Oncogene (2001) 20, 1135 ± 1141 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc SHORT REPORT Transcription factor ATF3 partially transforms chick embryo ®broblasts by promoting growth factor-independent proliferation

Sandrine Perez1, Emmanuel Vial1, Hans van Dam2 and Marc Castellazzi*,1

1Unite de Virologie Humaine, Institut National de la Sante et de la Recherche MeÂdicale (INSERM-U412), Ecole Normale SupeÂrieure, 46 alleÂe d'Italie, 69364 Lyon Cedex 07, France; 2Department of Molecular Cell Biology, Leiden University Medical Center, Sylvius Laboratories, 2300 RA Leiden, The Netherlands

Activating Transcription Factor 3 (ATF3) is a member levels and activities of the various Jun, Fos, ATF and of the bZip family of transcription factors. Previous Maf monomers present. AP1 complexes mediate studies in mammalian cells suggested that like other cellular responses to a wide variety of extracellular bZip family members e.g. Jun and Fos, ATF3 might play stimuli, including growth factors, cytokines and agents a role in the control of cell proliferation and participate inducing genotoxic stress (Angel and Karin, 1991; in oncogenic transformation. To investigate this putative Karin et al., 1997; Wisdom, 1999). ATF3 function directly, the rat ATF3 protein was Comparison of the ATF3 sequences from rat (Hsu et compared with v-Jun for its ability to transform primary al., 1991), mouse (Drysdale et al., 1996) and man (Hai cultures of chick embryo ®broblasts (CEFs). Like CEFs et al., 1989) shows that the gene displays a high degree accumulating v-Jun, CEFs accumulating the ATF3 of conservation in vertebrates. Recently, an ATF3 protein displayed a typical, fusiform morphology, homolog has also been isolated in the Japanese associated with an enhanced capacity to grow in medium pu€er®sh Fugu rubripes (Trower et al., 1996; Venkatesh with reduced amount of serum. However, in contrast to et al., 2000). In rodents, ATF3 is expressed at relatively v-Jun-transformed CEFs, the ATF3 overexpressing cells low level in most cell types (Freeman et al., 1998) with could not promote colony formation from single cells in the exception of skeletal muscle, intestine, and stomach agar. Partial transformation induced by ATF3 was found tissues (Hsu et al., 1991). At present, most of the to be associated with repression of multiple cellular genes signalling pathways that activate ATF3, speci®c that are also down-regulated by v-Jun, including those functions of the various ATF3-containing dimers, and coding for the extracellular components ®bronectin, speci®c ATF3 target genes remain to be elucidated decorin, thrombospondin 2, and the pro-apoptotic protein (Hai et al., 1999). Par-4. These data demonstrate that, at least in primary ATF3 shares several properties with the oncoprotein avian cells, rat ATF3 possesses an intrinsic oncogenic Jun, suggesting a role in the control of cell prolifera- potential. Moreover, the results suggest that ATF3 might tion. ATF3 mRNA rapidly and transiently accumulates induce growth factor independence by down-regulating a in response to serum (Mohn et al., 1991), Epidermal subset of the genes repressed by v-Jun. Oncogene (2001) Growth Factor and Fibroblast Growth Factor (Weir et 20, 1135 ± 1141. al., 1994), by various genotoxic stresses (Amundson et al., 1999; Chen et al., 1996; Hai et al., 1999), and after Keywords: ATF3; Jun; AP1; chicken embryo ®bro- partial hepatectomy in the regenerating liver (Chen et blasts; cell transformation al., 1996; Hsu et al., 1992). Like Jun, ATF3 might also be functionally involved in oncogenic transformation, as: (i) transformation by adenoviral protein 12S-E1A is Introduction accompanied by transcriptional activation of both c-jun and atf3 (Hagmeyer et al., 1996); and (ii) ATF3 Activating Transcription Factor 3 (ATF3; also known expression appears to be required for the maintenance as Liver Regeneration Factor 1 or LRF1; Hsu et al., of a high-metastatic state in some cancer cell lines 1991) is a member of the ATF/CREB subfamily of (Ishiguro et al., 1996, 2000). bZip transcription factors (Hai et al., 1989). ATF3 In the present study we examined whether ATF3 speci®cally binds to the 8 bp ATF/CREB consensus possesses intrinsic oncogenic potential by itself. We, motif 5'-TGACGTCA and related sequences (Hai and therefore, stably overexpressed rat ATF3 in primary Curran, 1991) and therefore contributes to the dimeric cultures of chick embryo ®broblasts (CEFs) using the transcription factor AP1 complexes. The total AP1 retroviral, self-replicating vector Rcas. In this avian binding activity in the cell is determined by the relative model of oncogenesis, single, virally-expressed, AP1 components from avian and mammalian origin can eciently transform primary cells without the need for (an)other cooperating oncoprotein(s) (Bos et al., 1990; *Correspondence: M Castellazzi Received 27 September 2000; revised 7 December 2000; accepted 19 Castellazzi et al., 1990; Vogt, 1994; Maki et al., 1987), December 2000 in contrast to mammalian cells (Hahn et al., 1999; ATF3-induced cell transformation S Perez et al 1136 SchuÈ tte et al., 1989). CEF cultures constitutively dependent promoters 56jun2-tata-luciferase, 56coll- overexpressing either ATF3 or v-Jun were compared TRE-tata-luciferase, or the corresponding tata-lucifer- with respect to their oncogenically transformed features ase control. 56jun2-tata contains ®ve copies of the in vitro and in vivo and, in addition, for the expression high-anity c-Jun:ATF2 binding site from the human levels of v-Jun target genes. c-jun promoter which eciently binds ATF3 (Hag- meyer et al., 1996). 56collTRE-tata contains ®ve copies of the proximal, high anity Fos : Jun binding Results and Discussion site from the human collagenase promoter (Materials and methods; van Dam et al., 1998). As shown in To investigate the putative oncogenic potential of Figure 2a, the activity of the 56jun2-tata and ATF3, cultures of chick embryo ®broblasts were 56collTRE-tata promoters was reduced in R-ATF3- chronically infected with either R-ATF3, a retroviral infected CEFs, respectively by 80 and 20%, when expression vector encoding rat ATF3, or, with the compared to the control R-infected CEFs. This positive and negative control vectors R-v-Jun or R. In repression by ATF3 on `jun2' ATF sites rather than the R-ATF3 infected CEFs, two major ATF3 proteins on `collTRE' Fos:Jun sites was con®rmed when were detected with an apparent molecular weight of 27 increasing amounts of ATF3 were transfected in non- and 28 kDa, which is in agreement with the size of the infected CEFs (Figure 2b). In some independently ATF3 proteins detected in mammalian cells (Hagmeyer generated primary cultures ATF3 slightly induced the et al., 1993; Hai et al., 1989) (Figure 1). In addition, a 56collTRE Fos:Jun containing model promoter, faster migrating band of about 17 kDa was observed. explaining the high standard deviation depicted in The intensity of this minor band varied from one cell Figure 2b. This induction is likely to be due to extract to another and is likely to correspond to a ATF3:Jun heterodimers, as they have been reported degradation product in CEFs. As described previously, to be able to bind to and transactivate transcription via the endogenous c-Jun levels are down-regulated in v- a consensus TRE (Hai and Curran, 1991; Hsu et al., Jun expressing CEFs (Castellazzi et al., 1990; Gao et 1991). We conclude from these data that in CEFs, like al., 1996; Kilbey et al., 1996). in mammalian cells, excess of ATF3 can repress ATF3 has been found to function as a transcrip- transcription via ATF sites. tional repressor on various promoters containing ATF We next examined the e€ect of ectopic expression of binding sites (Chen et al., 1994; Wolfgang et al., 1997, ATF3 on CEF proliferation and morphology. As 2000). To verify that the rat ATF3 protein was reported previously for R and R-v-Jun (Bos et al., functional in CEFs, transient transfection experiments 1990; Jurdic et al., 1995), infection with R-ATF3 did not were performed using the minimal ATF- or AP1- a€ect cell viability. When grown in normal medium (6% serum), all cultures became senescent after 1 ± 1.5 months in culture. However R-ATF3 and R-v-Jun-infected CEFs exhibited a more fusiform shape when compared to R-infected and to non-infected CEFs (not shown). When plated at low density (16103 cells per 100 mm plate), non-infected, R-, and R-ATF3-infected CEFs developed small foci at a frequency of 14.5, 10 and 9.5%, respectively, whereas R-v-Jun-infected CEFs gave rise to large, dense foci at an average frequency of 39.5% (Figure 4; left). When plated at a higher density (1.56105 cells per 100 mm plate), the plating eciency was the same for all cultures (80 ± 100%). Under these condi- tions, cell proliferation was measured over the following 10 days (Figure 3). The rat ATF3 expressing CEFs were found to proliferate faster than the non-transformed cultures but slower than the highly transformed, v-Jun- Figure 1 Detection of the virally-expressed v-Jun and ATF3 expressing cells. This became apparent both from the proteins in CEF cells by immunoblotting. The arrows point to the virally-expressed v-Jun, the endogenous c-Jun, and the three culture doubling times (26, 19 and 16 h for R, R-ATF3, forms of the rat ATF3. Preparation of cell extracts for Western and R-v-Jun-infected CEFs, respectively) and from the blotting was performed as described (Castellazzi et al., 1990). saturation densities (36106,56106, and 106106 con- Brie¯y, 10 ± 30 mg protein from cell extract was resolved by SDS/ tact-inhibited cells per plate). These data indicate that 10%-polyacrylamide gel electrophoresis and blotted onto nitro- ectopic expression of ATF3 in CEF induces both a cellulose membranes. For detection of rat ATF3 a commercial antibody against the C terminal fragment of mammalian ATF3 change in cell morphology as well as enhanced was used (Santa Cruz; sc # 188x). A rabbit anti-Jun antibody proliferation potential in normal medium. recognizing both avian c-Jun and v-Jun has been generated Next, the R-, R-ATF3 and R-v-Jun infected CEF against a GST-v-Jun fusion protein (Baguet and Castellazzi; cultures were analysed for their capacity to proliferate unpublished). The peroxidase-coupled secondary anti-rabbit anti- body was purchased from Rockland (PA, USA). Quanti®cation in low-serum medium and to form colonies in soft of the peroxidase activity was done by using the `BM agar, two main characteristics of in vitro transforma- chemiluminescence blotting substrate' (cat no. 1 500 708; Roche) tion. As shown in Figure 3, infection with R-ATF3

Oncogene ATF3-induced cell transformation S Perez et al 1137

Figure 2 ATF3 represses ATF-dependent promoters in CEFs. (a) CEFs chronically infected either with R or R-ATF3 were transiently transfected with the reporter constructs 56jun2-tata-luciferase, 56collTRE-tata-luciferase, or tata-luciferase. Data were from three independent experiments with di€erent primary cultures. (b) Normal CEFs were transiently transfected with the same constructs together with increasing amounts of the expression vector pDP or pDP-ATF3 (0, 0.5, 1 and 2 mg per 60 mm plate) as indicated. Data were from ®ve independent experiments with di€erent primary cultures. To generate the pDP-ATF3 expression vector, a SacI±SalI fragment containing the ATF3 cDNA was recovered from pE-ATF3 and introduced downstream of the Rous sarcoma virus LTR sequence into pDP (Vandel et al., 1995). In these experiments, CEFs were seeded at a density of 3.56105 per 60 mm-diameter plate in normal medium, and transfected 24 h later with 2 mg of either the 56collTRE-tata-luciferase, the 56jun2- tata-luciferase, or the tata-luciferase reporter plasmids (van Dam et al., 1998) using Fugene-6 transfection reagent (Roche). Cell lysates were prepared 40 h after transfection, and luciferase activity was measured by using the `luciferase assay system' (Promega) and the Bradford protein assay (Bio-Rad). To normalize for transfection eciencies, independent plates from the di€erent cultures were routinely transfected with 2 mg pRSV-b gal plasmid, a pBSK derivative in which the b-galactosidase enzyme was under the control of the Rous sarcoma virus 5' LTR. Plates were stained in order to individually visualize and count the b-galactosidase expressing cells as described (Vial et al., 2000). Fold activation represents the ratio 56jun2-tata-luciferase/tata-luciferase and 56collTRE-tata-luciferase/tata-luciferase induced sustained CEF proliferation in low (1%) can induce a partial transformed phenotype in CEF; it serum medium reaching 2.36106 cells per plate after promotes a fusiform morphology and sustained 8 days, instead of 0.56106 cells in the case of the R- proliferation in low serum medium, but cannot induce infected control culture. v-Jun-expressing CEFs pro- anchorage-independent growth. liferated most eciently, reaching 3.56106 per plate We next examined whether R-ATF3-infected CEFs after 6 days. Under these low serum conditions cells can grow out into tumours upon injection in chickens displayed a typical, fusiform shape and were arranged in vivo. For this purpose, the rat ATF3 expressing in parallel arrays (Figure 4), a state originally described CEFs were compared with CEFs infected with R-v- as `fusiform transformation' (Temin, 1960) and typical Jun-m1, a highly tumorigenic variant of v-Jun of Jun transformation (Bos et al., 1990; Castellazzi et (Huguier et al., 1998). The infected CEFs were injected al., 1990). subcutaneously into the wing web of 1-day-old chicks. For the analysis of anchorage-independent growth, As shown in Table 2, R-ATF3-infected cells did not single cells were plated in semisolid medium at di€erent form tumours in contrast to the R-v-Jun-m1-infected cell densities (Table 1). As reported previously (Bos et CEFs. al., 1990; Jurdic et al., 1995), R-v-Jun induced agar As ATF3 and v-Jun were found to induce similar colony formation very eciently, whereas no colonies e€ects on CEF morphology and serum-dependence, we were observed with uninfected (not shown) and R- were interested whether they also had similar e€ects on infected control CEFs. Importantly, the ATF3-expres- CEF gene expression. For this purpose, we analysed sing CEFs formed agar colonies only very ineciently. genes whose mRNA levels were either down- or up- In plates seeded with 16103 cells, 1.7% of the R- regulated in v-Jun(-m1)-transformed CEFs (Vial and ATF3-infected cells formed colonies versus 37% of the Castellazzi, 2000) and which are thought to contribute R-v-Jun-infected cells. Moreover, R-ATF3 solely to oncogenic transformation. These genes encode: (i) induced microcolonies (consisting of no more than 10 the extracellular matrix components ®bronectin (Nor- cells and only visible under the microscope), whereas ton and Hynes, 1987), decorin (Li et al., 1992), the colonies induced by R-v-Jun were large, compact, thrombospondin 2 (Tucker, 1993), collagen XII and clearly visible by eye (Figure 4). Taken together (WaÈ lchli et al., 1994), clusterin (Michel et al., 1989), these data show that ectopic expression of ATF3 only SPARC (Bassuk et al., 1993), a2 (I) collagen (Lehrach

Oncogene ATF3-induced cell transformation S Perez et al 1138 et al., 1978), and lysyl oxidase (Jourdan-Le Saux et al., 1994); (ii) the pro-apoptotic factor Par-4 (Barradas et al., 1999; Qiu et al., 1999; Perez, Herrlich and Castellazzi, unpublished); and (iii) Heparin-Binding Epidermal Growth Factor (Fu et al., 1999). Northern blot analysis for R-, R-ATF3- and R-v-Jun-m1- infected CEFs showed that these genes can be divided in three di€erent groups (Figure 5). The ®rst group consists of ®bronectin, decorin, thrombospondin 2, and par-4, which are down-regulated by both v-Jun and ATF3. The second group contains collagen XII, a2 (I) collagen, clusterin, and SPARC, which are repressed by v-Jun but not by ATF3. The third group contains lysyl oxidase and HB-EGF which are activated by v-Jun but more or less repressed by ATF3. Thus, as could be anticipated from the partial CEF transformation program induced by ATF3, there is not a strict coincidence between genes repressed or activated by ATF3 and genes repressed or activated by v-Jun. Interestingly, HB-EGF is only activated by v-Jun. This observation is in line with the fact that over- expression of HB-EGF induces soft agar growth (Fu et al., 1999) whereas ATF3 does not. The fact that ATF3 does not repress SPARC is in agreement with the ®nding that repression of SPARC is involved in tumour formation in the animal (Vial and Castellazzi, 2000) whereas ATF3 is non tumorigenic. To our knowledge this is the ®rst report demonstrat- ing that rat ATF3 per se possesses oncogenic potential in primary cells. Previous studies in mammalian cells only indirectly suggested a role for ATF3 in cell proliferation and oncogenesis. When compared to v- Jun, the transformed phenotype triggered by ATF3 in CEFs is only partial and restricted to an altered morphology and enhanced capacity to proliferate under reduced serum concentration. ATF3 does not signi®cantly stimulate anchorage-independent growth in agar, nor does it promote tumour development in vivo. Recently, we have proposed a two-pathway hypothesis for transformation by Jun in CEFs, i.e. Figure 3 Representative growth curves of CEF cultures chroni- cally infected by the empty Rcas-envA (=R) vector, or by the uncontrolled Jun:Fra2 activity causing anchorage- Rcas vectors expressing v-Jun or ATF3. Cultures were grown independent growth via genes regulated through 7 bp either in normal medium (supplemented with 6% serum; upper Jun:Fos binding sites and uncontrolled Jun:ATF2 ®gure) or in medium with reduced amount of serum (supple- activity triggering autocrine growth via genes regulated mented with 1% serum; lower ®gure) as indicated. Initial plating by 8 bp ATF sites (Huguier et al., 1998; van Dam et was always at 1.56105 cells per plate. Identical results were obtained in ®ve experiments with di€erent primary cultures. al., 1998). The ability of rat ATF3 to speci®cally Primary CEF cultures were obtained from 8-day-old, virus-free O activate growth in low serum, and not growth in agar, line chicken embryos (Institute for Animal Health, Compton, might, therefore, be related to its ability to di€eren- Berks, UK) and grown in regular medium made of Ham's F10 tially regulate Jun:Fos- and Jun:ATF2-dependent medium (Eurobio) supplemented with 10% tryptose phosphate genes. Moreover, in mammalian cells the ATF3 gene broth (Difco), 5% foetal calf serum (Roche) and 1% chicken can in fact be induced by Jun:ATF2 via an ATF serum (Sigma), 0.196% NaCO3, 1% penicillin and streptomycin, and 1.25 g ml71 amphotericin B. ATF3 and v-Jun expressing binding site in its promoter region (Liang et al., 1996). cultures were obtained by chronic infection with the Rcas Although an avian version of the ATF3 gene has not derivatives. Routinely, transfections with R (no insert), R-ATF3 and R-v-Jun plasmid DNAs were performed after the ®rst passage using the Fugene-6 transfection reagent (Roche) and viruses were allowed to spread through the entire population over the following week to generate fully infected CEF cultures. To generate R-ATF3 plasmid DNA, a 712 base pair NaeI±XbaI retroviral vector Rcas envA (designated R) (Hugues et al., 1987). fragment containing the rat ATF3 cDNA (Hsu et al., 1991) was The R-v-Jun plasmid has already been described (Jurdic et al., inserted into SmaI±XbaI digested pE plasmid (Huguier et al., 1995). Growth curve experiments in normal medium and in low 1998) after which the ATF3 coding sequence was cloned as a serum medium were conducted as previously described (Vial and ClaI±ClaI pE-ATF3 fragment into the replication competent, Castellazzi, 2000)

Oncogene ATF3-induced cell transformation S Perez et al 1139

Figure 4 Focus formation after plating of R-, R-v-Jun-, and R-ATF3-infected cells at low density (left). 16103 cells were plated per 100 mm dish in normal medium, incubated for 10 days, then ®xed with methanol and stained with a mixture of 10% Giemsa and 1% Wright stain (Sigma Accustain) in water to visualize colony growth. Representative microscopic ®elds from R-, R-v-Jun-, and R-ATF3-infected cells grown in agar (middle) or in low-serum containing liquid medium (right). The 2-week-old colonies shown on the middle part resulted from single cells plated at a density of 16104 per 60 mm petri dish, corresponding to the data in Table 1. The pictures depicted on the right part were taken at day 5 after medium change into 1% serum, corresponding to the data in Figure 3 (lower). The bars represent 0.1 mm

Table 1 Colony formation of CEF cultures fully infected with the various Rcas derivatives Number of colonies per plate after plating with Plating efficiency CEFs chronically infected with 16104 cells 56103 cells 26103 cells 16103 cells (%)

R 7/77/77/77/750.01 R-ATF3 202/192* 86* 44/24* 16/14* 1.7 R-v-Jun 42990/43760 1954/1434 980/712 312/450 37

Colonies were counted under the microscope after 2 weeks. Each number represents the number of colonies in a given 60 mm petri dish. Plating eciency refers to the percentage of single cells that developed into colonies. The following symbols were used: *for microcolonies not visible with the maked eye; 7, for absence of colonies

Table 2 Tumour formation from CEF cultures fully infected with been reported so far, preliminary biochemical studies various Rcas derivatives indicate the presence of a mitogen- and stress-inducible Wing tumoursa ATF3-like factor in extracts of CEF cells (Perez, van CEFs chronically Number of chickens habouring a wing Dam and Castellazzi, unpublished observations). infected with tumour/total number of chicks injected Characterization of this avian ATF3-like gene and R 0/3 analysis of its promoter region might provide further R-ATF3 0/3 clues on the mechanism by which Jun and ATF protein R-v-Jun-m1b 3/3 mediate Jun-induced oncogenesis. Finally, an important ®nding of this study is that a16106 infected cells were resuspended in 100 ml PBS and injected subcutaneously into the wing web of 1-day-old chicks and tumour ATF3 transformed CEFs show reduced expression of development was followed over 1.5 months (Garcia and Samarut, some of the cellular mRNAs that are repressed in v- 1990). bThe R-v-Jun-m1 retroviruses were described previously Jun-transformed cells. These data strongly suggest that (Huguier et al., 1998)

Oncogene ATF3-induced cell transformation S Perez et al 1140

Figure 5 Northern blot analysis of v-Jun-target genes using total mRNA from CEFs chronically infected by the retroviruses R, R- v-Jun-ml, and R-ATF3. The arrows indicate the relevant transcripts. The various cDNA used as a probe were from avian origin, except for lysyl oxidase which was from mouse origin (see Vial and Castellazzi, 2000). The avian par-4 probe has been isolated in a search for cellular genes whose expression is altered upon transformation by v-Jun in CEFs (Perez, Herrlich and Castellazzi, unpublished). The avian decorin probe has been isolated in a di€erential screening by PCR-substraction technique between cDNAs from non-transformed and Jun-transformed CEFs (Vial and Castellazzi, unpublished). Total RNA was extracted according to a previously published procedure (Vial and Castellazzi, 2000). Radioactive probes were coding sequences from the various v-Jun target genes as indicated and from the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) as a control

ATF3 transforms CEFs by repressing a subset of genes the Fondation pour la Recherche Me dicale (to S Perez and also repressed by v-Jun. Future research should learn E Vial). It was also funded by grants from the Association whether repression of these genes by v-Jun and ATF3 pour la Recherche sur le Cancer, from the Mutuelle is essential for v-Jun and ATF3-transformed cells to Ge ne rale de l'Education Nationale, from the European grow in an autocrine fashion. Economic Community Biomedicine and Health Program (Biomed II contract no. BMH4 CT98 3505 to M Castellazzi) and from the Netherlands Organization for Abbreviations Scienti®c Research (NWO 901-02-221 to H van Dam). We The abbreviations used are: ATF3, activating transcription thank the following persons for the gift of various avian factor 3; AP1, activating protein 1; CEF(s), chicken cDNA probes: Helene Sage for SPARC, Gilbert Brun for embryo ®broblast(s). clusterin, Beat Trueb for collagen XII, Sherrill L Adams and Fre de ric Mallein-Ge rin for a2 (I) collagen, Jack Lawler for thrombospondin 2, Richard Hynes for ®bro- Acknowledgments nectin, and Peter K Vogt for HB-EGF. We also thank This work was supported by fellowships from the MinisteÁ re Pascal Sommer for the gift of the mouse lysyl oxidase de la Recherche et de la Technologie (to S Perez) and from cDNA.

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