Myc represses the growth arrest gene gadd45 - Nature

Oncogene (1997) 14, 2825 ± 2834  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Myc represses the growth arrest gene gadd45 Wilson W Marhin1,3, Shaojun Chen3, Linda M Facchini1,3, Albert J Fornace Jr4 and Linda Z Penn1,2,3 1

Department of Molecular and Medical Genetics and 2Medical Biophysics, University of Toronto, 3Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9; 4Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA

The c-Myc protein strongly stimulates cellular proliferation, inducing cells to exit G0/G1 and enter the cell cycle. At a molecular level, Myc prevents growth arrest and drives cell cycle progression through the transcriptional regulation of Myc-target genes. Expression of the growth arrest and DNA damage inducible gene 45 (gadd45) is elevated in response to DNA damaging agents, such as ionizing radiation via a p53-dependent mechanism, upon nutrient deprivation, or during dierentiation. Gadd45 holds a vital role in growth arrest as ectopic expression confers a strong block to proliferation. Exposure of quiescent cells to mitogen stimulates a rapid increase in c-Myc expression which is followed by the subsequent reduction in gadd45 expression. The kinetics of these two regulatory events suggest that Myc suppresses the expression of gadd45, contributing to G0/ G1 phase exit of the cell cycle. Indeed, ectopic Myc expression in primary and immortalized ®broblasts results in the suppression of gadd45 mRNA levels, by a mechanism which is independent of cell cycle progression. Using an inducible MycERTM system, rapid suppression of gadd45 mRNA is ®rst evident approximately 0.5 h following Myc activation. The reduction in gadd45 mRNA expression occurs at the transcriptional level and is mediated by a p53-independent pathway. Moreover, Myc suppression and p53 induction of gadd45 following exposure to ionizing radiation are noncompetitive co-regulatory events. Myc suppression of gadd45 de®nes a novel pathway through which Myc promotes cell cycle entry and prevents growth arrest of transformed cells. Keywords: gadd45; Myc; cell cycle; p53; growth arrest

Introduction The ability of Myc to relinquish cellular growth arrest and promote G0/G1 to S phase transition likely contributes to its strong oncogenic potential. Physiologically, c-myc expression is tightly linked to the growth state of the cell (Dean et al., 1986; Kelly and Siebenlist, 1986; Waters et al., 1991). Expression is nearly undetectable in growth-arrested cells and is transiently elevated in response to mitogens, yet remains evident throughout the entire cell cycle (Eilers et al., 1989, 1991; Hann et al., 1985; Kaczmarek et al., 1985). Exposure of non-transformed

Correspondence: LZ Penn Received 20 December 1996; revised 6 March 1997; accepted 6 March 1997

cells to growth arrest signals such as serum withdrawal or inducers of dierentiation, triggers a rapid drop in Myc expression which results in cellular growth arrest. This responsiveness to extracellular signals is lost with c-myc deregulation, such that a Myc-activated cell which encounters a growth-arrest signal can no longer exit the cell cycle, and will undergo programmed cell death (apoptosis) (Askew et al., 1991; Evan et al., 1992). Thus, Myc elicits its eects in a dose-dependent manner; elevated levels stimulate cell proliferation and apoptosis, whereas the absence of Myc may be required for growth arrest and dierentiation. How Myc can so eectively and universally control the balance between cell growth arrest and proliferation remains unclear. Evidence to date suggests c-Myc mediates such diverse activities by functioning as a transcriptional activator when complexed with Max, another basic region helix ± loop ± helix leucine zipper protein (Amati et al., 1992; Blackwell et al., 1990, 1992; Blackwood and Eisenman, 1991; Prendergast et al., 1991; Prendergast and Zi, 1991). Myc/Max heterodimers bind to the canonical DNA hexamer CACGTG as well as to speci®c non-canonical elements (Blackwell et al., 1993; Blackwood and Eisenman, 1991; Fisher et al., 1993; Grandori et al., 1996). Despite Myc being wellde®ned as an activator of gene transcription, only a few direct genetic targets of Myc transactivation have been identi®ed in vivo, such as ornithine decarboxylase (ODC) (Bello-Fernandez et al., 1993; Wagner et al., 1993), carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase (cad) (Miltenberger et al., 1995), cdc25A (Galaktionov et al., 1996); eIF-4E (Rosenwald et al., 1993), a DEAD box-related gene (MrDb) (Grandori et al., 1996); a-prothymosin (Desbarats et al., 1996; Eilers et al., 1991; Gaubatz et al., 1995) and ECA39 (Benvenisty et al., 1992). As well as a transcriptional activator, Myc plays an important role as a repressor of gene expression (Desbarats et al., 1996; Inghirami et al., 1990; Lee et al., 1996; Li et al., 1994; Penn et al., 1990a; Philipp et al., 1994; Versteeg et al., 1988; Yang et al., 1993). Recent structure-function analysis of the aminoterminus of the c-Myc protein shows Myc repression and Myc transactivation are separable, with Myc repression being more closely associated with neoplastic transformation (Brough et al., 1995; Lee et al., 1996; Li et al., 1994; Penn et al., 1990b). Myc can repress C/EBPa (Antonson et al., 1995; Li et al., 1994), cyclin D1 (Philipp et al., 1994), the adenovirus 5 major-late promoter (AdMLP) (Li et al., 1994), and cmyc (Facchini et al., 1997) promoter activities. Myc suppression appears to be mediated through core regulatory elements and does not require the con-

gadd45 is suppressed by Myc WW Marhin et al

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sensus Myc/Max binding sites within the target promoter sequence (Antonson et al., 1995; Facchini et al., 1997; Li et al., 1994; Lucas et al., 1993; Philipp et al., 1994). Interestingly, Max heterodimerization is not required for cyclin D1 suppression (Philipp et al., 1994), yet it is obligatory for Myc autosuppression (Facchini et al., 1997) and remains unexplored for C/ EBPa and AdMLP suppression. It remains unclear whether Myc repression of these target genes occurs by direct or indirect pathways; however, Myc can clearly repress gene expression by more than one mechanism (Marhin et al., 1996). By identifying cellular Myc-regulated gene targets, the molecular mechanism(s) of Myc as a regulator of gene transcription can be delineated. Moreover, the link between Myc repression and transformation suggests the identi®cation of genes whose expression is suppressed in response to Myc will also prove crucial to understanding the role of Myc in neoplastic transformation. Intuitively, candidate Myc-repressed genes could include those whose pattern of expression contrasts that of c-Myc and are highly expressed during cellular growth arrest or quiescence. Known genes belonging to this category include the growth arrest-speci®c (gas) genes (Schneider et al., 1988), the serum deprivation response gene (sdr) (Gustincich and Schneider, 1993), the homeobox gax gene (Weir et al., 1995), and the growth-arrest and DNA damage inducible (gadd) genes (reviewed in Fornace, 1992; Papathanasiou and Fornace, 1991). With the exception of gas1 and gadd45, the functional role of these genes in eecting growth arrest is not clear. The gadd genes are coordinately expressed in response to growth arrest conditions as well as to a variety of DNA damaging agents (Fornace et al., 1988, 1989). Elevated gadd45 expression has also been linked to growth arrest triggered either by developmental signals (Constance et al., 1996) or to nutrientdeprivation associated with G0 phase of the cell cycle (Fornace et al., 1989; Smith and Fornace, 1996; Zhan et al., 1994b). Upon exposure to ionizing radiation gadd45 is induced (Hollander et al., 1993; Papathanasiou et al., 1991), and this induction is dependent upon the p53 transcription factor (Zhan et al., 1993). Functionally, Gadd45 confers a strong block to proliferation as shown through ectopic expression in tumorigenic carcinoma cell lines such as HeLa and RKO as well as non-transformed NIH3T3 cells (Vairapandi et al., 1996; Zhan et al., 1994b). Gadd45 is thought to mediate growth arrest through its interaction with two cell cycle components: the cyclin dependent kinase inhibitor p21, and proliferating cell nuclear antigen (PCNA) (Chen et al., 1995; Hall et al., 1995; Kearsey et al., 1995). The mechanism to overcome the growth-inhibitory aects of genes such as gadd45 and exit G0, in response to mitogen-stimulation or Myc-activation, remains unclear. In this manuscript we show gadd45 gene transcription is repressed in response to Myc activation. Ectopic Myc expression in both primary and immortalized ®broblasts results in a reduction in gadd45 mRNA expression. Myc suppression of gadd45 mRNA corresponds to a similar reduction in Gadd45 protein levels in Rat-1 cells expressing ectopic Myc protein. Although basal levels of gadd45 are low in subcon-

¯uent asynchronous cultures, expression of ectopic Myc can further down-regulate gadd45 expression, demonstrating that Myc suppression of gadd45 is not a simple consequence of cellular transition from quiescence into the cell cycle. The suppression of gadd45 by Myc occurs with rapid kinetics and occurs at the level of gene transcription in a p53-independent manner. Myc suppression aects the overall dose of Gadd45 expression as cells expressing an activated cmyc allele do not show maximal induction of gadd45 expression in response to ionizing radiation. The suppression of gadd45 in response to Myc represents a novel and biologically critical pathway, elucidating one of the mechanisms via which Myc stimulates cells to exit growth arrest, enter the cell cycle and contribute to transformation.

Results Sequential induction of c-myc and suppression of gadd45 mRNA levels following serum stimulation of quiescent Rat-1 ®broblasts To determine the relative kinetics of c-myc and gadd45 expression during cell cycle progression, we assayed the endogenous mRNA expression of these two genes in quiescent Rat-1 ®broblasts following serum stimulation. Con¯uent Rat-1 ®broblasts were cultured in low serum (0.1% FBS/aMEM) for 2 days to induce quiescence and then mitogen stimulated by changing the medium to 10% FBS/aMEM. RNA was harvested at 0, 0.5, 1, 2, 3, 6, 12, 18, 24 h post serum-stimulation and assayed by RNase Protection using single-stranded probes complementary to mRNA of endogenous rat cmyc, rat gadd45 and rat glyceraldehyde-3-phosphate dehydrogenase (gapdh). Mitogen-stimulation of quiescent Rat-1 ®broblasts resulted in a transient increase in c-myc mRNA expression ®rst evident at approximately 0.5 h after serum-stimulation (Figure 1, compare lane 2 with lane 1). This was followed by a gradual suppression in endogenous gadd45 mRNA levels which was clearly evident approximately 1 h postserum (Figure 1, compare lane 3 with lane 1). To

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Figure 1 Serum stimulation of con¯uent, quiescent Rat-1 ®broblasts results in the induction of c-myc mRNA expression followed by a suppression of gadd45 mRNA levels. Rat-1 cells were maintained in low serum 0.1% FBS/aMEM for 2 days then left untreated (lane 1) or exposed to 10% FBS (lanes 2 to 9) or 10% FBS and 50 mg/ml cycloheximide (lane 10). At the indicated intervals RNA was extracted and analysed by RNase Protection using single stranded probes complementary to endogenous cmyc, gadd45, and gapdh-speci®c sequences


ascertain whether the suppression of gadd45 mRNA levels was dependent upon de novo protein synthesis, con¯uent quiescent Rat-1 cells were treated simultaneously with 10% FBS/aMEM and 50 mg/ml cycloheximide for 3 h. Expression of the endogenous gadd45 gene was not suppressed in the presence of cycloheximide, showing gadd45 suppression was dependent upon de novo protein synthesis following serum stimulation. The timing of these two mechanistic events suggested a role for Myc in eecting the suppression of gadd45 mRNA expression. Ectopic Myc expression in primary cell cultures and immortalized cell lines results in a suppression of endogenous gadd45 mRNA levels To determine whether ectopic Myc expression can suppress gadd45 mRNA levels, primary rat embryo ®broblasts (REF) and the immortal non-tumorigenic Rat-1 cell line, were infected with control retrovirus (DOR/neo) carrying the neomycin resistance gene or retrovirus (Dok/v-mycneo) also carrying the v-myc gene. Primary mouse embryo ®broblast cultures (MEF) were similarly infected with either a control retrovirus (Babe/puro) or a retrovirus (Babe/v-mycpuro) carrying the v-myc gene. Drug resistant colonies were pooled and analysed as asynchronous proliferating cultures. Subcon¯uent-growing cells were analysed to ensure changes in endogenous gadd45 gene expression were not due to an indirect eect of Myc on cell cycle (Evan et al., 1992). Under the conditions of these experiments, populations of both control cells and cells expressing ectopic Myc protein showed similar rates of growth and a similar distribution of cells throughout all phases of the cell cycle (data not shown). RNA was extracted and analysed by RNase Protection assay for the expression of the endogenous gadd45 and gapdh mRNAs. Expression of ectopic Myc protein in primary cultures as well as in Rat-1 cells resulted in a 4 ± 5-fold repression in endogenous gadd45 mRNA levels compared to controls as determined by densitometry (Figure 2a, compare lanes 2, 4, 6 to lanes 1, 3, 5, respectively). Suppression of gadd45 was also clearly evident in Myc-expressing cell cultures which were grown to con¯uence (data not shown). The ectopic vmyc gene was expressed as demonstrated by RNase Protection assay (Figure 2b). Thus, constitutive ectopic Myc expression leads to a suppression in gadd45 mRNA levels. To determine if the suppression in gadd45 mRNA levels results in a corresponding decrease in Gadd45 protein expression, whole cell lysates were prepared from subcon¯uent asynchronous cultures of both control Rat-1 and Rat-1 cells expressing ectopic v-Myc protein and analysed by immunoprecipitation followed by immunoblot analysis. Rat-1 cells constitutively expressing ectopic Myc protein exhibited a ®vefold decrease in Gadd45 protein expression compared to control cells (Figure 2c). This level of suppression correlated with the observed 4 ± 5-fold decrease in gadd45 mRNA expression. Western blot analysis clearly demonstrated that the ectopic v-Myc protein is expressed in Rat-1 Myc cells compared to the control Rat-1 cells. The weak signal in the control lanes of the Western blot probed for v-Myc expression represents a non-speci®c cross-reactive band. Thus

constitutive ectopic Myc expression results in a suppression in gadd45 mRNA levels with a corresponding decrease in Gadd45 protein expression. The suppression of gadd45 in response to Myc-activation occurs with rapid kinetics To determine the kinetics of suppression of gadd45 in response to Myc we employed an estrogen-inducible MycERTM system in Rat-1 cells (Littlewood et al.,

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Figure 2 Ectopic Myc expression in primary and immortalized ®broblasts results in the suppression of gadd45 mRNA and protein levels. (a) 10 mg of RNA from subcon¯uent proliferating rat embryo ®broblasts (REF), Rat-1 ®broblasts (Rat-1), and mouse embryo ®broblasts (MEF) infected with control retrovirus (7) (lanes 1, 3, 5), or retrovirus carrying the v-myc gene (+) (Lanes 2, 4, 6) was analysed by RNase Protection assay to detect the presence of endogenous gadd45 and gapdh-speci®c sequences; (b) 10 mg of RNA from subcon¯uent proliferating rodent ®broblasts infected with control retrovirus (7) (lanes 1, 3, 5), or retroviruses carrying the v-myc gene (+) (Lanes 2, 4, 6) was analysed by RNase Protection assay to detect the presence of ectopic v-myc and gapdh-speci®c sequences; (c) 2 mg of total cell lysate from subcon¯uent Rat-1 ®broblasts infected with a control retrovirus (7), or retroviruses carrying the v-myc gene (+) was immunoprecipitated using a polyclonal anti-Gadd45 antibody (Sc797). Immunoprecipitated proteins were resolved on a 10% polyacrylamide gel and Gadd45 protein detected by Western blot analysis using antibody Sc797. The weak signal in the control lanes of the Western blot probed for exogenous Myc expression represents a cross-reactive band evident with this batch of panmyc antibody

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1995). Rat-1 cells were infected with a retrovirus carrying a chimeric gene consisting of the human cmyc cDNA linked in frame to the estrogen binding regulatory domain of the estrogen receptor, and individual drug-resistant clones were isolated. These Rat-1 wtMycERTM cells constitutively express an inactive MycERTM fusion protein, which is rapidly activated following exposure of cells to hydroxytamoxifen (OH-T). By this approach, Myc can be induced in the absence of mitogen-stimulation. Con¯uent Rat-1 wtMycERTM cells were cultured in low serum (0.1% FBS/aMEM) for 2 days to induce quiescence and then treated with OH-T to induce ectopic Myc activity. RNA was extracted at 0, 0.5, 1, 2, 3, 6, 9, 12, 18, 24 h following exposure of cells to OHT and expression of gadd45 and gapdh mRNA determined by RNase Protection assay (Figure 3). Induction of ectopic Myc activity triggered a reduction in gadd45 mRNA levels with a twofold reduction at 3 h and a maximal ®vefold suppression after approximately 12 h, as determined by densitometry. Similar results were observed in multiple independent Rat-1 wtMycERTM cell clones. To determine whether Myc directly or indirectly suppresses gadd45, Rat-1 wtMycERTM cells were exposed to cycloheximide at the time of MycER activation; however, the short 20 ± 30 min half-life of MycER quickly depleted the cell of eector protein making results uninterpretable (data not shown). As the half-life of gadd45 mRNA is approximatley 20 to 80 min (Jackman et al., 1994), Myc repression of gadd45 RNA was rapid and ®rst evident as early as 0.5 h following OH-T treatment. gadd45 repression was not evident in OH-T treated control Rat-1 DMycERTM cells expressing a nonfunctional MycERTM fusion protein harbouring a deletion in amino acids 106 ± 143 of the Myc portion of the fusion protein (data not shown). This region of the c-Myc protein is required for all known biological activities of Myc (Evan et al., 1992; Freytag et al., 1990; Garte, 1993; Penn et al., 1990b; Stone et al., 1987). This cell line serves as a control for the expression and hormone-activation of the MycERTM fusion protein. Thus, our results show gadd45 mRNA expression is suppressed in response to Myc with rapid kinetics.

The suppression of gadd45 in response to Myc does not require wildtype p53 activity It is well established that induction of the gadd45 gene following exposure to ionizing radiation, is mediated by the transcription factor p53 (Kastan et al., 1992; Zhan et al., 1993, 1994a). To determine whether Myc may indirectly repress gadd45 expression by aecting wildtype p53 expression, RNA isolated from subcon¯uent control Rat-1 and Rat-1 cells expressing ectopic v-Myc protein was assayed for endogenous p53 mRNA and protein expression. There was no change in endogenous p53 mRNA expression in Rat-1 Myc cells compared to control Rat-1 cells (Figure 4a). Furthermore, comparable levels of endogenous wildtype p53 protein were expressed in control and Mycexpressing Rat-1 cells (Figure 4b). Thus, constitutive ectopic Myc expression does not aect the expression of endogenous p53 mRNA or protein in Rat-1 cells. To determine whether wildtype p53 activity is required for the suppression of gadd45 by Myc, control Rat-1 and Rat-1 Myc cells were infected with either a control retrovirus (SVX.1) carrying only the neomycin resistance gene, or a retrovirus (SVX.6) also carrying the SV40 large T antigen (LTag) gene. Following selection, drug-resistant colonies were pooled and analysed as subcon¯uent proliferating cultures. The expression of SV40 LTag results in the inactivation of wildtype p53 activity (Jiang et al., 1993; Lenahan and Ozer, 1996; Mietz et al., 1992). Constitutive expression of ectopic SV40 LTag had no signi®cant eect on gadd45 expression in control Rat-1 cells or in Rat-1 Myc cells (Figure 5a). To ensure that the products of the ectopic genes were expressed, whole cell lysates from each of the Rat-1 cultures were assayed by Western blot analysis for the expression of ectopic v-Myc and LTag proteins. Ectopic v-Myc protein was clearly expressed in the appropriate cell lines (Figure 5b, compare lanes 3 and 4 with 1 and 2). Ectopic SV40 LTag protein expression was also highly expressed in Rat-1 cells which were infected with the SVX.6 retrovirus carrying the LTag gene (Figure 5b, compare lanes 2 and 4 with 1 and 3). Thus wildtype p53 activity was not required for either basal level gadd45 expression nor Myc suppression of gadd45.

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Figure 3 gadd45 mRNA expression is suppressed with rapid kinetics in response to Myc induction. Con¯uent Rat-1 wtMycERTM cells were maintained in low serum 0.1% FBS/aMEM for 2 days and then exposed to 100 nM OH-T to induce exogenous MycERTM activity. At the indicated intervals after OH-T treatment RNA was extracted and 10 mg total RNA analysed by RNase Protection assay using single stranded probes complementary to endogenous gadd45, and gapdh-speci®c sequences

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Figure 4 p53 mRNA and protein expression is not responsive to ectopic Myc expression in Rat-1 cells. (a) 10 mg of RNA from subcon¯uent Rat-1 cells infected with a control retrovirus (7), or retrovirus carrying the v-myc gene (+) was analysed by RNase Protection assay to detect the presence of endogenous p53 and gapdh-speci®c sequences. (b) Immunoblot analysis of 200 mg of total cell lysates from subcon¯uent Rat-1 cells infected with a control retrovirus (7), or retroviruses carrying the v-myc gene (+) to detect endogenous p53 protein expression


Myc and p53 co-regulate gadd45 transcription gadd45 mRNA expression is suppressed in response to Myc activation and induced by p53 following exposure to ionizing radiation (Kastan et al., 1992; Zhan et al., 1993, 1994a). To determine whether these two path-

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ways interact, subcon¯uent proliferating control Rat-1 and Rat-1 Myc cells were exposed to 10 Gy of ionizing radiation. Following treatment of cells, total RNA was extracted at 0, 1, 3, 8, 12 and 18 h and expression of endogenous gadd45 and gapdh mRNA was analysed by RNase Protection assay. gadd45 mRNA expression in unirradiated Rat-1 Myc cells was suppressed compared to unirradiated control Rat-1 cells (Figure 6, compare lane 7 to 1). Furthermore, exposure of either of the two cell lines to ionizing radiation lead to the gradual induction of gadd45 mRNA expression (Figure 6, compare lanes 1 to 6 with lanes 7 to 12). Importantly, maximal achievable levels of gadd45 expression evident in the control cells were not reached in the Myc-activated cells after 18 h (Figure 6). Extended time courses show similar results (data not shown). Hence both mechanisms; the suppression of gadd45 by Myc and the induction of gadd45 by p53, are independent regulatory mechanisms which combine to co-regulate gadd45 expression. Myc regulates transcription of the gadd45 promoter

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To ascertain whether Myc represses gadd45 expression at the level of gene transcription, we conducted transient-transfection reporter assays in both Rat-1 wtMycERTM and control Rat-1 DMycERTM cells. The gadd45 promoter lacks both TATA and Inr elements and contains few putative transcription factor binding sites. There are two potential OCT and two CAT sites located within 100 bp upstream of the transcription start site, and an AP1 site as well as a p53 binding site in intron 3 which are conserved in the human, mouse and hamster genes (Figure 7a) (Hollander et al., 1993). As our results show that Myc suppression is p53 independent, we focused our analysis on upstream regulatory sequences using a hamster gadd45 promoter luciferase construct consisting of the sequences from 71675 to +149 relative to the transcription start site. This construct was transiently transfected into subcon¯uent proliferating Rat-1 wtMycERTM and control Rat-1 DMycERTM cells. To control for transfection eciency a vector carrying the lacZ gene, was cotransfected with the promoter luciferase construct. Cells were treated with OH-T and at 0, 3, 6, 9, 12, 18, 24 h thereafter, whole cell lysates were prepared and then assayed for both luciferase and b-galactosidase activity. Treatment of Rat-1 DMycERTM cells with OH-T had no eect on luciferase activity (Figure 7b). However, OH-T induction of ectopic Myc activity in Rat-1 wtMycERTM cells resulted in the down-regula-

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Figure 5 The suppression of gadd45 by Myc does not require wildtype p53 activity. (a) 10 mg of RNA from subcon¯uent Rat-1 cells infected with a control retrovirus (7), retrovirus carrying the v-myc gene and/or retrovirus carrying the SV40 large T antigen (+) was analysed by RNase Protection assay to detect the presence of endogenous gadd45 and gapdh-speci®c sequences. Histograms represent gadd45 mRNA abundance relative to the control as quantitated by densitometry. All data was normalized to the level of gapdh mRNA expression. (b) Immunoblot analysis of total cell lysates from subcon¯uent Rat-1 ®broblasts as described in a, using a pan myc polyclonal antibody to detect ectopic v-Myc protein, and the monoclonal antibody Pab419 to detect ectopic SV40 LTag expression

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Figure 6 Myc and p53 co-regulate gadd45 transcription. Subcon¯uent Rat-1 cells infected with a control retrovirus (Rat-1 Control) (lanes 1 to 6), or retrovirus carrying the v-myc gene (Rat-1 Myc) (lanes 7 to 12) were exposed to 10 Gy of ionizing radiation. At the indicated intervals after treatment RNA was extracted and 10 mg of total RNA was analysed by RNase Protection using single stranded probes complementary to endogenous gadd45, and gapdh-speci®c sequences

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Figure 7 gadd45 transcription is suppressed in response to Myc. (a) Schematic diagram of the genomic structure of the hamster gadd45 gene, showing known and putative transcription factor binding sites. Numbers indicate position relative to the transcriptional start site (+1). Rat-1 DMycERTM and Rat-1 wtMycERTM cells were transfected with a 1.8 kb hamster gadd45 promoter luciferase construct, containing gadd45 sequences from nucleotides 71675 to +149. To assay transfection eciency, cells were co-transfected with a plasmid carrying the b-galactosidase gene under the control of the CMV promoter. Cell lines were subsequently treated with 100 nM OH-T for the times indicated to induce ectopic MycERTM activity. Whole cell lysates were prepared and analysed for luciferase and bgalactosidase activity. (b) Histograms represent the measured luciferase activity relative to the untreated control. Data was normalized to b-galactosidase activity to adjust for minor dierences in transfection eciency amongst the samples. (c) Histograms represent the bgalactosidase activity. Data are a representative out of three repeated experiments. Error bars denote standard deviations


tion of luciferase activity. As the half-life of luciferase is 3 to 6 h (Thompson et al., 1991), the level and kinetics of repression as measured in the gadd45 promoter-luciferase assay (Figure 7b) closely mimicked the suppression of endogenous gadd45 mRNA in response to MycERTM activation (Figure 3). The repression of the gadd45 promoter by Myc is speci®c, as induction of Myc activity had no signi®cant eect on b-galactosidase activity (Figure 7c). Thus, Myc can suppress transcription from the gadd45 promoter, and this suppression activity can be localized to nucleotide sequences found between 71675 and +149, relative to the transcription start site. Discussion Identifying the mechanisms by which Myc can overcome growth arrest and stimulate cells to enter the cell cycle is crucial to our understanding of how Myc promotes cell proliferation and contributes to cellular transformation. Our results show that the suppression of the potent growth arrest gene gadd45 by Myc may constitute one such mechanism. The reduction in gadd45 mRNA levels in response to mitogen stimulation of quiescent cells coincided with the induction of endogenous c-Myc expression. Moreover, simultaneous treatment of growth arrested cells with both cycloheximide and serum abrogated the reduction in gadd45 mRNA expression, demonstrating that the downregulation of gadd45 RNA levels was dependent upon de novo synthesis of a protein which was expressed soon after serum stimulation. To investigate the role of Myc in gadd45 suppression, we ectopically expressed Myc protein in primary cultures as well as in immortalized non-tumorigenic cell lines. Our results showed a reduction of gadd45 expression in Mycactivated cells. The 4 ± 5-fold suppression of endogenous gadd45 mRNA levels in Rat-1 cells expressing ectopic Myc protein, correlated with a similar reduction in Gadd45 protein expression. Importantly, this suppression was evident in subcon¯uent proliferating cells where the cell population was asynchronous, showing that Myc suppression of gadd45 was not an indirect result of the G0/G1 to S phase transition of the cell cycle. To determine the kinetics of gadd45 suppression in response to Myc induction we employed Rat-1 MycERTM cells. The MycERTM fusion protein is composed of the human c-Myc protein (Myc) fused in frame at its carboxyl terminus to the hormone regulatory domain of the estrogen receptor (ERTM). Rat-1 cells stably and constitutively express the inactive MycERTM fusion protein and exposure of cells to 4Hydroxy-Tamoxifen (OH-T) rapidly activates MycERTM in the absence of the broader response to mitogen. Activation of the MycERTM protein resulted in the suppression of gadd45 mRNA levels; suppression was ®rst evident as early as 0.5 h following OH-T stimulation and half-maximal suppression occurred after 3 h. The kinetics of suppression were rapid, as the half-life of gadd45 mRNA is 20 to 80 min (Jackman et al., 1994), showing gadd45 expression is a downstream target of Myc regulation. Transcription of the gadd45 gene is directly induced by the p53 transcription factor. Exposure to ionizing

radiation results in an increase in p53 protein levels which in turn activates transcription through a p53 responsive element in intron 3 of the gadd45 gene (Hollander et al., 1993; Kastan et al., 1992; Zhan et al., 1993, 1994a). Rat-1 cells express wildtype p53 protein as determined by immunoblot analysis, hence it is plausible that Myc suppression of gadd45 is either mediated through p53 or requires wildtype p53 activity. Our results demonstrated that constitutive expression of ectopic Myc protein in Rat-1 cells did not aect the expression of the endogenous p53 mRNA or protein. To determine if wildtype p53 activity is required for the suppression of gadd45 by Myc, SV40 LTag was ectopically expressed in both control and Rat-1 Myc cells to inhibit wildtype p53 activity (Jiang et al., 1993; Lenahan and Ozer, 1996; Mietz et al., 1992). We observed that gadd45 mRNA expression was suppressed in Rat-1 cells which are expressing ectopic Myc protein, irrespective of the p53 status of the cell. We further show the suppression of gadd45 by Myc and the induction of gadd45 by p53 following exposure to ionizing radiation were non-competitive regulatory mechanisms which co-regulated gadd45 expression. Thus the suppression of gadd45 by Myc is p53 independent. The results of the transient transfection reporter assays demonstrate that gadd45 transcription was suppressed in response to Myc activation. This response was speci®c to Myc as it was not evident in the control Rat-1 DMycERTM cells. The region of the gadd45 gene which was sucient to confer suppression in response to Myc encompassed 1.8 kb of the hamster gadd45 promoter containing nucleotides 71675 to +149, relative to the transcription start site. This region of the promoter also lacks the p53 binding site (Hollander et al., 1993), further supporting our observation that the eects of Myc and p53 on gadd45 regulation are separable. Sequence analysis of the gadd45 promoter revealed that this regulatory region lacks canonical Myc binding sites; a characteristic which is common among genes which are repressed by Myc (Antonson et al., 1995; Facchini et al., 1997). Recent studies suggest that Myc may repress gene expression either by directly interacting with components of the basal transcription machinery or by inhibiting transcription through an initiator element (Li et al., 1994; Philipp et al., 1994; Roy et al., 1993). Interestingly, the gadd45 gene lacks both an initiator site and a TATA box. The suppression of gadd45 transcription by Myc appears to be universal, as this mechanism is evident in both rodent and murine cell lines. This suggests that the suppression mechanism may be eected through shared enhancer elements found in the promoters of the gadd45 gene from dierent species. Indeed this region of the gadd45 gene possesses only two putative enhancers which are conserved between human, hamster, and mouse (Hollander et al., 1993). These include two OCT sites and two CAT sites located within 100 bp upstream of the transcription start site. The CAT sites are of particular interest as earlier studies suggest Myc can repress gene transcription by inhibiting the activities of CAT-binding transcription factors belonging to the CTF/NF1 family and the C/EBP family of trans-acting factors (Martin, 1991; Wedel and Ziegler-Heitbrock, 1995). Freytag et al., suggest that Myc is able to

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mediate a change in the phosphorylation state of CTF/ NF1, modifying its potential to activate transcription of the pro alpha 2(I) collagen promoter (Yang et al., 1991, 1993). Recently, transient transfection reporter assays demonstrate that in 3T3-L1 cells, ectopic Myc expression can abrogate the C/EBPa mediated activation of gadd45 transcription associated with adipocyte dierentiation (Constance et al., 1996). In addition, Mink et al. show ectopic v-Myc can suppress the expression of myelomonocytic speci®c genes by inhibiting both the expression and the function of C/ EBPa transcription factors (Mink et al., 1996) C/EBPa is predominantly expressed in dierentiated cells (Birkenmeier et al., 1989), hence the role of C/EBPa in the G0/G1-associated up-regulation, or Mycmediated repression of gadd45 in cycling cells, remains unclear. Indeed, Myc may repress gadd45 directly by interacting with components of the core transcription complex, indirectly in an enhancer-dependent manner or by a yet unknown mechanism of action. With the identi®cation of gadd45 as a Myc-suppressed gene target, the molecular mechanism of Myc-repression of this uncomplicated promoter can now be delineated and is in progress. The identi®cation of Myc-suppressed target genes is of fundamental importance, as recent studies have linked Myc repression of gene transcription with Myc induced transformation (Brough et al., 1995; Lee et al., 1996; Li et al., 1994). Indeed, we show that the region of the human cMyc protein, amino acids 106 to 143, required for Myc-suppression of gadd45, is also required for Myc-induced cell cycle progression, and cellular transformation (Evan et al., 1992; Freytag et al., 1990; Garte, 1993; Penn et al., 1990b; Stone et al., 1987). Interestingly this region of the c-Myc protein was dispensable for Myc suppression of cyclin D1 (Philipp et al., 1994), suggesting the molecular mechanisms of Myc suppression of gadd45 and cyclin D1 are likely non-overlapping and Myc may repress gene transcription by a number of pathways. gadd45 expression is strongly associated with growth arrest, and identifying gadd45 as a Mycsuppressed gene target contributes to our understanding of how Myc can stimulate cells to exit growth arrest and enter the cell cycle. Myc suppression of gadd45 likely contributes to its tumorigenic potential, precluding transformed cells from entering a growth arrested state. In this report we propose that gadd45 is a target of Myc repression, but may not be the sole target of Myc repression to permit the G0 to G1/S transition. Indeed we have observed that the growth arrest genes gas1, and gadd153 are also suppressed in response to Myc, while the gas genes 3 and 6 are not regulated by Myc (WWM, LZP, unpublished data). We propose that mitogen-stimulation induces Myc which in turn plays an important role in co-ordinating the suppression of speci®c growth arrest genes, thereby permitting cells to exit G0/G1 and enter the cell cycle. Materials and methods Cell culture Primary rat embryonic ®broblasts (REF) and primary mouse embryonic ®broblasts (MEF) were prepared as

previously described (Jacks et al., 1992; Land et al., 1983). Rat-1 ®broblasts are a subclone of the Fischer rat embryo ®broblast line F2408 (Lania et al., 1980). Fibroblasts were cultured in alpha modi®ed Eagle's medium (aMEM) supplemented with 10% fetal bovine serum (FBS, Gibco/ BRL), 100 mg/ml kanamycin (Sigma) and 2 mg/ml gentamycin (Roussel). To activate ectopic Myc activity, Rat-1 wtMycERTM and DMycERTM cells were exposed to 100 nM 4-Hydroxy, (Z) Tamoxifen (OH-T) (Research Biochemical International, Natick, MA). Rat-1 ®broblasts were exposed to 10 Gy of ionizing radiation using a Cesium137 source at 1.13 Gy/min. Early passage cell cultures were used for all assays. Unless otherwise stipulated all analyses were performed on asynchronous, subcon¯uent, exponentially growing cells. Retroviral infection The pDOR/neo, pBabe/hygro, pBabe/puro, pDok/v-mycneo, pBabe/v-mychygro, pBabe/v-mycpuro, pSVX.1, pSVX.6, pBpuro c-mycERTM, or pBpuro D106-143cmycERTM retroviral vectors were constructed as previously described (Facchini et al., 1997; Littlewood et al., 1995; Morgenstern and Land, 1990; Ridley et al., 1988). To produce infectious replication-defective ecotropic retroviral particles, recombinant retroviral constructs were transfected, using calcium phosphate precipitation (Graham and Eb, 1973), into the GP+E packaging cell line (Markowitz et al., 1988), and selected in 500 mg/ml G418 sulphate (Sigma), 2 mg/ml puromycin (Sigma) or 300 mg/ml hygromycin B (Sigma). Drug resistant clones were pooled and expanded for virus production. Fibroblast cultures were subsequently infected with retrovirus and stable drugresistant colonies were combined generating pooled populations. To generate clonal populations of Rat-1 wtMycERTM and Rat-1 DMycERTM cells, individual drug resistant colonies were isolated. RNase protection and luciferase assays RNA was prepared by the guanidinium isothiocyanate method (Chirgwin et al., 1979) and assayed by RNase Protection assay as described (Penn et al., 1990a). The probes were generated using T3 RNA polymerase (Stratagene) from linearized Bluescript KS and SK cloning vectors (Stratagene) containing: rat c-myc exon 1, the gag gene derived from the avian myelocytomatosis virus MC29, murine and rat gapdh cDNA (Facchini et al., 1994; Penn et al., 1990b). The probe for the rat gadd45 gene was transcribed using Sp6 RNA polymerase (Stratagene) from a linearized pGEM3ZF+ vector (Promega) containing a 450 bp PstI fragment of the rat gadd45 cDNA (kind gift of T Yoshida). The protected probes were resolved by electrophoresis on 6% denaturing polyacrylamide gels and visualized by autoradiography on X-OMAT ®lm (Kodak). Luciferase and b-galactosidase assays were performed as previously described (Facchini et al., 1997). Immunoblotting and immunoprecipitation Antibodies used in this study include the monoclonal antibodies, anti-p53 Pab421 and anti-SV40 Large T antigen Pab419 (kind gifts of S Benchimol), as well as polyclonal anti-gadd45 antibody Sc797 (Santa Cruz), and a polyclonal pan-myc antibody (kind gift of G Evan). Whole cell extracts were prepared as recommended (Santa Cruz), and total cell lysates (56105 cells) were immunoblotted as previously described (Daksis et al., 1994). Proteins were detected by incubating the membrane with their respective antibody for 1 h in TBS-T containing 1% non-fat milk. The membrane was washed, and incubated with a 1/2000


dilution of either horseradish peroxidase-conjugated goat anti-mouse IgG (Biorad) or swine anti-rabbit IgG antibody (Dako) in TBS-T containing 1% non-fat milk for 0.5 h. After washing, signals were detected using the ECL (Amersham) detection system and visualized by autoradiography (Kodak). Immunoprecipitations using the antigadd45 polyclonal antibody (Sc797) were performed as recommended by the supplier (Santa Cruz). Immune complexes were resolved on a 10% SDS/polyacrylamide gel, transferred to PVDF membrane (Millipore), developed with the same antibody, and then visualized by autoradiography (Kodak).

Acknowledgements We thank J Lear, M Shago, C Ho, J Dimitroulakos and S Lee for helpful discussions and critical reading of the manuscript; S Benchimol for SV40 large T antigen and p53 antibodies; and T Yoshida for the rat gadd45 cDNA. This work was supported by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (LZP), as well as the Leukemia Research Fund (LZP). Studentship support was kindly provided through the Ontario Graduate Scholarship Program (WM) and the Medical Research Council of Canada (LF).

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