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Endocrinology Vol. 138, No. 6 2219-2226
Copyright © 1997 by The Endocrine Society

ARTICLES

Growth Factor-Induced Transcription via the Serum Response Element Is Inhibited by Cyclic Adenosine 3',5'-Monophosphate in MCF-7 Breast Cancer Cells1

William L. Lowe, Jr., Ronggen Fu and Michelle Banko

Department of Medicine, Veterans Administration Chicago Healthcare System and Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Dr. William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15–703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effect of increased intracellular cAMP on MCF-7 breast cancer cell growth was examined by treating cells with either forskolin, an activator of adenylate cyclase, or 8-[4-chlorophenylthio]-cAMP (8-CPT-cAMP), a cAMP analog. Compared to cells maintained in control medium, treatment with either 1 or 10 µM forskolin decreased cell growth by 17% and 68%, respectively, whereas treatment with 250 µM 8-CPT-cAMP decreased cell growth by 29%. To determine whether this effect of cAMP on cell growth was mediated by inhibition of the activity of extracellular signal-regulated kinases 1 and 2 (ERK1 and -2), two mitogen-activated protein kinases, the effect of cAMP on growth factor-induced ERK activity in MCF-7 cells was examined. Treatment with either insulin-like growth factor I (IGF-I) or epidermal growth factor (EGF) for 10 min stimulated a 4- to 8-fold increase in ERK1 and -2 activity. This effect of IGF-I and EGF was not inhibited by increased intracellular cAMP generated by pretreatment of the cells with 10 µM forskolin. Similarly, 10 µM forskolin had no effect on IGF-I- or EGF-induced ERK activity in cells treated with growth factor for 30 min. To determine whether cAMP inhibits other growth factor-mediated effects, its effect on the activity of the serum response element (SRE), a DNA promoter element whose activity is regulated by a variety of growth-promoting events, was examined. For these assays, MCF-7 cells were transiently transfected with pTK81-SRE-Luc, a luciferase fusion gene that contains the SRE cloned 5' to a minimal thymidine kinase promoter and the luciferase gene. Treatment with either IGF-I or EGF increased pTK81-SRE-Luc activity in a dose-dependent fashion. Pretreatment of cells with 10 µM forskolin decreased IGF-I- and EGF-stimulated luciferase activity by ~75%. An intermediate effect was observed using 1 µM forskolin. When intracellular cAMP levels were increased using 8-CPT-cAMP, similar results were obtained. SRE activity is dependent upon the activation by phosphorylation of a ternary complex factor; included among the ternary complex factors is Elk-1. When MCF-7 cells were cotransfected with a vector that expresses a Gal4/Elk-1 fusion protein and UAS-TK-Luc, a plasmid that contains two Gal4 DNA recognition sites cloned 5' to a thymidine kinase promoter and the luciferase gene, treatment with forskolin partially inhibited the activation of Elk-1 by IGF-I and EGF. These data demonstrate that in MCF-7 breast cancer cells, cAMP has no effect on IGF-I- or EGF-induced ERK activity, but it inhibits growth factor-induced transcription. Taken together with the effects of cAMP on IGF-I- and EGF-induced Elk-1 activation, these data suggest that the effect of cAMP on SRE activity occurs distal to ERK activation, possibly via inhibition of an ERK-independent pathway. Finally, these data indicate that the effect of increased intracellular cAMP on breast cancer growth may be mediated through inhibition of specific growth factor-induced effects, including gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INCREASED intracellular cAMP has complex effects on cell growth, inhibiting the growth of some cell types and stimulating the growth of others (1, 2). Among the cell types whose growth is stimulated by cAMP is normal human breast epithelial cells in culture (3). In contrast, although there are some contradictory data (4, 5), several studies have demonstrated that cAMP inhibits the growth of breast cancer cells, as determined using both established cell lines and cells in primary culture (2, 3, 6, 7, 8, 9). The mechanism for this growth inhibitory effect of cAMP in breast cancer cells has not been determined, but in some cell types, cAMP is able to inhibit the activation of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1 and -2) (10, 11, 12, 13, 14, 15, 16). The ERKs are cytoplasmic protein kinases that phosphorylate and activate a variety of substrates, including phospholipase A2, p90rsk, and the ternary complex factor (TCF) that binds to the serum response element (SRE) (17, 18, 19). Moreover, the ERKs appear to be important in the mitogenic effect of a variety of growth factors, given that dominant negative ERK mutants, ERK antisense RNA, and overexpression of an ERK phosphatase, MAPK phosphatase 1, inhibit growth factor-induced mitogenesis (20, 21).

A family of growth factors that regulates breast cancer growth is the insulin-like growth factors (IGFs) (reviewed in Refs. 22 and 23). IGF-I treatment stimulates a ~3-fold increase in the growth of two different breast cancer cell lines, MCF-7 and T47D cells (24, 25). Moreover, treatment of MCF-7 cells with either IGF-binding protein-1 (IGFBP-1), a peptide that binds to and, in some cases, inhibits IGF action, or {alpha}-IR3, an antibody that blocks ligand binding to the IGF-I receptor, inhibits serum-induced cell growth (25, 26). A role for IGF-I in the growth of breast cancers in vivo has also been postulated based upon the observation that IGF-I is produced by stromal cells in breast cancers (22). IGF-I produced by stromal cells can then act in a paracrine fashion on malignant epithelial cells, as these cells express IGF-I receptors on their cell surface (23, 27). A second growth factor that is important for breast cancer growth is epidermal growth factor (EGF) (22, 28). The receptor for EGF is overexpressed in ~40% of breast cancers (29, 30, 31), and monoclonal antibodies directed against the EGF receptor inhibit basal and ligand-stimulated breast cancer cell growth (32, 33, 34, 35). One of the ligands that mediates its effects via the EGF receptor is transforming growth factor-{alpha} (TGF-{alpha}) (28). TGF-{alpha} overexpression, either in vitro or in vivo in transgenic mice, transforms mammary epithelial cells (36, 37), further suggesting a role for EGF receptor activation in breast cancer development.

As noted, the mechanisms responsible for the inhibition of breast cancer cell growth by increased intracellular cAMP levels are unknown. One potential mechanism for its growth inhibitory effects in breast cancer cells would be inhibition of growth factor-induced signal transduction, including activation of the ERKs. The present studies were designed to address that issue by examining the effect of cAMP on IGF-I- and EGF-induced ERK activation and gene transcription in MCF-7 breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
MCF-7 cells were kindly provided by Dr. Craig Jordan (Lurie Cancer Center, Northwestern University Medical School). The cells were maintained in 75-cm2 flasks in MEM supplemented with 5% calf serum (CS), 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (50 U/ml) at 37 C in 5% CO2. Upon reaching confluence, the cells were replated at a dilution of 1:4. In all experiments the cells were preincubated for the indicated period of time in phenol red-free MEM with 0.1% charcoal-stripped calf serum (CSCS).

Cell proliferation assay
Cell proliferation assays were performed using the CellTiter 96 Nonradioactive Cell Proliferation Assay kit (Promega Corp., Madison, WI) according to the manufacturer’s instructions. This assay is based upon the ability of viable cells to bioreduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium to formazon in the presence of phenazine methosulfate, an electron-coupling reagent. Formazon production is quantified by measuring absorbance at 490 nm, which is directly proportional to the number of living cells. For these assays, MCF-7 cells were plated at a density of 2 x 103 cells/well on a 96-well plate and maintained in MEM and 5% CS for 24 h. At that time the cells were treated with either forskolin or 8-\[4-chlorophenylthio\]-cAMP (8-CPT-cAMP) as indicated. Formazon production was determined 24 h (day 0), 72 h (day 2), 120 h (day 4), and 168 h (day 6) after plating using an EL 312e Bio-Kinetics Reader (Bio-Tek Instruments, Winooski, VT). In each of three independent experiments, the mean absorbance in six individual wells was determined for each condition and time point.

Plasmid constructions
The plasmid pTK81-SRE-Luc was constructed by cloning a 28-bp fragment of the SRE from the human c-fos gene into the SmaI site of the parent vector pTK81-Luc. The vector pTK81-Luc contains 81 bp of the thymidine kinase promoter cloned 5' to the coding region of the luciferase gene. The sequence of the SRE was as follows: 5'-ACAGGATGTCCATATTAGGACATCTGCG-3'. The vector c-fos-Luc was constructed as described previously (38). Briefly, it contained the region from -361 to +157 of the human c-fos gene, which had been subcloned 5' to the coding region of the luciferase gene in the vector pA3Luc. The plasmid -846 {alpha}Luc contained 846 bp of 5'-flanking region and 44 bp of exon 1 of the human glycoprotein hormone {alpha}-subunit gene, which had been subcloned 5' to the coding region of the luciferase gene in the vector pA3Luc (39). The plasmid UAS-TK-Luc contains two copies of the Gal4 recognition sequence (UAS) cloned 5' to a 109-bp fragment of the thymidine kinase promoter and a luciferase reporter gene in the vector pA3Luc. The vector p GAL-Elkc was kindly provided by Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School, Boston, MA). This vector is a derivative of the murine leukemia virus (MLV) enhancer/ß-globin-based expression plasmid MLVßplink, and its construction has been described previously (40). Briefly, it expresses a fusion protein that contains the activation domain of Elk-1 (residues 307–428) fused to the Gal4 DNA-binding domain (residues 1–147).

Transient transfection and luciferase assays
MCF-7 cells were transfected by calcium phosphate precipitation using standard methods (41). For transfection assays, the cells were plated onto 12-well plates at a density of 2 x 105 cells/well. During transfection, the cells were incubated initially with the calcium phosphate-DNA mixture alone for 20 min, followed by incubation for 5 h with the calcium phosphate-DNA mix in the presence of MEM and 5% CS. The cells were then washed and placed in phenol red-free MEM with 0.1% CSCS for 16 h. After this preincubation in medium with reduced serum, the cells were treated with growth factors for the indicated periods of time. The cells were then harvested, and luciferase assays were performed as described previously, except that the assay buffer contained 330 µM coenzyme A, and light emission was measured with an AutoLumat LB953 luminometer (EG&G Berthold, Bad Wildbad, Germany) using a 60-sec integration mode (42, 43). The luciferase activity present in each sample was normalized using the protein content of the sample, as determined using the method of Lowry et al. (44). All luciferase assays were performed in triplicate.

MAPK assays
MAPK assays were performed using an immune complex kinase assay as described previously with minor modifications (38, 45). Briefly, 24 h after plating, the cells were placed onto phenol red-free MEM and 0.1% CSCS for 48 h. After this 48-h incubation, the cells were treated as described with growth factors and lysed in 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 0.12 mM phenylmethylsulfonylfluoride, 0.7 µg/ml leupeptin, and 50 mM Tris, pH, 7.5. After clarification, 250 µg cell lysate protein were incubated with either polyclonal anti-ERK1 or -ERK2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) that had been prebound to protein A-agarose beads (Pierce Chemical Co., Rockford, IL). The beads were then collected, washed, and resuspended in kinase buffer (25 mM HEPES, pH 7.4; 10 mM MgCl2; 10 mM MnCl2; 1 mM dithiothreitol; and 15 µM ATP). To the reaction mix were added the substrate, myelin basic protein (Sigma Chemical Co., St. Louis, MO), at a final concentration of 167 µg/ml and 500 µCi/ml [{gamma}-32P]ATP. The reaction was allowed to proceed for 20 min at 22 C and was terminated by the addition of Laemmli sample buffer. The reaction products were then separated by SDS-PAGE. The resulting gel was dried and exposed to x-ray film or used in the PhosphorImager (Fuji Medical Systems, USA, Inc., Stanfield, CT) to calculate 32P incorporation into myelin basic protein. All assays were performed in duplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An initial series of studies was performed to examine the effect of increased intracellular cAMP on MCF-7 cell growth. Intracellular cAMP levels were increased using either forskolin, an activator of adenylate cyclase, or 8-CPT-cAMP, a cAMP analog that increases intracellular cAMP levels by a mechanism distinct from that of forskolin. In these studies, cells were grown for 6 days in normal growth medium (MEM plus 5% CS) in the absence or presence of either 1 or 10 µM forskolin or 250 or 500 µM 8-CPT-cAMP (Fig. 1Go). Treatment with 1 or 10 µM forskolin decreased cell growth by 17 ± 8% and 68 ± 15% (mean ± SEM; n = 3), respectively, compared to that of control cells grown in MEM and 5% CS. Treatment with 250 µM 8-CPT-cAMP decreased cell growth by 28 ± 9% (mean ± SEM; n = 3). Interestingly, when cells were grown in the presence of 500 µM 8-CPT-cAMP, cell growth was inhibited by 20 ± 7% (mean ± SEM; n = 3), an effect that was slightly less than that of 250 µM 8-CPT-cAMP. These effects of increased intracellular cAMP on MCF-7 cell growth are consistent with previous reports demonstrating inhibition of breast cancer cell growth by increased intracellular cAMP (3, 6, 7, 8, 9).



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  		Figure 1. Effect of cAMP on proliferation of MCF-7 breast cancer cells. MCF-7 cells were plated at a density of 2 x 103 cells/well in a 96-well plate, and 24 h after plating of the cells, which was defined as day 0, the cells were treated with medium alone (MEM and 5% CS) or medium with the indicated concentrations of either forskolin or 8-CPT-cAMP. Medium was changed daily, and cell number was determined as described in Materials and Methods on days 0, 2, 4, and 6. Values represent the relative absorbance at 490 nm compared to the absorbance on day 0, which was defined as 1.0, and are the mean of the values from six wells. The results shown here are representative of one experiment and were reproduced in three independent experiments.

 
To determine whether the inhibition of breast cancer cell growth by cAMP may be mediated via the inhibition of growth factor-induced ERK activity, a series of studies was performed to examine the effect of increased intracellular cAMP on growth factor-induced ERK activity in MCF-7 breast cancer cells. Initially, the time course of ERK activation by IGF-I and EGF was examined using an immune complex assay to determine ERK activity, as measured by 32P incorporation into myelin basic protein. Activation of ERK2 by IGF-I was seen as early as 5 min after the addition of IGF-I and was sustained for up to 30 min (Fig. 2Go, left panel). Similarly, EGF increased the activity of ERK2 (Fig. 2Go, right panel), an effect that was present as early as 5 min after the addition of EGF and was sustained for as long as 30 min. Similar results were obtained when the time-course effect of IGF-I and EGF on ERK1 activation was examined (data not shown).



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  		Figure 2. Time-course effect of IGF-I and EGF on ERK2 activity. After incubation for 48 h in phenol red-free MEM and 0.1% CSCS, cells were treated with either 50 ng/ml IGF-I (left panel) or 50 ng/ml EGF (right panel) for the indicated time periods. An immune complex assay was used to measure activity of ERK2 with myelin basic protein as a substrate. 32P incorporation into myelin basic protein was quantified using a PhosphorImager. The values represent the relative 32P incorporation into myelin basic protein compared to the incorporation stimulated by cells treated with MEM and 0.1% CSCS alone, which was defined as 1.0, and are the mean ± SD of two independent experiments performed in duplicate.

 
To determine the effect of increased intracellular cAMP on growth factor-induced ERK activity, MCF-7 cells were incubated for 30 min in the absence or presence of 10 µM forskolin, followed by treatment with either IGF-I or EGF (Fig. 3Go). Growth factor treatment was conducted for either 10 or 30 min to examine the effect of forskolin on both early and late activation of the ERKs by IGF-I and EGF. IGF-I again increased ERK2 activity at 10 min (Fig. 4AGo) and 30 min (Fig. 4BGo). In cells maintained in phenol red-free MEM and 0.1% CSCS alone, forskolin had little to no effect on the basal activity of ERK2 at either 10 or 30 min. Similarly, treatment with forskolin had no effect on IGF-I-induced activation of ERK2 at both 10 and 30 min (Fig. 4Go, A and B). Similar effects were seen when cells were treated with forskolin and EGF. Treatment with forskolin had little or no effect on basal activity of ERK2 at either 10 min (Fig. 4CGo) or 30 min (Fig. 4DGo), and forskolin treatment had no effect on EGF-induced activity of ERK2 at 10 or 30 min (Fig. 4Go, C and D). Treatment with forskolin also had no effect on either IGF-I- or EGF-induced ERK1 activity at 10 and 30 min (data not shown).



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  		Figure 3. Autoradiogram demonstrating the effect of forskolin on IGF-I- and EGF-stimulated ERK2 activity. After incubation for 48 h in phenol red-free MEM and 0.1% CSCS, cells were treated for 10 min with either 50 ng/ml IGF-I (left panel) or 50 ng/ml EGF (right panel) in the presence or absence of 10 µM forskolin. ERK2 activity was determined using an immune complex assay with myelin basic protein as a substrate. The arrows indicate bands that represent myelin basic protein.

 


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  		Figure 4. The effect of forskolin on IGF-I- and EGF-stimulated ERK2 activity. After incubation for 48 h in phenol red-free MEM and 0.1% CSCS, cells were treated with 50 ng/ml IGF-I for either 10 min (A) or 30 min (B) or with 50 ng/ml EGF for either 10 min (C) or 30 min (D) in the presence or absence of 10 µM forskolin. ERK2 activity was determined using an immune complex assay with myelin basic protein (MBP) as a substrate. For each panel, the values represent the relative 32P incorporation into myelin basic protein compared to the incorporation stimulated by cells treated with MEM and 0.1% CSCS (SF) alone, which was defined as 1.0, and are the mean ± SD of two independent experiments performed in duplicate.

 
Given the lack of effect of cAMP on ERK activity, the effect of cAMP on growth factor-induced gene transcription was examined to determine whether cAMP may inhibit other growth factor-mediated effects in MCF-7 cells. To address this issue, a model of growth factor-induced gene transcription was established. In MCF-7 cells stably transfected with a human c-fos-luciferase fusion gene (c-fos-Luc), treatment of the cells with either 50 ng/ml IGF-I or 5 ng/ml EGF increased luciferase activity 13.7 ± 1.5- and 11.5 ± 2.3-fold (mean ± SEM; n = 3 for IGF-I and n = 4 for EGF), respectively, compared to the luciferase activity present in extracts from cells maintained in phenol red-free MEM and 0.1% CSCS alone. As the c-fos promoter contains a cAMP response element in addition to other promoter elements, including the SRE, a sis-inducible element, and an activating protein-1 site (46), a simplified reporter system was established by subcloning a 28-bp fragment of the SRE 5' to a minimal thymidine kinase promoter and luciferase reporter gene to generate pTK81-SRE-Luc. The SRE was used because it is a growth factor-responsive promoter element that is activated in response to a variety of growth-promoting events (19, 47, 48).

To establish the growth factor responsiveness of pTK81-SRE-Luc, it was transiently transfected into MCF-7 cells, followed by treatment of the cells with either IGF-I or EGF. Time-course studies demonstrated a maximal effect of IGF-I and EGF on pTK81-SRE-Luc activity after 4–6 h of treatment (data not shown). The effect of increasing doses of either IGF-I or EGF on luciferase activity was then determined by maintaining the cells in phenol red-free MEM with 0.1% CSCS for 16 h after transfection and then treating the cells for 4 h with increasing doses of either IGF-I or EGF. Increasing doses of IGF-I stimulated a dose-dependent increase in luciferase activity (Fig. 5Go, left panel). The maximum effect was achieved with 100 ng/ml IGF-I, which increased luciferase activity 46.9 ± 10.6-fold. In contrast to the effect of IGF-I on luciferase activity in cells transfected with pTK81-SRE-Luc, treating cells that had been transfected with pTK81-Luc alone for 4 h with 50 ng/ml IGF-I had no effect (1.1 ± 0.1-fold increase) on luciferase activity. Thus, the effect of IGF-I on luciferase activity was mediated by the SRE and not by vector sequences. To confirm that the stimulatory effect of IGF-I was being mediated by the IGF-I receptor, the cells were also treated for 4 h with increasing doses of insulin. Treatment with insulin increased luciferase activity, although the effect of insulin was much less dramatic than that of IGF-I (Fig. 5Go, left panel). Treating cells with 100 ng/ml insulin increased luciferase activity 9.2 ± 1.7-fold, which is similar to the effect achieved with 5 ng/ml IGF-I. In contrast, 10 µg/ml insulin stimulated luciferase activity to a similar degree as IGF-I (data not shown). These data are consistent with the effect of IGF-I being mediated by the IGF-I receptor. EGF also stimulated SRE promoter activity, as demonstrated by the ability of increasing doses of EGF to increase luciferase activity in a dose-dependent fashion (Fig. 5Go, right panel). A maximum effect of EGF was obtained with 1 ng/ml EGF, which increased luciferase activity 72.5 ± 6.1-fold.



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  		Figure 5. Dose-response effects of IGF-I, insulin, and EGF on SRE activity. MCF-7 cells were transfected with pTK81-SRE-Luc as described in Materials and Methods and maintained in phenol red-free MEM and 0.1% CSCS for 14 h after transfection. The cells were then treated with the indicated doses of either IGF-I and insulin (left panel) or EGF (right panel). After a 4-h treatment period, luciferase activity was determined. The values represent the relative luciferase activity compared to the activity in cells maintained in phenol red-free MEM and 0.1% CSCS, which was defined as 1.0, and are the mean ± SEM of five individual experiments performed in triplicate.

 
To determine the effect of increased intracellular cAMP on growth factor-induced gene expression, MCF-7 cells were pretreated for 30 min with increasing doses of forskolin, followed by treatment for 4 h with either 50 ng/ml IGF-I or 50 ng/ml EGF. Treatment with 10 µM forskolin decreased the effects of IGF-I and EGF on luciferase activity by ~75% (Fig. 6Go, top panel), whereas treatment with 1 µM forskolin had an intermediate effect on IGF-I- and EGF-stimulated luciferase activity. In the absence of added growth factors, forskolin had a small stimulatory effect on the basal activity of pTK81-SRE-Luc. To demonstrate that the observed effect of forskolin on IGF-I- and EGF-induced SRE activity was not specific to forskolin, a second series of studies was performed using 8-CPT-cAMP. After pretreatment of the cells for 30 min with increasing doses of 8-CPT-cAMP, the cells were treated for 4 h with either 50 ng/ml IGF-I or 10 ng/ml EGF (Fig. 6Go, bottom panel). In the absence of 8-CPT-cAMP, IGF-I and EGF increased luciferase activity 42.6 ± 9.7- and 52.7 ± 16.8-fold, respectively, compared to the luciferase activity present in cells maintained in phenol red-free MEM and 0.1% CSCS alone. In contrast, in cells treated with 500 µM 8-CPT-cAMP, IGF-I and EGF increased luciferase activity 5.0 ± 0.5- and 4.9 ± 1.3-fold, respectively, compared to the luciferase activity that was present in cells treated with 500 µM 8-CPT-cAMP in the absence of added growth factors. Treating cells with 500 µM 8-CPT-cAMP alone increased the basal activity of pTK81-SRE-Luc by 3.1 ± 0.4-fold (Fig. 6Go, bottom panel). These data demonstrate that increased levels of intracellular cAMP attenuate growth factor-induced SRE activity.



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  		Figure 6. Effects of forskolin and 8-CPT-cAMP on growth factor-induced SRE activity. MCF-7 cells were transfected with pTK81-SRE-Luc as described in Materials and Methods and maintained in phenol red-free MEM and 0.1% CSCS for 14 h after transfection. The cells were then treated with the indicated doses of forskolin (top panel) or 8-CPT-cAMP (bottom panel) for 30 min followed by treatment for 4 h with medium alone, 50 ng/ml IGF-I, or 50 ng/ml EGF in the presence or absence of the indicated dose of forskolin or 8-CPT-cAMP. The cells were then harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared to the level in cells maintained in MEM and 0.1% CSCS in the absence of forskolin or 8-CPT-cAMP, which was defined as 1.0, and are the mean ± SEM of either 8 (top panel) or 3 (bottom panel) independent experiments performed in triplicate.

 
As a control to demonstrate that increasing intracellular cAMP levels with either forskolin or 8-CPT-cAMP did not have a toxic effect on the cells that resulted in a general inhibition of gene transcription, MCF-7 cells were also transiently transfected with -846 {alpha}Luc, a fusion gene that contains 846 bp of the 5'-flanking region of the glycoprotein hormone {alpha}-subunit gene cloned 5' to the luciferase gene. Expression of the glycoprotein hormone {alpha}-subunit gene is known to be cAMP responsive (49). After transient transfection of MCF-7 cells with -846 {alpha}Luc, treatment for 4 h with either 1 or 10 µM forskolin increased luciferase activity 79.7 ± 9.5- and 90.1 ± 14.7-fold, respectively (mean ± SEM; n = 5). Similarly, treatment of cells with 100, 250, or 500 µM 8-CPT-cAMP increased luciferase activity 16.4 ± 1.5-, 62.8 ± 8.7-, and 75.9 ± 6.3-fold (mean ± SEM; n = 3), respectively. These data demonstrate the specificity of the inhibitory effect of forskolin and 8-CPT-cAMP on growth factor-induced gene transcription.

SRE activity is modulated by activation of a TCF that binds to the SRE. TCF is one of several Ets domain DNA-binding proteins that include Elk-1 (19, 48). Phosphorylation of Elk-1 by the MAPKs, including ERK1 and -2, increases Elk-1 activity (19, 48). To determine whether the inhibition of growth factor-induced SRE activity by cAMP may occur at the level of Elk-1 activation, MCF-7 cells were transfected with pGAL/Elkc, an expression vector that expresses a Gal4/Elk-1 fusion protein that contains the DNA-binding domain of Gal4 and the transactivation domain of Elk-1. To measure activation of the fusion protein by phosphorylation of the transactivation domain of Elk-1, the cells were cotransfected with a reporter plasmid, UAS-TK-Luc, that contains two copies of the Gal4 DNA recognition site cloned 5' to a thymidine kinase promoter and a luciferase reporter gene. Initially, the ability of IGF-I to increase Elk-1 activity was examined. In cells transfected with UAS-TK-Luc alone, treatment with 50 ng/ml IGF-I stimulated only a modest increase in luciferase activity, presumably due to a direct effect on UAS-TK-Luc (Fig. 7Go). Cotransfection of both UAS-TK-Luc and pGAL/Elkc resulted in a modest increase in basal luciferase activity, whereas treatment of these cells with 50 ng/ml IGF-I markedly increased luciferase activity (Fig. 7Go). These data indicate that IGF-I activates Elk-1 in MCF-7 breast cancer cells. The effect of cAMP on IGF-I-stimulated Elk-1 activity was then examined. Treatment of cells that had been transfected with UAS-TK-Luc alone with 10 µM forskolin increased basal luciferase activity, again due to a direct effect of cAMP on UAS-TK-Luc activity. IGF-I had only a minimal effect on luciferase activity in these cells. When cells were cotransfected with both p GAL/Elkc and UAS-TK-Luc and treated with 10 µM forskolin, treatment with 50 ng/ml IGF-I increased luciferase activity, but the increase in luciferase activity was attenuated compared to that stimulated in the absence of forskolin (Fig. 7Go). A similar pattern of results was obtained in cells that had been transfected with UAS-TK-Luc with or without p GAL/Elkc and treated with EGF and/or forskolin (data not shown). In cells transfected with both UAS-TK-Luc and p GAL/Elkc, 10 ng/ml EGF stimulated a 15.9 ± 3.1-fold (mean ± SD; n = 2) increase in luciferase activity compared to the activity in cells maintained in phenol red-free MEM and 0.1% CSCS. In contrast, in the presence of 10 µM forskolin, 10 ng/ml EGF stimulated only a 3.7 ± 0.4-fold (mean ± SD; n = 2) increase in luciferase activity compared to the activity in cells maintained in phenol red-free MEM and 0.1% CSCS and 10 µM forskolin. These data suggest that the effect of cAMP on growth factor-induced SRE activity occurs in part through an attenuation of Elk-1 activation.



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  		Figure 7. Effects of IGF-I and forskolin on Gal4/Elk-1 activity. MCF-7 cells were transfected with UAS-TK-Luc with or without pGAL/Elkc as described in Materials and Methods and maintained in MEM and 5% CS for 14 h after transfection. The cells were then placed in phenol red-free MEM and 0.1% CSCS for 24 h. After this 24-h incubation, the cells were treated with phenol red-free MEM and 0.1% CSCS without or with 10 µM forskolin for 30 min followed by treatment for 4 h with medium alone or 50 ng/ml IGF-I in the presence or absence of the indicated dose of forskolin. The cells were harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared to the level in cells that had been transfected with UAS-TK-Luc alone and maintained in MEM plus 0.1% CSCS in the absence of forskolin and IGF-I, which was defined as 1.0. The values are the mean ± SEM of three independent experiments performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Increased intracellular cAMP has been shown previously to inhibit breast cancer cell growth (2, 3, 6, 7, 8, 9). The present study confirms the results of these previous studies by demonstrating that treatment with either forskolin or 8-CPT-cAMP decreases MCF-7 cell growth. Inhibition of cell growth by cAMP has been shown to be associated with a block of cells in different phases of the cell cycle, including G2 and G1 (6, 8, 50). In one study of MCF-7 breast cancer cells, increased intracellular cAMP levels were associated with a block in progression of cells through G1 of the cell cycle (8), whereas in a second study, cAMP decreased the proportion of breast cancer cells in S, G2, and M phases of the cell cycle and increased the proportion of noncycling cells (6). A variety of growth factors, including IGF-I and EGF, have been shown to play an integral role in progression through G1 (51). To address the possible interaction of cAMP with growth factor action, we established a reporter system based upon the activity of a DNA promoter element, the SRE, which is present in a variety of immediate early genes that are expressed in response to growth-promoting stimuli (19, 47, 48). Both IGF-I and EGF increased SRE activity in MCF-7 cells. Moreover, increased intracellular cAMP levels achieved with either forskolin or 8-CPT-cAMP inhibited IGF-I- and EGF-induced SRE activation. Taken together with the ability of forskolin and 8-CPT-cAMP to inhibit MCF-7 cell growth, these data suggest that the growth-inhibiting effects of increased intracellular cAMP in breast cancer cells may be mediated in part through an inhibition of growth factor-induced effects, including gene transcription.

The mechanism by which increased intracellular cAMP levels inhibit IGF-I- and EGF-induced SRE activity in MCF-7 cells is still undefined. The SRE in the c-fos gene has been extensively studied, and the proteins that bind to it have been well characterized (19, 47, 48). A ternary complex forms on the SRE that consists of the serum response factor and a second protein, the TCF. The TCF is one of several Ets domain-binding proteins, including Elk-1, Sap-1, and Erp/Net (19, 48). TCF is a substrate for and activated by ERK1 and -2 (19, 48). Phosphorylation by the ERKs does not, however, represent the sole mechanism for activation of TCF. For example, in macrophages, although Elk-1 is phosphorylated in response to colony-stimulating factor-1 by a signaling pathway that is dependent upon Ras and the ERKs, SAP-1 is phosphorylated by a Ras- and ERK-independent pathway (52). Moreover, in Chinese hamster ovary and HeLa cells, Elk-1 and SAP-1 can be phosphorylated by both the ERKs and the Jun kinases (JNKs), MAPKs that are distinct from the ERKs (53, 54). Thus, distinct signaling pathways are capable of converging upon the SRE. In the present studies, both IGF-I and EGF were able to activate ERK1 and ERK2 in MCF-7 cells. Interestingly, however, doses of forskolin that markedly inhibited IGF-I- and EGF-induced SRE activity had no effect on IGF-I- and EGF-stimulated ERK activity. In contrast, IGF-I and EGF-induced activation of the TCF Elk-1 was decreased by cAMP, suggesting that the inhibitory effect of cAMP occurred in part through inhibition of growth factor-induced Elk-1 activation. Taken together with the differential sensitivity of IGF-I- and EGF-induced activation of ERKs and SRE to inhibition by cAMP, these data suggest that the effect of cAMP on growth factor-induced SRE activity was mediated distal to ERK activation, possibly via inhibition of an ERK-independent pathway.

The effect of cAMP on ERK activation is complex, as increased intracellular cAMP has been shown to have diverse effects on ERK activity that are, to a large extent, cell type specific. In some cell types, including Rat-1 and NIH-3T3 fibroblasts, rat adipocytes, human arterial smooth muscle cells, and osteoblasts, increased intracellular cAMP levels stimulate phosphorylation of Raf-1, which inhibits its activation by Ras (10, 11, 12, 13, 14, 15, 16). As Raf-1 functions as a MAPK kinase kinase, this prevents ERK activation. This inhibitory effect of cAMP has been demonstrated for EGF-induced ERK activation in several cell types (10, 11, 14, 15). Similarly, in a pituitary cell line and Rat-1 fibroblasts, increased intracellular cAMP levels are able to inhibit IGF-I-induced ERK activation (55). In contrast to the inhibitory effect of cAMP on ERK activity, under certain experimental conditions cAMP activates the ERKs in PC-12 cells (56, 57), and in cells from the pars tuberalis in primary culture, both forskolin and IGF-I are able to increase ERK activity, with the effects of each being additive (58). The effect of cAMP on basal and growth factor-induced ERK activity in breast cancer cells had not been previously examined, but in our studies, increased intracellular cAMP levels had no effect on either basal or growth factor-stimulated ERK activity. The discrepancy between the effects of cAMP on growth factor-induced ERK activity and other growth factor-induced effects in MCF-7 cells is similar to the results of recent studies in Chinese hamster lung fibroblasts (CCL39 fibroblasts) (59). In these cells, cAMP inhibits cell growth but has no effect on the magnitude of growth factor-induced ERK activation, although it does delay the peak of ERK activity.

The signal transduction pathways that mediate the effect of growth factors on SRE activation and are sensitive to the inhibitory effect of cAMP on MCF-7 cell growth remain to be defined. As noted, in addition to being a substrate for the ERKs, TCF can be phosphorylated by other pathways, e.g. the JNKs (53, 54). Interestingly, in T cells stimulated by either Con A or phorbol esters, cAMP has little or no effect on ERK activity, but it does inhibit JNK activity (60). Similarly, in vascular smooth muscle cells, inhibition of thrombin-induced DNA synthesis by cAMP is accompanied by inhibition of JNK activity, whereas cAMP has no effect on thrombin-induced ERK activity (61). These data support the concept that the effect of cAMP on growth factor-induced gene transcription in MCF-7 cells may be mediated via effects on an ERK-independent pathway. The potential role of the JNKs and other signal transduction pathways in the inhibition of breast cancer cell growth are currently under investigation.


   Acknowledgments
 
The authors thank Drs. Larry Jameson and Peter Kopp for critical reading of the manuscript, Dr. Larry Jameson and Ms. Vidya Sundaresan for providing c-fos-Luc and UAS-TK-Luc, Dr. Laird Madison for providing -846 {alpha}Luc, and Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School) for providing p GAL/Elkc.


   Footnotes
 
1 This work was supported by grants from the Northwestern Memorial Foundation and the Butz Foundation. Back

Received September 6, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Seuwen K, Pouyssegur J 1992 G protein-controlled signal transduction pathways and the regulation of cell proliferation. Adv Cancer Res 58:75–94[Medline]
  2. Cho-Chung YS 1992 Suppression of malignancy targeting cyclic AMP signal transducing proteins. Biochem Soc Trans 20:425–430[Medline]
  3. Starzec AB, Spanakis E, Nehme A, Salle V, Veber N, Mainguene C, Planchon P, Valette A, Prevost G, Israel L 1994 Proliferative responses of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast pathogenesis. J Cell Physiol 161:31–38[CrossRef][Medline]
  4. Kung W, Roos W, Eppenberger U 1983 Growth stimulation of human breast cancer MCF-7 cells by dibutyryl cAMP. Cell Biol Int Rep 7:345–351[CrossRef][Medline]
  5. Sheffield LG, Welsch CW 1985 Cholera-toxin-enhanced growth of human breast cancer cell lines in vitro and in vivo: interaction with estrogen. Int J Cancer 36:479–483[Medline]
  6. Revillion F, Vandewalle B, Lassalle B, Lefebvre J 1992 cAMP effect on extracellular matrix synthesis in human breast cancer cells. Cell Prolif 25:633–642[Medline]
  7. Drees M, Zimmermann R, Eisenbrand G 1993 3',5'-Cyclic nucleotide phosphodiesterase in tumor cells as potential targets for tumor growth inhibition. Cancer Res 53:3058–3061[Abstract/Free Full Text]
  8. Vintermyr OK, Boe R, Brustugun OT, Maronde E, Aakvaag A, Doskeland SO 1995 Cyclic adenosine monophosphate (cAMP) analogs 8-Cl- and 8-NH2-cAMP induce cell death independently of cAMP kinase-mediated inhibition of the G1/S transition in mammary carcinoma cells (MCF-7). Endocrinology 136:2513–2520[Abstract]
  9. Planchon P, Veber N, Magnien V, Prevost G, Starzec AB, Israel L 1995 Evidence for separate mechanisms of antiproliferative action of indomethacin and prostaglandin on MCF-7 breast cancer cells. Life Sci 57:1233–1240[CrossRef][Medline]
  10. Cook SJ, McCormick F 1993 Inhibition of Ras-dependent activation of Raf. Science 262:1069–1072[Abstract/Free Full Text]
  11. Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW 1993 Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 262:1065–1069[Abstract/Free Full Text]
  12. Sevetson BR, Kong X, Lawrence Jr JC 1993 Increasing cAMP attenuates activation of mitogen-activated protein kinase. Proc Natl Acad Sci USA 90:10305–10309[Abstract/Free Full Text]
  13. Graves LM, Bornfeldt KE, Raines EW, Potts BC, MacDonald SG, Ross R, Krebs EG 1993 Protein kinase A antagonizes platelet-derived growth factor-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci USA 90:10300–10304[Abstract/Free Full Text]
  14. Burgering BMT, Pronk GJ, van Weeren PC, Chardin P, Bos JL 1993 cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 12:4211–4220[Medline]
  15. Hordijk PL, Verlaan I, Jalink K, van Corven EJ, Moolenaar WH 1994 cAMP abrogates the p21ras-mitogen activated protein kinase pathway in fibroblasts. J Biol Chem 269:3534–3538[Abstract/Free Full Text]
  16. Siddhanti SR, Hartle II JE, Quarles LD 1995 Forskolin inhibits protein kinase C-induced mitogen-activated protein kinase activity in MC3T3–E1 osteoblasts. Endocrinology 136:4834–4841[Abstract]
  17. Davis RJ 1993 The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553–14556[Free Full Text]
  18. Seger R, Krebs EG 1995 The MAPK signaling cascade. FASEB J 9:726–735[Abstract]
  19. Treisman R 1994 Ternary complex factors: growth factor regulated transcriptional activators. Curr Opin Genet Dev 4:96–101[CrossRef][Medline]
  20. Pages G, Lenormand P, L’Allemain G, Chambard JC, Meloche S, Pouyssegur J 1993 Mitogen-activated protein kinase p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90:8319–8323[Abstract/Free Full Text]
  21. Brondello JM, McKenzie FR, Sun H, Tonks NK, Pouyssegur J 1995 Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibroblasts. Oncogene 10:1895–1904[Medline]
  22. Dickson RB, Lippman ME 1995 Growth factors in breast cancer. Endocr Rev 16:559–589[CrossRef][Medline]
  23. Yee D 1994 The insulin-like growth factor system as a target in breast cancer. Breast Cancer Res Treat 32:85–95[CrossRef][Medline]
  24. Karey KP, Sirbasku DA 1988 Differential responsiveness of human breast cancer cell lines MCF-7 and T47D to growth factors and 17ß-estradiol. Cancer Res 48:4083–4092[Abstract/Free Full Text]
  25. Arteaga CL, Osborne CK 1989 Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor. Cancer Res 49:6237–6241[Abstract/Free Full Text]
  26. Figueroa JA, Sharma J, Jackson JG, McDermott MJ, Hilsenbeck SG, Yee D 1993 Recombinant insulin-like growth factor binding protein-1 inhibits IGF-I, serum, and estrogen-dependent growth of MCF-7 breast cancer cells. J Cell Physiol 157:229–236[CrossRef][Medline]
  27. Jammes H, Peyrat J-P, Ban E, Vilain M-O, Haour F, Dijiane J, and Bonneterre J 1992 Insulin-like growth factor 1 receptors in human breast tumour: localisation and quantification by histo-autoradiographic analysis. Br J Cancer 66:248–253[Medline]
  28. Normanno N, Ciardiello F, Brandt R, Salomon DS 1994 Epidermal growth factor-related peptides in the pathogenesis of human breast cancer. Breast Cancer Res Treat 29:11–27[CrossRef][Medline]
  29. Toi M, Tominaga T, Osaki A, Toge T 1994 Role of epidermal growth factor receptor expression in primary breast cancer: results of a biochemical and an immunocytochemical study. Breast Cancer Res Treat 29:51–58[CrossRef][Medline]
  30. Fox SB, Smith K, Hollyer J, Greenall M, Hastrich D, Harris AL 1994 The epidermal growth factor receptor as a prognostic marker: results of 370 patients and review of 3009 patients. Breast Cancer Res Treat 29:41–49[CrossRef][Medline]
  31. Earp HS, Dawson TL, Li X, Yu H 1995 Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research. Breast Cancer Res Treat 35:115–132[CrossRef][Medline]
  32. Arteaga CL, Coronado E, Osborne CK 1988 Blockade of the epidermal growth factor receptor inhibits transforming growth factor {alpha}-induced but not estrogen-induced growth of hormone-dependent human breast cancer. Mol Endocrinol 2:1064–1069[CrossRef][Medline]
  33. Ennis BW, Valverius EM, Bates SE, Lippman ME, Bellot F, Kris R, Schlessinger J, Masui H, Goldenberg A, Mendelsohn J, Dickson RB 1989 Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells. Mol Endocrinol 3:1830–1838[CrossRef][Medline]
  34. Baselga J, Mendelsohn J 1994 The epidermal growth factor receptor as a target for therapy in breast cancer. Breast Cancer Res Treat 29:127–138[CrossRef][Medline]
  35. Ciardello F, Damiano V, Bianco C, di Isernia G, Ruggiero A, Caraglia M Tagliaferri P, Baselga J, Mendelsohn J, Bianco AR, Tortora G 1995 Cooperative antiproliferative effects of 8-chloro-cyclic AMP and 528 anti-epidermal growth factor receptor monoclonal antibody on human cancer cells. Clin Cancer Res 1:161–167[Abstract]
  36. Shankar V, Ciardiello F, Kim N, Derynck R, Liscia DS, Merlo G, Langton B, Sheer D, Callahan R, Bassin R, Lippman R, Hynes NE, Salomon DS 1989 Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA. Mol Carcinog 2:1–11[Medline]
  37. Sandgren EP, Luetteke NC, Palmiter R, Brinster R, Lee DC 1990 Overexpression of TGF{alpha} in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia and carcinoma of the breast. Cell 61:1121–1135[CrossRef][Medline]
  38. Sundaresan S, Colin IM, Pestell RG, Jameson JL 1996 Stimulation of mitogen-activated protein kinase by gonadotropin releasing hormone: evidence for involvement of protein kinase C. Endocrinology 137:304–311[Abstract]
  39. Kay TWH, Jameson JL 1992 Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone {alpha}-subunit promoter. Mol Endocrinol 6:1767–1773[Abstract]
  40. Marais R, Wynne J, Treisman R 1993 The SRF accessory protein lk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73:381–393[CrossRef][Medline]
  41. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Abstract/Free Full Text]
  42. Lowe Jr WL, Teasdale RM 1992 Characterization of a rat insulin-like growth factor I gene promoter. Biochem Biophys Res Commun 189:972–978[CrossRef][Medline]
  43. An MR, Lowe Jr WL 1995 The major promoter of the rat insulin-like growth factor-I gene binds a protein complex that is required for basal expression. Mol Cell Endocrinol 114:77–89[CrossRef][Medline]
  44. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–272[Free Full Text]
  45. Fabian JR, Morrison DK, Daar IO 1993 Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes. J Cell Biol 122:645–652[Abstract/Free Full Text]
  46. Hipskind RA, Nordheim A 1991 Functional dissection in vitro of the human c-fos promoter. J Biol Chem 266:19583–19592[Abstract/Free Full Text]
  47. Treisman R 1992 The serum response element. Trends Biol Sci 17:423–426
  48. Johansen F-E, Prywes R 1995 Serum response factor: transcriptional regulation of genes induced by growth factors and differentiation. Biochim Biophys Acta 1242:1–10[Medline]
  49. Deutsch PJ, Jameson JL, Habener JF 1987 Cyclic AMP responsiveness of human gonadotropin-alpha gene transcription is directed by a repeated 18-base pair enhancer. Alpha-promoter receptivity to the enhancer confers cell-preferential expression. J Biol Chem 262:12169–12174[Abstract/Free Full Text]
  50. Vittet D, Duperay C, Chevillard C 1995 Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line. J Cell Physiol 163:645–655[CrossRef][Medline]
  51. Lowe Jr WL 1991 Biological actions of the insulin-like growth factors. In: LeRoith D (ed) Insulin-Like Growth Factors: Molecular and Cellular Aspects. CRC Press, Boca Raton, pp 49–85
  52. Hipskind RA, Buscher D, Nordheim A, Baccarini M 1994 Ras/MAP kinase-dependent and -independent signaling pathways target distinct ternary complex factors. Genes Dev 8:1803–1816[Abstract/Free Full Text]
  53. Whitmarsh AJ, Shore P, Sharrocks AD, Davis RJ 1995 Integration of MAP kinase signal transduction pathways at the serum response element. Science 269:403–407[Abstract/Free Full Text]
  54. Zinck R, Cahill MA, Kracht M, Sachsenmaier C, Hipskind RA, Nordheim A 1995 Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1. Mol Cell Biol 15:4930–4938[Abstract]
  55. Webster J, Prager D, Melmed S 1994 Insulin-like growth factor-1 activation of extracellular signal related kinase-1 and -2 in growth hormone-secreting cells. Mol Endocrinol 8:539–544[Abstract]
  56. Frodin M, Peraldi P, van Obberghen E 1994 Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J Biol Chem 269:6207–6214[Abstract/Free Full Text]
  57. Erhardt P, Troppmair J, Rapp UR, Cooper GM 1995 Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells. Mol Cell Biol 15:5524–5530[Abstract]
  58. Hazlerigg DG, Thompson M, Hastings MH, Morgan PJ 1996 Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin. Endocrinology 137:210–218[Abstract]
  59. McKenzie FR, Pouyssegur J 1996 cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition. cAMP-dependent protein kinase induces a temporal shift in growth factor-stimulated MAP kinases. J Biol Chem 271:13476–13483[Abstract/Free Full Text]
  60. Hsueh Y-P, Lai M-Z 1995 c-Jun N-terminal kinase but not mitogen-activated protein kinase is sensitive to cAMP inhibition in T lymphocytes. J Biol Chem 270:18094–18098[Abstract/Free Full Text]
  61. Rao GN, Runge MS 1996 Cyclic AMP inhibition of thrombin-induced growth in vascular smooth muscle cells correlates with decreased JNK1 activity and c-Jun expression. J Biol Chem 271:20805–20810[Abstract/Free Full Text]



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