Transformation Blocks Differentiation-Induced Inhibition of Serum Response Factor Interactions with Serum Response Elements1

[CANCER RESEARCH 59, 3795–3802, August 1, 1999] Transformation Blocks Differentiation-induced Inhibition of Serum Response Factor Interactions with Serum Response Elements1

Wei Ding, Michael M. Witte, and Robert E. Scott2 Department of Pathology, University of Tennessee Medical Center, Memphis, Tennessee 38163

ABSTRACT responsiveness to all mitogens as they undergo TD (3, 6). In contrast, the differentiation of SV40-transformed 3T3T cells (CSV3-1 cells) The differentiation of nontransformed 3T3T mesenchymal stem cells is into adipocytes does not repress mitogenic responsiveness but rather a multistep process that is associated with the progressive repression of increases it (7, 8). For example, at the NTD state, 5% serum is mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The sufficient to induce the majority of CSV3-1 adipocytes to undergo repression of serum-induced mitogenesis by differentiation correlates with DNA synthesis, whereas 30% serum is required for 3T3T cells. repression of the serum-inducible transcription of junB and c-fos.In Therefore, CSV3-1 adipocytes cannot efficiently undergo TD. contrast, the differentiation of neoplastically transformed cells does not The molecular mechanisms that mediate the progressive loss of repress mitogenic responsiveness or junB or c-fos inducibility. Because the serum-induced mitogenic responsiveness during adipocyte differenti- junB and c-fos promoters both contain serum response elements (SREs), ation in nontransformed 3T3T cells but not in transformed cells has the current studies tested the possibility that differentiation might repress not been definitively established. We have shown that adipocyte the ability of serum response factor (SRF) to bind to the SRE in normal differentiation represses AP-1 DNA binding activity and the serum- cells but not in transformed cells. We now report that differentiation inducible transcription of AP-1 factors including junB and c-fos (9). In represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyl- this regard, cellular senescence also decreases the inducibility of c-fos transferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF- transcription by serum (10). Because the junB and c-fos promoters binding domain. Adipocyte differentiation of nontransformed cells also both contain SREs (11), it is possible that adipocyte differentiation markedly represses the ability of SRF to bind to the junB SRE, the c-fos represses proliferation by blocking the ability of the SRE to interact SRE, and other SREs, as determined by mobility shift and gel supershift with the SRF that is required for its transactivation. The studies assays, without affecting the DNA binding characteristics of the nuclear reported here test this possibility. protein SP-1. By comparison, the ability of SRF to bind SRE is not An intact SRE is necessary for the transcriptional activation of junB repressed by the differentiation of SV40 large T antigen-transformed and c-fos expression by serum stimulation, and the SRE alone is 3T3T cells. The results further establish that adipocyte differentiation sufficient to confer the serum-dependent transcriptional activation of blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells. heterologous promoters (12). The sequence -CC[A/T]6GG-, which constitutes the core SRE domain, is the binding site for SRF (13). This

Mr 67,000 nuclear phosphoprotein contains multiple domains: (a)a INTRODUCTION SRE DNA binding domain; (b) a transactivation domain; and (c) several phosphorylation domains (12). SRF binds to the SRE as a Cellular proliferation and differentiation are coordinately regulated biological processes in most nontransformed cells. For example, cel- homodimer, and SRF can form a complex with TCFs, including Elk1, lular proliferative potential is repressed as basal epithelial keratino- Sap1, Sap2, FLI1, and EWS-FLI1 in association with the Ets domain cytes differentiate (1), and differentiation of skeletal myoblasts into that commonly adjoins a SRE (12, 14, 15). X-ray crystallographic contractile myotubes results in their permanent withdrawal from the studies also suggest that the interaction of SRE and SRF involves cell cycle (2). The differentiation of murine mesenchymal 3T3T stem mutual structural changes (16). cells into adipocytes also progressively represses cellular proliferative SRF transcription and expression are induced when quiescent un- potential via a multistep process that ultimately leads to terminal differentiated cells are treated with serum because the SRF promoter differentiation. contains a SRE, thus establishing the possibility of autoregulation Three distinct steps are involved in the differentiation of 3T3T cells (17). Serum treatment of cells can also induce SRF phosphorylation at into adipocytes: (a) PGA;3 (b) NTD; and (c) TD (3). Cells first sites near a unique SRF nuclear localization signal within its NH2- undergo PGA associated with the expression of several transacting terminal region (18). Control of SRF nuclear localization is also factors including PPAR-␥ and C/EBP-␥ and -␤ (4). In this state, cells indirectly influenced by protein kinase A (19). SRF phosphorylation show mitogenic responsiveness to 10 ng/ml PDGF and 5–10% serum. modulates the association versus dissociation characteristics of SRF Subsequently, cells modulate C/EBP expression so that C/EBP-␣ is with the SRE (20). Furthermore, repression of SRF DNA binding the dominant form (4) as they enter a nonterminal state of differen- activity in senescent human fibroblasts is mediated by the hyperphos- tiation in which they lose their responsiveness to most mitogens phorylation of SRF (21). except high concentrations of serum (5). Thereafter, cells lose their From this perspective, it is important to emphasize that two general classes of signaling mechanisms involving the SRF can regulate the

Received 10/1/98; accepted 6/4/99. activity of the SRE. A TCF-dependent pathway mediates the effects of The costs of publication of this article were defrayed in part by the payment of page PDGF, colony-stimulating factor 1, and 12-O-tetradecanoylphorbol- charges. This article must therefore be hereby marked advertisement in accordance with 13-acetate on SRE via the ras 3 raf 3 mitogen-activated protein/ 18 U.S.C. Section 1734 solely to indicate this fact. 3 1 Supported by the Muirhead Chair of Excellence (to R. E. S.). The data in this paper extracellular signal-regulated kinase kinase extracellular signal- represent part of the Ph.D. thesis research of Wei Ding. regulated kinase cascade (22). Both phosphorylation of TCFs and the 2 To whom requests for reprints should be addressed, at Department of Pathology, 800 binding of TCFs to SRF are required for activation of the SRE by this Madison Avenue, 576 Baptist Main Building, Memphis, TN 38163. 3 The abbreviations used are: PGA, predifferentiation growth arrest; SRE, serum pathway. A TCF-independent pathway mediates signals induced by response element; SRF, serum response factor; NTD, nonterminal differentiation; TD, serum to the SRE via the SRF, and these pathways involve the Rho terminal differentiation; C/EBP, CAAT/enhancer-binding protein; PDGF, platelet-derived growth factor; TCF, ternary complex factor; BCS, bovine calf serum; CAT, chloramphen- family of GTPases (23). It currently appears that the SRF DNA icol acetyltransferase. binding activity is required for both of these pathway. For example, 3795

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. DIFFERENTIATION EFFECTS ON SRE-SRF INTERACTIONS microinjection of anti-SRF antibodies into rat fibroblasts blocks the centrifugation at 2000 ϫ g for 10 min at 4°C, the supernatant containing response of the SRE to serum and growth factor stimulation (12). nuclear proteins was collected, divided into aliquots, and frozen at Ϫ80°C for The studies reported here focus on the effects of adipocyte differ- subsequent assays. The protein concentrations were determined using a protein entiation in nontransformed and transformed 3T3T cells on SRE-SRF assay kit (Bio-Rad Laboratories) according to the supplier’s instructions. interactions. The results show that adipocyte differentiation in non- Electrophoretic Mobility Shift and Gel Supershift Assays. Multiple oligonucleotide pairs were used in these experiments: (a)5Ј-CTTCCTGTGC- transformed cells represses SRE-SRF interactions concomitant with CCTAATATGGATGCTGG-3Ј and its complimentary partner that contains the the differentiation-induced repression of mitogenic responsiveness junB SRE (Biosynthesis); (b)5Ј-GGATGTCCATATTAGGACATC-3Ј and its and the loss of junB and c-fos serum inducibility. The data further complimentary partner that contains the c-fos SRE (Santa Cruz Biotechnology, show that in neoplastically transformed cells, differentiation fails to Inc.); (c)5Ј-GGATGTCCATATTATTACATC-3Ј and its complimentary part- repress mitogenic responsiveness, the inducibility of junB and c-fos, ner that contains a mutant c-fos SRE (Santa Cruz Biotechnology, Inc.); (d) and SRE-SRF interactions. Our final discovery that differentiation 5Ј-CTTCCTGTGCCCTTATATGGATGCTGG-3Ј and its complimentary part- represses SRF-SRE interactions by restricting SRF nuclear localiza- ner that contains the ␤-actin SRE; (e)5Ј-CTTCCTGTGCCCTTTTTTGGAT- tion only in nontransformed cells suggests that neoplastic transforma- GCTGG-3Ј and its complimentary partner that contains the EGR2 SRE; (f) tion can abrogate this key regulatory mechanism. 5Ј-CTTCCTGTGCCCTTATTTGGATGCTGG-3Ј and its complimentary partner that contains the thrombospondin-1 SRE; and (g)5Ј-CTTCCTGTGC- CCTATTATGGATGCTGG-3Ј and its complimentary partner that contains the MATERIALS AND METHODS dystrophin SRE. The latter four preparations were synthesized by the St. Jude Children’s Cell Lines and Growth and Differentiation Conditions. Nontransformed Research Hospital Center for Biotechnology. One oligonucleotide from each 32 murine 3T3T mesenchymal stem cells, SV40 large T antigen-transformed pair was radioactively end-labeled using [␥- P]ATP (3000 Ci/mmol; Amer- 3T3T cells (designated CSV3-1 cells), and spontaneously transformed 3T3T sham) and T4 polynucleotide kinase and annealed to its complimentary part- cells (designated DW1 cells) were used in these experiments. They were ner. Binding reactions were carried out by mixing 10 ␮g of nuclear proteins and 0.035 pmol (in 1 ␮l) of 32P-labeled oligonucleotide pairs with 1 ␮gof routinely cultured at 37°C in 5% CO2/95% air in DMEM supplemented with 10% BCS unless otherwise stated. poly(deoxyinosinic-deoxycytidylic acid) in a total volume of 25 ␮l of binding To induce quiescence and subsequent adipocyte differentiation, growing buffer [2 mM Tris-HCl (pH 7.5), 8 mM NaCl, 0.2 mM EDTA, 0.2 mM 3T3T or CSV3-1 cells were dissociated with 0.1% EDTA in PBS and plated ␤-mercaptoethanol, and 0.8% glycerol]. The binding reactions were allowed to onto 100-mm, ethylene oxide-sterilized, bacteriological Petri dishes at low proceed at room temperature for 20 min. Thereafter, 2 ␮l of 0.1% bromphenol density in heparinized DMEM containing 25% (v/v) human plasma. In this blue were added, and the reaction mixture was subjected to electrophoresis on medium, 3T3T and CSV3-1 cells become quiescent within 3–4 days and nondenaturing 5% polyacrylamide gels followed by visualization using auto- subsequently express the nonterminal adipocyte phenotype between days 6 and radiography as described previously (9). 8. The terminal differentiation phenotype is expressed thereafter between days For gel supershift assays, 1 ␮g (in 1 ␮l) of specific supershift antibodies 10 and 15 only in 3T3T cells; under the same condition, most CSV3-1 cells against SRF (Santa Cruz Biotechnology, Inc.) or of other control antibodies retain their ability to undergo DNA synthesis when restimulated with serum was added to the reaction mixture containing the nuclear protein extract and (3, 9). The extent of differentiation in such cultures was routinely characterized incubated for an additional 30 min at room temperature before the performance by phase microscopic examination, and it exceeded 75%. of mobility shift assays (9). In selected experiments, quiescence in nontransformed 3T3T cells in an Indirect Immunofluorescence. For indirect immunofluorescence, undif- undifferentiated state was induced by culture in DMEM containing 0.5% BCS ferentiated or differentiated cells were allowed to attach to slides overnight at for 3–4 days. In all studies, quiescence was established by observation of 37°C in culture media. Cells were rinsed briefly with PBS and then fixed for secession of growth and/or by quantitation of 3[H]thymidine incorporation into 20 min with 4% (w/v) paraformaldehyde in 100 mM sodium phosphate (pH DNA (3). 7.4). Fixed cells were washed three times in PBS containing 10 mM glycine for Plasmids, Transfection, and CAT Assays. The plasmids pjunB SREtk/ 5 min each, permeablized for 5 min with 1% NP40 in PBS and glycine, and CAT and pc-fos SREtk/CAT were prepared by insertion of a junB SRE washed as described previously. Slides were exposed to various dilutions (1:25 (5Ј-CTAGACTTCCTGTGCCCTAATATGGATGCTGGG-3Ј)orac-fos SRE to 1:200) of rabbit anti-SRF or anti-SP-1 antibody (Santa Cruz Biotechnology, Ј Ј Inc.) for 60 min. Cells then were rinsed as described previously and incubated (5 -CTAGAGGATGTCCATATTAGGACATCTG-3 ) into pBLCAT2, which contains the thymidine kinase gene promoter upstream of the CAT gene (24). with fluorescein-conjugated goat anti-rabbit IgG diluted 1:200 (Santa Cruz For transient transfection using Lipofectamine (Life Technologies, Inc.), mu- Biotechnology, Inc.) for 45 min. Slides were washed with three changes of rine 3T3T cells were grown in DMEM-10% BCS before transfection with 20 PBS, and then coverslips were mounted with mounting medium containing ␮g of pSREtk/CAT DNAs. Transfected cells were refed 24 h later with either 4Ј,6-diamidino-2-phenylindole (Vector Laboratories, Inc.). The cells were DMEM-0.5% BCS (to induce quiescence) or heparinized DMEM-25% human examined using a Zeiss fluorescence microscope with a Plano ϫ40 objective plasma (to induce differentiation). Five days later, CAT assays were performed and photographed using Kodak black and white ASA 200 film. on cells before and after stimulation with 10% BCS for 7 h, which was Western Blotting Analysis. Western immunoblotting procedures were predetermined to be the time required for maximum stimulation. performed as described previously (9). Nuclear proteins were mixed with SDS For CAT assays, cell lysates were incubated at 37°C for 15 h in 0.25 M sample buffer, boiled for 5 min, separated by electrophoresis in a 7.5% Tris-HCl (pH 8.0), 1.5 ␮Ci/ml [14C]chloramphenicol, and 500 ␮M acetyl-CoA. SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted [14C]Chloramphenicol (Amersham) and its acetylated products were then with rabbit anti-SRF or anti-SP-1 antibodies (Santa Cruz Biotechnology, Inc.). separated by TLC, and the extent of conversion of chloramphenicol to its Bound antibody was detected using goat anti-rabbit IgG conjugated to perox- acetylated forms was quantitated using a radioanalytic imaging computer idase (Sigma) and visualized using enhanced chemiluminescence Western system. blotting analysis kit (Amersham). Preparation of Nuclear Proteins. Nuclear extracts were prepared as described previously (9). After washing cells twice with 4°C PBS (pH 7.4), the cells were harvested in 4°C PBS with a cell scraper. After centrifugation at RESULTS 500 ϫ g for 5 min at 4°C, cell pellets were resuspended and incubated on ice Repression of junB and c-fos SRE Transcriptional Activation for 5 min in 5 ml of 4°C STM buffer [20 mM Tris-HCl (pH 7.85), 250 mM by Differentiation. To initiate our investigation of the mechanisms sucrose, 1.1 mM MgCl2, and 0.2% Triton X-100]. Nuclei were then sedimented by centrifugation at 800 ϫ g for 10 min, washed once with STM buffer, and by which adipocyte differentiation represses the transcription of junB washed once with STM buffer lacking Triton X-100. Nuclei were then resus- and c-fos, a 5300-bp junB promoter fused to the bacterial CAT gene pended in STM buffer lacking Triton X-100, 0.4 M KCl, and 5 mM ␤-mercap- was stably transfected into 3T3T cells, and the effect of serum toethanol and incubated on ice for 10 min to facilitate nuclear lysis. After induction was tested. Initial studies showed that serum induced CAT 3796

Table 1 Repression of the serum-induced transcriptional activation of junB and c-fos SRE Nontransformed 3T3T cells were transiently transfected with pjunB SREtk/CAT or c-fos SREtk/CAT. Transfected cells were refed 24 h later with either 0.5% BCS (to induce quiescence) or 25% human plasma (to induce differentiation). Five days later, cells were incubated with 10% BCS for 7 h. Cell lysates then were prepared and analyzed for CAT activity. The CAT activities of serum-induced cells relative to those of uninduced cells are presented. Induction of CAT Induction of CAT Transfected activity in activity of plasmid quiescent cells differentiated cells pc-fos SREtk/CAT 10.0ϫ 1.5ϫ pjunB SREtk/CAT 11.5ϫ 1.3ϫ

activity in quiescent undifferentiated cells, but not in differentiated cells (data not shown). This suggested that differentiation exerts its inhibitory activity on junB transcription via an effect on the junB promoter. Because SREs exist in both the junB and c-fos promoters, we hypothesized that they might represent the target domain for such repression. To determine whether the function of the junB and c-fos SREs is specifically repressed by adipocyte differentiation, oligonucleotides corresponding to the junB SRE and the c-fos SRE were synthesized and ligated upstream of the herpes simplex virus thymidine kinase promoter in the CAT reporter plasmid pBLCAT2 (24). When they were transiently transfected into murine 3T3T cells, both reporter genes were established to be serum inducible. Table 1 shows that CAT activity regulated by either the junB SRE or the c-fos SRE was induced Ͼ10-fold when quiescent undifferentiated cells were stimu- Fig. 2. Repression of the SRF DNA binding activity in differentiated 3T3T cells. ␮ lated with 10% BCS. In contrast, serum treatment of NTD adipocytes Electrophoretic mobility shift assays were performed using 10 g of nuclear extracts prepared from either growing (Lanes 2, 5, and 8) or differentiated (Lanes 3, 6, and 9) 3T3T induced only minimal CAT activity using either the pjunB SREtk/ cells and 0.035 pmol of 32P end-labeled oligonucleotides containing either c-fos SRE (Lanes 1–3)orjunB SRE (Lanes 4–6) or a single SP-1 binding site (Lanes 7–9). Lanes 1, 4, and 7 contain the DNA probe without nuclear proteins. The arrow indicates the marks a unique (ء) SRF-SRE complexes present within the boxed area. The asterisk differentiation-induced complex with the junB SRE; this band was not detected with other SREs. RG, rapidly growing, undifferentiated cells; NTD, NTD cells.

CAT or pc-fosSREtk/CAT reporter plasmids. This indicates that the function of both junB and c-fos SREs is repressed by adipocyte differentiation. Because neither SRE in these plasmids contains an intact TCF Ets binding domain, adipocyte differentiation must repress SRE activity in a TCF-independent manner. Adipocyte Differentiation Represses the Ability of SRF to Bind to the junB and c-fos SREs in Nontransformed 3T3T Cells. The binding characteristics of the SRF transcription factor to the junB and c-fos SREs were investigated by electrophoretic mobility shift assays using junB SRE oligonucleotides or c-fos SRE oligonucleotides as binding targets. First, nuclear proteins extracted from rapidly growing 3T3T cells were incubated with labeled oligonucleotides, and the identity and specificity of shifted complexes were determined by competition assays using different unlabeled DNA competitors and by supershift assays using SRF supershift antibodies. Fig. 1 shows that a comparable SRF band was evident in lanes containing junB or c-fos SRE probes when nuclear extracts of undifferentiated cells were used. This complex was established to contain both SRE and SRF using several approaches. The binding Fig. 1. Specific binding of the SRF to the c-fos and junB SRE. Electrophoretic mobility is specifically eliminated by the addition of a 50-fold excess of shift assays were performed using 10 ␮g of nuclear extracts prepared from growing 3T3T unlabeled junB or c-fos SRE oligonucleotides, whereas no inhibi- cells and 0.035 pmol of 32P-end-labeled oligonucleotide probes containing either the c-fos SRE (Lanes 1–6)orthejunB SRE (Lanes 7–12). Lanes 1 and 7 show the binding pattern tion was detected when an oligonucleotide with point mutations in to the DNA probes obtained with nuclear extracts alone; the major SRF-SRE complex is the GG sites of the c-fos SRE, which abolishes its binding with indicated by the arrow marked SRF. For competition assays, a 50-fold molar excess of SRF, was used in competition assays. Moreover, this band is unlabeled c-fos SRE (Lanes 3 and 10), junB SRE (Lanes 4 and 9), or c-fos SRE mutant (c-fos SREm; Lanes 2 and 8) was added to the binding reactions. For other lanes, 1 ␮gof supershifted by a SRF supershift antibody, but not by a JunD the antibodies indicated at the top of the panel was added to the binding reactions. supershift antibody. Incubation with anti-SRF antibody (Lanes 5 and 11) supershifts a SRE-SRF band to show a slower mobility (ss-SRF), whereas incubation with anti-JunD antibody has no supershift In contrast, when junB SRE and c-fos SRE oligonucleotides were effect. The SRF-SRE complexes are presented within the boxed area. incubated with proteins extracted from the nuclei of differentiated 3797

Fig. 3. Evidence of the SRF binding activity to multiple SREs. The CC[A/T]6GG sequences of six oligonucleotide pairs used as targets in these electrophoretic mobility shift assays include c-fos SRE (-CCATATTAGG-), junB SRE (-CCTAATATGG-), ␤-actin SRE (-CCTTATATGG-), EGR2 SRE (-CCTTTTTTGG-), dystrophin SRE (-CCTATTATGG-), and thrombospondin-1 SRE (-CCTTATTTGG-). In A and B, electrophoretic mobility shift assays were performed using 10 ␮g of nuclear extracts prepared from growing 3T3T cells and 0.035 pmol of 32P end-labeled oligonucleotides containing the ␤-actin SRE (A, Lanes 1–6), EGR2 SRE (A, Lanes 7–12), dystrophin SRE (B, Lanes 1–6), or thrombospondin-1 SRE (B, Lanes 7–12), respectively. In both A and B, Lanes 2 and 8 show the binding pattern to the DNA probes obtained with nuclear extracts alone; the SRF-SRE complexes are indicated by the arrow marked SRF. For competition assays, a 50-fold molar excess of unlabeled oligonucleotide was added to the reactions. Lanes 3 and 9 contain the unlabeled SRE oligonucleotide as a competitor. Lanes 4 and 10 contain the c-fos SRE as a competitor. Lanes 5 and 11 contain the c-fos SRE mutant as a competitor. In Lanes 6 and 12,1␮gofthe anti-SRF antibody was added to the binding reactions, generating a supershifted SRE-SRF band with slower mobility (ss-SRF). Lanes 1 and 7 contain the DNA probes without nuclear extracts. The SRF-SRE complexes are presented within the boxed area. cells, mobility shift analysis showed a Ͼ90% reduction in the binding differentiation. It is important to note that differentiation induces a activity of SRF to both junB and c-fos SREs (Fig. 2). Repression of general 35–40% decrease in total protein expression, including both SRF binding activity to the SRE is specific in that the binding of the SP-1 and SRF, in whole cell extract preparations. Therefore, de- SP-1 transcription factor to its DNA domain shows no repression by creased binding activity of the SRF to the junB and c-fos SREs

Fig. 4. Repression of the SRF DNA binding activity to multiple SREs in differentiated 3T3T cells. SRF DNA binding activity of nuclear extracts prepared from either growing 3T3T cells (A and B, Lanes 2, 5, 8, and 11) or differentiated 3T3T adipocytes (A and B, Lanes 3, 6, 9, and 12) and 0.035 pmol of 32P end-labeled oligonucleotides is presented. More specifically, A is c-fos SRE (Lanes 1–3), ␤-actin SRE (Lanes 4–6), EGR2 SRE (Lanes 7–9), and a single SP-1 binding site (Lanes 10–12); B is junB SRE (Lanes 1–3), dystrophin SRE (Lanes 4–6), thrombospondin-1 SRE (Lanes 7–9), or a single SP-1 binding site (Lanes 10–12). Lanes 1, 4, 7, and 10 in A and B contain the DNA probes without nuclear proteins. The arrow indicates the major SRF-SRE complex that is present within the boxed area. RG, rapidly growing cells; NTD, NTD cells. 3798

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. DIFFERENTIATION EFFECTS ON SRE-SRF INTERACTIONS correlates with the previously described loss of transcriptional activation function of these elements during adipocyte differentiation (9). Adipocyte Differentiation Represses SRF Binding Activity to

Multiple SRE Motifs. The sequence motif CC[A/T]6GG, which is the binding site for SRF, is also called the CArG box (12). It is found not only in the promoters of serum-inducible genes involved in controlling proliferation, but also in the promoter regions of some muscle-specific genes. We therefore investigated whether the repression of SRF binding during adipocyte differentiation is characteristic of only a unique subset of SREs or of a broader array of SREs. We synthesized four additional SRE-containing oligonucleotides characteristic of the EGR2, ␤-actin, thrombospondin-1, and dystrophin promoters and used them as the binding targets for mobility shift assays.

It is noteworthy that the [A/T]6 sequence in each of the six SREs is different (25–28). When mobility shift assays were performed using these SRE- oligonucleotides and nuclear proteins extracted from growing 3T3T cells, a common SRF complex formed with each SRE, and the yield of the complex was reduced comparably by excess, unlabeled c-fos SRE, but not by a c-fos SRE mutant. The complex was also supershifted by SRF supershift antibody. These results suggest that all four SREs are recognized by SRF and have binding activities similar to that of the c-fos SRE (Fig. 3). Fig. 5. The kinetics of repression of the SRF DNA binding activity during adipocyte differentiation. Electrophoretic mobility shift assays were performed by using 10 ␮gof Mobility shift assays were next performed to determine whether nuclear extracts and 0.035 pmol of 32P end-labeled oligonucleotides containing either adipocyte differentiation represses the binding activity of SRF to the c-fos SRE (Lanes 1–6)orjunB SRE (Lanes 7–12). Nuclear extracts were prepared from ␤-actin, EGR2, thrombospondin-1, and dystrophin SREs in a manner growing 3T3T cells (Lanes 1, 2, 7, and 8) or differentiating 3T3T cells that were cultured in differentiation-promoting medium for 2 days (Lanes 3 and 9), 4 days (Lanes 4 and 10), comparable to the effect of differentiation on the binding of SRF to 6 days (Lanes 5 and 11), and 8 days (Lanes 6 and 12). In Lanes 1 and 7,1␮gofthe the junB and c-fos SREs. Fig. 4 shows that differentiation indeed anti-SRF antibody was added to the binding reactions, generating a supershifted SRE-SRF complex with lower mobility (ss-SRF) than that of the native SRE-SRF complex marked represses SRF binding to all tested SREs. This suggests that repres- with the arrow labeled SRF. All of these SRF-containing complexes are included in the sion of SRF binding activity by adipocyte differentiation is not limited boxed area. The differentiation status of cells is indicated at the top of the panel (RG, to the c-fos and junB SREs but rather involves a global effect on rapidly growing). multiple SRF binding sites. These results further suggest that the [A/T] portion of the CC[A/T] GG domain is not critically important 6 6 serum and repression of the ability of serum to induce the transcrip- to the effects of differentiation because the [A/T] sequence differs in 6 tion of junB and c-fos. each of the six SREs tested. Differentiation Does Not Repress SRF Binding Activity to SREs Kinetics of Repression of the SRF Binding Activity Correlates in Transformed 3T3T Cells. The CSV3-1 transformed cell line with Expression of the Adipocyte Phenotype. Because adipocyte serves as an outstanding murine model for human cancer because it differentiation is a complex process that involves the sequential mod- expresses multiple defects in the multistep process of differentiation. ulation of many transacting factors that regulate many genes leading A most significant defect is the inability of differentiation to repress to irreversible TD, studies next characterized the kinetics of repression mitogenic responsiveness that is required for TD. In this regard, we of SRF and SRE interactions by differentiation. It has been well have demonstrated that in CSV3-1 cells, differentiation not only fails documented that after the initial induction of the adipocyte differen- to repress mitogenic responsiveness to serum, but it also fails to tiation pathway, 10–15 days are typically required for TD to occur (3, repress the inducibility of the AP-1 factors by serum (7, 8). 5). The first step in the differentiation process involves the establish- We therefore performed a series of experiments on transformed ment of PGA, which requires 3–4 days after the addition of differ- CSV3-1 cells and a spontaneously transformed 3T3T cell line desig- entiation-promoting medium to growing cells. Thereafter, between nated DW1 to analyze SRE-SRF interactions. To assure that differ- days 6 and 8, expression of the nonterminal adipocyte phenotype and entiation-inducing culture conditions per se do not affect the interac- repression of serum inducibility of junB and c-fos develop. Although tions of the SRF and SRE in transformed cells, we first studied the they are highly differentiated, these adipocytes, retain mitogenic re- DW1 transformed 3T3T cell line that cannot differentiate. After such sponsiveness to selected combinations of mitogens (3, 5). Continuous cells are cultured in differentiation medium for more than 8 days, the culture of such adipocytes in differentiation medium to days 10–15 majority of the cells remain undifferentiated. Nuclear extract prepared results in the expression of the TD phenotype wherein adipocytes are from these cells was used in mobility shift assays, and the result shows unresponsive to all physiological mitogens. no reduction of SRF binding activity to the junB or c-fos SREs (Fig. Accordingly, Fig. 5 presents the results of kinetic analysis of the 6). This result rules out the possibility that some factors in the repression of SRF binding to the SRE during the process of adipocyte differentiation-promoting medium influence SRF binding to the SRE. differentiation. Repression of SRF binding to the SRE is not obvious We next investigated the effects of differentiation of CSV3-1 cells within the first 4 days after the addition of differentiation-promoting on the binding of SRF to junB and c-fos SREs. Fig. 7 presents the data medium, when the majority of cells reside in the PGA state. Only after showing that differentiation in CSV3-1 cells fails to repress SRF the expression of the nonterminal adipocyte phenotype on days 6–8 binding to the junB and c-fos SREs. For these assays, which were is a dramatic repression of SRF binding to the SRE evident. This reproduced multiple times, CSV3-1 cell specimens showed Ͼ75% correlates with the expression of an adipocyte differentiation pheno- adipocyte differentiation. Therefore, aberrant differentiation as ex- type that is associated with repression of mitogenic responsiveness to pressed in CSV3-1 cells fails to repress (a) mitogenic responsiveness; 3799

differences between the effect of differentiation on SRF-SRE interactions of nontransformed and transformed cells.

DISCUSSION The multistep process of adipocyte differentiation in nontransformed 3T3T cells progressively represses mitogenic responsiveness (3, 5, 9). NTD adipocytes specifically show a significant repression in their mitogenic responsiveness to serum and PDGF. Whereas 5–10% serum induces Ͼ95% of quiescent undifferentiated cells to undergo mitogenesis, Ͻ20% of NTD cells are induced to proliferate under these conditions. Similarly, treatment with 10 ng/ml PDGF and 50 ␮g/ml insulin induces Ͼ70% of quiescent undifferentiated cells to grow, but Ͻ15% of NTD cells are induced to undergo DNA synthesis. Nevertheless, NTD adipocytes can be efficiently induced to proliferate when they are treated with high concentrations of combinations of mitogens. Therefore, reduced mitogenic responsiveness can be used to clearly distinguish NTD cells from TD cells, which, by definition, cannot be induced by mitogens to proliferate. A major research focus of our laboratory for the past several years has been to establish the molecular mechanisms that mediate the progressive repression of mitogenic responsiveness by differentiation. We established previously that adipocyte differentiation repressed the AP-1 DNA binding activity of the Jun and Fos transcription factors

Fig. 6. Culture of transformed DW1 3T3T cells in differentiation-promoting medium and that differentiation represses the serum-inducible transcription of does not repress SRF DNA binding activity. Electrophoretic mobility shift assays were junB and c-fos genes (9). Previous reports also established that neo- performed by using 10 ␮g of nuclear extracts and 0.035 pmol of 32P end-labeled plastic transformation blocks the ability of differentiation to repress oligonucleotides containing either junB SRE (Lanes 1–5)orc-fos SRE (Lanes 6–10). Nuclear extracts were prepared from growing 3T3T cells (Lanes 2 and 7) or differentiated mitogenic responsiveness (7, 8). 3T3T cells (Lanes 3 and 8) and from transformed DW1 cells that were cultured in 10% BCS (Lanes 4 and 9) or in differentiation-promoting medium for 8 days (Lanes 5 and 10). Lanes 1 and 6 contain the DNA probes without nuclear extracts. The SRF-SRE complexes are presented within the boxed area and indicated by the arrow marked SRF. RG, growing 3T3T cells; NTD, NTD 3T3T cells; G, transformed DW1 cells growing in 10% BCS; D, DW1 cells cultured in differentiation-promoting medium.

(b) the serum-dependent inducibility of Jun and Fos expression; and (c) the interaction of SRF and SRE. Differentiation Blocks SRF Nuclear Localization in Nontrans- formed Cells but not in Transformed Cells. There are many possible molecular mechanisms that could explain how differentiation represses SRF-SRE interactions. However, our results suggest that adipocyte differentiation restricts SRF-SRE interactions by blocking the nuclear localization of SRF in 3T3T cells, but not in CSV3-1 cells. Fig. 8 documents this fact using immunofluorescence methods. The data specifically show that SRF is clearly localized to the nucleus in undifferentiated 3T3T cells, but not in differentiated 3T3T cells. Differentiation-induced inhibition of SRF nuclear localization is not a generalized phenomenon because Fig. 8 also shows that differentiation does not block the nuclear localization of the SP-1 transacting factor. Because the staining of differentiated 3T3T adipocytes with the anti-SRF antibody shows a high immunofluorescence background that makes it difficult to illustrate the clear lack of nuclear staining, Western blotting methods were also used to characterize the relative amount of SRF in whole cell versus nuclear preparations of undifferentiated and differentiated nontransformed 3T3T cells and neoplasti- Fig. 7. Differentiation of SV40-transformed CSV3-1 cells does not repress SRF DNA binding activity. Electrophoretic mobility shift assays were performed using 10 ␮gof cally transformed CSV3-1 cells. Fig. 9 clearly shows that differenti- nuclear extracts prepared from either growing (Lanes 2, 3, 7, and 8) or differentiated ation of nontransformed 3T3T cells blocks the nuclear localization of (Lanes 4, 5, 9, and 10) SV40-transformed CSV3-1 cells and 0.035 pmol of 32P end-labeled SRE, whereas differentiation fails to block SRF nuclear localization in oligonucleotides containing either junB SRE (Lanes 1–5)orc-fos SRE (Lanes 6–10). Lanes 1 and 6 contain the DNA probe without nuclear proteins. In Lanes 3, 5, 8, and 10, transformed CSV3-1 cells. 1 ␮g of the anti-SRF antibody was added to the binding reactions, generating a super- These results strongly suggest that differentiation represses SRF- shifted SRE-SRF complex with lower mobility (ss-SRF) than the native SRE-SRF complex marked with the arrow labeled SRF. All of these SRF-containing complexes are SRE interactions in nontransformed 3T3T cells by blocking the ability included in the boxed area. The differentiation status of cells is indicated at the top of the of SRF to localize to the nucleus. In addition, these results explain the panel (RG, rapidly growing cells). 3800

Fig. 8. Decreased nuclear localization of SRF in differentiated cells demonstrated by indirect immunofluorescence microscopy. The localization of SRF and SP-1 in growing undifferentiated 3T3T cells and in differentiated 3T3T adipocytes was evaluated using rabbit anti-SRF or anti-SP-1 antibodies and fluorescence-labeled secondary antibodies. As determined by immunofluorescence microscopy, the nuclear localization of SP-1 can be clearly detected in undifferentiated growing 3T3T cells (A) and in differentiated 3T3T adipocytes (B). In contrast, SRF shows a strong nuclear localization in undifferentiated 3T3T cells (C), but minimal nuclear localization in differentiated 3T3T adipocytes (D). Due to significant background staining with the anti-SRF antibody in 3T3T adipocytes that contain numerous fat droplets, the repression of SRF nuclear localization is difficult to illustrate, but it is easily documented using biochemical methods (see Fig. 9).

The current studies were designed to test the hypothesis that an interactions accounts, at least in part, for the differentiation-induced essential mechanism underlying the ability of differentiation of non- transcriptional repression of growth response genes and might explain transformed 3T3T cells to repress mitogenic responsiveness and the the loss of mitogenic responsiveness that is seen in differentiated cells. serum-inducible transcription of junB and c-fos factors involves mod- This conclusion is supported by the fact that differentiation repressed ulation of the interaction of the SRE and the SRF. We first tested the ability of SRF to bind to the SREs from three other immediate whether adipocyte differentiation represses the activity of SREs in early genes: (a) EGR2; (b) thrombospondin-1; and (c) ␤-actin. Be- transient transfection assays using 3T3T cells and showed that adipo- cause many additional immediate early genes carry SREs within their cyte differentiation markedly represses the serum inducibility of regulatory regions, such as EGR1, cyr61, pip92, and zif-268 (29), pjunB SREtk/CAT and pc-fos SREtk/CAT activities. We next estab- their serum inducibility might also be repressed by adipocyte differ- lished that adipocyte differentiation represses the junB and c-fos entiation. In addition to its role in regulating the activation of many SRE-SRF interactions using mobility shift and gel supershift assays. immediate early genes, some studies suggest that SRF-SRE interac- The effects of differentiation on SRE-SRF interactions were also tion might be important and necessary in regulating cell cycle pro- established not to reflect a global repression of DNA-binding factors gression (30). It has been shown that inhibiting the interaction of because differentiation does not significantly alter the interaction of SRF-SRE through microinjection of anti-SRF antibodies or injection SP-1 with its DNA binding site. Furthermore, the ability of differen- of SRE oligonucleotides efficiently blocks the entry of stimulated tiation to repress SRE-SRF interaction correlates with the kinetics of fibroblasts into S phase (30). In fact, DNA synthesis is inhibited even the transcriptional repression of the serum inducibility of junB and when cells are injected 8–15 h after serum stimulation. This suggests c-fos by differentiation. This suggests that the disruption of SRF-SRE that SRF-SRE interactions are continuously involved and required in the proliferation pathway. SRF-SRE interactions are also involved in regulating the expression of several muscle-specific genes. Therefore, the data showing the repression of SRF binding to the muscle-specific dystrophin gene SRE by adipocyte differentiation is not surprising because myogenic differentiation and adipocyte differentiation are two highly distinct biological processes. How does nonterminal adipocyte differentiation repress SRE-SRF interactions? A series of possibilities exists. Differentiation could modify SRF by phosphorylation to restrict SRE-SRF interactions, as has been demonstrated in other biological systems (18, 20, 21). Next, differentiation could induce the expression of a SRE-binding factor to compete with SRF for its DNA binding site. Such factors could include YY1, myogenin, Nkx-2.5, or E12 (31–34). Differentiation could also repress SRF transcription or translation so that it would not be available to bind SRE. In addition, differentiation could restrict the Fig. 9. Differentiation-induced repression of the nuclear localization of SRF in 3T3T nuclear localization of SRF so that it could not physically bind to cells but not in SV40-transformed 3T3T cells as determined by Western blotting. Equal SREs. In this regard, cell differentiation has been previously shown to amounts of cellular or nuclear protein from growing undifferentiated cells (RG)onNTD cells (NTD) were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and to modulate subcellular localization of N-myc; it is usually localized to Western blotting. SRF was then identified by probing the nitrocellulose membrane with the nucleus of embryonic neural tissues, but it accumulates in the anti-SRF antibody. The data show that differentiation has only a minimal effect on total cellular SRF expression and that differentiation blocks SRF nuclear localization in cytoplasm upon differentiation of specific classes of neurons (35). nontransformed 3T3T cells, but not in transformed CSV3-1 cells. Similar effects have been reported for human D4S234 (36). The data 3801

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. DIFFERENTIATION EFFECTS ON SRE-SRF INTERACTIONS in this study further suggest that adipocyte differentiation in 3T3T 14. Hill, C. S., Wynne, J., and Treisman, R. Serum-regulated transcription by serum cells represses SRF-SRE interactions by blocking the nuclear local- response factor (SRF): a novel role for the DNA binding domain. EMBO J., 13: 5421–5432, 1994. ization of SRF. How this differentiation-dependent effect is mediated 15. Watson, D. K., Robinson, L., Hodge, D. R., Kola, I., Papas, T. S., and Seth, A. FLI1 remains unknown, but it might involve modification of the nuclear and EWS-FLI1 function as ternary complex factors and ELK1 and SAP1a function as ternary and quaternary complex factors on the EGR1 promoter serum response localization signal sequence that exists within SRF (37). elements. Oncogene, 14: 213–221, 1997. Our discovery that differentiation of transformed CSV3-1 cells 16. Pellegrini, L., Tan, S., and Richmond, T. J. Structure of serum response factor core cannot repress SRF-SRE interactions has the potential to be an im- bound to DNA. Nature (Lond.), 376: 490–498, 1995. 17. Spencer, J. A., and Misra, R. P. Expression of the serum response factor gene is portant discovery, especially from the perspective of previously pub- regulated by serum response binding sites. J. Biol. Chem., 271: 16535–16543, 1996. lished data showing that differentiation of CSV3-1 cells fails to 18. Misra, R. P., Rivera, V. M., Wang, J. M., Fan, P. D., and Greenberg, M. E. The serum repress the serum inducibility of AP-1 factors (38), whereas treatment response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts. Mol. Cell. Biol., 11: 4545–4554, 1991. of CSV3-1 cells with atypical mitogens can induce junB expression 19. Gauthier-Rouviere, C., Vandromme, M., Lautredou, N., Cai, Q. Q., Girard, R., (39). The discovery that differentiation of transformed CSV3-1 cells Fernandez, S., and Lamb, N. The serum response factor nuclear localization signal: does not block the nuclear localization of SRF as occurs in nontrans- general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation. Mol. Cell. Biol., 15: 433–444, 1995. formed cells suggests that neoplastically transformed cells may ex- 20. Marais, R. M., Hsuan, J. J., McGuigan, C., Wynne, J., and Treisman, R. Casein kinase press lesions in the molecular mechanisms that regulate the nuclear II phosphorylation increases the rate of serum response factor-binding site exchange. EMBO J., 11: 97–105, 1992. transport of critical regulatory factors. Our findings are in accord with 21. Atadja, P. W., Stringer, K. F., and Raibowol, K. T. Loss of serum response element- other reports showing that the nuclear localization of other transacting binding and hyperphosphorylation of serum response factor during cellular aging. factors can be modified during carcinogenesis. These include the Mol. Cell. Biol., 14: 4991–4999, 1994. ␬ 22. Johansen, F-E., and Prywes, R. Two pathways for serum regulation of the c-fos serum glucocorticoid receptor, ISGF-3, nuclear factor B, and AP-1 factors response element require specific sequence elements and a minimal domain of serum (37). It has also been reported that the tumor suppressor effects of response factor. Mol. Cell. Biol., 14: 5920–5928, 1994. wild-type p53 can be inactivated by blocking its nuclear localization 23. Hill, C. S., Wynne, J., and Treisman, R. The Rho family GTPase RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell, 81: 1159–1170, 1995. in selected cases of breast cancer and neuroblastoma (40). We there- 24. Subramaniam, M. L., Schmidt, L. H., Crutchfield, C. E., and Getz, M. J. Negative fore favor the concept that many cancers may express defects in the regulation of serum-response enhancer elements. Nature (Lond.), 340: 64–66, 1989. ability of the nuclear membrane and/or nuclear pores to correctly 25. Chavrier, P., Janssen-Timmen, U., Mattei, M-G., Zerial, M., Bravo, R., and Charnay, P. Structure, chromosome location and expression of the mouse zinc finger gene control and maintain the critical balance of key regulatory factors in Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos. the nucleus. Mol. Cell. Biol., 9: 787–797, 1989. 26. Frederickson, R. M., Micheau, M. R., Iwamoto, A., and Miyamoto, N. 5Ј flanking and first intron sequences of the human ␤-actin gene required for efficient promoter ACKNOWLEDGMENTS activity. Nucleic Acids Res., 17: 253–270, 1989. 27. Galvagni, F., Lestingi, M., Cartocci, E., and Oliviero, S. Serum response factor and We thank Wanda Patrick and Joyce Baker for assistance in typing the protein-mediated DNA bending contribute to transcription of the dystrophin muscle- specific promoter. Mol. Cell. Biol., 17: 1731–1743, 1997. manuscript and preparing the figures and Robin Cox for technical advice. 28. Framson, P., and Bornstein, P. A serum response element and a binding site for NF-Y mediate the serum response of the human thrombospondin 1 gene. J. Biol. Chem., 268: 4989–4996, 1993. REFERENCES 29. Spencer, J. A., and Misra, R. P. Expression of serum response factor gene is regulated 1. Wilke, M. S., Hsu, B. M., Wille, J. J., Pittelkow, J. R., and Scott, R. E. Biologic by serum response factor binding sites. J. Biol. Chem., 271: 16535–16543, 1996. mechanisms for the regulation of normal human keratinocyte proliferation and 30. Gauthier-Rouviere, C., Cavadore, J-C., Blanchard, J-M., Lamb, N. C., and Fernandez, differentiation. Am. J. Pathol., 131: 171–181, 1988. A. p67 SRF is a constitutive nuclear protein implicated in the modulation of genes 2. Schneider, J. W., Gu, W., Zhu, L., Mahdavi, V., and Nadal-Ginard, B. Reversal of required throughout the G1 period. Cell Regul., 2: 575–588, 1991. terminal differentiation mediated by p107 in RbϪ/Ϫ muscle cells. Science (Wash- 31. Chen, C-Y., and Schwartz, R. J. Competition between negative acting YY1 versus ington DC), 264: 1467–1471, 1994. positive acting serum response factor and tinman homologue Nkx-2.5 regulates ␣ 3. Scott, R. E., Hoerl, B. J., Wille, H. H., Florine, D. L., Krawisz, B. R., and Yun, K. cardiac -actin promoter activity. Mol. Endocrinol., 11: 812–822, 1997. Coupling of proadipocyte growth arrest and differentiation: a cell cycle model for the 32. Gualberto, A., Page, D., Pons, G., Mader, S. L., Park, K., Atchison, M. L., and Walsh, physiological control of cell proliferation. J. Cell Biol., 94: 400–405, 1982. K. Functional antagonism between YY1 and the serum response factor. Mol. Cell. 4. McKnight, L. S. CCATT/enhancer binding protein. In: S. L. McKnight and K. R. Biol., 12: 4209–4214, 1992. Yamamoto (eds.), Transcriptional Regulation, pp. 771–795. Cold Spring Harbor, NY: 33. Groisman, R., Masutani, H., Leibovitch, M-P., Robin, P., Soudant, I., Trouche, D., Cold Spring Harbor Laboratory Press, 1992. and Harel-Bellan, A. Physical interaction between the mitogen-responsive serum 5. Hoerl, B. J., and Scott, R. E. Nonterminally differentiated cells express decreased response factor and myogenic basic-helix-loop-helix proteins. J. Biol. Chem., 271: growth factor responsiveness. J. Cell. Physiol., 139: 68–75, 1989. 5258–5264, 1996. 6. Wier, M. L., and Scott, R. E. Regulation of the terminal event in cellular differenti- 34. Chen, C. Y., and Schwartz, R. J. Recruitment of the tinman homolog Nkx-2.5 by ation: biological mechanisms of the loss of proliferative potential. J. Cell Biol., 102: serum response factor activate cardiac ␣-actin gene transcription. Mol. Cell. Biol., 16: 1955–1964, 1986. 6372–6384, 1996. 7. Estervig, D. N., Minoo, P., Tzen, C-Y., and Scott, R. E. Three distinct effects of 35. Wakamatsu, Y., Watanabe, Y., Shimono, A., and Kondoh, H. Transition of localiza- SV40-T antigen gene transfection on cellular differentiation. J. Cell. Physiol., 142: tion of the N-Myc protein from nucleus to cytoplasm in differentiating neurons. 552–558, 1990. Neuron, 10: 1–9, 1993. 8. Wang, H., and Scott, R. E. Unique and selective mitogenic effects of vanadate on 36. Carlock, L., Vo, T., Lorincz, M., Walker, P. D., Bessert, D., Wisniewski, D., and SV40 transformed cells. Mol. Cell. Biochem., 153: 59–67, 1995. Dunbar, J. C. Variable subcellular localization of a neuron-specific protein during 9. Wang, H., and Scott, R. E. Adipocyte differentiation selectively represses the serum NTera2 differentiation into post-mitotic human neurons. Mol. Brain Res., 42: 202– inducibility of c-jun and jun-B by reversible transcription-dependent mechanisms. 212, 1996. Proc. Natl. Acad. Sci. USA, 91: 4649–4653, 1994. 37. Jans, D. A., and Hobner, S., Regulation of protein transport to the nucleus: central role 10. Seshadri, T., and Campisi, T. Repression of c-fos transcription and an altered genetic of phosphorylation. Physiol. Rev., 76: 651–685, 1996. program in senescent human fibroblasts. Science (Washington DC), 247: 205–209, 38. Wang, H. L., Xie, Z., and Scott, R. E. Differentiation modulates the balance of 1990. positive and negative Jun/AP-1 DNA binding activities to regulate cellular prolifer- 11. Kitabayashi, I., Kawakami, Z., Matsuoka, T., Chiu, R., Gachelin, G., and Yokoyama, ative potential: different effects in nontransformed and transformed cells. J. Cell Biol., K. Two cis-regulatory elements that mediate different signaling pathways for serum- 135: 1151–1162, 1996. dependent activation of the jun-B gene. J. Biol. Chem., 268: 14482–14489, 1993. 39. Wang, H. Wang, J-Y., Johnson, L. R., and Scott, R. E. Selective induction of c-jun 12. Treisman, R. The serum response element. Trends Biochem. Sci., 17: 423–426, 1992. and junB but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the 13. Treisman, R. Structure and function of serum response factor. In: S. L. McKnight and predifferentiation growth arrest state. Cell Growth Differ., 2: 645–652, 1991. K. R. Yamamoto (eds.), Transcriptional Regulation, pp. 881–905. Cold Spring 40. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323–331, Harbor, NY: Cold Spring Harbor Laboratory Press, 1992. 1997.

3802

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1999 American Association for Cancer Research. Transformation Blocks Differentiation-induced Inhibition of Serum Response Factor Interactions with Serum Response Elements

Wei Ding, Michael M. Witte and Robert E. Scott

Cancer Res 1999;59:3795-3802.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/59/15/3795

Cited articles This article cites 37 articles, 19 of which you can access for free at: http://cancerres.aacrjournals.org/content/59/15/3795.full#ref-list-1

Citing articles This article has been cited by 4 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/59/15/3795.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/59/15/3795. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.