©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of 12-O-Tetradecanoylphorbol-13-acetate on Estrogen Receptor Activity in MCF-7 Cells (*)

(Received for publication, June 12, 1995; and in revised form, August 3, 1995)

Mary Beth Martin (1) (3)(§) Pilar Garcia-Morales (1) (3) Adriana Stoica (1) (3) Harrison B. Solomon (3) (2) Meredith Pierce (3) (2) Deborah Katz (3) (2) Shimin Zhang (1) Mark Danielsen (1) Miguel Saceda (1) (3)

From the  (1)Departments of Biochemistry and Molecular Biology and (2)Medical Oncology, the (3)Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.


INTRODUCTION

Breast cancer is one of the most prevalent of all cancers and is characterized by hormonal growth control. The proliferation and phenotype of breast cancer cells is determined to a great extent by estrogen, and elevated concentrations of estrogen may contribute to an increase in the risk of breast cancer(1) . Because of the hormonal dependence of breast cancer, the presence of estrogen receptor (ER) (^1)and progesterone receptor (PgR) in breast tumors is used to predict those patients who will benefit from hormonal therapy(2, 3, 4, 5) . Although significant concentrations of estrogen receptor have been detected in approximately 60% of human breast cancers, only 60% of these ER-positive tumors respond to endocrine therapy(2, 3, 4, 5) . Absence or loss of estrogen responsiveness in breast tumors correlates with a more malignant form of the disease. Several mechanisms may be responsible for the loss of estrogen responsiveness in human breast cancer; including the loss of ER expression, the presence of subpopulations of cells which are ER negative, and the presence of ER variants with altered activity. Several ER variants, including point mutations and alternately spliced forms, have been described in tumors and in breast cancer cell lines (6, 7) . Some of these variants lack the ability to bind estradiol, whereas other variants are active in the absence of ligand. In addition to the above mechanisms, treatment of breast carcinoma cell lines with the anti-estrogen tamoxifen has been shown to result in the accumulation of ER that has lost its ability to bind estradiol(8) . Phosphorylation of the estrogen receptor has also been shown to regulate its function. In the case of hormone binding, it has been suggested that dephosphorylation of a tyrosine residue in the hormone binding domain of the receptor blocks estradiol binding(9) . Interactions with other factors have also been shown to modulate steroid receptor function. The unregulated expression of c-Jun or c-Fos proteins inhibits ER activity in human breast cancer-derived cells(10, 11) .

Several lines of evidence suggest that protein kinase C may play a role in the loss of estrogen responsiveness in breast cancer. In human mammary carcinoma cells, an inverse relationship exists between the levels and activity of protein kinase C and the level of estrogen receptor(12) , that is, the activity of protein kinase C is several times higher in ER-negative breast cancer cell lines than in ER-positive cell lines(13) . We and others have also shown that treatment of the breast carcinoma cell line MCF-7 with TPA, an activator of protein kinase C, results in a decrease in the level of ER (14, 15) . This effect is mediated by a post-transcriptional destabilization of the ER mRNA(14) .

The data presented in this report demonstrate dramatic effects of TPA treatment on ER activity which are independent of its effect on the level of ER. Following long term treatment, TPA inhibits the activity of the ER as shown by the lack of estradiol induction of PgR and the inability of estradiol to activate an estradiol-inducible reporter gene. The inhibitory effect of TPA on ER activity is due to the induction/activation of a factor that blocks the binding of estradiol to the ER.


MATERIALS AND METHODS

Tissue Culture

Monolayer cultures of MCF-7 breast cancer cells were grown in improved minimal essential medium (IMEM) supplemented with 5% (v/v) fetal calf serum (FCS). When the cells were 70-80% confluent, the medium was replaced with phenol red-free IMEM containing 5% charcoal-treated calf serum (CCS)(16) . To remove endogenous steroids, serum was treated first with sulfatase followed by the addition of dextran-coated charcoal. After 2 days in estrogen-depleted medium, TPA, estradiol, or vehicle was added, and the cells were harvested at the times indicated. In long term experiments, cells were treated with TPA for 24 h. The medium containing TPA was removed. The cells were washed three times with phosphate-buffered saline (PBS), and fresh phenol red-free IMEM supplemented with 5% CCS was added. Cells were harvested 2-3 days after the change of medium.

Estrogen Receptor and Progesterone Receptor Assays

To measure total ER and PgR content, the cells were homogenized by sonication in a high salt buffer (10 mM Tris, 1.5 mM EDTA, 5 mM Na(2)MoO(4), 0.4 M KCl, 1 mM monothioglycerol, and 2 mM leupeptin). The homogenate was incubated on ice for 30 min and centrifuged at 10,000 times g for 1 h. Aliquots of total extracts were then analyzed using enzyme immunoassay kits containing monoclonal antibodies to ER and PgR from Abbott according to the manufacturer's instructions.

Estrogen Receptor-binding Assays

To determine the binding of [^3H]estradiol to the ER, several protocols were employed. In the cytosol-binding assay, a cell extract was prepared as described for the enzyme immunoassay. Aliquots of extract were incubated with increasing amounts of [^3H]estradiol (0.1-6.0 nM) in the presence or absence of 100 times excess diethylstilbestrol to determine nonspecific binding. The incubation was conducted in TED buffer (0.01 M Tris, pH 7.5, 5 mM EDTA, 0.5 mM dithiothreitol). In some experiments, the time and temperature of the incubation were varied. Binding assays were performed in triplicate at 37 °C for 1 h or at 4 °C for 4-16 h. In some assays, samples were divided in two. One aliquot was assayed for [^3H]estradiol-bound receptor in the presence of 100 nM TPA; the other aliquot was assayed in the absence of TPA. Activated dextran-coated charcoal (800 µl) was added, and the samples were incubated on ice for 10 min. After centrifugation, 400 µl of the supernatant was counted. The data were analyzed by the Scatchard method(17) .

A whole cell-binding assay was also used to measure the binding capacity of the estrogen receptor. 80,000 cells per well were plated in 6-well plates. Following treatment with TPA, cells were washed with PBS and [^3H]estradiol (0.1-6.0 nM) was added in the presence or absence of a 100 times excess diethylstilbestrol to determine nonspecific binding. Binding assays were done in triplicate at 37 °C for 1 h. Cells were washed three times with PBS and disrupted by sonication in Hanks' solution. Samples were counted, and the data were analyzed by the method of Scatchard.

Simultaneous Determination of Estrogen Receptor Protein and Binding Capacity

After treatment with TPA (2.5-100 nM), a whole cell extract was prepared as described for the enzyme immunoassay. Aliquots of the cell extract were analyzed either for ER protein by the enzyme immunoassay or for [^3H]estradiol binding by a single point analysis. For the single point analysis, aliquots were incubated with 2 nM [^3H]estradiol in the presence and absence of 100 times diethylstilbestrol. After a 4-h incubation in TED buffer at 4 °C, dextran-coated charcoal was added, and [^3H]estradiol binding was determined as described above. The data are presented as either percent of control values of ER protein or binding.

Glucocorticoid Receptor Hormone-binding Assay and Western blot Analysis

Cells were harvested by scraping from flasks using a rubber policeman and then washed three times with phosphate-buffered saline. The following manipulations were carried out at 0-4 °C unless indicated otherwise. The cell pellets were suspended in 3 volumes of binding buffer (20 mM HEPES, pH 7.3, 20 mM molybdate, 5 mM EDTA), placed on ice for 5 min, and then ruptured by 13 strokes with the A pestle of a Dounce homogenizer. The homogenate was centrifuged at 15,000 times g for 30 min. The resulting supernatant was centrifuged at 110,000 times g for 1 h. The clear supernatant was defined as cytosol which was used for hormone-binding assays and Western blots.

Hormone-binding assays were performed by the charcoal adsorption method (18) . Cytosol was incubated with [^3H]triamcinolone acetonide in an ice water bath overnight in a total volume of 100 µl containing 0.6 mg of protein. Nonspecific binding was determined by incubating the mixture with 1000-2000-fold molar excess of nonradioactive triamcinolone acetonide. After 18 h, 300 µl of 2.5% dextran-coated charcoal were added followed by mixing for 10 s. The tubes were placed at room temperature for 10 min and then centrifuged for 8 min. [^3H]Triamcinolone acetonide present in the supernatant was determined using a scintillation counter. Specific hormone binding was obtained by subtracting nonspecific binding from total binding. The binding assays were performed in triplicate. Dissociation constants were determined by computer analysis of the data based on the Michaelis-Menton equation [HR] = [H][R]/[H] + K(d) where HR = hormone-receptor complex, H = free hormone concentration, R = free and complexed receptor.

For Western blot analysis, electrophoresis was performed in 12% slab gels according to Laemmli (19) as described previously by Blackshear (20) . Lysozyme (15.3 kDa), beta-lactoglobulin (18.3 kDa), carbonic anhydrase (27.8 kDa), ovalbumin (44.2 kDa), bovine serum albumin (71 kDa), phosphorylase B (105.7 kDa), and H-chain of myosin (196 kDa) from Life Technologies, Inc., were used as molecular mass standard markers. Cytosol was mixed with an equal volume of SDS-polyacrylamide gel electrophoresis sample buffer, boiled for 4 min, and loaded onto the gel. After electrophoresis, the gels as well as nitrocellulose membranes were immersed in transfer buffer (48 mM Tris, 39 mM glycine, pH 9.2, 20% methanol, 0.00375% SDS) for 20 min. Proteins were transferred from gels to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) overnight at room temperature on an orbital shaker. The membranes were washed three times with TBST and incubated with primary antibody against the glucocorticoid receptor (GR49) at room temperature for 1 h with shaking. Unbound antibody was removed by washing as described above. The membrane was then incubated for 1 h with peroxidase-conjugated rabbit anti-mouse antibody. The unbound antibody was removed as described above. The membranes were then incubated with detection solution (Amersham Corp.) for 1 min and exposed to Hyperfilm for 2-5 s.

Measurement of Estrogen Receptor, Progesterone Receptor, and pS2 mRNA Levels

Total cellular RNA was extracted from MCF-7 cells by the method of Chomizynsk and Sacchi(21) . The levels of ER, PgR, and pS2 mRNA were determined by RNase protection analysis as described previously(22) . Homogeneously P-labeled antisense RNAs (cRNA) were synthesized in vitro from pOR300 (22) for the ER riboprobe, pPgR250 (23) for the PgR riboprobe, pS2 riboprobe(24) , and p36B4 (22) and hybridized to total RNA. After digestion with RNase A, protected probes were resolved on denaturing polyacrylamide gels. The bands were visualized by autoradiography and quantified by optical densitometry. Data were normalized to the internal control 36B4(22, 25, 26) .

Transient Transfection Assays

MCF-7 cells were plated in 100-mm culture dishes at a density of 500,000 cells/plate in IMEM supplemented with 10% FCS. After 24 h, the cells were transiently transfected with 30 µg/plate of the reporter plasmid Vit-TK-CAT (27) in 1 ml of BES-buffered saline (50 mM Bes, pH 6.96, 280 mM NaCl, 1.5 mM Na(2)HPO(4)) containing 125 mM CaCl(2)(28) . The cells were incubated at 35 °C in 2% CO(2) for 16 h. Media were removed and the cells were washed twice with PBS. Fresh phenol red-free IMEM supplemented with 5% CCS was added. After 48 h, cells were treated as described in the text. Following treatment, cells were harvested and the cellular pellet was resuspended in 0.25 M Tris, pH 7.5. Cells were disrupted by several cycles of freeze-thawing. After centrifugation, supernatants were analyzed for CAT activity by thin layer chromatography(29) . In a number of experiments, the level of CAT protein was analyzed by a commercial enzyme-linked immunosorbent assay (Promega). The efficiency of transfection was determined by a Hirt (30) analysis.

A second transfection assay system was also employed. In these experiments, the reporter plasmid ERE-TK-Luc (31) was used. Cells were plated in 6-well plates in IMEM supplemented with 10% FCS. The next day, cells were transfected with 1 µg of plasmid DNA using Lipofectamine (Promega) in serum-free IMEM. After 5 h at 37 °C in serum-free IMEM, medium supplemented with 10% FCS was added to achieve a final concentration of 5% FCS. The cells were incubated overnight at 37 °C. The next day, the medium was removed, and the cells were washed with PBS. Cells were maintained in phenol red-free IMEM supplemented with 5% CCS for 48 h. Treatment with TPA and subsequent induction with estradiol were performed as described above. After treatment, cells were harvested, and a cellular extract was obtained as described above. The cellular extract was analyzed for luciferase activity using a luminometer.

DNA Mobility Shift Assay

Nuclear extracts from control and treated cells were isolated following a protocol previously described (32) . Briefly, cells were harvested, and the cell pellet was washed twice with 0.1 volume of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl(2), 10 mM KCl, and 0.5 mM dithiothreitol). Pellets were resuspended in 100 µl of buffer A containing 0.01% Nonidet P-40, incubated on ice for 10 min, mixed by vortexing, and pelleted by centrifugation at 10,000 rpm at 4 °C for 10 min in a microcentrifuge. The nuclear pellet was resuspended in 75 µl of buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol), incubated on ice for 15 min, and pelleted by centrifugation as above. The nuclear supernatant was diluted with 375 µl of buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.05 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and stored at -70 °C in 100-µl aliquots. Protein concentration was determined prior to storage.

For the DNA mobility shift assay, a P-labeled estrogen-responsive element (ERE; 5`-GATCCTCACGGTCACAGTGACCTGCCCGGGATT) was incubated with nuclear extracts from MCF-7 cells for 20 min at room temperature in a buffer containing 10 mM Tris, pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and 6% glycerol. After incubation, 1 µg of antibody to the ER was added to the appropriate samples. After a 20-min incubation at room temperature, samples were loaded onto a 6% polyacrylamide gel in 1 times TBE (50 mM Tris borate, pH 8.3, and 0.5 mM EDTA) and run at 20 mA/gel at 4 °C. The gel was dried, and the bands were visualized by autoradiography.

Mixing Experiments

For these experiments, whole cell extracts were made from cells treated with TPA for various times or from cells treated with TPA for 24 h and then maintained in fresh medium for 2-3 days. Extracts were isolated by the protocol described for the ER and PgR assays. Extracts were made in the presence and absence of 5 mM Na(2)MoO(4). Aliquots of the extracts were mixed together at 4 °C, and the ability of the ER to bind [^3H]estradiol was determined using the single point binding analysis described above. The results of the mixing experiments are presented as percent of the expected binding.


RESULTS

Effects of TPA on Estradiol Binding to the Estrogen Receptor

We and others have demonstrated that treatment of MCF-7 cells with TPA results in a decrease in the concentration of ER protein and ER mRNA(14, 15) . Following treatment with 100 mM TPA, the level of ER protein decreases to 20% of control values by 24 h as determined by an enzyme immunoassay. The decrease in protein is preceded by a decrease in ER mRNA. After removal of TPA from the medium, the levels of ER protein and mRNA return to control values by 72 h (data summarized in Fig. 1A).


Figure 1: Effect of TPA on the steady state level of ER protein and mRNA. MCF-7 cells were grown in IMEM supplemented with 5% fetal calf serum. When cells were 70% confluent, the medium was changed to phenol red-free IMEM containing 5% CCS. Cells were grown in this medium for 2-3 days and then treated with 100 nM TPA. Twenty-four hours later, medium containing TPA was removed, and the cells were maintained in fresh medium for 3 days. A, the levels of ER protein (box) and mRNA () were determined by an enzyme immunoassay and a RNase protection assay, respectively, as described under ``Materials and Methods.'' B, the levels of ER protein (bullet) and ER binding capacity (box) were determined simultaneously by an enzyme immunoassay and a whole cell assay, respectively, as described under ``Materials and Methods.''



To determine whether the decrease and subsequent recovery of ER protein corresponds to a similar change in estradiol-binding sites, the concentration of ER protein and binding capacity were measured simultaneously (Fig. 1B). The binding of estradiol to the ER was determined by a whole cell-binding assay using increasing amounts of [^3H]estradiol. Following treatment with TPA, a significant decrease in the estradiol binding capacity was observed. The number of estrogen-binding sites decreased from 592 fmol/mg of protein in control cells to 119 fmol/mg of protein in cells treated with TPA for 24 h. These results are consistent with the results obtained with the enzyme immunoassay. However, following removal of TPA from the medium, the number of binding sites did not increase. Although the concentration of ER protein returned to control values, the number of estrogen-binding sites remained at approximately 20% of control values.

The discrepancy between the concentration of receptor protein and hormone binding capacity was unexpected. To confirm that TPA did indeed induce a decrease in the number of estradiol-binding sites which did not return to control values upon TPA removal, a number of conditions were employed in the cytosol-binding assay. To rule out the possibility that TPA directly interfered with estradiol binding to its receptor, the ability of the ER to bind [^3H]estradiol in the presence of added TPA was determined. Binding experiments were also performed at 4 and 37 °C. The data from the different binding assays are summarized in Table 1. Independent of the assay conditions, the level of [^3H]estradiol binding was approximately 20% of the expected values when the cells were treated with TPA. TPA added to the binding reaction of untreated cells did not affect hormone binding. Scatchard analysis of the data indicated that treatment of cells with TPA did not affect the dissociation constant of the estrogen receptor. These data indicate that, although the levels of ER protein and ER mRNA returned to control levels 72 h after the change of medium, the ER was not capable of binding estradiol.



Effects of TPA on Estrogen Receptor Function

The discrepancy between the levels of ER protein and ER binding capacity following removal of TPA from the media suggested that the estrogen receptor was not functional. To test the transcriptional activity of the ER, the ability of estradiol to induce the progesterone receptor was determined following treatment with TPA. In this study, the level of PgR was determined by an enzyme immunoassay. The data in Fig. 2demonstrate a dramatic increase in the level of PgR in response to estradiol treatment. By 24 h there was a 4-fold increase in PgR expression; by 48 h there was a 7-fold increase in progesterone receptor. In cells treated with TPA for 24 h prior to the addition of estradiol, the induction of PgR was completely blocked. In cells treated with TPA for 24 h and placed in fresh medium for 2-3 days prior to the addition of estradiol, only a modest induction of PgR (1.5-fold by 24 h) was achieved, even though the level of ER protein had returned to control values in these cells. Treatment of cells with TPA alone had no effect on PgR expression (data not shown). 4-alpha-Phorbol (100 nM), a compound related to TPA but lacking the biological effects of TPA, had no effect on PgR expression (data not shown).


Figure 2: Effect of TPA on induction of progesterone receptor. MCF-7 cells were grown as described in Fig. 1. Prior to the addition of estradiol, cells were treated with TPA for 24 h (TPA) or treated with TPA for 24 h and allowed to recover in fresh medium for 48 h (R). The level of progesterone receptor protein was determined using an enzyme immunoassay.



The effect of TPA on estrogen receptor activity was also studied in a transient transfection assay with the reporter gene Vit-TK-CAT(27) . In this construct, the thymidine kinase promoter is under the control of an ERE. The level of CAT activity was determined by thin layer chromatography. The data from several independent experiments are summarized in Fig. 3. In control cells, the addition of estradiol produced a 2-fold increase in CAT activity. In cells treated with TPA for 24 h, estradiol failed to induce CAT activity. In cells treated with TPA for 24 h and subsequently maintained in fresh medium for 72 h, no increase in CAT activity was seen following estradiol treatment. These data suggest that the ER, although present in these cells, is not functional. To rule out the possibility that the lack of induction of CAT activity in the latter cells was due to the length of time of the recovery experiment, cells were also maintained in culture for 4 days after transfection, and the induction of CAT activity by estradiol was determined. The longer time course did not diminish the estradiol response.


Figure 3: Effects of TPA on ER activation of an estrogen-responsive reporter gene. MCF-7 cells were transiently transfected with the estrogen-responsive reporter gene, Vit-TK-CAT, as described under ``Materials and Methods.'' Following transfection, the cells were maintained in phenol red-free IMEM supplemented with 5% CCS for 2-3 days. Cells were treated with 100 nM TPA and 1 nM estradiol for 24 h or treated with TPA for 24 h and allowed to recover in fresh medium for 48 h (R) prior to the addition of estradiol. Cells were harvested, and CAT activity was measured. Data represent the mean ± S.D. (n = 3-5).



To show that the effects of TPA were not Vit-TK-CAT vector-specific, a number of transfection experiments also were performed using a second reporter gene ERE-TK-Luc(31) . In MCF-7 cells, transiently transfected with the ERE-TK-Luc reporter plasmid, treatment with estradiol produced a 3-fold increase in luciferase activity from 120 units/mg of protein in control cells to 360 units/mg of protein in the cells stimulated with estradiol. Treatment of cells with TPA for 24 h prior to the addition of estradiol blocked estradiol induction of luciferase activity (data not shown).

Other studies have shown that treatment of MCF-7 cells with TPA activates the estrogen receptor(33) . To explain the discrepancy between our results and previously reported results, we studied the short term effects of TPA on ER activity in transient transfection assays using the Vit-TK-CAT reporter. Treatment of cells with 1 nM estradiol for 3 h resulted in a 2-fold increase in the level of CAT protein. Pretreatment of cells with TPA for 24 h prior to the addition of estradiol blocked the induction of CAT protein. However, simultaneous treatment with TPA and estradiol for 3 h resulted in a greater increase (4-fold) in the level of CAT protein than in cells treated with estradiol alone (data not shown). To determine if the synergistic effect of short term treatment with the phorbol ester was also observed on the expression of an endogenous gene, the effects of TPA and estradiol on pS2 expression were studied 6 h after treatment using a RNase protection assay. Short term treatment with TPA for 6 h resulted in a 20% increase in pS2 mRNA, whereas treatment with estradiol resulted in a 40% increase in the level of pS2 mRNA. However, the combined treatment of TPA and estradiol resulted in a 175% increase in the level of pS2 mRNA (data not shown). These results demonstrate that the effects of TPA on ER activity are time-dependent. The initial effects of TPA are to activate the receptor, whereas the long term effects are to block receptor function.

Alternately spliced mutants of the estrogen receptor have been identified in breast cancer tumors and cell lines(6, 34, 35, 36, 37) . Mutants which have been identified include deletion of exons 2, 3, 5, and 7. To determine if inactivation of estrogen receptor by TPA was due to the induction of an aberrant ER species by alternative splicing, ER mRNA was analyzed by a RNase protection assay using riboprobes for the entire coding sequence (Fig. 4C). In the case of the pOR-C probe, there was no evidence of an alternately spliced product. Deletion of exons would have resulted in the loss of the 652-bp protected fragment and the appearance of smaller protected fragments. Deletion of exon 1 would result in the appearance of a protected fragment of 568 bp; deletion of exon 2 would give protected fragments of 84 and 377 bp; and deletion of exon 3 would result in the appearance of fragments of 275 and 260 bp. The deletion of both exons 3 and 4 would result in the appearance of a protected fragment of 275 bp in the pOR-C lane and a protected fragment of 229 bp in the pOR-B lane. The fragments protected by the pOR-C probe are not consistent with an alternately splice product. They are, however, consistent with truncated forms of the probe (probe not shown). In the case of deletion of exons 5 and 7, alternate splicing would have resulted in the loss of probes pOR-B and pOR-R and the appearance of several fragments of less than 100 bp. No protected fragments of the expected sizes (44, 76, and 90 bp) appeared after TPA treatment (Fig. 4). These data suggest that inactivation of the estrogen receptor was not due to the induction of an aberrant ER species.


Figure 4: Effect of TPA on the structure of ER mRNA. Total RNA was isolated from MCF-7 cells treated with 100 nM TPA for 24 h. Four different riboprobes for the coding sequence of the ER were employed in RNase protection assays. A, total RNA from control cells; B, total RNA from TPA-treated cells; C, map of the ER riboprobes.



Effect of TPA Concentration on Estrogen Receptor Expression

Due to the hydrophobic nature of TPA it is possible that residual levels of the phorbol ester are present during the recovery phase of these experiments. It is possible that very low concentrations of TPA are responsible for the inhibition of estradiol binding and the subsequent loss of estrogen receptor activity but are insufficient to decrease the steady-state levels of ER protein and mRNA. To test this hypothesis, MCF-7 cells were treated for 24 h with concentrations of TPA from 2.5 to 100 nM. The level of ER protein was determined by the enzyme immunoassay, and the ability of the ER to bind [^3H]estradiol was determined by the hormone binding assay. The results of the enzyme immunoassay and the hormone binding assay are presented in Fig. 5as the percent of ER protein and hormone binding in control cells. Low concentrations of TPA, 2.5 nM, substantially decreased the binding capacity of the ER to 25% of control values but had no effect on receptor concentration, whereas higher concentrations of TPA decreased both the concentration and binding capacity of the ER. These results suggest that the effect of TPA on ER binding is independent of its effect on ER levels as these effects can be separated at very low concentrations of TPA.


Figure 5: Concentration effect of TPA on the level of ER protein and binding capacity. MCF-7 cells were treated with TPA, 2.5-100 nM, for 24 h as described in Fig. 1. ER protein was determined by enzyme immunoassay as described under ``Materials and Methods.'' () The ability of ER to bind estradiol was determined using the single point analysis as described under ``Materials and Methods.'' (box) The experiment was performed in triplicate and repeated with similar results.



Effects of Protein Kinase C Inhibitors on the TPA-induced Inhibition of Estrogen Receptor Activity

To study the role of protein kinase C in the TPA-induced inhibition of estrogen receptor activity, the protein kinase C inhibitors, H-7 and bryostatin, were employed. The effects of TPA on estradiol induction of progesterone receptor were determined after treatment with 40 uM H-7 or 100 nM bryostatin. The concentration of TPA (2.5 nM) chosen for this study blocked estradiol binding to the receptor but had no effect on ER concentration. Estradiol treatment resulted in a 6-fold increase in progesterone receptor, and this induction was significantly blocked by treatment with TPA (Fig. 6). Bryostatin and H-7 had no effect on PgR expression and blocked the inhibitory effects of TPA. Similar but more dramatic effects were observed at the level of PgR mRNA. Estradiol treatment resulted in a 9-fold induction of PgR mRNA. In cells treated with TPA and estradiol, there was only a 1.6-fold induction of progesterone receptor mRNA. However, when cells were treated with TPA and estradiol in the presence of the inhibitor, H-7, there was a 7.6-fold induction of PgR mRNA (data not shown). The ability of bryostatin and H-7 to block the inhibitory effects of TPA suggests that the effects of the phorbol ester are mediated by protein kinase C.


Figure 6: Effects of H-7 and bryostatin on the TPA-induced inhibition of ER activity. MCF-7 cells were grown as described in Fig. 1. Cells were treated with 1 nM estradiol, 2.5 nM TPA, 100 mM bryostatin (BRYO), or 40 µM H-7. Progesterone receptor protein was determined by enzyme immunoassay as described under ``Materials and Methods.'' The results shown are the mean of two experiments.



Effects of TPA on Glucocorticoid Receptor Expression

To determine whether the effects of TPA are specific for the estrogen receptor or are universal for steroid receptors, the effects of TPA on glucocorticoid receptor expression were studied. MCF-7 cells were treated with 2.5 nM TPA for 24 h. The level of glucocorticoid receptor was determined by an immunoblot analysis, and the binding capacity of the receptor was determined by a charcoal absorption assay. The results are presented in Fig. 7. Treatment of cells with TPA had no effect on the level of glucocorticoid receptor protein or its ability to bind triamcinolone acetonide. These results suggest that the effects of TPA on hormone binding are specific for the estrogen receptor and are not universal for all steroid hormone receptors.


Figure 7: Effect of TPA on glucocorticoid receptor protein and binding capacity. MCF-7 cells were grown as described in Fig. 1and treated with 2.5 nM TPA for 24 h. Glucocorticoid receptor (GR) hormone binding activity was determined as described under ``Materials and Methods.'' The graph shows the specific hormone binding activity of cytosol from control (box) and TPA-treated (circle) cells. Insert, cytosol from TPA-treated and control cells was subjected to Western blot analysis using the glucocorticoid receptor-specific antibody GR49. The positions of the glucocorticoid receptor, molecular mass markers, and a nonspecific band (ns) are shown.



Effect of TPA on Estrogen Receptor Interaction with an Estrogen Response Element

Since the effects of estradiol are mediated, in part, through the interaction of the activated ER and sequences in DNA termed ERE, the effects of TPA treatment on the interaction of the ER with the ERE were studied. To measure the ability of the ER to bind to an ERE, DNA mobility shift assays were performed using nuclear extracts isolated from MCF-7 cells treated with 100 nM TPA for 24 h or from cells treated with TPA for 24 h and placed in fresh medium for 2-3 days to allow recovery of ER protein to control values. The concentration of ER was determined with the enzyme immunoassay and equivalent amounts of ER were incubated with a P-labeled ERE. The ability of the estrogen receptor to bind DNA was indicated by the retardation of the P-labeled ERE on a polyacrylamide gel. The results of a typical DNA mobility shift experiment are shown in Fig. 8. In control cells, the ER binds to the consensus ERE as demonstrated by the supershift in the retarded band upon addition of a monoclonal antibody to the ER. In cells treated with TPA for 24 h or in cells treated with TPA and subsequently placed in fresh medium for 2-3 days, the ER does not bind to the ERE as indicated by the absence of a specific retarded DNA band. This is an interesting result, as the estrogen receptor in this assay binds to the ERE in the presence and absence of estradiol and anti-estrogens. These data indicate that the effects of TPA treatment on ER binding to DNA are hormone-independent and suggest the possibility that TPA may induce/activate a factor which blocks binding of the ER to a consensus ERE.


Figure 8: Effect of TPA on ER binding to an estrogen-response element. Nuclear extracts were isolated from MCF-7 cells treated with 100 nM TPA for 24 h and subsequently maintained in fresh medium for 3 days. The ability of ER to bind to a P-labeled ERE was determined in a DNA mobility shift assay as described under ``Materials and Methods.'' Lane 1, control; lane 2, control plus and anti-ER antibody; lane 3, TPA; lane 4, TPA plus an anti-ER antibody; lane 5, recovery; lane 6, recovery plus an anti-ER antibody. Several independent experiments were performed with similar results.



Effect of TPA on Estradiol Binding in an in Vitro Mixing Experiment

To investigate the possibility that TPA treatment induces or activates a factor that inhibits the ability of the ER to bind estradiol, the binding of [^3H]estradiol was determined after mixing nuclear extracts from control cells, cells treated with TPA (2.5 and 100 nM), or cells treated with TPA (100 nM) and subsequently maintained in fresh medium. The number of estradiol-binding sites of individual nuclear extracts were determined by Scatchard analysis prior to and following mixing. The expected binding is defined as the number of binding sites in control extracts measured prior to mixing. Mixing experiments were conducted at 4 °C. The data in Fig. 9are presented as the percent of expected binding. When nuclear extracts from cells treated with 2.5 and 100 nM were mixed with nuclear extracts from control cells, there was a 50 and 95% decrease in the expected binding, respectively, suggesting that the effect of TPA was dose-dependent. When increasing amounts of control nuclear extracts were added to extracts from TPA-treated cells, the inhibitory activity was blocked. The inhibitory activity was also present in nuclear extracts from cells treated with TPA for 24 h and subsequently maintained in fresh medium for 72 h. The addition of the phosphatase inhibitor, molybdate, to these experiments had no effect on estradiol binding. The inhibitor present in TPA-treated extracts appeared to be temperature-labile as heating the extracts partially blocked the effect. Taken together these data suggest that nuclear extracts isolated from cells treated with TPA contain a factor that interacts with the ER to inhibit its ability to bind estradiol.


Figure 9: Effect of TPA on ER binding capacity in mixing experiments. Nuclear extracts from MCF-7 cells treated with TPA, 2.5 or 100 nM, for 24 h were isolated as described under ``Materials and Methods.'' The ability of these extracts to inhibit binding of [^3H]estradiol to the ER in control extracts was determined by a single point binding analysis. The results are presented as the percent of the expected binding. The expected binding is defined as the amount of binding in the individual extracts prior to mixing. C, control extract; 2C and 3C, 2 and 3 times the amount of control extract; TPA, extract after treatment with TPA for 24 h; 3TPA, 3 times the amount of TPA extract; R, extract after treatment with TPA for 24 h followed by a 48-h recovery; 2R, 2 times the amount of R extract.




DISCUSSION

The results presented demonstrate that treatment of MCF-7 cells with TPA has a dual effect on estrogen receptor activity. The initial effects of treatment are to enhance ER activity, whereas the long term effects are to block activity of the receptor. The loss of estrogen receptor activity appears to be due to the induction/activation of a factor which interacts with the ER to block binding of estradiol.

Regulation of ER expression in breast cancer cell lines is a complex process involving transcriptional as well as post-transcriptional events controlled by estradiol, anti-estrogens, and activators of protein kinase C(14, 22, 38) . Treatment of MCF-7 cells with TPA results in a decrease in the concentration of ER(14, 15) . This decrease in ER levels is due to post-transcriptional destabilization of ER mRNA(14) . In addition to the effects on the level of ER, TPA has been shown to modulate ER activity in MCF-7 cells. However, these reports are contradictory. It has been shown that TPA treatment of MCF-7 cells transiently transfected with different estrogen-inducible reporter genes results in a synergistic activation of the ER(33) , whereas other reports suggest that TPA and protein kinase C negatively regulate estrogen receptor activity in MCF-7 as well as other cell lines(10, 11) . The results of this study demonstrate an increase in estrogen receptor activity following short term treatment with TPA and an inactivation of the ER following long term treatment with the phorbol ester. The observed differences appear to be due to treatment conditions.

Although the inhibitory activity present in TPA-treated breast cells remains to be identified, c-Jun and c-Fos are possible candidates. Many interactions between the protein kinase C-mediated signal transduction pathway and steroid receptors have been reported(10, 11, 39, 40, 41, 42) . c-Jun and c-Fos have been shown to inhibit ER function(10, 11) . In these studies, loss of estrogen responsiveness was demonstrated by transient transfection assays with reporter genes or by DNA mobility shift assays where ER and c-Jun were mixed in vitro. In contrast to these results, activation of the ovalbumin gene by estrogen receptor has been shown to require the fos/jun complex(41) . The reason for the discrepancy between these reports is not clear but may be due to different transfection systems and methodologies. Inhibition of ER function/activity in the former studies may be due to the high copy number of the transfected plasmids and competition for factors required for efficient transcription. In addition to c-Fos and c-Jun, a protein has been identified in HeLa cells which translocates to the nucleus after TPA treatment(43) . This protein interacts with hsp90 which interacts with the ER; hsp90 has shown to be important for estrogenic activity(44) . The ability of this protein to modulate estradiol binding to the ER remains to be defined.

It has been proposed that estradiol binding to its receptor is regulated by a phosphorylation/dephosphorylation process (for review, see (45) ). In this model, dephosphorylation of the ER inhibits estradiol binding, whereas phosphorylation is require for hormone binding. It has been suggested that a nuclear phosphatase is responsible for the removal of phosphate from the receptor and the subsequent loss of hormone binding(46, 47) . In our study, the addition of the phosphatase inhibitor molybdate to the mixing experiments had no effect on hormone binding, suggesting the inhibitory activity in TPA-treated cells was not due to the induction/activation of a phosphatase.

Treatment of MCF-7 cells with TPA has been shown to inhibit growth and change the morphology of the cells, suggesting a differentiation effect in these cells(48, 49, 50) . This ``differentiation'' may result in a change in the pattern of gene expression in these cells. We suggest that one of these changes is the expression of a factor(s) that interacts with the ER. This factor inhibits hormone binding and consequently inactivates the estrogen receptor. In an osteosarcoma cell line stably transfected with ER, loss of ability of ER to bind estradiol has also been observed(51) . These cells are responsive to estrogenic stimulation at subconfluence but become refractory when confluence is reached. The concentration of ER protein and mRNA are similar in the subconfluent and postconfluent cells. However, a decrease in the number of ER-binding sites and loss of estrogen responsiveness is observed in postconfluent cells.

In conclusion, this study demonstrates that activation of the protein kinase C signal transduction pathway modulates ER activity. In short term treatment, TPA synergistically activates the ER in the presence of estradiol. The mechanism for this effect is unknown. In contrast to the short term effects, long term treatment with TPA inactivates the ER. This effect is due to the induction/activation of a factor that interacts with the estrogen receptor to inhibit both the binding of estradiol to the ER and binding of the ER to its response element. This factor may explain, in part, the role of protein kinase C in the loss of estrogen responsiveness in breast cancer.


FOOTNOTES

*
This work was supported in part by the National Institutes of Health Grants UO1 CA51908 (to M. B. M.) and RO1 DK42552 and K04 DK02105 (to M. D.), by the Cancer Research Foundation of America and the Milheim Foundation (to M. S.), and by the Susan Komen Breast Cancer Foundation (to S. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vincent T. Lombardi Cancer Research Center, E411 Research Bldg., 3970 Reservoir Rd., NW, Washington, DC 20007. Tel.: 202-687-3768; Fax: 202-687-7505.

(^1)
The abbreviations used are: ER, estrogen receptor; PgR, progesterone receptor; IMEM, improved minimal essential medium; TPA, 12-O-tetradecanoylphorbol-13-acetate; CCS, charcoal-treated calf serum; FCS, fetal calf serum; PBS, phosphate-buffered saline; ERE, estrogen-responsive element; CAT, chloramphenicol acetyltransferase; bp, base pair; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. M. E. Lippman for helpful discussions and H. M. Westphal for GR49 antibody.


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