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Overexpression of C-Jun, Junb,Or Jund Affects Cell Growth Differently

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Overexpression of C-Jun, Junb,Or Jund Affects Cell Growth Differently Proc. Nail. Acad. Sci. USA Vol. 88, pp. 8890-8894, October 1991 Cell Biology Overexpression of c-jun, junB, or junD affects cell growth differently (APi factor/growth suppression/in vitro transformation/transcription factor/retroviral vector) MARC CASTELLAZZI*, GIANNIS SPYROUt, NATHALIE LA VISTA*, JEAN-PIERRE DANGY*, FABRICE PIU*, MOSHE YANIVt, AND GILBERT BRUN* *Laboratoire de Biologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique-Unite Mixte de Recherche 49, Ecole Normale Sup6rieure, 46, Allke d'Italie 69364, Lyon, France; and tUnite des Virus Oncogenes, Centre National de la Recherche Scientifique-Unitd de Recherche Associde 1149, Ddpartement des Biotechnologies, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris, France Communicated by Andre Lwoff, June 21, 1991 (receivedfor review March 21, 1991) ABSTRACT The coding sequences of murine c-jun, junB, When quiescent fibroblasts are stimulated to enter the cell or junD, which code for proteins with practically identical cycle, transcription of c-jun and junB is rapidly and tran- dimerization and DNA binding properties, were introduced siently activated in early G1 phase. They are therefore into a nondefective retroviral vector, and the phenotype of considered as "immediate early genes" (9, 10), like the primary avian fibroblasts chronically infected with each of members ofthefos family.junD transcription is only weakly these viruses was studied. Cells expressing c-jun grew in activated under these conditions (11). Furthermore, the three low-serum medium and developed into colonies in agar, two protooncogenes respond differently to activation of protein properties characteristic of in vitro transformation. Cells ex- kinase A- or protein kinase C-dependent signal transduction pressing junB grew in agar, with a reduced efficiency as pathways (11). Differences between the threejun genes were compared to c-jun, but did not grow in low-serum medium. also observed in their interactions with hormone receptors. Finally, no effect of junD expression on cell growth was Although excess c-Jun strongly suppressed estrogen- observed. These different phenotypes suggest that these three dependent transcriptional activation in MCF7 human breast closely related transcription factors play distinct roles during cancer cells, excess JunB only partially repressed estrogen normal cell growth. Analysis of c-jun deletion mutants and of activity, and JunD had no effect at all (12). These properties, c-jun/junB and c-jun/junD chimeric genes showed that the in addition to differential tissue distribution in adult orga- N-terminal portion (amino acids 2-168) of the c-Jun protein nisms (7) and during embryonic development (13), suggest that is involved in transcriptional activation is required for distinct functional roles for the three Jun proteins. efficient transformation. On the contrary, cells expressing a Human c-jun, and to a lesser extent humanjunB, have been truncated mouse c-Jun lacking this N-terminal domain grew shown to transform primary rat embryo cells, but only in slower than normal embryo fibroblasts. The reduced growth cooperation with an activated ras gene (14, 15). In contrast, rate may be related to the finding that expression of the intact overexpression of v-jun or of c-jun from avian, murine, or or the truncated mouse c-jun repressed the endogenous avian human origin was shown to be sufficient for the transforma- c-Jun homologue, suggesting that functional c-Jun product is tion of primary chicken embryo fibroblasts (CEFs) (16-18). required for normal cell growth. In the present study, we took advantage of this avian system to compare directly the oncogenic potential of each of the The c-jun protooncogene was first characterized as the murinejun genes. cellular counterpart of the v-jun oncogene carried by the avian sarcoma virus ASV17 (1, 2). The c-Jun protein together MATERIALS AND METHODS with c-Fos were shown to be components of the activator Vector Construction. The various jun coding sequences protein 1 (AP1) transcriptional complex. c-Jun can form were introduced into the RCAS retroviral vector (noted R) either Jun/Jun homodimers or Jun/Fos heterodimers via the using the intermediate CLA12 adaptor plasmid (19). R-cJUN leucine repeat present in both proteins. Homo- and het- carries a 1148-base-pair (bp) Fsp I-Sca I fragment of the erodimers bind to the same consensus sequence, TGACTCA, murine c-jun gene, R-JUNB carries a 1036-bp Stu I-Ava I present in numerous promoters, initially defined as the phor- fragment of the mouse junB gene, and R-JUND carries a bol 12-myristate 13-acetate response element (3, 4). Two 1065-bp EcoRI-HindIII fragment ofjunD. R-cJUN^169 and other genes closely related to c-jun were characterized in R-cJUNCDL carry, respectively, the CJ169 and the CDL mouse and man, junB (5) and junD (6, 7). The three Jun defective c-jun genes described previously (20). CJ169 is proteins are almost identical in their C-terminal regions, deleted from amino acid 2 to 168. The leucine repeat region which are involved in dimerization and DNA binding, involved in dimerization (amino acids 284-311) has been whereas their N-terminal parts, which are involved in tran- deleted in CDL. c-jun/junB and c-jun/junD chimeric genes scriptional activation, diverge. All three form heterodimers were constructed using the internal Acc I site present in the among themselves or with c-Fos or other members of the three genes (Fig. 6). The corresponding viruses were named c-Fos family. The existence ofgene families with identical or R-JUNCB (with the N-terminal part of c-Jun and the C-ter- very similar DNA binding specificities is not restricted to the minal part ofJunB) and R-JUNBC (opposite configuration) as jun or fos families. It is rather frequent among nuclear well as R-JUNCD and R-JUNDc. proteins (8). One of the interesting issues will be to under- CEF Culture Conditions and Generation of Fully Infected stand the reasons for this diversity. Studies on the three jun Cultures. Cell culture conditions, preparation of primary genes bring some hints in this direction. CEFs, growth curve experiments, DNA transfections, and viral infections were as described (17). To measure plating The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CEF, chicken embryo fibroblast; RSV, Rous sar- in accordance with 18 U.S.C. §1734 solely to indicate this fact. coma virus. 8890 Downloaded by guest on September 25, 2021 Cell Biology: Castellazzi et al. Proc. Natl. Acad. Sci. USA 88 (1991) 8891 efficiency, 1 x 103 cells from each culture were seeded per 108 100-mm dish in regular medium and incubated for 2 weeks. The plates were then fixed with MeOH and stained with Giemsa, and the foci were counted. For the colony-forming - R-JUNB assay, cells were plated in 0.36% Difco agar in regular A R-JUND medium per 60-mm dish, on top of a 0.72% hard agar layer. uninfected Low-serum medium contained 0.5% fetal bovine serum as the only serum supply. To generate primary viral stocks, freshly prepared CEFs were transfected with the various plasmid DNAs carrying RCAS or the R-JUN constructs, expanded 106 for 1 week in regular medium, and the cell culture superna- tants were harvested. These primary stocks (5-10 x 104 infectious particles per ml on average) were used to infect freshly prepared CEFs and to generate within a week fully 0. .0o 105 infected cultures. In all experiments presented in this paper, 8 only such fully infected cultures were analyzed. The per- 8) centage of infected CEFs as well as the number of infectious co particles in the supernatant were routinely estimated using an E 107 -0C immunochemical assay with anti-p27915 antibodies (17). 7 When individual cell clones were isolated from infected o0 cultures and probed for integrated R-JUN genomes, we found no evidence for rearrangements or deletions ofthejun coding sequences (unpublished observations; ref. 17). Antisera. Affinity-purified polyclonal rabbit antisera react- ing against the mouse c-Jun (anti-Jun), JunB, or JunD were R-JUNB prepared as described (12). Anti-Jun antibodies were gener- R-JUND ated against the 143 C-terminal amino acids of the mouse uninfected c-Jun protein beginning at the AAGLAFP sequence. Anti- bodies against JunB (anti-PepB) or against JunD (anti-PepD) were generated, respectively, against the peptides ISYLPHAPPFAGG (amino acids 210-222) and GC- QLLPQHQVPAY (amino acids 329-341). 1 0 20 Western Blotting and Immunofluorescence. Recovery of days cells, Western blotting, and immunofluorescence were done as described (12, 17). FIG. 1. Growth curves of CEF cultures, uninfected or infected with RCAS, R-cJUN, R-JUNB, or R-JUND, in regular medium (A) or in low-serum medium (B). Cell number corresponds to the average RESULTS value of two 100-mm Petri dishes. Phenotypes of CEFs Infected with Retroviruses Carrying c-junjunR, orjunD. CEF cultures fully infected with RCAS, low serum was usually more pronounced with RCAS- R-cJUN, R-JUNB, or R-JUND were generated (see Mate- infected cultures and in elder CEFs, as already reported (17). rials andMethods). They were healthy cultures, growing well Taken together, these data indicate that the R-JUN viruses in regular medium (Fig. lA). CEFs infected with R-cJUN alter to different degrees the growth capacities ofthe infected exhibited slightly enhanced growth potential. This was ob- CEFs. R-cJUN transforms CEFs, by promoting strong cell vious from (i) a shorter latency period after plating (between growth in agar and in low-serum medium. R-JUNB also day 0 and day 2), (ii) a higher saturation density (reaching 20 transforms CEFs, at a lower efficiency, promoting growth in x 106 cells per 100-mm dish compared with 10-15 x 106 with agar but not in low serum.
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