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Endocrinology Vol. 144, No. 5 1812-1824
Copyright © 2003 by The Endocrine Society

Estrogen-Dependent Rapid Activation of Protein Kinase C in Estrogen Receptor-Positive MCF-7 Breast Cancer Cells and Estrogen Receptor-Negative HCC38 Cells Is Membrane-Mediated and Inhibited by Tamoxifen

B. D. Boyan, V. L. Sylvia, T. Frambach, C. H. Lohmann, J. Dietl, D. D. Dean and Z. Schwartz

Wallace H. Coulter Department of Biomedical Engineering (B.D.B., Z.S.), Georgia Institute of Technology, Atlanta, Georgia 30332; Departments of Periodontics (Z.S.) and Orthopaedics (V.L.S., D.D.D.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229; Department of Gynecology (T.F., J.D.), University of Würzburg, 97070 Würzburg, Germany; Department of Orthopedics (C.H.L.), University of Eppendorf, 20255 Hamburg, Germany; and Department of Periodontics (Z.S.), Hebrew University Hadassah, Jerusalem 91120, Israel

Address all correspondence and requests for reprints to: Barbara D. Boyan, Ph.D., Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, 315 Ferst Drive NW, Atlanta, Georgia 30332. E-mail: Barbara.Boyan{at}bme.gatech.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined protein kinase C (PKC) in the regulation of breast cancer cells by estrogen. Estrogen receptor (ER)- positive (+) MCF-7 and ER-negative (-) HCC38 cells were treated with 17ß-estradiol (E2) or E2-BSA, which cannot enter the cell. E2 and E2-BSA rapidly increased PKC-{alpha} in both cells via phosphatidylinositol-dependent phospholipase C and G protein, but not phospholipase A2 or arachidonic acid. In MCF-7 cells, E2 and E2-BSA had comparable effects, maximal at 90 min. In HCC38 cells, PKC was maximal at 9 min, with E2-BSA more than E2. Tamoxifen blocked estrogen-dependent PKC in MCF-7 cells and reduced it in HCC38 cells. ER-antagonist ICI 182780, ER-agonist diethylstilbestrol, and antibodies to ER{alpha} and ERß had no effect. E2 stimulated [3H]thymidine incorporation in MCF-7 only; E2-BSA had no effect. Tamoxifen did not alter E2-dependent increases in MCF-7 cells, whereas ICI 182780 reduced DNA synthesis in control and E2-treated cultures. PKC activity was positively correlated with tumor severity in 133 breast cancer specimens and was greater in ER(-) tumors. Tamoxifen treatment reduced recurrence, and recurrent tumors had higher PKC activity. This indicates that E2 rapidly increases PKC activity via membrane pathways not involving ER{alpha} or ERß and suggests that tamoxifen works by reducing PKC activity through non-ER{alpha}/ERß-dependent mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTEIN KINASE C (PKC) activity is elevated in breast cancer tissues (1, 2), suggesting an association with tumorigenicity. Most studies examining the relationship between PKC and breast cancer measured activity of the {alpha} isoform of the enzyme (PKC{alpha}; Refs.3 and4). MCF-7 breast cancer cells transfected with PKC{alpha} are more neoplastic (5), supporting the hypothesis that this isoform is related to tumorigenicity. Other PKC isoforms may play a role as well. NIH 3T3 fibroblasts transfected with PKC-I cDNA display enhanced tumorigenicity (6), and rat embryos overproducing PKCß1 have growth abnormalities (7). Recent studies have shown that PKC{epsilon} is also regulated by estrogen in human breast cancer cells (8).

PKC is involved in intracellular signaling. It has been associated with epidermal growth factor signaling in both estrogen receptor (ER)-positive (+) and ER-negative (-) breast cancer cell lines (9). In chondrocytes (10) and human colon cancer cells (11), 17ß-estradiol (E2) rapidly increases PKC specific activity without new gene expression or protein synthesis. In rat costochondral cartilage cells, E2-dependent increases in PKC{alpha} activity involve G protein and phosphatidylinositol-specific phospholipase C (PI-PLC; Ref. 12); activated PKC is then translocated to the plasma membrane. Plasma membrane fluidity is altered by E2 as well (13). Neither PKC activation nor altered membrane fluidity is caused by the stereoisomer 17{alpha}-estradiol, indicating that these rapid effects of E2 are stereospecific and suggesting that they are receptor-mediated.

Although recent reports suggest that ER{alpha} and ERß may be present in plasma membranes (14), they may not be responsible for the effect of E2 on PKC. In rat costochondral growth plate cartilage cells, neither the ER agonist diethylstilbestrol nor the ER antagonist ICI 182780 blocks the action of E2 on enzyme activity (15). Moreover, ERs are present in chondrocytes from female and male rats (16), but rapid changes in membrane fluidity and increased PKC are only observed in cells from female rats (10, 13). Others have reported that rapid responses to E2 in MCF-7 breast cancer cells may also not be due to traditional ER-mediated events (17, 18, 19).

The membrane action of E2 is supported by studies using E2-BSA, because the conjugated form of the hormone cannot enter the cell (20). In rat chondrocytes (21) and human osteoblasts (22), E2-BSA elicits many of the same effects as E2, including PKC activation. In addition, inhibition of PKC blocks some of the physiological responses of chondrocytes to E2 and E2-BSA (21), indicating the importance of this signaling pathway in the mechanism of estrogen’s action.

The existence of a membrane-associated receptor for E2 may explain the success of agents like tamoxifen in treatment of both ER(+) and ER(-) breast cancers (23), because tamoxifen is a potent PKC inhibitor (24). Additional support for this hypothesis is found in the vitamin D literature, where tamoxifen was shown to inhibit the rapid effect of 1{alpha},25(OH)2D3 and 24R,25(OH)2D3 on PKC in rat chondrocytes in a gender-neutral manner (25). Thus, the effect of tamoxifen may not be specific to traditional ERs, but to PKC{alpha}, which is the isoform sensitive to the action of these regulatory agents (10). Whether this is the case for breast cancer cells is not known.

To better understand the relationship between the ability of tamoxifen to inhibit PKC and its success in the treatment of breast cancer, we used a cell culture model. We examined E2 and E2-BSA regulation of PKC activity in ER(+) MCF-7 cells (26) and ER(-) HCC38 cells (27) in the presence and absence of tamoxifen. We also measured PKC activity in extracts of breast cancer tissue from 133 female patients. Data were stratified as a function of tumor size and type, degree of malignancy, histological evidence of nuclear ERs, and treatment regimen and outcome. Tamoxifen may also exert its effects through calmodulin (28, 29), although the importance of this pathway is not yet clear (30). The role of calmodulin in PKC regulation in breast cancer cells, in response to either tamoxifen or estrogen, was not examined in the present study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture model
Two breast cancer cell lines were used as models. MCF-7 cells express traditional nuclear ERs (26). HCC38 cells lack these ERs (27). Both cells lines were purchased from the American Type Culture Collection (Manassas, VA). The presence or absence of ER{alpha} and ERß was verified by RT-PCR using total RNA isolated from confluent cultures with TRIzol reagent (Life Technologies, Inc., Carlsbad, CA). Primer sets were based on published sequences (31): ER{alpha} sense, 5'-AAGGAGACTCGCTACTGT-3' (nt 762–769), and antisense, 5'-TCAAAGATCTCCACCATGCC-3' (nt 1482–1501); ERß sense, 5'-GTTGTGCCAGCCCTGTTACT-3' (nt 683–702), and antisense, 5'-CGTTTCTCTTGGCTTTGCTC-3' (nt 1014–1033). Primers were synthesized by the Center for Advanced DNA Technologies (The University of Texas Health Science Center, San Antonio, TX). RNA was reverse-transcribed using the First Strand CDNA synthesis kit (Pharmacia Biotech, Piscataway, NJ) and sequenced by the Center for Advanced DNA Technologies. Rat ovary total RNA (1 µg; Ambion, Inc., Austin, TX) was used as a positive control template for both receptors. The expected product sizes were 740 bp (ER{alpha}) and 351 bp (ERß). Sequences of the RT-PCR products were confirmed by direct comparison with published rat ER{alpha} and ERß sequences. Northern blot analysis was performed to confirm the RT-PCR findings and to provide a quantitative assessment of the amounts of mRNA for each receptor in MCF-7 and HCC38 cells.

Western blot analysis of cell culture lysates determined whether the cells synthesized ER{alpha} and/or ERß. For these experiments, cells were lysed in sample buffer, and the proteins present in the lysates were separated on 4–20% gradient acrylamide gels. Blots of the gels were probed with either rabbit polyclonal antibodies to ER{alpha} (H-184, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:200 dilution) or with goat polyclonal antibodies to ERß1 (N-19, Santa Cruz Biotechnology, Inc.; 1:100 dilution). Control blots were probed with nonspecific rabbit IgG or goat IgG at the same dilution used for the antibodies. Immunoreactive bands were visualized using alkaline phosphatase-conjugated polyclonal antirabbit or antigoat secondary antibodies. Recombinant human ER{alpha} was used as a control.

MCF-7 breast cancer cells were confirmed by RT-PCR to express the nuclear ERs, ER{alpha} and ERß (Fig. 1Go). Northern blot analysis confirmed that ER{alpha} mRNA was more abundant than that for ERß (data not shown). Western blot analysis demonstrated the presence of both receptors in cell layer lysates (Fig. 2Go). In contrast, HCC38 breast cancer cells were negative for ER{alpha} and ERß by Northern blot analysis, and ER{alpha} or ERß protein was absent on Western blots (data not shown). However, RT-PCR (35 cycles) detected mRNA for ERß in these cells (Fig. 1Go).



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  		Figure 1. RT-PCR analysis of ER{alpha} and ERß expression in MCF-7 and HCC38 breast cancer cells. Total RNA was isolated from confluent cultures and subjected to RT-PCR as described in Materials and Methods. The amplified fragments were separated by 1% agarose gel electrophoresis. The primer for ER{alpha} amplified a cDNA of 740 bp. Primers for ERß amplified a cDNA of 351 bp. In all cases, the amplified cDNAs were sequenced and confirmed to be the expected amplicon. Reactions were carried out in the presence of active and inactive RT to ensure the specificity of the reaction; only data for active RT are shown. Rat ovary total RNA was used as a positive control.

 


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  		Figure 2. Western blot of ER{alpha} in lysates of MCF-7 and HCC38 breast cancer cells. Cell lysates were electrophoresed on sodium dodecyl sulfate polyacrylamide gels and then transferred for Western blot analysis. Blots were incubated with polyclonal rabbit antibodies to ER{alpha}, and immunoreactive bands were visualized using an alkaline phosphatase-linked second antibody. Recombinant human ER{alpha} was used as a control. MW, Molecular weight.

 
Cells were cultured in T-75 flasks in DMEM containing 10% fetal bovine serum, 1% antibiotics, and 0.01 mg/ml insulin at 37 C in an atmosphere containing 5% CO2 and 100% humidity. When third- passage cells were confluent, they were subcultured into 24-well plates using seeding densities of 10,000 cells/cm2 and incubated using the same culture conditions as above. At confluence, culture media were replaced with media containing vehicle, E2, or E2-BSA (Sigma, St. Louis, MO), as indicated below. Previous studies in our laboratory (21) showed that part of the effect of E2-BSA on chondrocytes was due to free E2. However, prefiltration of the E2-BSA to remove any free E2 eliminated this problem. Therefore, before the E2-BSA was diluted in media, it was filtered through a 3000 molecular weight cut-off filter (Millipore Corp., Bedford, MA; Ref. 22). All cell culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD).

PKC activity
Initial experiments determined the time course of E2 and E2-BSA regulation of PKC activity. Confluent cultures were treated for 9, 90, or 270 min with vehicle alone or with 10-8 and 10-7 M E2 or E2-BSA. Dose response was determined by treating MCF-7 cells and HCC38 cells for 90 min with 10-11 to 10-7 M E2 or E2-BSA. To determine whether tamoxifen altered the PKC response to estrogen, one half of the cultures were treated with 10-9 M tamoxifen (Sigma). Alternatively, the cultures were treated for 90 min with 10-8 M E2 or E2-BSA plus 10-11, 10-10, or 10-9 M tamoxifen.

The contribution of nuclear ERs to any change in PKC activity was determined by treating confluent cultures with 10-8 M E2 or E2-BSA as above, together with the ER antagonist ICI 182780 (Tocris Cookson, Ballwin, MO; Refs.32 and33) or the ER agonist diethylstilbestrol (Sigma; Ref. 34). MCF-7 cells and HCC38 cells were treated for 90 min with 10-8 M E2 or E2-BSA and either ICI 182870 (10-8, 10-7, or 10-6 M) or diethylstilbestrol (10-9, 10-8, or 10-7 M). In addition, MCF-7 cells and HCC38 cells were incubated for 90 min with 10-8 M E2 or E2-BSA in the presence of specific antibodies to ER{alpha} and ERß (GeneTex, Inc., San Antonio, TX). Antibodies were added to the cultures at dilutions of 1:500, 1:1000, and 1:5000. Although these antibodies are specific for intracellular ERs, the anti-ER{alpha} antibody has been used successfully to label proteins in the plasma membrane (35).

Isoform-specific antibodies were used to determine which PKC isoform is sensitive to E2 or E2-BSA as described previously (21). After treatment with 10-8 M E2 or E2-BSA for 90 min, HCC38 cells were lysed as described below, and the lysates were incubated with nonspecific IgG or with antibodies to one of the following isoforms: PKC{alpha}, PKCß, PKC{delta}, PKC{epsilon}, and PKC{zeta} (Santa Cruz Biotechnology, Inc.). After precipitating the antigen-antibody complexes, PKC specific activity was determined in the supernatant.

To assess the mechanisms involved in the increase in PKC specific activity by E2 or E2-BSA, HCC38 cells were treated for 90 min with 10-8 M E2 or E2-BSA in the presence of specific inhibitors of signaling pathways associated with membrane-mediated PKC activation (all inhibitors were purchased from Calbiochem (La Jolla, CA). The PI-PLC inhibitor U73122 (36) was used to determine the role of PLC in the mechanism. Cells were treated with 0, 0.1, 1, or 10 µM U73122. The role of G proteins was examined using the general G protein inhibitor GDPßS and the G protein activator GTP{gamma}S (37) at concentrations of 0, 1, 10, and 100 µM. To assess whether PKC was increased via a phospholipase A2 (PLA2)-dependent mechanism, HCC38 cells were treated with the PLA2 inhibitor quinacrine (38) at concentrations of 10-9 M to 10-7 M. In addition, HCC38 cells were treated with 10-9 to 10-7 M indomethacin to prevent the metabolism of arachidonic acid, thereby testing the potential role of prostaglandin in the pathway.

All experiments included vehicle-only control cultures. After treatment, PKC activity was measured in cell culture lysates as described previously for rat chondrocytes (25). At the termination of the incubation with E2 or E2-BSA, cell layers were rinsed with PBS, removed from the wells with a cell scraper, and lysed in solubilization buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and 1% Nonidet P-40] for 30 min on ice. Lysates containing equivalent amounts of protein, determined using the bicinchoninic protein assay reagent kit from Pierce Chemical Co. (Rockford, IL), were mixed for 20 min with a lipid preparation containing phorbol-12- myristate-1-acetate, phosphatidylserine and Triton X-100 mixed micelles, thereby providing the necessary cofactors and conditions for optimal activity. To this mixture, a high-affinity myelin basic protein peptide and [32P]ATP (25 µCi/ml) were added to a final assay volume of 50 µl. After a 10-min incubation in a 30 C waterbath, samples were spotted onto phosphocellulose disks, which were then washed twice with 1% phosphoric acid and once with distilled water to remove unincorporated label before placement in a scintillation counter.

DNA synthesis
Effects of E2 and E2-BSA on proliferation were determined as a function of [3H]thymidine incorporation, as described previously for chondrocytes (15). Nearly confluent cultures of MCF-7 cells and HCC38 cells were treated for 24 h with 10-11 to 10-7 M E2 or E2-BSA. Tamoxifen (10-9 M) was added to one half of the cultures. Four hours before harvest, [3H]thymidine was added to the cultures. To determine whether DNA synthesis was regulated via nuclear ERs, cells were treated with E2 and E2-BSA in the presence of ICI 182780 or diethylstilbestrol. For these experiments, cells were cultured for 24 h with 10-8 M E2 or E2-BSA plus ICI 182780 (10-8, 10-7, or 10-6 M) or diethylstilbestrol (10-9, 10-8, or 10-7 M). Vehicle-only controls were included in all experiments.

Local production of estrogen
To determine whether MCF-7 cells or HCC38 cells are able to synthesize and secrete estrogen, we examined the ability of the cultures to convert testosterone to 17ß-estradiol, as described previously for chondrocytes (39).

Statistical analysis
For each experiment, each variable was tested in six independent cultures. Data presented in Results are from one set of experiments and are means ± SE of the mean for n = 6. To assess statistical significance, data were first analyzed using ANOVA. Individual groups were compared using Bonferroni’s modification of Student’s t test. All experiments were repeated a minimum of two times to ensure validity of the observations. Because the number of patients in each experiment was sufficiently great in terms of power, we opted to show actual values from one representative experiment, rather than data from multiple experiments that had been normalized as a percentage of control values.

PKC in human breast cancer tissue
Breast tumor tissue was obtained from 133 patients who presented for treatment at the Department of Obstetrics and Gynecology, University of Würzburg, under a protocol approved by the University’s Institutional Review Board. A pathologist classified the tissues as fibroadenoma, invasive lobular carcinoma, or invasive ductal carcinoma. Carcinomas were scored as ER(+) or ER(-) on the basis of immunohistochemistry using an antibody to ER{alpha}. ER(+) tumors exhibited nuclear antibody staining. Cells in ER(-) tumors exhibited no nuclear, cytosolic, or plasma membrane immunoreactivity. Carcinomas (116 of 118) were also assigned a differential receptor score from 0–12 based on the classification system of Remmele and Stegner (40). Scores from 0–1 are considered ER(-); scores from 2–12 are considered to be ER(+). Tumors were sized according to the TNM classification system (41).

Tissues were also separated into groups on the basis of the treatment protocol selected for each patient: excision of the tumor or ablation of the breast tissue. We noted whether the patient received tamoxifen after surgery and whether there was any tumor recurrence or metastasis. Six patients were lost to follow-up, reducing the number of patients to 112 for these analyses. Because this was a retrospective study using banked tissues, it was not possible to assess prospectively whether patients receiving tamoxifen developed recurrent tumors with lower levels of PKC than their primary tumor exhibited.

Tissue was also frozen immediately after surgical removal and stored at -70 C in the Breast Cancer Tissue Bank at the University of Würzburg. Before determining PKC activity, frozen samples were thawed, their wet weights were determined, and they were then homogenized in RIPA buffer (100 mg tissue wet weight/ml) using a Polytron device (Brinkmann Instruments, Inc., Westbury, NY). PKC in the supernatant obtained after centrifugation at 15,000 x g for 2 min was measured as previously described using assay conditions optimized for phospholipid- and calcium-dependent PKC (10). Protein content was determined as described previously (10).

Data were analyzed by ANOVA, and post hoc comparisons were made using Bonferroni’s modification of Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro studies using MCF-7 and HCC38 cells
E2 and E2-BSA caused a time- and dose-dependent increase in PKC specific activity in MCF-7 and HCC38 cells, indicating that PKC specific activity was regulated by a membrane-mediated mechanism in both ER(+) and ER(-) breast cancer cells. However, the effects were cell-specific. In MCF-7 cells, a 3.2-fold increase was evident at 9 min, and there was a 5.4-fold increase at 90 min in response to E2 (Fig. 3AGo). E2-BSA caused a 3.1-fold increase at 9 min and a 5.7-fold increase at 90 min (Fig. 3BGo). In HCC38 cells, the greatest increases in PKC specific activity were seen at 9 min. E2 caused a 3.4-fold increase at 9 min and a 2.5-fold increase at 90 min (Fig. 3CGo). E2-BSA caused an 8.1-fold increase at 9 min, and the effect was reduced to 4.9-fold at 90 min (Fig. 3DGo). Although effects of E2 and E2-BSA were comparable in MCF-7 cells, the stimulatory effect of E2-BSA in HCC38 cells was more than twice that of E2. Concentrations of E2 or E2-BSA as low as 10-11 M were stimulatory in the MCF-7 cells (Fig. 4Go, A and B). HCC38 cells were less sensitive to E2, exhibiting increases in PKC specific activity only at concentrations of 10-9 M and greater, whereas E2-BSA stimulated PKC at 10-11 M (Fig. 4Go, C and D).



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  		Figure 3. Time course of estrogen-dependent regulation of PKC specific activity in ER(+) MCF-7 and ER(-) HCC38 breast cancer cells. Cells were treated with E2 (A and C) or E2-BSA (B and D) for 9, 90, or 270 min. PKC specific activity was measured in cell layer lysates. Values are the mean ± SEM for six independent cultures for each variable. #, P < 0.05, treatment vs. control at each time point; {bullet}, P < 0.05, 10-7 M vs. 10-8 M; *, P < 0.05, vs. 9 min for each treatment.

 


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  		Figure 4. Dose-dependent effects of estrogen on PKC specific activity in cultures of MCF-7 and HCC38 breast cancer cells and regulation by trans-tamoxifen. Cells were treated with E2 (A and C) or E2-BSA (B and D) for 90 min in the presence or absence of 10-9 M trans-tamoxifen. PKC specific activity was measured in cell layer lysates. Values are the mean ± SEM for six independent cultures for each variable. #, P < 0.05, tamoxifen vs. control; *, P < 0.05, E2 or E2-BSA vs. control.

 
The PKC isoform that was sensitive to both E2 and E2-BSA was PKC{alpha}. Only when lysates of estrogen-treated cells were incubated with isoform-specific antibodies to PKC{alpha} was PKC activity reduced (Table 1Go). In contrast, antibodies to PKCß, PKC{delta}, PKC{epsilon}, and PKC{zeta}, as well as nonspecific IgG, had no effect.


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  		Table 1. Isoform specificity of E2 and E2-BSA-dependent PKC activation in HCC38 cells

 
PKC activity was regulated by tamoxifen in both control and estrogen-stimulated cultures. In MCF-7 cells, 10-9 M tamoxifen reduced PKC activity in control cultures by 64% and blocked the effects of both E2 and E2-BSA at all concentrations tested (Fig. 4Go, A and B). In HCC38 cells, 10-9 M tamoxifen reduced PKC activity in control cultures by 25–50%, but it only partially decreased the stimulatory effect of E2 and E2-BSA (Fig. 4Go, C and D). The effect of tamoxifen was dose-dependent, completely abolishing the stimulatory effect of E2 and E2-BSA at 10-10 M in MCF-7 cells (Fig. 5Go, A and B). In HCC38 cells, even a 10-fold higher concentration of tamoxifen only partially blocked the estrogen-dependent increase in PKC activity (Fig. 5Go, C and D). However, an extended dose-response study showed that 10-8 M tamoxifen reduced PKC in E2-treated HCC38 cells to levels seen in untreated control cultures, and 10-7 M tamoxifen reduced PKC to levels seen in control cultures treated with tamoxifen (data not shown).



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  		Figure 5. Dose-dependent effect of tamoxifen on estrogen-dependent PKC specific activity in cultures of MCF-7 and HCC38 breast cancer cells. Cells were treated with 10-8 M E2 (A and C) or E2-BSA (B and D) for 90 min in the presence of 10-11 to 10-9 M trans-tamoxifen. PKC specific activity was measured in cell layer lysates. Values are the mean ± SEM for six independent cultures for each variable. #, P < 0.05, E2 or E2-BSA vs. control at each dose of tamoxifen; {bullet}, P < 0.05, 10-9 M tamoxifen vs. 10-10 or 10-11 M tamoxifen; *, P < 0.05, tamoxifen vs. control.

 
Nuclear ERs did not play a role in the PKC response to E2 or E2-BSA. PKC was activated by both forms of estrogen in HCC38 cells, which lacked evidence of ER{alpha} and ERß on Western blots or of mRNA for either form of receptor by Northern analysis (data not shown). Neither the ER antagonist ICI 182780 nor the ER agonist diethylstilbestrol altered the increase caused by E2 or E2-BSA in MCF-7 cells (data not shown) or in HCC38 cells (Fig. 6Go, A and B). In addition, antibodies to ER{alpha} and ERß had no effect on PKC activity in cultures treated with E2 or E2-BSA (data not shown).



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  		Figure 6. Effect of ER antagonist ICI 182780 (A) and ER agonist diethylstilbestrol (B) on PKC specific activity in HCC38 breast cancer cells. HCC38 cells were incubated with 10-8 M E2 or E2-BSA for 90 min in the presence of 10-8 to 10-6 M ICI 182780 or 10-9 to 10-7 M diethylstilbestrol. PKC specific activity was measured in cell layer lysates. Values are the mean ± SEM for six independent cultures for each variable. #, P < 0.05, E2 or E2-BSA vs. control at each dose of ICI 182780 or diethylstilbestrol; {bullet}, P < 0.05, E2 vs. E2-BSA.

 
E2 and E2-BSA increased PKC activity by a mechanism that involved PI-PLC (Fig. 7Go). The PI-PLC inhibitor U73122 had no effect on PKC activity of control cultures. However, U73122 caused a dose-dependent decrease in the estrogen-stimulated increase in PKC specific activity. At 1 µM, U73122 reduced the stimulatory effect of the estrogens by more than 50%, and at 10 µM, U73122 blocked any estrogen-dependent increase.



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  		Figure 7. Effect of inhibition of PI-PLC on PKC specific activity in HCC38 breast cancer cells. HCC38 cells were incubated with 10-8 M E2 or E2-BSA for 90 min in the presence of 0, 0.1, 1, or 10 µM U73122. PKC specific activity was measured in cell layer lysates. Values are the mean ± SEM for six independent cultures for each variable. #, P < 0.05, E2 or E2-BSA vs. control at each dose of U73122; *, P < 0.05, with U73122 vs. without U73122.

 
G protein was also involved in the mechanism. The G protein inhibitor GDPßS did not affect PKC in control cultures, but it caused a dose-dependent decrease in PKC in estrogen-treated cultures (Fig. 8AGo). At 100 µM, the effect of estrogen was abolished. In contrast, GTP{gamma}S caused a dose-dependent increase in PKC activity in HCC38 cells (Fig. 8BGo). At 10 µM GTP{gamma}S, the G protein activator alone caused a 2.1-fold increase that was comparable to the effect of 10-8 M E2 and E2-BSA alone. At 100 µM GTP{gamma}S, the G protein activator alone caused a 3.1-fold increase in PCK activity, which was comparable to the effect of GTP{gamma}S plus 10-8 M E2. The effect of 100 µM GTP{gamma}S was synergistic with the effect of 10-8 M E2-BSA.



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  		Figure 8. Role of G protein in the increase in PKC specific activity by E2 and E2-BSA. HCC38 cells were incubated with 10-8 M E2 or E2-BSA for 90 min in the presence of 0, 1, 20, or 100 µM GDPßS to inhibit G proteins (A) or in the presence of 0, 1, 10, or 100 µM GTP{gamma}S to stimulate G proteins (B). PKC specific activity was measured in cell layer lysates. Values are the mean ± SEM for six independent cultures for each variable. #, P < 0.05, E2 or E2-BSA vs. control at each dose of GDPßS or GTP{gamma}S; *, P < 0.05, with G protein regulator vs. without G protein regulator.

 
PLA2 did not play a role in the mechanism by which either estrogen activated PKC. Inhibition of PLA2 with quinacrine had no effect on PKC activity in control cultures or in cultures treated with E2 or E2-BSA (data not shown). Arachidonic acid metabolites also had no effect because the general cyclooxygenase inhibitor indomethacin did not alter the ability of E2 or E2-BSA to increase PKC activity (data not shown).

Proliferation of the breast cancer cells was regulated by estrogen via the nuclear ERs. E2 affected DNA synthesis in MCF-7 cells in a dose-dependent manner, but E2-BSA had no effect on [3H]thymidine incorporation (Fig. 9Go, A and B). ICI 182780 reduced DNA synthesis in control cultures and blocked [3H]thymidine incorporation in response to E2 (Fig. 9AGo). The effects of the ER antagonist were dose-dependent (Fig. 10AGo). At the highest concentration, ICI 182780 completely abolished any increase due to E2. In addition, the inhibitory effect of ICI 182780 on DNA synthesis in control cultures of MCF-7 cells was maintained in the E2-BSA-treated cultures, regardless of the E2-BSA concentration used (Fig. 10AGo). Neither form of estrogen affected [3H]thymidine incorporation by HCC38 cells (Fig. 10BGo). ICI 182780 caused a small decrease in [3H]thymidine incorporation in control cultures of HCC38 cells and in all cultures treated with E2 or E2-BSA (Fig. 10BGo). Tamoxifen (10-9 M) had no effect on the E2-stimulated increase in DNA synthesis in the MCF-7 cells (data not shown) and it had no effect on proliferation, whether or not the HCC38 cells were treated with E2 or E2-BSA (data not shown).



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  		Figure 9. Effect of 182780 on E2- and E2-BSA-dependent [3H]thymidine incorporation by MCF-7 breast cancer cells. Confluent cultures of MCF-7 cells were treated for 24 h with 10-11 to 10-7 M E2 (A) or E2-BSA (B) in the presence of 10-6M ICI 182780. Values are the mean ± SEM for six independent cultures for each variable. *, P < 0.05, E2 vs. control; #, P < 0.05, ICI 182780 vs. control.

 


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  		Figure 10. Effect of ICI 182780 on [3H]thymidine incorporation by MCF-7 (A) and HCC38 (B) breast cancer cells. Confluent cultures were treated for 24 h with E2 or E2-BSA in the presence of 10-8 to 10-6 M ICI 182780. Values are the mean ± SEM for six independent cultures for each variable. *, P < 0.05, ICI 182780 vs. control; #, P < 0.05, E2 vs. E2-BSA.

 
There was no evidence of aromatase activity in either of the two cell types (data not shown).

PKC in human breast carcinomas
Classification of the 133 tumors used in this study showed that 15 of the tumors were fibroadenomas, 33 were invasive lobular carcinomas, and 85 were invasive ductal carcinomas. As noted previously (27), tumor severity correlated with patient age (fibroadenoma, 43.9 ± 3.4 yr; invasive lobular carcinoma, 62.2 ± 1.4 yr; and invasive ductal carcinoma, 64.7 ± 2.5 yr). There was a direct correlation between tumor type and PKC specific activity. PKC in fibroadenomas was less than half that measured in invasive lobular carcinomas, and this was approximately half the specific activity measured in invasive ductal carcinomas (Fig. 11AGo). PKC in carcinomas was more than twice that found in normal breast tissue from the same patient (Fig. 11BGo). PKC also varied with tumor severity and was higher in larger carcinomas. Values ranged from 26.1 ± 3.7 pmol PO4/µg protein·min for T1 tumors to 50.4 ± 1.2 pmol PO4/µg protein·min for T4 tumors (Table 2Go). PKC varied with ER status as well. ER(-) breast carcinomas had twice the specific activity of ER(+) tissues (Fig. 11CGo). Enzyme activity varied in a biphasic manner with receptor score (Table 3Go). As receptor score increased from 0 to 5–7, PKC decreased, with the lowest levels being observed in specimens with scores of 5–7. As receptor score increased from 8–12, PKC specific activity increased, with greatest PKC activity found in tissue specimens with receptor scores of 11–12.



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  		Figure 11. PKC specific activity in human breast tumor tissue. A, PKC activity in fibroadenoma, invasive lobular carcinoma, and invasive ductal carcinoma. B, PKC activity in breast cancer tissue (n = 4) and adjacent normal tissue in the same patient (n = 4). C, PKC activity in ER(-) (n = 39) or ER(+) (n = 79) tissue. *, P < 0.05, carcinoma vs. fibroadenoma (A); breast cancer vs. normal tissues (B); or ER(-) vs. ER(+) carcinomas (C); #, P < 0.05, lobular carcinoma vs. ductal carcinoma.

 

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  		Table 2. Relationship between breast cancer size (TNM classification) and PKC specific activity (pmol PO4/µg protein·min)

 

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  		Table 3. Relationship between ER score and PKC specific activity

 
Carcinoma patients (n = 118) had a mean age of 63.1 ± 1.2 yr. ER(-) patients (n = 39) averaged 60.7 ± 3.4 yr (Table 4Go). Twelve were treated by tumor excision, and 27 were treated by breast tissue ablation. ER(+) patients (n = 79) averaged 64.6 ± 1.5 yr. Twenty-eight had their tumors excised, and 51 had their breast tissue ablated. In patients receiving tamoxifen treatment, there was no recurrence or metastasis in 47 of 56 patients with ER(+) tumors (5 patients were lost to follow-up), and similarly, 8 of 11 patients with ER(-) tumors (no patients were lost to follow-up) had no recurrence or metastasis. In the groups not receiving tamoxifen, 15 of 18 patients with ER(+) tumors had no recurrence, and 10 of 28 patients with ER(-) tumors had no recurrence.


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  		Table 4. Summary of carcinoma patient profiles by ER status and treatment regime

 
Tissue from patients experiencing recurrence or metastasis of their carcinoma (n = 32) had higher levels of PKC activity than seen in tissue from patients without a recurrence (n = 80; 56.2 ± 11.7 vs. 34.2 ± 5.8 pmol PO4/µg protein·min), although statistical significance was not achieved (P = 0.064; Table 5Go). When data were stratified with respect to ER status, PKC was related in a positive manner with metastatic disease in ER(+) tumors (no recurrence, 27.2 ± 3.8 pmol PO4/µg protein·min, n = 62; recurrence, 57.2 ± 18.6 pmol PO4/µg protein·min, n = 12; P = 0.015). In contrast, there was no association of PKC activity with metastatic disease in ER(-) tumors (no recurrence, 57.5 ± 18.0 pmol PO4/µg protein·min, n = 18; recurrence, 63.5 ± 21.6 pmol PO4/µg protein·min, n = 20), although PKC activity in all ER(-) tumors was higher than that in ER(+) tumors not displaying recurrence.


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  		Table 5. PKC specific activity (pmol PO4/µg protein·min) in breast carcinoma tissue from patients that experienced a tumor recurrence during the follow-up period

 
In 72 tamoxifen patients, 55 experienced no recurrence of their tumor, whereas 12 patients had a recurrence during the follow-up period (five patients lost to follow-up; Table 6Go). Tissue from those patients with recurrent carcinoma or metastasis had twice as much PKC activity (66.4 ± 17.6 pmol PO4/µg protein·min vs. 33.3 ± 6.2 pmol PO4/µg protein·min; P = 0.036). There was also a greater incidence of recurrence in patients not receiving tamoxifen (20 of 45 patients) than in those receiving tamoxifen (12 of 67 patients). PKC activity in carcinomas from patients not receiving tamoxifen and who did not experience recurrence of their tumor during follow-up was lower than that observed in tissue from patients experiencing recurrence (36.0 ± 12.8 pmol PO4/µg protein·min, n = 25; vs. 50.0 ± 15.7 pmol PO4/µg protein·min, n = 20), but the difference was not significant. Whether or not tamoxifen was prescribed, however, enzyme activity in the tissues was comparable if stratified on the basis of carcinoma recurrence or metastasis.


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  		Table 6. PKC specific activity (pmol PO4/µg protein·minute) in breast carcinoma tissue from patients receiving tamoxifen during the follow-up period

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies indicate that breast cancer cells respond to estrogen with a rapid increase in PKC activity, even in the absence of nuclear ERs. Both ER(+) MCF-7 cells and HCC38 cells, which lack ER{alpha} or ERß, exhibited dose-dependent increases in PKC activity within 9 min in response to E2 and E2-BSA. At least part of this response is membrane mediated, because E2-BSA elicited comparable effects on PKC as were seen in MCF-7 cells treated with E2.

The effect of E2-BSA was not due to free E2. Previous studies had shown that part of the response of chondrocytes to E2-BSA was due to free E2, although less than 5% of the E2 in E2-BSA was unconjugated and therefore able to enter the cell and bind to nuclear ERs (21). By prefiltering the E2-BSA to remove unconjugated estrogen (22), the effect of free E2 was eliminated (21). For this reason, only prefiltered E2-BSA was used in the present study.

Neither ER{alpha} nor ERß was present in Western blots of HCC38 cells, making it unlikely that the receptor responsible for PKC activation by E2 or E2-BSA was a membrane form of these receptors (15, 21). Moreover, antibodies specific for ER{alpha} and ERß did not block the effects of E2 or E2-BSA in MCF-7 cells, which do possess both receptors. Although it could be argued that the antibodies could not detect nuclear receptors, if these receptors were present in the plasma membrane they would have been seen, as has been reported by others (35). This suggests that an alternate receptor may be involved. Whether the receptor is a splice variant of ER{alpha} or ERß that is not recognized by the antibodies or primers used cannot be ruled out.

Another possibility is that E2 and E2-BSA exert their effects via separate membrane receptor-mediated mechanisms in MCF-7 cells and HCC38 cells. Studies using vascular endothelial cells as the model have provided evidence that E2 binds ER{alpha} in the plasma membranes but E2-BSA binds three non-ER proteins (42). This latter hypothesis is supported by the enhanced sensitivity of PKC to E2-BSA noted in the ER(-) HCC38 cells.

Our results indicate that if separate receptors are used by E2 and E2-BSA, they signal by the same pathways in HCC38 cells. E2 and E2-BSA cause an increase in PKC specific activity via a PI-PLC-dependent mechanism that involves G proteins, based on the specific inhibition of activation by U73122 and GDPßS, as well as the increase in activation by GTP{gamma}S. Neither form of estrogen signals via a PLA2-dependent mechanism, nor are arachidonic acid metabolites involved. Moreover, both forms of estrogen affect the {alpha} isoform of PKC in HCC38 cells as well as in MCF-7 cells, as demonstrated by the reduction in PKC activity in the presence of antibodies to PKC{alpha}, but not to PKCß, PKC{delta}, PKC{epsilon}, or PKC{zeta}. E2 and E2-BSA also function in the same manner in rat growth plate chondrocytes, specifically activating PKC{alpha} via PI-PLC and G protein (10), suggesting that this pathway is common across cell types and species. Species and/or cell-type differences may exist, however. Others have reported that in MCF-7 cells, PKC{epsilon} (8) is translocated to the membrane. In rat chondrocytes, PKC translocation to the plasma membrane also occurs in response to E2 and E2-BSA (10). However, PKC{epsilon} was not affected by either form of estrogen in the chondrocytes, nor was it affected in the HCC38 cells in the present study.

Although our results demonstrate definitively that non-ER mechanisms are involved in the rapid activation of PKC by estrogen, traditional ER-mediated mechanisms may also play a role. In MCF-7 cells, in which ER{alpha} and ERß are both present, E2 and E2-BSA elicit rapid increases in PKC, as well as a more robust increase at 90 min. In rat chondrocytes, E2/E2-BSA sensitive PKC translocation to the plasma membrane accounts for the rapid response, but new PKC expression and synthesis are required for the delayed increase (10). Whether the newly expressed PKC is a consequence of classical ER-dependent gene activation is not known. PKC- dependent pathways can also lead to gene expression, and several studies have now shown that E2 and E2-BSA can lead to MAPK activation (43, 44, 45).

Our results indicate that estrogen-dependent regulation of DNA synthesis in breast cancer cells is not via membrane receptors. Only E2 increased [3H]thymidine incorporation by MCF-7 cells, and neither form of estrogen modulated DNA synthesis in HCC38 cells. However, ER-dependent and ER-independent pathways may both play a role. Several studies have suggested that E2 exerts its effects on growth through non-ER mediated mechanisms (17, 18, 19). At least part of the response is via the ER, however, because the ER antagonist ICI 182780 decreased [3H]thymidine incorporation in ER(+) MCF-7 cultures treated by E2 and E2-BSA. ICI 182780 reduced DNA synthesis in HCC38 cells, which lack ER{alpha} and ERß protein, suggesting that it may exert antiestrogenic effects by mechanisms unrelated to traditional nuclear ERs. ICI 182780-dependent reduction of DNA synthesis in control cultures reflects the fact that the cells were conditioned by low levels of estrogen in the culture media (<10-12 M). The goal of our study was not to study the effects of E2 and E2-BSA under conditions in which the cells were starved of such conditioning factors present in serum, but to study the incremental effects of estrogen in healthy cells. The fact that ICI 182780 was able to reduce DNA synthesis under these culture conditions supports the hypothesis that E2 in the control medium and ICI 182780 mediated their effects via a mechanism other than ER{alpha} or ERß. The possibility cannot be ruled out that small levels of ERß, undetectable on Western blots, were sufficient to mediate the antiestrogenic effect of ICI 182780 in HCC38 cells because RT-PCR was able to detect low levels of ERß mRNA.

Our cell culture studies suggest that mechanisms other than growth arrest may contribute to the success of tamoxifen clinically. In the present study, tamoxifen did not inhibit the stimulatory effect of E2 on DNA synthesis in MCF-7 cells, nor did it alter DNA synthesis in control cultures. Similarly, tamoxifen did not alter DNA synthesis in HCC38 cells, whether or not they were treated with E2 or E2-BSA. This was an unexpected finding for which we do not yet have a comprehensive explanation. Tamoxifen is a PKC inhibitor, and DNA synthesis is mediated by a PKC-dependent mechanism in MCF-7 cells because phorbol esters, which activate PKC (46), also regulate growth in these cells (47). Tamoxifen has cytotoxic effects in breast cancer cells (48) and blocks the stimulatory effects of E2 on DNA synthesis in some MCF-7 cell lines (49). However, other studies using MCF-7 cells show that tamoxifen can also stimulate DNA synthesis (50), whereas others report that the effect of tamoxifen can vary (49, 51). Tamoxifen concentrations comparable to those we used have not been shown to be cytotoxic, although they are inhibitory to PKC. This suggests that tamoxifen-sensitive PKC, which is membrane-mediated on the basis of its stimulation by E2-BSA, is not involved in growth regulation of breast cancer cells, at least with respect to DNA synthesis. This hypothesis is supported by the observation that PCK{eta} but not PKC{alpha}, PKC{delta}, or PKC{epsilon} is specifically involved in protection of MCF-7 cells against TNF cytotoxicity (52).

The failure of tamoxifen to reduce DNA synthesis in breast cancer cells, although it inhibits PKC activity, differs from colon tumor cells in which inhibition of PKC with chelerythrine or treatment with ICI 182870 blocked E2-dependent DNA synthesis (11). PKC inhibition by chelerythrine and tamoxifen are not necessarily the same, however. In rat costochondral cartilage cells, chelerythrine causes a complete reduction in PKC in control cultures and reduces [3H]thymidine incorporation to a greater extent than is seen with E2 (10). Chelerythrine primarily targets PKC{alpha} (53), but it also has inhibitory effects on other PKC isoforms (54) as well as PKA (55), which may also regulate proliferation. Tamoxifen inhibits PKC in rat chondrocytes (25) as well as in other cells (24, 56), and it has been reported to inhibit calmodulin-dependent cAMP phosphodiesterase activity in rat brain (57). In addition, tamoxifen has been shown to specifically affect PKC{epsilon} (8), whereas E2 affects PCK{alpha} (12). The effects of tamoxifen on PKC activity as well as on proliferation of MCF-7 cells also depend on the configuration of the molecule. Cis-tamoxifen is a more potent inhibitor of Ca2+ and phosphatidylserine-dependent rat brain PKC than trans- tamoxifen (58). Although others have reported that cis-tamoxifen promotes MCF-7 cell growth and trans-tamoxifen inhibits growth (59), we failed to demonstrate an effect of trans-tamoxifen on DNA synthesis. This may be due to differences in experimental design. Our studies were done using confluent cultures to correlate the effects of estrogen on PKC activity with the regulation of cell physiology by E2 and E2-BSA. It may be that culture conditions, such as confluence, modulate the effects of estrogen on the cells.

Recent studies examining local production of steroid hormones suggest that many cells use these agents as autocrine modulators. E2 is secreted by rat chondrocytes (39), human osteoblasts (60), and colon and breast cancer cells (61, 62) at concentrations up to 1000-fold greater than circulating hormone levels. By acting via membrane receptors coupled to rapid action signaling pathways, locally produced steroids can fine-tune the response of the cell to conditioning levels of the systemically circulating hormone. We failed to find evidence of aromatase activity based on estradiol production via aromatization in either cell line in the present study. Because the fetal bovine serum used in the culture media was not charcoal-stripped, there were low levels of E2 present even in the control cultures (<10-12 M), which may have suppressed local E2 production by the MCF-7 and HCC38 cells.

Our results confirm and extend the relationship between PKC activity and tumorigenicity suggested by previous studies (1, 2, 3, 5). Although earlier reports examined relatively small numbers of samples, we examined 133 specimens, enabling us to establish with a reasonable degree of certainty that tumor severity and PKC specific activity are correlated in a positive manner. Moreover, PKC activity is greater in carcinoma vs. fibroadenoma, particularly in ER(-) carcinomas, suggesting that the PKC signaling pathway may be up-regulated in these cells. We did not specifically assess whether a particular PKC isoform(s) was responsible for the increased enzyme activity in the breast cancer samples. Our assay system is optimized for PKC{alpha}, which is both Ca2+- and phospholipid-dependent (63). Recent studies examining expression of PKC{zeta} failed to show a correlation between PKC{zeta} and total PKC activity in cultures of breast cancer cells from 12 patients, nor were they able to show that PKC{zeta} expression was influenced by a number of commonly used antineoplastic agents (64). PKC{zeta} is an atypical form of the enzyme and is insensitive to both Ca2+ and phospholipid (65). Using antiisoform antibodies, we showed that PKC{zeta} is not regulated by estrogen in human breast cancer cells, as was shown previously in rat chondrocytes (10). Changes in PKC{eta} may contribute to increased PKC in the breast carcinoma tissues we examined because others have shown by immunohistochemistry that distribution of PKC{eta} is altered in in situ and invasive lesions in comparison with normal breast tissue (66). Taken together, our in vitro and in vivo results suggest that PKC activity in aggressive neoplasia is related to aspects of the tumors in addition to DNA synthesis, and it is these features that are tamoxifen sensitive.


   Acknowledgments
 
We thank Carol Heston and Wanda Whitfield for their help in the preparation of this manuscript.


   Footnotes
 
This research was funded by the Susan G. Komen Foundation and United States Public Health Service Grants DE-05937 and DE-08603.

Abbreviations: E2, 17ß-estradiol; ER, estrogen receptor; PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; PLA2, phospholipase A2.

Received October 1, 2002.

Accepted for publication January 21, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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