be that the computed normal figure substitute for the last paragraph: represents a summation of the myeloid (M) and The data in Table 3 permit several interesting calcu lymphoid (L) cells that make up the normal leukocyte lations. If one compares the amount of glucose actually population. Thus, if M = +0.27 and L = â€”0.16 and disappearing with the sum of the amount equivalent to the normal differential is 65 per cent M and So per cent lactic acid produced plus that equivalent to 02 con L, then sumption, it is seen that the amount of glucose â€œcleav 0.65 (+0.27) + 0.35 (â€”0.16) = +0.12 age productsâ€•exceedsthe amount of glucose utilized b a figure identical to the observed +0.12 for normal 12 per cent in N and 27 per cent in CML and is exceeded leukocytes.

308 Mutant p53 Protects Cells from 12-O -Tetradecanoylphorbol-13-Acetate−Induced Death by Attenuating Activating Transcription Factor 3 Induction

Yosef Buganim, Eyal Kalo, Ran Brosh, et al.

Cancer Res 2006;66:10750-10759.

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