0013-7227/91/1292-1033$03.00/0 Endocrinology Copyright u? 1991 by The Endocrine Society
Vol. 129, No. 2 Printed in U.S.A.
Antimitogenic Effects of Dexamethasone in Chemically Transformed Mouse Fibroblasts DOMINIQUE FAGOT, CHRISTINE BUQUET-FAGOT*, AND JAN MESTER INSERM U.55, 75571 Paris Cedex 12, France
at quiescence certain genes related to the GO/Gl transition in the original nontransformed A31 cell line. Of the transcripts corresponding to these genes, dexamethasone caused a rapid elimination of the JE mRNA, coding for a protein of the family of cytokines. The cell content of c-jun mRNA was also strongly reduced in the cells incubated at quiescence with dexamethasone (in the absence of mitogen). The presence of TPA along with dexamethasone prevented the elimination of c-jun, but not of JE mRNA. Short (30-min; together with the inducers) or long (24-h) treatment of the cells with dexamethasone did not prevent the induction of the c-fos gene expression by either TPA or basic fibroblast growth factor, indicating that dexamethasone does not interfere with mitogenic signal transduction. We conclude that in TPA-stimulated cells, the antiproliferative effect of dexamethasone is not due to interference with the expression of the c-jun gene, but may be related to the decreased level of the JE cytokine mRNA as well as to the synthesis of growth inhibitory protein(s). (Endocrinology 129: 1033-1041, 1991)
ABSTRACT. Dexamethasone (a synthetic glucocorticoid) inhibited the entry into the S-phase of quiescent chemically transformed mouse fibroblasts (BP-A31) stimulated with 12-O-tetradecanoyl 13-acetate (TPA; a protein kinase-C activator) or with basic fibroblast growth factor. The basal rate of DNA synthesis was also strongly reduced by dexamethasone. In contrast, the mitogenic activity of insulin (acting via the insulin-like growth factor-I receptor) was little or not at all affected by dexamethasone. The antimitogenic activity of dexamethasone was enhanced when the steroid was included in the culture medium 24 h before the addition of mitogens. The effects of dexamethasone were glucocorticoid specific, partially reversed by the antiglucocorticoid RU 486, and prevented by cycloheximide (suggesting the involvement of glucocorticoid-induced protein synthesis in the antimitogenic activity of dexamethasone). Under the conditions of exponential growth in serum-free medium as well as in the presence of TPA, dexamethasone arrested the proliferation of sparsely seeded cells after a delay of 24-48 h. The BPA31 cells are known to be constitutively competent and express
G
LUCOCORTICOIDS are potent antiinflammatory agents used in the treatment of chronic inflammatory diseases, both systemically and locally. At the same time, they are known to slow down wound healing, and several studies in man or animals have shown that glucocorticoids inhibit fibroblast proliferation (1). However, results from in vitro studies are contradictory, since both inhibitory (1, 2) and stimulatory (3, 4) effects of glucocorticoids on cell proliferation have been reported (5, 6). In the present study we have analyzed the action of dexamethasone, a synthetic glucocorticoid, on the proliferation of the chemically transformed mouse fibroblasts (BP-A31). The BP-A31 cell line has been obtained from BALB/c mouse 3T3 fibroblasts (clone A31) by transformation with benzo-«-pyrene (7). When placed in a serum-free medium, these cells become quiescent after about 48 h, as evidenced by the low rate of [3H]thymidine incorporation (8, 9). At quiescence, the BP-A31 cells continue to express a number of genes, such as c-myc, Received January 22, 1991. Address requests for reprints to: Dr. Dominique Fagot, INSERM U.55,184 rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France. * Supported by a grant from the Ministere de la Recherche et de la Technologic.
JE, as well as c-jun (8-12), which are characteristic of the state of competence in the nontransformed A31 cells. [Competence can be induced in the quiescent A31 cells by platelet-derived growth factor (PDGF) and is required for the completion of Gl-phase under the effect of progression factors, such as insulin-like growth factor-I (IGF-I) (13).] In contrast, the c-fos gene behaves alike in both the BP-A31 and A31 cell lines; its expression is absent at quiescence and can be induced by serum, 12O-tetradecanoyl 13-acetate (TPA), or basic fibroblast growth factor (bFGF) (11, 12). The product of the protooncogene c-fos forms a complex with the product of the protooncogene c-jun (14-16). This complex (transcription factor API) binds with high affinity to a specific region of DNA found in the promoters of numerous genes [TPA response element (TRE)] and leads to stimulation of transcription under appropriate conditions {e.g. stimulation with TPA). Several groups have recently demonstrated that the glucocorticoid receptor-hormone complex binds to the components of the API transcription factor and interferes with the transactivation of AP1dependent transcription (17-20). The quiescent serumdeprived BP-A31 cells respond to serum by resumption
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DEXAMETHASONE AND CELL GROWTH
of the cell division cycle and entry into the S-phase after a delay of about 10 h. Moreover, individual growth factors [IGF-I or insulin at 10"7 M or greater concentrations, at which it interacts with IGF-I receptors, bFGF, as well as phorbol esters (activators of protein kinase-C)] also assure the progression of the BP-A31 cells through the entire cell division cycle (9-12). We show that dexamethasone inhibits the growth response of BP-A31 cells to TPA and bFGF, but does not affect the mitogenic activity of insulin. At the same time, dexamethasone inhibits the expression of certain genes associated with the early Gl phase. The antimitogenic effects of dexamethasone are prevented by cycloheximide, suggesting that they require protein synthesis and, therefore, may not be exclusively the consequence of inhibition of the API-dependent gene expression.
Materials and Methods Cell culture Benzo-cv-pyrene-transformed BALB/c 3T3 mouse fibroblasts (BP-A31 cells) were cultured in a-Minimum Essential Medium supplemented with 6% fetal calf serum (FCS) in a humidified atmosphere containing 5% CO2. For the study of mitogenic effects, the cells were seeded in 24-well boxes (2-4 x 104 cells/well). After 24 h, the medium was replaced with 1 ml a-Minimum Essential Medium (serum free) plus 2.5 nM FeSO4, and the cells were allowed to enter quiescence during the next 2-3 days. For RNA preparation, the cells were grown in 100-
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DEX [nil] FlG. 2. Concentration dependance of the effect of dexamethasone (DEX). Quiescent BP-A31 cells (M) were restimulated with TPA (100 ng/ml; • ) in the presence of different concentrations of dexamethasone, as indicated. The rate of [3H]thymidine incorporation was measured as described in Fig. 1. Data are expressed as a percentage of the maximal [3H]thymidine incorporation. The means of triplicates ± SEM are represented. Binding of [3H]dexamethasone (O) was measured, as described in Materials and Methods, and is shown as a percentage of the maximum (detected with 1 nM [3H]dexamethasone). Scatchard plot analysis of these data (not shown) indicated an apparent Kd of 2 X 10"8 M.
mm petri dishes to approximately 2 X 106 cells/dish before serum starvation. Incorporation of PH]thymidine Cells were incubated with 2 ^Ci [3H]thymidine/ml (Amersham, Les Ulis, France). Incorporation was allowed to proceed during 2-h pulses or for the entire period following the restimulation of serum-deprived cells and was terminated by acidification with 1 M ascorbic acid (three drops per ml). The cells were fixed with 5% trichloroacetic acid, solubilized in 0.1 N NaOH, and the incorporated radioactivity was determined by liquid scintillation counting. Analysis of RNA
Control
TPA
FGF
INS
FCS
FIG. 1. Effects of mitogens in BP-A31 cells: selective inhibition by dexamethasone. BP-A31 cells rendered quiescent by serum starvation were restimulated with TPA (200 ng/ml), bFGF (10 ng/ml), insulin (INS; 1 nM), or FCS (10%) in a-Minimum Essential Medium. [3H] Thymidine incorporation for 24 h after stimulation was determined, as described in Materials and Methods. Cells were restimulated in the absence (•) or presence of dexamethasone (2 x 10'7 M; H) or were preincubated (24 h) with 2 x 10'7 M dexamethasone before restimulation with mitogens and dexamethasone (2 x 10'7 M; • ) . The data shown are means of triplicates ± SEM. The differences were evaluated by Student's t test. *, P < 0.01; **, P < 0.001).
Cells in P100 dishes were chilled on ice and washed, and total RNA was isolated using the urea-lithium chloride precipitation procedure (21). Poly(A)+ RNA was selected by chromatography on oligo(dT)-cellulose. Twenty micrograms of total or 5 Mg poly(A)+ RNA were size-fractionated on agarose (1%)formaldehyde (2.2 M) gel, transferred onto nitrocellulose paper, fixed by heating at 80 C, and hybridized at 42 C with 32Plabeled probes in a solution containing 50% formamide, 0.45 M NaCl, 3 mM EDTA, and 75 mM phosphate buffer, pH 7.4. The filters were washed at 45 C in 0.15 M NaCl and 15 mM sodium citrate, and exposed with XAR 5 Kodak film and intensifying screen (Eastman Kodak, Rochester, NY; for more details, see Ref. 22). Probes
The probes were the 3' exon (Clal-EcoRl fragment) of the human c-myc gene (23), rat a-tubulin cDNA (24) used as
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DEXAMETHASONE AND CELL GROWTH
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TABLK 1. Steroid specificity of the effect of dexamethasone 3
H]Thymidine incorporation (cpm)
Steroid Basal 14,340 ± 730 6,250 ± 370° 8,570 ± 380° 9,600 ± 760* 12,570 ± 400 13,110 ± 1,220
None Dexamethasone Hydrocortisone Aldosterone Testosterone Progesterone
FGF
TPA 80,690 ± 28,670 ± 32,930 ± 62,510 ± 70,740 ± 73,430 ±
Insulin 99,830 ± 6,890 98,440 ± 4,480 98,090 ± 5,530 99,500 ± 3,680 96,100 ± 1,970 97,790 ± 1,460
59,120 ± 3,040 39,730 ± 1,400" 39,650 ± 430" 63,900 ± 1,920 65,200 ± 2,950 61,140 ± 2,750
3,830 170° 1,360° 2,0606 1,520 1,060
Serum-deprived BP-A31 cells were restimulated with TPA (100 ng/ml), FGF (50 ng/ml), or insulin (1 nM) in the presence or absence of steroids (2 X 10"7 M), as indicated. The data shown are means of triplicates ± SEM. The statistical significance of the effect of the different steroids was evaluated by Student's t test. " P < 0.001.
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FIG. 3. Reversal of the glucocorticoid effect by RU486. Quiescent BPA31 cells were restimulated by TPA (100 ng/ml; • ) or bFGF (50 ng/ ml; D) in the presence or absence of dexamethasone (DEX; 4 x 10"8 M) and different concentrations of RU486, as indicated. [3H]Thymidine incorporation was measured as described in Fig. 1. The data shown are means of triplicates ± SEM. internal standard, since the level of this mRNA does not vary
early in the cell cycle (25), JE cDNA corresponding to a PDGFinduced mRNA in BALB/c 3T3 cells (26), mouse c-jun cDNA (27), and the Bglll-Pvull fragment of the FBJ provirus {v-fos) (28).The probes were 3aP-labeled by the Multiprime DNA labeling system (Amersham). Evaluation of the glucocorticoid receptor contents Cells in P100 dishes were incubated for 30 min at 37 C with 10 nM [3H]triamcinolone acetonide (29 Ci/mmol; total binding) or [;iH]triamcinolone acetonide and 2 nM dexamethasone (nonsaturable binding). The cells were harvested and lysed with 0.5% Nonidet P-40 (NP40), and the nuclei were extracted with 0.5 M NaCl. To separate the receptor-bound and unbound steroid, the extracts were treated with 0.25% active charcoal suspension and centrifuged, and the radioactivity remaining in the supernatant was determined by liquid scintillation count-
TPA: + + + + + + Dex added a t ( h ) : 0 0 3 7 9 15 FIG. 4. Effect of delayed addition of dexamethasone (Dex). Quiescent BP-A31 cells were restimulated with TPA (200 ng/ml) at time zero, and dexamethasone (2 x 10'7 M) was added at various times thereafter. Incorporated radioactivity was measured 24 h after a continuous exposure to [3H]thymidine, as described in Fig. 1. The data shown are means of triplicates ± SEM.
ing. The same fractionation was also applied in the experiments in which nuclear binding of [3H]dexamethasone (92 Ci/mmol) was evaluated after incubation with different concentrations of the hormone. The nonsaturable binding was subtracted from the total binding value to calculate the receptor-bound [3H] dexamethasone. Growth factors and chemicals Bovine insulin was obtained from Boehringer; [3H]thymidine, [3H]triamcinolone acetonide, [3H] dexamethasone, and [32P]dCTP were purchased from Amersham. bFGF from two producers was used: Amersham (recombinant at 10 ng/ml) and Collaborative Research (Bethesda, MD; purified from bovine pituitary gland at 50 ng/ml). TPA and dexamethasone were obtained from Sigma (St. Louis, MO). Other compounds (reagent grade) were purchased from the usual commercial sources.
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DEXAMETHASONE AND CELL GROWTH
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TIME (hours) FIG. 5. Reversibility of the dexamethasone effect. Quiescent BP-A31 cells (•) were restimulated with TPA (100 ng/ml; • ) or pretreated with dexamethasone (2 x 10'7 M) during the last 10 h of quiescence, washed, and either immediately restimulated with TPA (•) or restimulated with TPA after 14 h of recovery in a-Minimum Essential Medium (0). [3H]Thymidine incorporation was measured during 2-h pulses every 3 h. The data shown are means of duplicate determinations represented at the mean times of the pulses.
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FIG. 6. Inhibition by dexamethasone (DEX) of the mitogenic response to TPA is prevented by cycloheximide. Quiescent BP-A31 cells were pretreated with cycloheximide (1 fig/m\), dexamethasone (2 X 10'7 M), or a combination of both, as indicated, for 12 h and then placed in fresh (serum-free) medium or restimulated with TPA (100 ng/ml). [3H] Thymidine incorporation was measured between 0-26 h. The data shown are means of triplicates ± SEM.
Results Effects of dexamethasone on I3H] thymidine incorporation in mitogen-stimulated BP-A31 cells As previously described (9-12), the quiescent serumstarved BP-A31 fibroblasts resume the cell division cycle
Endo«1991 Vol 129 • No 2
when placed in a medium containing either 10% FCS or individual growth factors, such as insulin, bFGF, or TPA. Dexamethasone (2 X 10~7 M), added simultaneously with the mitogens, caused a decrease in the rate of [3H] thymidine incorporation induced by TPA or bFGF (55% and 30%, respectively, in the experiment shown in Fig. 1). In contrast, the mitogenic effects of insulin or FCS were not or only slightly affected. The antimitogenic potency of dexamethasone was enhanced when dexamethasone was added 24 h before restimulation with TPA or bFGF (and maintained in the medium during incubation with the mitogens). In this case, the inhibition of [3H]thymidine incorporation was 80% and 60%, respectively. The presence of dexamethasone also led to a large decrease in the basal rate of [3H]thymidine incorporation in control cells fed fresh (serum-free) medium after serum starvation (Fig. 1). The antimitogenic effects of dexamethasone were concentration dependent. The 50% effective concentration evaluated on the basis of inhibition of the mitogenic effect of TPA was approximately 5 x 10"9 M (Fig. 2). In comparison, the apparent Kd of the receptor-mediated tight nuclear binding of [3H]dexamethasone was approximately 2 x 10 s M. [Similarly, Cook et al. (29) reported that in rat hepatoma Fu5 cells, the dose-response for growth inhibition by dexamethasone is shifted to concentrations of hormone lower than those required for comparable levels of receptor binding.] At 2 x 10'6 M, dexamethasone caused cell death, as observed by optical microscopy. The hypothesis of the involvement of the glucocorticoid receptor in the antimitogenic effects of dexamethasone was supported by the fact that steroids known to activate this receptor's function (hydrocortisone and aldosterone) were also inhibitors of TPA- and FGF-induced [3H]thymidine incorporation, in contrast with, for instance, testosterone (not bound by glucocorticoid receptors) or progesterone (forming a complex with the glucocorticoid receptor, but lacking agonist activity), which did not have a significant effect at 2 X 10'7 M (Table 1). Moreover, RU 486 (a synthetic antiglucocorticoid which competes for the glucocorticoid receptorbinding site) (30) antagonized the antimitogenic activity of dexamethasone in a concentration-dependent manner (Fig. 3). The reversal by RU 486 of the inhibitory effect of dexamethasone was apparently more effective with bFGF than with TPA. This may be related to the fact that TPA-induced DNA synthesis is more sensitive to inhibition by dexamethasone, so that the low proportion of agonist-receptor complexes present in the cells incubated with dexamethasone plus RU 486 (0.2 /IM) is sufficient to reduce the mitogenic response to TPA, but not that to FGF. At concentrations of 1 ixM or more, RU 486 was toxic to the cells.
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DEXAMETHASONE AND CELL GROWTH
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TABLE 2. Nuclear content of the glucocorticoid receptor in the BP-A31 cells: effect of pretreatment with dexamethasone 3
1. Control 2. Control after 15-min incubation with 2 X 10' M dexamethasone 3. Pretreatment (10-h) with cycloheximide (1 tig/ml), then incubation (15-min) with 2 X 1CT7 M dexamethasone 4. Pretreatment (10-h) with cycloheximide (1 /ig/ml) + dexamethasone (2 x 10"7 M), then incubation (15-min) with 2 x 10"7 M dexamethasone
H]Triamcinoline acetonide in the nuclear extract (cpm)
Determined nuclear binding (sites/cell)
Total
Nonsaturable
402 163
22 20
3800 1430
238
33
2050
175
24
1500
Serum-deprived BP-A31 cells were treated as indicated before incubation with [3H]triamcinolone acetonide (10 8 M) in the absence (total binding) or presence (nonsaturable binding) of 2 nM dexamethasone. The content of receptor-bound radioactive ligand was measured as described in Materials and Methods. Treatment of the nuclear extracts with 0.25% charcoal suspension did not reduce the radioactivity, confirming that the a H-labeled steroid was macromolecule bound. In the cells exposed to 2 X 10"7 M dexamethasone, only about 40% of the total receptor content was detected (due to isotopic dilution or incomplete exchange; compare lines 1 and 2).
The antimitogenic activity of dexamethasone may take place in the late Gl phase, as illustrated by the experiment shown in Fig. 4 in which the hormone was added to the cells at different time intervals after restimulation with TPA. With increasing delay after the addition of TPA, a progressive decrease in the inhibitory activity of dexamethasone was observed, but even when added as late as 9 h after the mitogen, dexamethasone significantly inhibited DNA synthesis. Once the cells entered into the S-phase of the cell cycle, dexamethasone had no effect on [3H]thymidine incorporation. Antimitogenic effects of dexamethasone in TPAstimulated cells require protein synthesis The antimitogenic effects of dexamethasone were also observed when the hormone was present in the culture medium for 10 h before, but not during, restimulation of the cells with TPA (Fig. 5). However, when cycloheximide (a protein synthesis inhibitor) was included along with dexamethasone during the preincubation, the inhibitory effects of dexamethasone on the cell's response to TPA were abolished (Fig. 6), indicating that dexamethasone action requires the synthesis of an intracellular or secreted antimitogenic protein(s). An alternative interpretation of this result could be elimination of the glucocorticoid receptor (down-regulation) during the preincubation with dexamethasone, under conditions where synthesis of new receptor molecules is blocked. To test this possibility, we performed an experiment in which the level of the receptor was evaluated by whole cell binding of [3H]triamcinolone acetonide; the results showed that only about 30% of the glucocorticoid-binding sites were lost during the 10-h incubation of the cells with dexamethasone and cycloheximide (us. cycloheximide alone; Table 2).
The experiments in which conditioned medium harvested from dexamethasone-treated cells was tested (data not shown) did not favor the possibility that an antimitogenic protein was secreted into the medium. The postulated glucocorticoid-induced protein (or the corresponding mRNA) is apparently long-lived, since inhibition of the mitogenic response to TPA was also observed in the experiment in which the cells were first treated with dexamethasone (10 h) and then incubated for 14 h in the absence of the steroid before restimulation (Fig. 5). The basal response of quiescent cells to fresh (mitogenfree) medium is enhanced by cycloheximide pretreatment (Fig. 6) (11, 12). The potentiation of the basal response was abolished by the presence of dexamethasone together with cycloheximide during the pretreatment (Fig. 6). Effects of dexamethasone in exponentially growing cells To determine whether exponentially growing cells are sensitive to dexamethasone, mitogen-dependent proliferation was studied in the absence or presence of 2 x 10'7 M dexamethasone (Fig. 7). In a mitogen-free medium as well as in TPA-containing medium, dexamethasone arrested cell growth within 24-48 h. In contrast, dexamethasone had little or no effect on cell growth in the presence of insulin. Effects of dexamethasone on the expression of early cell cycle-related genes
A long (24-h) treatment of the quiescent BP-A31 cells with dexamethasone did not prevent the induction of cfos mRNA accumulation by TPA or by bFGF (Fig. 8A, lanes 7 and 8 us. lane 6), indicating that it did not interfere with the FGF- or TPA-initiated mitogenic signal. (The induction ofc-fos gene transcription is probably
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DEXAMETHASONE AND CELL GROWTH
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a consequence of the activation of the serum-response element (31)-binding factor.] The mRNA corresponding to the JE gene was virtually eliminated during the 24-h incubation with dexamethasone (Fig. 8A, lanes 6-8). A short exposure of the cells to dexamethasone (3 h) was sufficient to strongly reduce the content in the JE as well as c-jun mRNA (Fig. 8B, lanes 2 vs. 1). However, when the cells were incubated with both dexamethasone and TPA, the decrease in the content of c-jun mRNA was not observed (Fig. 8B, lanes 3 and 4). The effect of dexamethasone on the expression of the JE and the cjun genes is independent of protein synthesis, since it also takes place in the presence of cycloheximide (Fig. 9, lanes 2 vs. 1). When the cells had been incubated with dexamethasone (3 h), only a slight decrease in the content of c-m.;yc mRNA was observed (Fig. 8B, lanes 2 vs. 1), and there was no effect if cycloheximide was included in the culture medium (Fig. 9, lanes 1 and 2). All of these genes are actively transcribed in the quiescent BP-A31 cells, since blocking the transcription by the addition of actinomycin-D (10 jug/ml) led to a total elimination of the corresponding mRNAs within 3 h (Fig. 9, lane 3). These results suggest that dexamethasone regulates the level of JE and c-jun mRNA at the transcriptional level.
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Under cell culture conditions, glucocorticoids frequently exercise an antiproliferative effect (1, 2), but the contrary, i.e. growth-promoting action, has also been reported for human diploid fibroblasts (3, 4). Our results demonstrate that dexamethasone, a potent synthetic glucocorticoid agonist, exercises a negative control of proliferation of mouse fibroblasts BP-A31 when either TPA or bFGF is used as the mitogen, but has little or no effect on the insulin-dependent growth (mediated by the IGFI receptor). This observation is consistent with our earlier report, in which we have proposed that the TPAand FGF-initiated mitogenic signaling pathways are in part coincident and distinct from that initiated by the IGF-I receptor-ligand interaction (11, 12). The effect of dexamethasone is possibly related to contact inhibition, since under the conditions of growth of sparsely seeded cells in a TPA-containing medium, dexamethasone had little effect in the early period, but caused an arrest at a FIG. 7. Effect of dexamethasone on growth of sparsely seeded bp-A31 cells. BP-A31 cells were plated in 24-well boxes (104 cells/well; - - -) or (4 x 104cells/well; —) in medium containing 10% FCS. After 24 h, the cells were washed and placed in a-Minimum Essential Medium containing TPA (100 ng/ml; B) or insulin (1 nM; C) or without added mitogens (A) in the presence (•, A, and • ) or absence (O, A, and • ) of dexamethasone (2 x 10"7 M). The cells were trypsinized and counted at the times indicated. In A and B, medium was refreshed after 96 h. The points represent means and ranges (when greater than the symbols) of duplicates.
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DEXAMETHASONE AND CELL GROWTH lower cell density. The effect is steroid specific, and in particular, it is countered by the glucocorticoid antagonist RU 486, indicating that it is mediated by the glucocorticoid receptor. The addition of dexamethasone to TPA-stimulated serum-starved BP-A31 cells as late as 9 h after the addition of TPA has a marked inhibitory effect on [3H] thymidine incorporation, suggesting that dexamethasone acts (also) in the late G l phase. (There is no effect of dexamethasone during the S-phase itself.) The antiproliferative effects of dexamethasone are not due to an inhibition of the mitogenic signaling pathways leading to early changes in gene expression, since dexamethasone does not prevent, for instance, the induction of c-fos RNA accumulation by mitogens. Modulation of gene expression is another possible mechanism for the antiproliferative activity of dexamethasone. In fact, glucocorticoids are known to induce the transcription of numerous genes possessing the glucocorticoid response element in the promoter region (32, 33), but can also inhibit gene expression, either by direct interaction with a negative control element in the promoter region (34) or indirectly by forming a complex with the transcription factor A P I (17-20) and, thus, blocking the TRE-directed transcription. In BP-A31 cells, treatment with dexamethasone at quiescence reduces the contents of both J E and c-jun mRNAs, probably by inhibiting the transcription of the corresponding genes (both containing T R E sequences at their promoters) (35, 36). When quiescent BP-A31 cells are pretreated with cycloheximide and then placed in mitogenfree medium, the basal rate of DNA synthesis is en-
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Fir.. 8. Effect of dexamethasone on the expression of early cell cyclerelated genes. A, Quiescent BP-A31 cells were restimulated (30 min) as follows: lane 1, control; lane 2, TPA (200 ng/ml); lane 3, TPA plus dexamethasone (2 X 10"7 M); lane 4, bFGF (10 ng/ml); and lane 5, bFGF plus dexamethasone. In lanes 6-8, the quiescent cells were preincubated with dexamethasone (for 24 h) before restimulation (for 30 min) with: lane 6, medium change control; lane 7, TPA; and lane 8, bFGF. Oligo(dT)-selected RNA samples (5 /ig/lane) were analyzed by Northern blotting and hybridized with fos, JE, and a-tublin probes. B, Serum-deprived BP-A31 cells were incubated in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of dexamethasone (2 x 10'7 M) for 3 h with: lanes 3 and 4, TPA (200 ng/ml); and lane 5 and 6, insulin (1 jtM). Samples (20 ^g total RNA/lane) were analyzed, as described in Materials and Methods, and hybridized with the probes as shown.
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123 c-jun c-myc a-tub.
••
JE FIG. 9. Effect of dexamethasone on early cell cycle-related mRNA levels in the presence of cycloheximide. Serum-deprived BP-A31 cells were incubated for 3 h in the presence of 1 /ug/ml cycloheximide alone (lane 1), with dexamethasone (2 x 10"7 M; lane 2), or with actinomycinD (10 Mg/ml; lane 3). Oligo(dT)-selected RNA samples (5 /ng/lane) were analyzed, as described above, and hybridized with the probes as shown.
hanced (10), possibly as a result of the superinduction of certain early cell cycle-related genes, including c-myc and c-fos (11, 25, 37) as well as c-jun (Buchou, T., unpublished data). The inhibition by dexamethasone of basal DNA synthesis as well as its potentiation by cycloheximide may be (in part) a consequence of the inhibition oifos-jun (API) complex-dependent TRE-directed gene expression. The reduction by dexamethasone of c-jun mRNA content was countered by TPA and is, therefore, unlikely to account for the antimitogenic activity of the steroid in TPA-stimulated cells. On the other hand, the JE protein does have cellular signaling activity (it is the monocyte chemoattractant protein MCP1) (38) and may play a role in the regulation of fibroblast growth. In nontransformed fibroblasts, JE/MCP1 is induced by the competence factor platelet-derived growth factor and may participate in the subsequent stimulation of the cells by progression factors; in BP-A31 cells, the constitutive synthesis of the JE/MCP1 protein may favor the mitogenic activity of phorbol esters. JE/MCP1 may also be involved in the inflammatory processes associated with wound healing (39). If so, both the growth inhibitory and antiinflammatory effects of glucocorticoids may be (partly) the result of inhibition of expression of the JE gene. [Glucocorticoids have also been reported to inhibit the growth of Fu 5 hepatoma cells by blocking the synthesis of a secreted growth factor (40).] Inhibition of TRE-dependent gene expression is probably not the only mechanism by which glucocorticoids inhibit the mitogenic response of the cells to TPA. In fact, preincubation with dexamethasone before restimulation with TPA (in the absence of the steroid) leads to strongly reduced [3H]thymidine incorporation compared with that in TPA-stimulated cells not treated with dexamethasone; this inhibitory effect is abolished if protein
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DEXAMETHASONE AND CELL GROWTH
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synthesis is blocked by cycloheximide during preincubation with dexamethasone. These observations indicate the involvement of a glucocorticoid-induced growth inhibitory protein. Alternatively, incubation with cycloheximide for 10 h could lead to elimination of a protein necessary to mediate the action of dexamethasone, such as the glucocorticoid receptor. However, we have verified that under these conditions, the content of glucocorticoid receptor was reduced by only about 30%. [This result is compatible with that of an earlier study (41) in which the ti/2 of glucocorticoid receptor turnover in cells cultured with dexamethasone was found to be 9.5 h.] There is at present no evidence of the involvement of proteins other than glucocorticoid receptor in the mediation of the effects of glucocorticoids on gene expression. In summary, two different mechanisms appear to participate in the antimitogenic activity of dexamethasone in mouse fibroblasts (BP-A31): inhibition of expression of Gl progression-related genes (for instance, those having TRE sequences at their promoter), and induction of the synthesis of a growth inhibitory protein(s). These growth inhibitory proteins are probably intracellular and remain unknown at present. The lipocortin family offers possible candidates; these proteins have been reported to inhibit the intracellular activity of phospholipase-A2, thus interfering with the formation of metabolites of arachidonic acid. It has been suggested that these metabolites may participate in the autocrine mode of regulation of cell growth, particularly in the inflammatory situation (42, 43). The possibility of the involvement of such compounds in the antimitogenic effect of dexamethasone needs further study. Acknowledgments The authors thank Y. Issoulie for illustrations, and Dr. G. Rosselin for his interest in this work. References 1. Durant S, Duval D, Homo-Delarche F 1986 Factors involved in the control of fibroblast proliferation by glucocorticoids: a review. Endocr Rev 7:254-269 2. Young DV, Dean MC 1980 The suppression of cellular proliferation in SV 40-transformed 3T3 cells by glucocorticoids. J Cell Physiol 102:223-231 3. Cristofalo VJ, Rosner BA 1979 Modulation of cell proliferation and senescence of WI-38 cells by hydrocortisone. Fed Proc 38:18511856 4. Finlay CA, Cristofalo VJ 1987 Autocrine stimulation of W138 cell proliferation in the presence of glucocorticoids. Exp Cell Res 168:191-202 5. Finlay CA, Cristofalo VJ 1987 In: Boyton AL, Leffert HL (eds) In Vitro Control of Animal Cell Proliferation. Academic Press, New York, vol 2:203-217 6. Vedeckis WV, Eastman-Reks SB, Lapointe MC, Reker CE 1987 In: Spelsberg TC, Kumar R (eds) Steroid and Sterol Hormone Action. Martinus Nijhoff, Boston, pp 213-225 7. Dubrow DR, Riddle VGH, Pardee AB 1979 Different responses to drug and serum of cells transformed by various means. Cancer Res
Endo«1991 Voll29«No2
39:2718-2726 8. Campisi J, Gray HE, Pardee AB, Dean M, Sonnenshein GE 1984 Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell 36:241-247 9. Gray HE, Buchou T, Mester J 1987 Serum-induced G0/G1 transition in chemically transformed 3T3 cells. Exp Cell Res 169:95104 10. Buchou T, Charollais RH, Mester J 1988 Involvement of serum factor(s) adsorbed to the dish in the response of cycloheximidepretreated BP-A31 cells to serum pulses. Exp Cell Res 174:411420 11. Buchou T, Charollais RH, Fagot D, Mester J 1989 Mitogenic activity of phorbol esters and insulin-like growth factor 1 in chemically transformed mouse fibroblasts BP-A31: independent effects and differential sensitivity to inhibition by 3-isobutyl-l-methyl xanthine. Exp Cell Res 182:129-143 12. Buchou T, Mester J 1990 The fibroblast growth factor-dependent mitogenic signal transduction pathway in chemically transformed mouse fibroblast is similar to but distinct from that initiated by phorbol esters. J Cell Physiol 142:559-565 13. Pledger WJ, Stiles CD, Antoniades HN, Scher CD 1978 An ordered sequence of events is required before Balb/c 3T3 cells become committed to DNA synthesis. Proc Natl Acad Sci USA 75:28392843 14. Lee W, Mitchell P, Tjian R 1987 Purified transcription factor AP1 interacts with TPA-inducible enhancer elements. Cell 49:741752 15. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M 1987 Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729-739 16. Bohmann D, Bos TJ, Admon A, Nishimura T, Vogt PK, Tjian R 1987 Human protooncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor API. Science 238:1386-1392 17. Lucibello FC, Slater EP, Jooss KU, Beato M, Miiller R1990 Mutual transrepression of Fos and glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB. EMBO J 9:2827-2834 18. Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and anti inflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189-1204 19. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between cjun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:12051215 20. Schiile R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-jun and the glucocorticoid receptor. Cell 62:12171226 21. Auffray C, Rougeon F 1980 Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. EurJBiochem 10:303-314 22. Maniatis T, Fritch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 23. Dalla Favera R, Gelmann EP, Martinotti S, Franchini G, Papas TS, Gallo R, Wong-Staal F 1982 Cloning and characterization of different human sequences related to the one gene (c-myc) of avian myelocytomatosis virus (MC29). Proc Natl Acad Sci USA 79:64976501 24. Lemishka IR, Farmer S, Racaniello VR, Sharp PA 1981 Nucleotide sequence and evolution of a mammalian a-tubulin messenger RNA. J Mol Biol 51:101-121 25. Greenberg ME, Ziff EB 1984 Stimulation of 3T3 cells induces transcription of the c-fos protooncogene. Nature 311:433-438 26. Cochran BH, Reffel AC, Stiles CD 1983 Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell 33:939947 27. Ryseck RP, Hirai SI, Yaniv M, Bravo R 1988 Transcriptional
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DEXAMETHASONE AND CELL GROWTH
28. 29.
30.
31. 32.
33. 34.
35.
activation of c-jun during the GO/Gl transition in mouse fibroblasts. Nature 334:535-537 Curran T, Peters G, Van Beveren G, Teich NM, Verma IM 1982 FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA. J Virol 44:679-682 Cook PW, Swanson KT, Edwards CP, Firestone GL 1988 Glucocorticoid receptor-dependent inhibition of cellular proliferation in dexamethasone-resistant and hypersensitive rat hepatoma cell variants. Mol Cell Biol 8:1449-1459 Jung-Testas I, Baulieu EE 1983 Inhibition of glucocorticosteroid action in cultured L-929 mouse fibroblasts by RU-486, a new antiglucocorticosteroid of high affinity for the glucocorticosteroid receptor. Exp Cell Res 147:177-182 Treisman R 1987 Identification and purification of a polypeptide that binds the c-fos serum response element. EMBO J 6:2711-2717 Geisse S, Scheidereit C, Westphal HM, Hynes NE, Groner B, Beato M 1982 Glucocorticoid receptors recognize DNA sequences in and around murine mammary tumor virus DNA. EMBO J 1:1613-1619 Pfahl M 1982 Specific binding of the glucocorticoid-receptor complex to the mouse mammary tumor proviral promoter region. Cell 31:475-482 Drouin J, Trifiro MA, Plante KR, Nemer M, Ericksson P, Wrange 0 1989 Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent inhibition of pro-opiomelanocortin gene transcription. Mol Cell Biol 9:5305-5314 Timmers HTM, Pronk GJ, Bos JL, van der Eb AJ 1990 Analysis
36. 37.
38. 39. 40.
41. 42. 43.
1041
of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction. Nucleic Acids Res 18:23-34 Angel P, Hattori K, Smeal T, Karin M 1988 The jun protooncogene is positively autoregulated by its product, Jun/AP-1. Cell 55:875-885 Dani C, Blanchard JM, Piechaczyk M, El Sabouty S, Mathy L, Jeanteur P 1984 Extreme instability of c-myc mRNA in normal and transformed human cells. Proc Natl Acad Sci USA 81:70467050 Rollins BJ, Sher P, Ernst T, Wong GG 1989 The human homolog of the JE gene encodes a monocyte secretory protein. Mol Cell Biol 9:4687-4695 Leonard EJ, Yoshimura T 1990 Human monocyte chemoattractant protein-1 (MCP-1). Immunol Today 11:97-101 Cook PW, Edwards CP, Haraguchi T, Firestone GL 1989 Partial characterization of a glucocorticoid suppressible mitogenic activity secreted from rat hepatoma cell line hypersensitive to the antiproliferative effects of glucocorticoids. J Biol Chem 264:14151-14158 Mclntyre WR, Samuels HH 1985 Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-time of the activated nuclear receptor form. J Biol Chem 260:418-427 Taylor L, Polgar P 1977 Self regulation of growth by human diploid fibroblasts via prostaglandin production. FEBS Lett 79:69-72 Pentland AP, Needleman P 1986 Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis. J Clin Invest 77:246-251
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Vol. 129, No. 2 Printed in U.S.A.
Antimitogenic Effects of Dexamethasone in Chemically Transformed Mouse Fibroblasts DOMINIQUE FAGOT, CHRISTINE BUQUET-FAGOT*, AND JAN MESTER INSERM U.55, 75571 Paris Cedex 12, France
at quiescence certain genes related to the GO/Gl transition in the original nontransformed A31 cell line. Of the transcripts corresponding to these genes, dexamethasone caused a rapid elimination of the JE mRNA, coding for a protein of the family of cytokines. The cell content of c-jun mRNA was also strongly reduced in the cells incubated at quiescence with dexamethasone (in the absence of mitogen). The presence of TPA along with dexamethasone prevented the elimination of c-jun, but not of JE mRNA. Short (30-min; together with the inducers) or long (24-h) treatment of the cells with dexamethasone did not prevent the induction of the c-fos gene expression by either TPA or basic fibroblast growth factor, indicating that dexamethasone does not interfere with mitogenic signal transduction. We conclude that in TPA-stimulated cells, the antiproliferative effect of dexamethasone is not due to interference with the expression of the c-jun gene, but may be related to the decreased level of the JE cytokine mRNA as well as to the synthesis of growth inhibitory protein(s). (Endocrinology 129: 1033-1041, 1991)
ABSTRACT. Dexamethasone (a synthetic glucocorticoid) inhibited the entry into the S-phase of quiescent chemically transformed mouse fibroblasts (BP-A31) stimulated with 12-O-tetradecanoyl 13-acetate (TPA; a protein kinase-C activator) or with basic fibroblast growth factor. The basal rate of DNA synthesis was also strongly reduced by dexamethasone. In contrast, the mitogenic activity of insulin (acting via the insulin-like growth factor-I receptor) was little or not at all affected by dexamethasone. The antimitogenic activity of dexamethasone was enhanced when the steroid was included in the culture medium 24 h before the addition of mitogens. The effects of dexamethasone were glucocorticoid specific, partially reversed by the antiglucocorticoid RU 486, and prevented by cycloheximide (suggesting the involvement of glucocorticoid-induced protein synthesis in the antimitogenic activity of dexamethasone). Under the conditions of exponential growth in serum-free medium as well as in the presence of TPA, dexamethasone arrested the proliferation of sparsely seeded cells after a delay of 24-48 h. The BPA31 cells are known to be constitutively competent and express
G
LUCOCORTICOIDS are potent antiinflammatory agents used in the treatment of chronic inflammatory diseases, both systemically and locally. At the same time, they are known to slow down wound healing, and several studies in man or animals have shown that glucocorticoids inhibit fibroblast proliferation (1). However, results from in vitro studies are contradictory, since both inhibitory (1, 2) and stimulatory (3, 4) effects of glucocorticoids on cell proliferation have been reported (5, 6). In the present study we have analyzed the action of dexamethasone, a synthetic glucocorticoid, on the proliferation of the chemically transformed mouse fibroblasts (BP-A31). The BP-A31 cell line has been obtained from BALB/c mouse 3T3 fibroblasts (clone A31) by transformation with benzo-«-pyrene (7). When placed in a serum-free medium, these cells become quiescent after about 48 h, as evidenced by the low rate of [3H]thymidine incorporation (8, 9). At quiescence, the BP-A31 cells continue to express a number of genes, such as c-myc, Received January 22, 1991. Address requests for reprints to: Dr. Dominique Fagot, INSERM U.55,184 rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France. * Supported by a grant from the Ministere de la Recherche et de la Technologic.
JE, as well as c-jun (8-12), which are characteristic of the state of competence in the nontransformed A31 cells. [Competence can be induced in the quiescent A31 cells by platelet-derived growth factor (PDGF) and is required for the completion of Gl-phase under the effect of progression factors, such as insulin-like growth factor-I (IGF-I) (13).] In contrast, the c-fos gene behaves alike in both the BP-A31 and A31 cell lines; its expression is absent at quiescence and can be induced by serum, 12O-tetradecanoyl 13-acetate (TPA), or basic fibroblast growth factor (bFGF) (11, 12). The product of the protooncogene c-fos forms a complex with the product of the protooncogene c-jun (14-16). This complex (transcription factor API) binds with high affinity to a specific region of DNA found in the promoters of numerous genes [TPA response element (TRE)] and leads to stimulation of transcription under appropriate conditions {e.g. stimulation with TPA). Several groups have recently demonstrated that the glucocorticoid receptor-hormone complex binds to the components of the API transcription factor and interferes with the transactivation of AP1dependent transcription (17-20). The quiescent serumdeprived BP-A31 cells respond to serum by resumption
1033
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1034
DEXAMETHASONE AND CELL GROWTH
of the cell division cycle and entry into the S-phase after a delay of about 10 h. Moreover, individual growth factors [IGF-I or insulin at 10"7 M or greater concentrations, at which it interacts with IGF-I receptors, bFGF, as well as phorbol esters (activators of protein kinase-C)] also assure the progression of the BP-A31 cells through the entire cell division cycle (9-12). We show that dexamethasone inhibits the growth response of BP-A31 cells to TPA and bFGF, but does not affect the mitogenic activity of insulin. At the same time, dexamethasone inhibits the expression of certain genes associated with the early Gl phase. The antimitogenic effects of dexamethasone are prevented by cycloheximide, suggesting that they require protein synthesis and, therefore, may not be exclusively the consequence of inhibition of the API-dependent gene expression.
Materials and Methods Cell culture Benzo-cv-pyrene-transformed BALB/c 3T3 mouse fibroblasts (BP-A31 cells) were cultured in a-Minimum Essential Medium supplemented with 6% fetal calf serum (FCS) in a humidified atmosphere containing 5% CO2. For the study of mitogenic effects, the cells were seeded in 24-well boxes (2-4 x 104 cells/well). After 24 h, the medium was replaced with 1 ml a-Minimum Essential Medium (serum free) plus 2.5 nM FeSO4, and the cells were allowed to enter quiescence during the next 2-3 days. For RNA preparation, the cells were grown in 100-
Endo-1991 Voll29«No2
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100 200
DEX [nil] FlG. 2. Concentration dependance of the effect of dexamethasone (DEX). Quiescent BP-A31 cells (M) were restimulated with TPA (100 ng/ml; • ) in the presence of different concentrations of dexamethasone, as indicated. The rate of [3H]thymidine incorporation was measured as described in Fig. 1. Data are expressed as a percentage of the maximal [3H]thymidine incorporation. The means of triplicates ± SEM are represented. Binding of [3H]dexamethasone (O) was measured, as described in Materials and Methods, and is shown as a percentage of the maximum (detected with 1 nM [3H]dexamethasone). Scatchard plot analysis of these data (not shown) indicated an apparent Kd of 2 X 10"8 M.
mm petri dishes to approximately 2 X 106 cells/dish before serum starvation. Incorporation of PH]thymidine Cells were incubated with 2 ^Ci [3H]thymidine/ml (Amersham, Les Ulis, France). Incorporation was allowed to proceed during 2-h pulses or for the entire period following the restimulation of serum-deprived cells and was terminated by acidification with 1 M ascorbic acid (three drops per ml). The cells were fixed with 5% trichloroacetic acid, solubilized in 0.1 N NaOH, and the incorporated radioactivity was determined by liquid scintillation counting. Analysis of RNA
Control
TPA
FGF
INS
FCS
FIG. 1. Effects of mitogens in BP-A31 cells: selective inhibition by dexamethasone. BP-A31 cells rendered quiescent by serum starvation were restimulated with TPA (200 ng/ml), bFGF (10 ng/ml), insulin (INS; 1 nM), or FCS (10%) in a-Minimum Essential Medium. [3H] Thymidine incorporation for 24 h after stimulation was determined, as described in Materials and Methods. Cells were restimulated in the absence (•) or presence of dexamethasone (2 x 10'7 M; H) or were preincubated (24 h) with 2 x 10'7 M dexamethasone before restimulation with mitogens and dexamethasone (2 x 10'7 M; • ) . The data shown are means of triplicates ± SEM. The differences were evaluated by Student's t test. *, P < 0.01; **, P < 0.001).
Cells in P100 dishes were chilled on ice and washed, and total RNA was isolated using the urea-lithium chloride precipitation procedure (21). Poly(A)+ RNA was selected by chromatography on oligo(dT)-cellulose. Twenty micrograms of total or 5 Mg poly(A)+ RNA were size-fractionated on agarose (1%)formaldehyde (2.2 M) gel, transferred onto nitrocellulose paper, fixed by heating at 80 C, and hybridized at 42 C with 32Plabeled probes in a solution containing 50% formamide, 0.45 M NaCl, 3 mM EDTA, and 75 mM phosphate buffer, pH 7.4. The filters were washed at 45 C in 0.15 M NaCl and 15 mM sodium citrate, and exposed with XAR 5 Kodak film and intensifying screen (Eastman Kodak, Rochester, NY; for more details, see Ref. 22). Probes
The probes were the 3' exon (Clal-EcoRl fragment) of the human c-myc gene (23), rat a-tubulin cDNA (24) used as
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DEXAMETHASONE AND CELL GROWTH
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TABLK 1. Steroid specificity of the effect of dexamethasone 3
H]Thymidine incorporation (cpm)
Steroid Basal 14,340 ± 730 6,250 ± 370° 8,570 ± 380° 9,600 ± 760* 12,570 ± 400 13,110 ± 1,220
None Dexamethasone Hydrocortisone Aldosterone Testosterone Progesterone
FGF
TPA 80,690 ± 28,670 ± 32,930 ± 62,510 ± 70,740 ± 73,430 ±
Insulin 99,830 ± 6,890 98,440 ± 4,480 98,090 ± 5,530 99,500 ± 3,680 96,100 ± 1,970 97,790 ± 1,460
59,120 ± 3,040 39,730 ± 1,400" 39,650 ± 430" 63,900 ± 1,920 65,200 ± 2,950 61,140 ± 2,750
3,830 170° 1,360° 2,0606 1,520 1,060
Serum-deprived BP-A31 cells were restimulated with TPA (100 ng/ml), FGF (50 ng/ml), or insulin (1 nM) in the presence or absence of steroids (2 X 10"7 M), as indicated. The data shown are means of triplicates ± SEM. The statistical significance of the effect of the different steroids was evaluated by Student's t test. " P < 0.001.
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FIG. 3. Reversal of the glucocorticoid effect by RU486. Quiescent BPA31 cells were restimulated by TPA (100 ng/ml; • ) or bFGF (50 ng/ ml; D) in the presence or absence of dexamethasone (DEX; 4 x 10"8 M) and different concentrations of RU486, as indicated. [3H]Thymidine incorporation was measured as described in Fig. 1. The data shown are means of triplicates ± SEM. internal standard, since the level of this mRNA does not vary
early in the cell cycle (25), JE cDNA corresponding to a PDGFinduced mRNA in BALB/c 3T3 cells (26), mouse c-jun cDNA (27), and the Bglll-Pvull fragment of the FBJ provirus {v-fos) (28).The probes were 3aP-labeled by the Multiprime DNA labeling system (Amersham). Evaluation of the glucocorticoid receptor contents Cells in P100 dishes were incubated for 30 min at 37 C with 10 nM [3H]triamcinolone acetonide (29 Ci/mmol; total binding) or [;iH]triamcinolone acetonide and 2 nM dexamethasone (nonsaturable binding). The cells were harvested and lysed with 0.5% Nonidet P-40 (NP40), and the nuclei were extracted with 0.5 M NaCl. To separate the receptor-bound and unbound steroid, the extracts were treated with 0.25% active charcoal suspension and centrifuged, and the radioactivity remaining in the supernatant was determined by liquid scintillation count-
TPA: + + + + + + Dex added a t ( h ) : 0 0 3 7 9 15 FIG. 4. Effect of delayed addition of dexamethasone (Dex). Quiescent BP-A31 cells were restimulated with TPA (200 ng/ml) at time zero, and dexamethasone (2 x 10'7 M) was added at various times thereafter. Incorporated radioactivity was measured 24 h after a continuous exposure to [3H]thymidine, as described in Fig. 1. The data shown are means of triplicates ± SEM.
ing. The same fractionation was also applied in the experiments in which nuclear binding of [3H]dexamethasone (92 Ci/mmol) was evaluated after incubation with different concentrations of the hormone. The nonsaturable binding was subtracted from the total binding value to calculate the receptor-bound [3H] dexamethasone. Growth factors and chemicals Bovine insulin was obtained from Boehringer; [3H]thymidine, [3H]triamcinolone acetonide, [3H] dexamethasone, and [32P]dCTP were purchased from Amersham. bFGF from two producers was used: Amersham (recombinant at 10 ng/ml) and Collaborative Research (Bethesda, MD; purified from bovine pituitary gland at 50 ng/ml). TPA and dexamethasone were obtained from Sigma (St. Louis, MO). Other compounds (reagent grade) were purchased from the usual commercial sources.
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DEXAMETHASONE AND CELL GROWTH
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TIME (hours) FIG. 5. Reversibility of the dexamethasone effect. Quiescent BP-A31 cells (•) were restimulated with TPA (100 ng/ml; • ) or pretreated with dexamethasone (2 x 10'7 M) during the last 10 h of quiescence, washed, and either immediately restimulated with TPA (•) or restimulated with TPA after 14 h of recovery in a-Minimum Essential Medium (0). [3H]Thymidine incorporation was measured during 2-h pulses every 3 h. The data shown are means of duplicate determinations represented at the mean times of the pulses.
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FIG. 6. Inhibition by dexamethasone (DEX) of the mitogenic response to TPA is prevented by cycloheximide. Quiescent BP-A31 cells were pretreated with cycloheximide (1 fig/m\), dexamethasone (2 X 10'7 M), or a combination of both, as indicated, for 12 h and then placed in fresh (serum-free) medium or restimulated with TPA (100 ng/ml). [3H] Thymidine incorporation was measured between 0-26 h. The data shown are means of triplicates ± SEM.
Results Effects of dexamethasone on I3H] thymidine incorporation in mitogen-stimulated BP-A31 cells As previously described (9-12), the quiescent serumstarved BP-A31 fibroblasts resume the cell division cycle
Endo«1991 Vol 129 • No 2
when placed in a medium containing either 10% FCS or individual growth factors, such as insulin, bFGF, or TPA. Dexamethasone (2 X 10~7 M), added simultaneously with the mitogens, caused a decrease in the rate of [3H] thymidine incorporation induced by TPA or bFGF (55% and 30%, respectively, in the experiment shown in Fig. 1). In contrast, the mitogenic effects of insulin or FCS were not or only slightly affected. The antimitogenic potency of dexamethasone was enhanced when dexamethasone was added 24 h before restimulation with TPA or bFGF (and maintained in the medium during incubation with the mitogens). In this case, the inhibition of [3H]thymidine incorporation was 80% and 60%, respectively. The presence of dexamethasone also led to a large decrease in the basal rate of [3H]thymidine incorporation in control cells fed fresh (serum-free) medium after serum starvation (Fig. 1). The antimitogenic effects of dexamethasone were concentration dependent. The 50% effective concentration evaluated on the basis of inhibition of the mitogenic effect of TPA was approximately 5 x 10"9 M (Fig. 2). In comparison, the apparent Kd of the receptor-mediated tight nuclear binding of [3H]dexamethasone was approximately 2 x 10 s M. [Similarly, Cook et al. (29) reported that in rat hepatoma Fu5 cells, the dose-response for growth inhibition by dexamethasone is shifted to concentrations of hormone lower than those required for comparable levels of receptor binding.] At 2 x 10'6 M, dexamethasone caused cell death, as observed by optical microscopy. The hypothesis of the involvement of the glucocorticoid receptor in the antimitogenic effects of dexamethasone was supported by the fact that steroids known to activate this receptor's function (hydrocortisone and aldosterone) were also inhibitors of TPA- and FGF-induced [3H]thymidine incorporation, in contrast with, for instance, testosterone (not bound by glucocorticoid receptors) or progesterone (forming a complex with the glucocorticoid receptor, but lacking agonist activity), which did not have a significant effect at 2 X 10'7 M (Table 1). Moreover, RU 486 (a synthetic antiglucocorticoid which competes for the glucocorticoid receptorbinding site) (30) antagonized the antimitogenic activity of dexamethasone in a concentration-dependent manner (Fig. 3). The reversal by RU 486 of the inhibitory effect of dexamethasone was apparently more effective with bFGF than with TPA. This may be related to the fact that TPA-induced DNA synthesis is more sensitive to inhibition by dexamethasone, so that the low proportion of agonist-receptor complexes present in the cells incubated with dexamethasone plus RU 486 (0.2 /IM) is sufficient to reduce the mitogenic response to TPA, but not that to FGF. At concentrations of 1 ixM or more, RU 486 was toxic to the cells.
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DEXAMETHASONE AND CELL GROWTH
1037
TABLE 2. Nuclear content of the glucocorticoid receptor in the BP-A31 cells: effect of pretreatment with dexamethasone 3
1. Control 2. Control after 15-min incubation with 2 X 10' M dexamethasone 3. Pretreatment (10-h) with cycloheximide (1 tig/ml), then incubation (15-min) with 2 X 1CT7 M dexamethasone 4. Pretreatment (10-h) with cycloheximide (1 /ig/ml) + dexamethasone (2 x 10"7 M), then incubation (15-min) with 2 x 10"7 M dexamethasone
H]Triamcinoline acetonide in the nuclear extract (cpm)
Determined nuclear binding (sites/cell)
Total
Nonsaturable
402 163
22 20
3800 1430
238
33
2050
175
24
1500
Serum-deprived BP-A31 cells were treated as indicated before incubation with [3H]triamcinolone acetonide (10 8 M) in the absence (total binding) or presence (nonsaturable binding) of 2 nM dexamethasone. The content of receptor-bound radioactive ligand was measured as described in Materials and Methods. Treatment of the nuclear extracts with 0.25% charcoal suspension did not reduce the radioactivity, confirming that the a H-labeled steroid was macromolecule bound. In the cells exposed to 2 X 10"7 M dexamethasone, only about 40% of the total receptor content was detected (due to isotopic dilution or incomplete exchange; compare lines 1 and 2).
The antimitogenic activity of dexamethasone may take place in the late Gl phase, as illustrated by the experiment shown in Fig. 4 in which the hormone was added to the cells at different time intervals after restimulation with TPA. With increasing delay after the addition of TPA, a progressive decrease in the inhibitory activity of dexamethasone was observed, but even when added as late as 9 h after the mitogen, dexamethasone significantly inhibited DNA synthesis. Once the cells entered into the S-phase of the cell cycle, dexamethasone had no effect on [3H]thymidine incorporation. Antimitogenic effects of dexamethasone in TPAstimulated cells require protein synthesis The antimitogenic effects of dexamethasone were also observed when the hormone was present in the culture medium for 10 h before, but not during, restimulation of the cells with TPA (Fig. 5). However, when cycloheximide (a protein synthesis inhibitor) was included along with dexamethasone during the preincubation, the inhibitory effects of dexamethasone on the cell's response to TPA were abolished (Fig. 6), indicating that dexamethasone action requires the synthesis of an intracellular or secreted antimitogenic protein(s). An alternative interpretation of this result could be elimination of the glucocorticoid receptor (down-regulation) during the preincubation with dexamethasone, under conditions where synthesis of new receptor molecules is blocked. To test this possibility, we performed an experiment in which the level of the receptor was evaluated by whole cell binding of [3H]triamcinolone acetonide; the results showed that only about 30% of the glucocorticoid-binding sites were lost during the 10-h incubation of the cells with dexamethasone and cycloheximide (us. cycloheximide alone; Table 2).
The experiments in which conditioned medium harvested from dexamethasone-treated cells was tested (data not shown) did not favor the possibility that an antimitogenic protein was secreted into the medium. The postulated glucocorticoid-induced protein (or the corresponding mRNA) is apparently long-lived, since inhibition of the mitogenic response to TPA was also observed in the experiment in which the cells were first treated with dexamethasone (10 h) and then incubated for 14 h in the absence of the steroid before restimulation (Fig. 5). The basal response of quiescent cells to fresh (mitogenfree) medium is enhanced by cycloheximide pretreatment (Fig. 6) (11, 12). The potentiation of the basal response was abolished by the presence of dexamethasone together with cycloheximide during the pretreatment (Fig. 6). Effects of dexamethasone in exponentially growing cells To determine whether exponentially growing cells are sensitive to dexamethasone, mitogen-dependent proliferation was studied in the absence or presence of 2 x 10'7 M dexamethasone (Fig. 7). In a mitogen-free medium as well as in TPA-containing medium, dexamethasone arrested cell growth within 24-48 h. In contrast, dexamethasone had little or no effect on cell growth in the presence of insulin. Effects of dexamethasone on the expression of early cell cycle-related genes
A long (24-h) treatment of the quiescent BP-A31 cells with dexamethasone did not prevent the induction of cfos mRNA accumulation by TPA or by bFGF (Fig. 8A, lanes 7 and 8 us. lane 6), indicating that it did not interfere with the FGF- or TPA-initiated mitogenic signal. (The induction ofc-fos gene transcription is probably
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a consequence of the activation of the serum-response element (31)-binding factor.] The mRNA corresponding to the JE gene was virtually eliminated during the 24-h incubation with dexamethasone (Fig. 8A, lanes 6-8). A short exposure of the cells to dexamethasone (3 h) was sufficient to strongly reduce the content in the JE as well as c-jun mRNA (Fig. 8B, lanes 2 vs. 1). However, when the cells were incubated with both dexamethasone and TPA, the decrease in the content of c-jun mRNA was not observed (Fig. 8B, lanes 3 and 4). The effect of dexamethasone on the expression of the JE and the cjun genes is independent of protein synthesis, since it also takes place in the presence of cycloheximide (Fig. 9, lanes 2 vs. 1). When the cells had been incubated with dexamethasone (3 h), only a slight decrease in the content of c-m.;yc mRNA was observed (Fig. 8B, lanes 2 vs. 1), and there was no effect if cycloheximide was included in the culture medium (Fig. 9, lanes 1 and 2). All of these genes are actively transcribed in the quiescent BP-A31 cells, since blocking the transcription by the addition of actinomycin-D (10 jug/ml) led to a total elimination of the corresponding mRNAs within 3 h (Fig. 9, lane 3). These results suggest that dexamethasone regulates the level of JE and c-jun mRNA at the transcriptional level.
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Under cell culture conditions, glucocorticoids frequently exercise an antiproliferative effect (1, 2), but the contrary, i.e. growth-promoting action, has also been reported for human diploid fibroblasts (3, 4). Our results demonstrate that dexamethasone, a potent synthetic glucocorticoid agonist, exercises a negative control of proliferation of mouse fibroblasts BP-A31 when either TPA or bFGF is used as the mitogen, but has little or no effect on the insulin-dependent growth (mediated by the IGFI receptor). This observation is consistent with our earlier report, in which we have proposed that the TPAand FGF-initiated mitogenic signaling pathways are in part coincident and distinct from that initiated by the IGF-I receptor-ligand interaction (11, 12). The effect of dexamethasone is possibly related to contact inhibition, since under the conditions of growth of sparsely seeded cells in a TPA-containing medium, dexamethasone had little effect in the early period, but caused an arrest at a FIG. 7. Effect of dexamethasone on growth of sparsely seeded bp-A31 cells. BP-A31 cells were plated in 24-well boxes (104 cells/well; - - -) or (4 x 104cells/well; —) in medium containing 10% FCS. After 24 h, the cells were washed and placed in a-Minimum Essential Medium containing TPA (100 ng/ml; B) or insulin (1 nM; C) or without added mitogens (A) in the presence (•, A, and • ) or absence (O, A, and • ) of dexamethasone (2 x 10"7 M). The cells were trypsinized and counted at the times indicated. In A and B, medium was refreshed after 96 h. The points represent means and ranges (when greater than the symbols) of duplicates.
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DEXAMETHASONE AND CELL GROWTH lower cell density. The effect is steroid specific, and in particular, it is countered by the glucocorticoid antagonist RU 486, indicating that it is mediated by the glucocorticoid receptor. The addition of dexamethasone to TPA-stimulated serum-starved BP-A31 cells as late as 9 h after the addition of TPA has a marked inhibitory effect on [3H] thymidine incorporation, suggesting that dexamethasone acts (also) in the late G l phase. (There is no effect of dexamethasone during the S-phase itself.) The antiproliferative effects of dexamethasone are not due to an inhibition of the mitogenic signaling pathways leading to early changes in gene expression, since dexamethasone does not prevent, for instance, the induction of c-fos RNA accumulation by mitogens. Modulation of gene expression is another possible mechanism for the antiproliferative activity of dexamethasone. In fact, glucocorticoids are known to induce the transcription of numerous genes possessing the glucocorticoid response element in the promoter region (32, 33), but can also inhibit gene expression, either by direct interaction with a negative control element in the promoter region (34) or indirectly by forming a complex with the transcription factor A P I (17-20) and, thus, blocking the TRE-directed transcription. In BP-A31 cells, treatment with dexamethasone at quiescence reduces the contents of both J E and c-jun mRNAs, probably by inhibiting the transcription of the corresponding genes (both containing T R E sequences at their promoters) (35, 36). When quiescent BP-A31 cells are pretreated with cycloheximide and then placed in mitogenfree medium, the basal rate of DNA synthesis is en-
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c-jun
c-fos a-tub.
c-myc •— —'
a-tub.
Fir.. 8. Effect of dexamethasone on the expression of early cell cyclerelated genes. A, Quiescent BP-A31 cells were restimulated (30 min) as follows: lane 1, control; lane 2, TPA (200 ng/ml); lane 3, TPA plus dexamethasone (2 X 10"7 M); lane 4, bFGF (10 ng/ml); and lane 5, bFGF plus dexamethasone. In lanes 6-8, the quiescent cells were preincubated with dexamethasone (for 24 h) before restimulation (for 30 min) with: lane 6, medium change control; lane 7, TPA; and lane 8, bFGF. Oligo(dT)-selected RNA samples (5 /ig/lane) were analyzed by Northern blotting and hybridized with fos, JE, and a-tublin probes. B, Serum-deprived BP-A31 cells were incubated in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of dexamethasone (2 x 10'7 M) for 3 h with: lanes 3 and 4, TPA (200 ng/ml); and lane 5 and 6, insulin (1 jtM). Samples (20 ^g total RNA/lane) were analyzed, as described in Materials and Methods, and hybridized with the probes as shown.
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123 c-jun c-myc a-tub.
••
JE FIG. 9. Effect of dexamethasone on early cell cycle-related mRNA levels in the presence of cycloheximide. Serum-deprived BP-A31 cells were incubated for 3 h in the presence of 1 /ug/ml cycloheximide alone (lane 1), with dexamethasone (2 x 10"7 M; lane 2), or with actinomycinD (10 Mg/ml; lane 3). Oligo(dT)-selected RNA samples (5 /ng/lane) were analyzed, as described above, and hybridized with the probes as shown.
hanced (10), possibly as a result of the superinduction of certain early cell cycle-related genes, including c-myc and c-fos (11, 25, 37) as well as c-jun (Buchou, T., unpublished data). The inhibition by dexamethasone of basal DNA synthesis as well as its potentiation by cycloheximide may be (in part) a consequence of the inhibition oifos-jun (API) complex-dependent TRE-directed gene expression. The reduction by dexamethasone of c-jun mRNA content was countered by TPA and is, therefore, unlikely to account for the antimitogenic activity of the steroid in TPA-stimulated cells. On the other hand, the JE protein does have cellular signaling activity (it is the monocyte chemoattractant protein MCP1) (38) and may play a role in the regulation of fibroblast growth. In nontransformed fibroblasts, JE/MCP1 is induced by the competence factor platelet-derived growth factor and may participate in the subsequent stimulation of the cells by progression factors; in BP-A31 cells, the constitutive synthesis of the JE/MCP1 protein may favor the mitogenic activity of phorbol esters. JE/MCP1 may also be involved in the inflammatory processes associated with wound healing (39). If so, both the growth inhibitory and antiinflammatory effects of glucocorticoids may be (partly) the result of inhibition of expression of the JE gene. [Glucocorticoids have also been reported to inhibit the growth of Fu 5 hepatoma cells by blocking the synthesis of a secreted growth factor (40).] Inhibition of TRE-dependent gene expression is probably not the only mechanism by which glucocorticoids inhibit the mitogenic response of the cells to TPA. In fact, preincubation with dexamethasone before restimulation with TPA (in the absence of the steroid) leads to strongly reduced [3H]thymidine incorporation compared with that in TPA-stimulated cells not treated with dexamethasone; this inhibitory effect is abolished if protein
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synthesis is blocked by cycloheximide during preincubation with dexamethasone. These observations indicate the involvement of a glucocorticoid-induced growth inhibitory protein. Alternatively, incubation with cycloheximide for 10 h could lead to elimination of a protein necessary to mediate the action of dexamethasone, such as the glucocorticoid receptor. However, we have verified that under these conditions, the content of glucocorticoid receptor was reduced by only about 30%. [This result is compatible with that of an earlier study (41) in which the ti/2 of glucocorticoid receptor turnover in cells cultured with dexamethasone was found to be 9.5 h.] There is at present no evidence of the involvement of proteins other than glucocorticoid receptor in the mediation of the effects of glucocorticoids on gene expression. In summary, two different mechanisms appear to participate in the antimitogenic activity of dexamethasone in mouse fibroblasts (BP-A31): inhibition of expression of Gl progression-related genes (for instance, those having TRE sequences at their promoter), and induction of the synthesis of a growth inhibitory protein(s). These growth inhibitory proteins are probably intracellular and remain unknown at present. The lipocortin family offers possible candidates; these proteins have been reported to inhibit the intracellular activity of phospholipase-A2, thus interfering with the formation of metabolites of arachidonic acid. It has been suggested that these metabolites may participate in the autocrine mode of regulation of cell growth, particularly in the inflammatory situation (42, 43). The possibility of the involvement of such compounds in the antimitogenic effect of dexamethasone needs further study. Acknowledgments The authors thank Y. Issoulie for illustrations, and Dr. G. Rosselin for his interest in this work. References 1. Durant S, Duval D, Homo-Delarche F 1986 Factors involved in the control of fibroblast proliferation by glucocorticoids: a review. Endocr Rev 7:254-269 2. Young DV, Dean MC 1980 The suppression of cellular proliferation in SV 40-transformed 3T3 cells by glucocorticoids. J Cell Physiol 102:223-231 3. Cristofalo VJ, Rosner BA 1979 Modulation of cell proliferation and senescence of WI-38 cells by hydrocortisone. Fed Proc 38:18511856 4. Finlay CA, Cristofalo VJ 1987 Autocrine stimulation of W138 cell proliferation in the presence of glucocorticoids. Exp Cell Res 168:191-202 5. Finlay CA, Cristofalo VJ 1987 In: Boyton AL, Leffert HL (eds) In Vitro Control of Animal Cell Proliferation. Academic Press, New York, vol 2:203-217 6. Vedeckis WV, Eastman-Reks SB, Lapointe MC, Reker CE 1987 In: Spelsberg TC, Kumar R (eds) Steroid and Sterol Hormone Action. Martinus Nijhoff, Boston, pp 213-225 7. Dubrow DR, Riddle VGH, Pardee AB 1979 Different responses to drug and serum of cells transformed by various means. Cancer Res
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39:2718-2726 8. Campisi J, Gray HE, Pardee AB, Dean M, Sonnenshein GE 1984 Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell 36:241-247 9. Gray HE, Buchou T, Mester J 1987 Serum-induced G0/G1 transition in chemically transformed 3T3 cells. Exp Cell Res 169:95104 10. Buchou T, Charollais RH, Mester J 1988 Involvement of serum factor(s) adsorbed to the dish in the response of cycloheximidepretreated BP-A31 cells to serum pulses. Exp Cell Res 174:411420 11. Buchou T, Charollais RH, Fagot D, Mester J 1989 Mitogenic activity of phorbol esters and insulin-like growth factor 1 in chemically transformed mouse fibroblasts BP-A31: independent effects and differential sensitivity to inhibition by 3-isobutyl-l-methyl xanthine. Exp Cell Res 182:129-143 12. Buchou T, Mester J 1990 The fibroblast growth factor-dependent mitogenic signal transduction pathway in chemically transformed mouse fibroblast is similar to but distinct from that initiated by phorbol esters. J Cell Physiol 142:559-565 13. Pledger WJ, Stiles CD, Antoniades HN, Scher CD 1978 An ordered sequence of events is required before Balb/c 3T3 cells become committed to DNA synthesis. Proc Natl Acad Sci USA 75:28392843 14. Lee W, Mitchell P, Tjian R 1987 Purified transcription factor AP1 interacts with TPA-inducible enhancer elements. Cell 49:741752 15. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M 1987 Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729-739 16. Bohmann D, Bos TJ, Admon A, Nishimura T, Vogt PK, Tjian R 1987 Human protooncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor API. Science 238:1386-1392 17. Lucibello FC, Slater EP, Jooss KU, Beato M, Miiller R1990 Mutual transrepression of Fos and glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB. EMBO J 9:2827-2834 18. Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and anti inflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189-1204 19. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between cjun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:12051215 20. Schiile R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-jun and the glucocorticoid receptor. Cell 62:12171226 21. Auffray C, Rougeon F 1980 Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. EurJBiochem 10:303-314 22. Maniatis T, Fritch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 23. Dalla Favera R, Gelmann EP, Martinotti S, Franchini G, Papas TS, Gallo R, Wong-Staal F 1982 Cloning and characterization of different human sequences related to the one gene (c-myc) of avian myelocytomatosis virus (MC29). Proc Natl Acad Sci USA 79:64976501 24. Lemishka IR, Farmer S, Racaniello VR, Sharp PA 1981 Nucleotide sequence and evolution of a mammalian a-tubulin messenger RNA. J Mol Biol 51:101-121 25. Greenberg ME, Ziff EB 1984 Stimulation of 3T3 cells induces transcription of the c-fos protooncogene. Nature 311:433-438 26. Cochran BH, Reffel AC, Stiles CD 1983 Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell 33:939947 27. Ryseck RP, Hirai SI, Yaniv M, Bravo R 1988 Transcriptional
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of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction. Nucleic Acids Res 18:23-34 Angel P, Hattori K, Smeal T, Karin M 1988 The jun protooncogene is positively autoregulated by its product, Jun/AP-1. Cell 55:875-885 Dani C, Blanchard JM, Piechaczyk M, El Sabouty S, Mathy L, Jeanteur P 1984 Extreme instability of c-myc mRNA in normal and transformed human cells. Proc Natl Acad Sci USA 81:70467050 Rollins BJ, Sher P, Ernst T, Wong GG 1989 The human homolog of the JE gene encodes a monocyte secretory protein. Mol Cell Biol 9:4687-4695 Leonard EJ, Yoshimura T 1990 Human monocyte chemoattractant protein-1 (MCP-1). Immunol Today 11:97-101 Cook PW, Edwards CP, Haraguchi T, Firestone GL 1989 Partial characterization of a glucocorticoid suppressible mitogenic activity secreted from rat hepatoma cell line hypersensitive to the antiproliferative effects of glucocorticoids. J Biol Chem 264:14151-14158 Mclntyre WR, Samuels HH 1985 Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-time of the activated nuclear receptor form. J Biol Chem 260:418-427 Taylor L, Polgar P 1977 Self regulation of growth by human diploid fibroblasts via prostaglandin production. FEBS Lett 79:69-72 Pentland AP, Needleman P 1986 Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis. J Clin Invest 77:246-251
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