Affiliations of authors: X. Chen, C. Danes, M. Lowe, T. W. Herliczek, Division of Molecular Medicine, Wadsworth Center, Albany, NY; K. Keyomarsi, Division of Molecular Medicine, Wadsworth Center, and Department of Biomedical Sciences, State University of New York, Albany.
Correspondence to and present address: Khandan Keyomarsi, Ph.D., Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 66, Houston, TX 77030-4095 (e-mail: kkeyomar{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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The ER is a 67-kd estrogen-modulated transcription factor that belongs to the superfamily of ligand-activated nuclear transcription factors, which includes receptors for steroids, thyroids, and retinoic acid (46). In the presence of estrogen (the ligand), the ER dimerizes and binds to specific sequences, termed estrogen-response elements (EREs), located in the 5` flanking region of ER-responsive genes (such as the progesterone receptor), activating their transcription and thus manifesting their growth-stimulating effects (7,8). At the onset, 46%77% of breast cancers (9) are ER positive and up to 70% of them respond to hormone manipulation (10). However, about 10% of receptor-negative breast cancers also exhibit hormonal responsiveness (11). The ER status of a breast tumor affects its intrinsic biologic character. For example, ER-positive tumors are more likely to be histologically well differentiated and diploid and to have a lower S-phase fraction (12). Consequently, the presence of a functional ER serves as a marker of differentiation and as a predictor of disease-free survival in patients with either lymph node-negative or lymph node-positive breast cancer (13,14).
As breast cancer progresses, molecular diversity in ER expressionalternative splicing, decreased transcription, or various mutations in the ER geneleads to resistance to antiestrogen therapy and to the broader problems of tumor progression and cellular heterogeneity, characteristics of advanced breast cancer (15). The hallmark of some hormone-resistant tumors is a reduction or the absence of ER expression, leading to a more aggressive metastatic phenotype (12,14,16).
In a cell with functional ERs, estrogen mediates the transition of cells from the G1 to S phase of the cell cycle; antiestrogens inhibit ER signaling and arrest cells in the G1 phase (17,18). Several studies (1922) have shown that cyclin D1, a G1 cyclin, can interact directly with the ligand-binding domain of ER and stimulate transactivation of ER in a ligand-independent and cyclin-dependent kinase (CDK)-independent fashion. However, the role of CDK inhibitors in inhibiting cell growth through the ER and the estrogen-signaling pathway has remained elusive. p21WAF1/CIP1 (hereafter referred to as p21) is a major negative regulator of the G1 checkpoint by binding to and inhibiting the activities of most cyclin/CDK complexes [reviewed in (2325)]. In addition, p21 and p27 can function as adaptor molecules that promote the association of CDK4 with D-type cyclins and that increase CDK4 kinase activity (2629). p21 may also be involved in G1-phase arrest mediated by antiestrogenic inhibition of ER. In fact, transfection of ER or p21 complementary DNAs (cDNAs) into breast cancer cells results in growth inhibition or apoptosis of the transfected cells (30,31). Because ER and p21 have growth-regulatory effects in normal and tumor cells, we have investigated whether p21 can act as a mediator of estrogenic actions in ER-negative breast cancer cells, sensitizing these cells to the growth inhibitory effects of antiestrogens.
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MATERIALS AND METHODS |
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17ß-Estradiol and MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were purchased from Sigma Chemical Co. (St. Louis, MO), ICI 182,780 was from Tocris Cookson Inc. (Ballwin, MO), charcoal-stripped, fetal bovine serum was from HyClone Laboratories, Inc. (Logan, UT), and cell culture medium was from Life Technologies, Inc. (GIBCO BRL) (Gaithersburg, MD). All of the other chemicals used were reagent grade. The culture conditions for normal 76N and 81N cells and breast cancer cell lines MCF-7, ZR75T, T47D, BT20, MDA-MB-157, Hs578T, MDA-MB-436, MDA-MB-231, and HBL100 were as described previously (32). All of the cells were cultured and treated at 37 °C in a humidified incubator in an atmosphere of 6.5% CO2/93.5% air and maintained free of Mycoplasma, as determined by Hoechst staining (33). Snap-frozen surgical specimens from patients diagnosed with breast cancer were obtained from the Quantitative Diagnostic Laboratories (Almhurst, IL). All of the tumor tissue samples were treatment naive because they were resected before any type of therapy was given. In addition, most of the ER-positive, p21-positive tumor samples had a well-differentiated phenotype. Tumor tissues were collected from discarded material with no patient identification. This study was approved by the Wadsworth Center (Albany, NY).
Plasmid Constructs
ER3500-230LUC, the ER promoter-reporter construct (ER-Luc; provided by Dr. Ronald J. Weigel, Stanford University, CA), contains a full-length ER promoter that controls expression of the Luciferase gene (34). The ERE2-tk-luciferase/SV-neo plasmid (herein termed ERE-tk-Luc) was provided by Drs. Nancy E. Davidson (The Johns Hopkins University School of Medicine, Baltimore, MD) (35) and Benita Katzenellenbogen (University of Illinois, Urbana) (36) and contains two EREs and the viral thymidine kinase (tk) promoter that drives expression of the Luciferase reporter gene (35). Expression of the reporter gene in this plasmid can be used to monitor 17ß-estradiol-induced transcription. The ER complementary DNA (cDNA) (HEGO [human wild-type estrogen receptor]), which contains the full-length ER, was provided by Dr. Myles Brown (Dana-Farber Cancer Institute, Boston, MA). The plasmid containing the p21 promoter-controlling expression of the Luciferase reporter gene (p21-Luc) was provided by Dr. Wafik el-Deiry (University of Pennsylvania School of Medicine, Philadelphia) (37). The pBSTR1 self-contained, one-plasmid, tetracycline-inducible vector where the transcription of the inserted gene is activated by the removal of tetracycline (i.e., a "Tet-off" system) was provided by Dr. Steven Reeves (Massachusetts General Hospital, Charleston) (38). The pBSTR-p21 vector was constructed by ligating a NotI-linked 559-base-pair fragment containing the coding region for p21 from the CIP-1 cDNA provided by Dr. Stephen Elledge (Baylor College of Medicine, Houston, TX) (39).
Transfection, Flow Cytometry, and Luciferase Assays
For stable transfections, 70% confluent MDA-MB-436 cultures, as described (40), were transfected by electroporation at 0.25 kV and 960 microfarad (µF). For transfection, 1 x 107 cells were suspended in 0.5 mL of serum-free -modified Eagle medium (MEM) containing 40 µg of plasmid (pBSTR1 or pBSTR1-p21) in a 0.4-cm gap cuvette. After transfection, cells were plated in complete medium (
-MEM medium containing 10% fetal bovine serum, epidermal growth factor at 25 µg/L, and insulin at 1 mg/L), allowed to attach and recover for 48 hours, and then cultured in selection medium containing tetracycline (1 µg/mL) and puromycin (0.5 µg/mL). Fresh medium was added every other day until the colonies were isolated (23 weeks). For each transfection, 25 stable clones and one pooled population were isolated. Inducible expression of p21 was monitored by western blot analysis (see below).
For transient transfections, 70% confluent cultures were transfected by electroporation at voltages ranging from 0.25 to 0.32 kV and a capacitance of 960 µF. For the determination of the transfection efficiency, cells were cotransfected with a vector containing a green fluorescent protein (GFP) cDNA (i.e., pEGFPC-1 [Clonetech Laboratories, Inc., Palo Alto, CA]). Briefly, 1 x 107 cells were suspended in 0.5 mL of serum-free medium containing 40 µg of plasmid (promoterLuciferase [i.e., ER-Luc, ERE-tk-Luc, or p21-Luc] plus pEGFPC-1) in a 0.4-cm gap cuvette, at a GFP vector to the promoterLuciferase vector ratio of 1 : 4. After electroporation, cells were plated with complete medium and harvested 48 hours after transfection for analysis.
Percent transfection efficiency by GFP expression was assayed two ways. First, we used immunohistochemistry with GFP to visualize and count the transfected cells and to determine the intensity of the transfection. With the use of fluorescent microscopy, cells expressing GFP were identified and counted in several fields (for accuracy), and the percentage of cells expressing GFP was calculated. Second, we subjected living cells to flow cytometry to determine transfection efficiency by measuring GFP fluorescence. Briefly, cells were centrifuged at 1000g for 10 minutes at 4 °C and suspended in phosphate-buffered saline (PBS). GFP expression was then measured on a Becton Dickinson FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). GFP has an excitation wavelength of 350 nm and an emission wavelength of 485 nm. Data were analyzed with the CellQuest program (Becton Dickinson Immunocytometry Systems); the intensity of GFP expression is presented on one axis and the number of cells expressing GFP is presented on the other axis from the scatter window. The percent efficiency is expressed as the percentage of cells with specific GFP fluorescence (greater than background fluorescence). The percent efficiencies calculated from the flow cytometry measurements were then compared with those values obtained from immunohistochemistry (i.e., enumeration of GFP-positive cells microscopically). In all cases, the measurements from both assays matched.
For some experiments, cells were transiently transfected by use of the GenePORTER transfection reagent because this method has a high (>60%) transfection efficiency in MDA-MB-436 cells. Briefly, 1 x 106 cells were plated in 100-mm dishes, and 40 µg of plasmid (promoterLuciferase plus pEGFPC-1), 30 µL of GenePORTER transfection reagent, and 5 mL of culture medium were added. Cells were harvested 24 hours after transfection for analysis.
For Luciferase assays, cells were washed in PBS, lysed in extraction buffer (100 mM potassium phosphate and 1 mM dithiothreitol [pH 8.0]), sonicated, and centrifuged at 100 000g for 45 minutes (all steps at 4 °C). The high-speed supernatant (50 µg) was then mixed with 500 µL of Luciferase buffer (1 mM dithiothreitol, 15 mM potassium phosphate, 25 mM Gly-Gly [pH 7.8], 4 mM ethylene glycol bis(ß-aminoethyl)-N,N,N`,N`-tetraacetic acid, and 15 mM MgSO4 [pH 7.8]). D-Luciferin (Sigma Chemical Co.) then was added at a saturating substrate concentration (0.2 mM in Luciferase buffer) after the supernatant was mixed with Luciferase buffer, and Luciferase activity was assayed in a LUMAT luminometer as described (41).
MTT Assay
Cell proliferation was measured with the MTT assay, as described previously (42). To ensure that the MTT assay measurements correspond to cell number and not just viable cell mass, we calibrated the MTT assay with enumeration of live cells after drug treatment. For each cell line used in this study (including MCF-7, MDA-MB-231, Hs578T, the MDA-MB-436 parental line, stable p21 clones derived from MDA-MB-436 cells, and mock-transfected [vector alone] MDA-MB-436 control lines), we initially measured cell proliferation by directly counting living cells and by performing the MTT assay side by side. In all cultures examined, the fold increase (or decrease) of cell numbers, as a result of treatment, was directly proportional (1 : 1) to the increase (or decrease) in the MTT dye reduction product. These results were reproduced in three different side-by-side determinations of the two assays with all different cell lines. On the basis of these results, we used the MTT assay for all cell proliferation studies described herein. To examine the growth-modulating effects of 17ß-estradiol or ICI 182,780, we counted exponentially growing cells with a Coulter Counter (Coulter Corp., Hialeah, FL) and plated 1500 cells per well in 96-well plates (in 200 µL of culture medium/well). Culture medium was estradiol-free Dulbecco's MEM (without phenol red) and was supplemented with fetal bovine serum that had been treated with 5% dextran-coated charcoal to bind steroid hormones (i.e., 5% dextran-coated, charcoal-stripped fetal bovine serum). After 24 hours, 17ß-estradiol or ICI 182,780 was added to cells as indicated, and fresh drug was added every other day for the duration of the experiment. At the end of each experiment, cell proliferation was measured with the MTT assay. Each data point represents the average of six determinations from at least three replicates of each experiment. Data are presented as the percent increase or decrease relative to data from untreated cells.
Cell and Tissue Extraction and Western Blot Analysis
Retinoblastoma protein (pRb), ER, p53, cyclin D1, cyclin D3, p21, and CDK2 were measured in cells and tissues by western blotting. Briefly, cell lysates from the indicated cell lines, transfected cells, and tissue homogenates (prepared from snap-frozen surgical specimens) were prepared and examined by western blot analysis, as described previously (28,43). All of the cell lines were harvested routinely when 70% confluent, and fresh medium was added to the cells 24 hours before harvesting. For each lysate, 50 µg of protein per lane was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. A 7% gel was used for pRb and ER; a 10% gel was used for p53, cyclin D1, and cyclin D3; and a 13% gel was used for p21, p27, and CDK2. All samples were transferred to an Immobilon P membrane overnight at 4 °C at a constant 35 mV. The blots were blocked overnight at 4 °C in Blotto (i.e., 5% nonfat dry milk in 20 mM TrisHCl, 137 mM NaCl, and 0.25% Tween 20 [pH 7.6]). After six 10-minute washes in TBST (i.e., 20 mM TrisHCl, 137 mM NaCl, and 0.05% Tween 20 [pH 7.6]), the blots were incubated with primary antibodies for 3 hours at room temperature. Primary antibodies were from the following sources: pRb monoclonal antibody from PharMingen (San Diego, CA), monoclonal antibodies to ER- from Novocastra-Vector Laboratories, Burlingame, CA [clone 6F11, ER-
(N)] and Santa Cruz (Santa Cruz Biochemicals, Santa Cruz, CA) [clone C-311, ER-
(SC)] (see below for antibody characterization), monoclonal antibodies to p27, cyclin D1, cyclin D3, and CDK2 from Transduction Laboratories (Lexington, KY), and monoclonal antibodies to p21 and p53 from Oncogene Research Products/Calbiochem (San Diego, CA). All of these antibodies were used at 0.1 µg/mL in Blotto. The primary actin monoclonal antibody was from Boehringer-Mannheim Biochemicals (Indianapolis, IN) and was used at 0.63 µg/mL in Blotto. After incubation with the primary antibody, the blots were washed and incubated with a goat anti-mouse antibodyhorseradish peroxidase conjugate (Pierce Chemical Co., Rockford, IL) at a dilution of 1 : 5000 in Blotto for 1 hour, washed, and developed with the Renaissance chemiluminesence system, as directed by the manufacturer (Du Pont NEN, Boston, MA).
ER Antibodies and Antigen Competition Assays
The antigen used to generate the ER-(N) mouse monoclonal antibody (immunoglobulin G [IgG]1; clone 6F11) is a recombinant protein corresponding to the full-length ER molecule. Although detailed epitope mapping has not yet been done with this antibody, it has been shown that ER-
(N) recognizes full-length ER-
as well as a truncated ER-
containing the first 184 amino-terminal amino acids of ER-
(Novocastra-Vector Laboratories, personal communications). The antigen used to generate the ER-
(SC) mouse monoclonal antibody (IgG2a) is a recombinant protein corresponding to amino acids 495595, located at the carboxyl terminus of ER-
.
For competition assays, we used the full-length recombinant ER- and western blots (similar to those presented in Fig. 3 , B
) and limiting amounts of ER-
(N) or ER-
(SC) antibody. After we determined the lowest concentrations of antibody that could detect the ER-related bands, we performed antibody competition assays. For these assays, new western blots were prepared (again similar to those presented in Fig. 3, B
) and were incubated with the limiting concentration of antibody and increasing amounts of full-length recombinant ER-
.
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All P values are from two-sided tests. All error bars presented are 95% confidence intervals of the population mean.
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RESULTS |
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In our initial analysis of the expression of CDK inhibitor in normal and tumor cells, we found a curious relationship between the presence of CDK inhibitors p21 and p27 and the ER status of breast tumor cell lines. We first examined the expression of p21 and p27 in nine breast cancer cell lines by western blot analysis and found that the four ER-positive breast cancer cell lines expressed both p21 and p27 at much higher levels than did the five ER-negative lines (Fig. 1, A). We then examined the expression of p21 and p27 in extracts from 60 breast cancer samples, 30 that were ER positive and 30 that were ER negative (Fig. 1, B
). We used western blot analysis to detect p21, p27, ER, and actin (as a loading control) and found that 25 of 30 ER-positive samples were also positive for p21 and that 27 of 30 ER-negative samples were also negative for p21 (P<.001; Fig. 1, B
). These results suggest a strong association between p21 and ER, but the association between p27 and ER was not as strong. In fact, most of the ER-negative tumor tissues expressed a moderate to high level of p27 (Fig. 1, B
). Consequently, p27 may not have a direct effect on the ER-signaling pathway, and so we concentrated our efforts on p21 and ER.
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The strong association between the expression of ER and p21, shown in Fig. 1, suggested that there could be "cross-talk" between these two proteins. Because ER is a transcription factor, we hypothesized that it might activate the expression of p21 either directly or indirectly. To test this hypothesis, we transiently transfected ER-negative, p21-negative MDA-MB-436 cells [Fig. 1
and (44)] with a full-length ER cDNA (HEGO) (Fig. 2
), assessed the expression and transcriptional activity of ER, and determined whether expression of the ER induced the expression of p21. For analysis of ER function in transactivation analysis, we cotransfected cells with an ER cDNA (HEGO) and a construct containing two EREs and a thymidine kinase promoter driving the expression of a Luciferase reporter gene (ERE-tk-Luc) (35). Although we observed that transient transfection of MDA-MB-436 cells with ER cDNA led to the expression of a transcriptionally active ER protein (Fig. 2, A
), as measured by the ability of ER to activate ERE-tk-Luc (Fig. 2, B
), we did not detect p21 expression. We also observed that expression of ER did not alter the expression of other cell cycle regulatory proteins (such as p27, CDK2, CDK4, cyclin D1, cyclin D3, pRb, or p53) (Fig. 2, A
).
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p21-Induced Expression of ER and ER-Related Proteins
We next asked whether p21 could induce the expression of ER and activate the estrogen-signaling pathway. Because ectopic expression of p21 could arrest cell cycle progression, we used an inducible system under the control of tetracycline-responsive promoters to express p21 (46). After transfection of MDA-MB-436 cells with pBSTR1-p21, a vector containing p21 under the control of a tetracycline-inducible promoter in one plasmid (38), 25 stable p21 clones were selected with puromycin, isolated, propagated, and analyzed by western blotting for p21 expression 72 hours after the removal of tetracycline (Fig. 3, A). Western blot analysis of five representative p21-positive clones (of 15 positive clones) and two p21-negative clones (of 10 negative clones) showed that p21 can be induced at very high levels in these clones (Fig. 3, A
). All 25 clones were examined, and the results shown in Fig. 3
, A, are consistent with the results from other clones (data not shown).
The expression of ER- RNA and protein was also examined in parental MDA-MB-436 cells and stable p21 clones cultured in the presence or absence of 17ß-estradiol. Neither northern blot analyses nor reverse transcription-coupledpolymerase chain reactions detected appreciable expression of the ER-
message RNA or any alternatively spliced ER-
transcript in stable p21 clones (data not shown). We used two ER antibodies with different epitope specificities to detect ER protein (Fig. 3, B
). Overexpression of p21 in the MDA-MB-436/p21 clones did not result in the expression of the wild-type 67-kd ER-
, which was detected in the MCF-7 cells by both antibodies. However, an 87-kd band that mapped to the amino terminus of ER was detected by the ER-
(N) antibody, and a 62-kd band that mapped to the carboxyl terminus of ER was detected by the ER-
(SC) antibody. Levels of these proteins were significantly higher in the stable p21 clones than in MCF-7 cells, and both proteins were induced by 17ß-estradiol in MCF-7 cells and in stable p21 clones (Fig. 3, B
). In these cells, levels of p21 and p27 were not affected by the presence or absence of 17ß-estradiol. MDA-MB-436 breast cancer cells, 81N normal breast epithelial cells, and mock-transfected cells were the negative controls; these cells do not express the wild-type ER-
, the 87-kd ER-
-related protein, or the 62-kd ER-
-related protein (Fig. 3, B
). Antigen competition studies showed that the full-length ER-
recombinant protein could specifically compete with wild-type ER-
in MCF-7 cells and the two ER-related proteins detected in the stable p21 clones in a dose-dependent fashion (data not shown). These results suggest that the antibody-reactive bands are, in fact, ER related and the antibody binding is specific.
Estrogen-Inducible Transactivation of the ER Promoter by p21
Because the p21 expression induced only the expression of ER--related proteins and not the 67-kd ER-
wild-type protein in stable p21 clones, we investigated whether ER transactivation was active in these cells. First, we transfected stable p21 clones and MDA-MB-436 parental cells with ER-Luc, a plasmid containing the ER promoter that controls the expression of the Luciferase reporter gene (34,38), or the vector alone (i.e., Mock) (Fig. 4, A
), and then we assessed whether p21 could modulate ER transactivation by measuring Luciferase activity in the presence or absence of 17ß-estradiol (Fig. 4, B
). We used 17ß-estradiol because ER can autoregulate its own expression in an estrogen-dependent pathway (47). p21 induces the ER promoter, as shown by the induction of Luciferase activity: 10-fold to 40-fold in the absence of 17ß-estradiol, and addition of 17ß-estradiol further increases Luciferase activity by 3.2-fold to 7.5-fold (Fig. 4
). The activity of ER-Luc in ER-Luc-transfected p21 clones 7 and 12 is up to 40-fold higher than the activity in the mock-transfected p21 clone 16 or the parental cells in the absence of 17ß-estradiol (Fig. 4, A
). The ER transcriptional activity in different clones depends on the amount of p21 expressed in each clone. For example, p21 clone 12 expresses four times more p21 than clone 7 (data not shown), and the basal transcriptional activity of ER is four times higher in clone 12 than it is in clone 7 (Fig. 4, A
). These results were our first indication that overexpression of p21 may have activated an estrogenic response in otherwise ER-
-negative breast cancer cells.
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Our finding of the estrogen-inducible transactivation of the ER promoter in the p21 clones (Fig. 4) prompted us to investigate whether ER-responsive elements (i.e., ERE sequences) transfected into these clones could be induced in an estrogen-responsive manner (35). We transiently transfected MCF-7 cells and stable p21 clones with the ERE-tk-Luc plasmid in the presence or absence of 17ß-estradiol and observed that ERE-tk-Luc (i.e., Luciferase activity) was induced by 17ß-estradiol in both cell types (Fig. 5, A
). However, when ERE-tk-Luc was inserted into mock-transfected clone 16 or p21-negative parental MDA-MB-436 cells in the presence or absence of 17ß-estradiol, Luciferase activity remained at basal or undetectable levels, indicating that ERE-tk-Luc was not induced by 17ß-estradiol. Compared with ERE-tk-Luc-transfected mock-transfected cells or parental cells, Luciferase activity in ERE-tk-Luc-transfected stable p21 clones was induced more than 100-fold (Fig. 5, A
), and 17ß-estradiol further stimulated Luciferase activity by threefold and sixfold in ERE-tk-Luc-transfected p21 clones 12 and 25, respectively (Fig. 5, B
). To further examine the difference in 17ß-estradiol sensitivity between MCF-7 cells and stable p21 clone 25, we transfected both cell lines with the ERE-tk-Luc plasmid and then incubated them with increasing concentrations of 17ß-estradiol (0.1500 nM). The doseresponse relationship of Luciferase activity to 17ß-estradiol in MCF-7 cells and p21 clone 25 showed that, in both cell types, the ERE was induced in a similar 17ß-estradiol-dependent manner (Fig. 5, C
). Last, we treated cells with the potent antiestrogen ICI 182,780 to determine whether this drug would inhibit the estrogen-sensitive ERE-tk-Luc activity. We found that, when cells were treated with both 17ß-estradiol and the ICI 182,780, 17ß-estradiol-stimulated ERE-tk-Luc activity was reduced in the p21 clones to approximately basal level, and it was reduced in ER-positive MCF-7 cells to slightly below basal level (Fig. 5, A and B
). Thus, the estrogen-signaling pathway appears to be functional in the p21 clones. The expression of p21 in MDA-MB-436 cells activates ERE transactivation through both 17ß-estradiol-independent (i.e., high basal activity of ERE-tk-Luc) and estradiol-dependent (i.e., ERE-tk-Luc activity induced by 17ß-estradiol) pathways: The 17ß-estradiol-dependent response in both MCF-7 cells and stable p21 clones is reduced by antiestrogen ICI 182,780.
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We next investigated whether the growth of p21-overexpressing ER-negative cells was more sensitive to 17 ß-estradiol and antiestrogens than the growth of the p21-negative, ER-negative parental MDA-MB-436 cells. We treated all cells with 0125 nM 17ß-estradiol, as indicated in Fig. 6, A. Proliferation of ER-positive MCF-7 cells was very responsive to 17ß-estradiol, showing a fairly consistent 75% increase [by the MTT assay (29)] at concentrations of 17ß-estradiol greater than 5 nM compared with cells in 17ß-estradiol-free culture medium. Stable p21 clones derived from MDA-MB-436 cells, although not as sensitive as MCF-7 cells to 17ß-estradiol, responded quite dramatically; 17ß-estradiol increased their rate of proliferation by 25%40%. 17ß-Estradiol did not stimulate the growth of MDA-MB-436 parental or mock-transfected cells.
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To determine whether the overexpression of p21 rendered the ER-negative, stable p21 clones sensitive to the antiestrogen ICI 182,780, we cultured these cells in 080 nM ICI 182,780 (Fig. 7). As controls, MCF-7, parental MDA-MB-436 cells, and mock-transfected clone cells were cultured under similar conditions. The growth of naturally ER-positive MCF-7 cells was inhibited 34% at 20 nM ICI 182,780 compared with untreated cells. The MDA-MB-436 parental cell line or a mock-transfected clone, both of which are ER negative and unresponsive to the transactivation effects of estrogen, showed little or no response to ICI 182,780 at concentrations up to 80 nM. However, the growth of stable p21 clones 9 and 12 was inhibited 15%28% at 80 nM ICI 182,780 (Fig. 7
). The response of the stable p21 clones was not as strong as that of MCF-7 cells; however, when higher concentrations of drug were used, the growth of the p21 clones was inhibited similarly (data not shown). These data also corroborate the results of our ERE-transactivation studies, suggesting that overexpression of p21 in ER-negative breast cancer cells can activate the estrogen-signaling pathway in these cells.
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DISCUSSION |
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The p21-mediated activation of the estrogen-signaling pathway occurs in the absence of wild-type ER-, as demonstrated by western blot analysis. However, two proteins of 87 and 62 kd detected by ER antibodies are expressed in the p21 clones. The expression of these ER-related proteins is induced by 17ß-estradiol in stable p21 clones and MCF-7 cells. Although the wild-type ER-
is not detected in stable p21 clones, 17ß-estradiol can activate the estrogenic response pathway and transactivate ERE activities, and the antiestrogen ICI 182,780 can inhibit these activities (Fig. 5
). Furthermore, proliferation of stable p21 clones is stimulated by 17ß-estradiol (Fig. 6
) and inhibited by the antiestrogen ICI 182,780 (Fig. 7
). In addition, in stable p21 clones, the ER-promoter activity is transactivated by 17ß-estradiol (Fig. 4
). Collectively, these observations suggest that the 87- and 62-kd ER-related proteins may be variants of ER-
that can activate the estrogen-signaling pathway but with lower potency than wild-type ER-
. This is a plausible hypothesis because, if the expression of the wild-type ER-
were induced by p21 to a level or activity similar to that in MCF-7 cells, it could result in a 17ß-estradiol-induced inhibition of cell proliferation (see Fig. 6, B
). In fact, one of the distinct characteristics of cells transfected with the ER cDNA is the estrogen-mediated inhibition of growth (30). Although the precise mechanism of this paradoxical response to estradiol is unknown, it is conjectured that overexpression of ER beyond a certain level alters the gene expression machinery of the cell by either turning on ER-dependent unknown lethal genes or switching off of important housekeeping genes (30). We also observed this phenomenon when we transiently transfected ER cDNA into three different ER-negative breast cancer cell lines. Treatment of these ER-overexpressing cells with 17ß-estradiol inhibited cell proliferation (Fig. 6, B
).
Our emerging hypothesis from these studies is that overexpression of p21 leads to the expression of ER-related proteins that can activate the estrogen-signaling pathway. However, because the activity of these ER-related proteins is lower than that of wild-type ER-, p21-overexpressing cells and ER-overexpressing cells exhibit different responses. Overexpression of p21 mediates an "MCF-7-like" estrogenic phenotype in which cell proliferation is stimulated by estrogen and inhibited by antiestrogens.
The novel role of p21 in mediating an ER-like response in otherwise ER-negative breast cancer cells is another example of the pleiotropic functions of p21 in cell growth and differentiation. p21 has multiple roles in regulating cell growth through activation or inhibition of CDKs [reviewed in (2325)], mediating differentiation (4850), and inhibiting apoptosis (51,52). A prerequisite for terminal differentiation is that cells exit the cell cycle, and p21 expression is induced during terminal differentiation in vitro (4850) and in vivo (53,54). For example, the ectopic expression of p21 promotes differentiation of the megakaryoblastic leukemia cell line CMK (55) and the myelomonocytic cell line U937 (56), and disruption of p21 in normal diploid fibroblasts results in an extended in vitro life span and a bypass of senescence (57). Herein we show another role for p21 in modulating the estrogen-signaling pathway in the absence of wild-type 67-kd ER-. Our results suggest that p21 can uncouple the ER from its downstream activation of the estrogen-signaling pathway. The mechanism of this p21-induced activation of the estrogen-signaling pathway is currently unknown; however, it is apparent that overexpression of p21 in an otherwise ER-negative cell line can confer an MCF-7-like, well-differentiated phenotype that is responsive to antiestrogens. Although the effect of p21 on the estrogen-signaling pathway may represent a novel function for p21, it is also possible that the putative general CDK-inhibitory properties of p21 (also shared by p27 and p57) may have a role in modulating the estrogen-signaling pathway.
The p21-mediated activation of the estrogen-signaling pathway in ER-negative tumor cells has important clinical implications. A number of agents currently used for the treatment of breast cancer result in the induction of p21. For example, paclitaxel (Taxol), a potent and highly effective antineoplastic agent for the treatment of advanced, drug-refractory, metastatic breast cancers, results in the induction of p21 in the ER-negative MDA-MB-435 cell line (58). Similarly, treatment of MCF-7 cells with paclitaxel (59), with doxorubicin in the presence or absence of neu differentiation factor (60), or with vinorelbine (a vinca alkaloid analogue) in combination with a progesterone analogue (61) results in the induction of p21. Last, treatment of a number of breast cancer cell lines and primary cultures from 20 different invasive ductal carcinoma of the breast with doxorubicin resulted in a substantial increase in the level of p21 (62). Similar to chemotherapeutic agents, several differentiation-inducing agents also result in the induction of p21. For example, p21 is induced in HL60 leukemia cells by agents, such as vitamin D3 (63), 12-O-tetradecanoylphorbol 13-acetate, retinoic acid, dimethyl sulfoxide (50,64), and butyrate (50). p21 is induced in melanoma cells when cells are terminally differentiated with recombinant human fibroblast interferon and mezerein (65). p21 is induced in leukemia cells by okadaic acid (66). Consequently, it follows that chemotherapy of ER-negative breast cancer may activate the estrogen-signaling pathway. The clinical translation of this hypothesis lies in the potential of p21-positive, ER-negative breast cancers to respond to antiestrogens. These results suggest a treatment strategy that combines chemotherapy and antihormonal therapy for ER-negative cancers that express p21. Because chemotherapeutic agents can induce p21 and p21 can activate the estrogen pathway, such a combination therapy could benefit patients with ER-negative breast cancer that do not respond to antiestrogens. Induction of the estrogen-signaling pathway in otherwise ER-negative tumors by increased levels of p21 could ultimately promote tumor cell differentiation and result in tumor regression and improved survival rates. This treatment strategy could also benefit the 10% of the patients with ER-negative, progesterone receptor-negative breast cancers that have high basal levels of p21.
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NOTES |
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We thank Drs. Stephen Elledge, Steven Reeves, Ronald J. Weigel, Nancy Davidson, Benita Katzenellenbogen, Myles Brown, and Wafik el-Deiry for reagents. We also acknowledge the use of the Wadsworth Center's Molecular Immunology, Tissue Culture, Molecular and Photography/Graphics core facilities.
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Manuscript received November 29, 1999; revised June 6, 2000; accepted June 20, 2000.
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