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Originally published In Press as doi:10.1074/jbc.M502278200 on March 8, 2005

J. Biol. Chem., Vol. 280, Issue 18, 17617-17625, May 6, 2005
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c-Myc Suppresses p21WAF1/CIP1 Expression during Estrogen Signaling and Antiestrogen Resistance in Human Breast Cancer Cells*

Shibani Mukherjee and Susan E. Conrad{ddagger}

From the Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824

Received for publication, March 1, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen rapidly induces expression of the proto-oncogene c-myc. c-Myc is required for estrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc expression has been implicated in antiestrogen resistance. In this report, we investigate the mechanism(s) by which c-Myc mediates estrogen-stimulated proliferation and contributes to cell cycle progression in the presence of antiestrogen. The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Using stable MCF-7 derivatives with inducible c-Myc expression, we demonstrated that in antiestrogen-treated cells, the elevated mRNA and protein levels of p21WAF1/CIP1, a cell cycle inhibitor, decreased upon either c-Myc induction or estrogen treatment. Expression of p21 blocked c-Myc-mediated cell cycle progression in the presence of antiestrogen, suggesting that the decrease in p21 is necessary for this process. Using RNA interference to suppress c-Myc expression, we further established that c-Myc is required for estrogen-mediated decreases in p21WAF1/CIP1. Finally, we observed that neither c-Myc nor p21WAF1/CIP1 is regulated by estrogen or antiestrogen in an antiestrogen-resistant MCF-7 derivative. The p21 levels in the antiestrogen-resistant cells increased when c-Myc expression was suppressed, suggesting that loss of p21 regulation was a consequence of constitutive c-Myc expression. Together, these studies implicate p21WAF1/CIP1 as an important target of c-Myc in breast cancer cells and provide a link between estrogen, c-Myc, and the cell cycle machinery. They further suggest that aberrant c-Myc expression, which is frequently observed in human breast cancers, can contribute to antiestrogen resistance by altering p21WAF1/CIP1 regulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A majority of estrogen receptor (ER)1-positive breast tumors require estrogenic steroids such as 17{beta}-estradiol (E2) for proliferation and can be treated with antiestrogens that antagonize the actions of E2 and inhibit tumor growth (1–3). However, many patients with initially responsive tumors experience recurrence, indicating the development of acquired antiestrogen resistance (2–4). Understanding the mechanisms by which E2 stimulates proliferation would provide insight into how antiestrogen resistance might arise, and suggest strategies to prevent or reverse its development.

E2 mediates passage from G1 to S phase by activating the ER, which functions as a transcription factor and induces the expression of gene products required for cell cycle progression (5, 6). Antiestrogens cause a G0/G1 arrest by binding the ER, thus preventing the activation of genes by E2 (7, 8). Cyclin-dependent kinases (CDKs) control the G1/S transition (9–11), and CDK activity is regulated by multiple mechanisms including phosphorylation, activation by binding of cyclins (cyclin D1-CDK4 or cyclin E/A-CDK2), and inhibition by binding of CDK inhibitors such as p21WAF1/CIP1 (p21) and p27KIP1 (p27) (12–14). Various proteins involved in CDK regulation have been identified as potential mediators of E2-induced mitogenesis (15–18), and deregulated expression of these key targets may enable cells to proliferate in the absence of E2 or the presence of antiestrogens (18–24).

One important target of E2 in breast cancer cells is the proto-oncogene c-myc (25, 26). Chromatin immunoprecipitation assays have demonstrated that E2 treatment of responsive breast cancer cells leads to ER binding and co-activator recruitment at the c-myc promoter, resulting in the rapid induction of c-Myc (27). In addition, transient transfections using c-myc promoter-reporter gene constructs have mapped the responsive region to within 116 bp upstream of the P2 promoter (28). The c-Myc protein is a transcriptional regulator, and c-myc antisense oligonucleotides inhibit E2-stimulated proliferation (29, 30). Moreover, ectopic expression of c-Myc is sufficient to induce proliferation of E2-dependent breast cancer cells in the presence of the antiestrogen ICI 182,780 (ICI; Faslodex) (19, 31). Thus, c-Myc plays a crucial role in mediating E2-regulated proliferation and may contribute to antiestrogen resistance.

Among multiple targets of c-Myc identified in diverse cellular systems, several are key cell cycle regulators (29). Specifically, c-Myc has been reported to increase expression of positive cell cycle regulators, including cyclin E and CDK4 (32, 33), and to decrease expression of CDK inhibitors, such as p21 and p27 (34–39). c-Myc could therefore be mediating its proliferative effects in breast cancer cells by regulating the levels of some or all of these proteins and their distribution among cyclin-CDK complexes. However, the target(s) through which c-Myc mediates E2-stimulated proliferation of breast cancer cells and can contribute to proliferation in the presence of antiestrogen have not been identified. In this study, we sought to identify these target(s) using the MCF-7 cell line, a well characterized model of estrogen-dependent and antiestrogen-sensitive human breast cancer (40, 41).

To conduct these experiments, we established stably transfected MCF-7 derivatives in which ectopic c-Myc expression could be induced and confirmed that c-Myc induction promoted cell cycle progression in the presence of ICI. We then examined several cell cycle proteins reported to be targets of c-Myc and established that c-Myc induction in ICI-treated cells decreased expression of the CDK inhibitor p21 to levels seen in E2-treated cells. We further showed that this decrease was a consequence of down-regulated p21 mRNA levels and that c-Myc could repress p21 promoter activity. Expression of p21 from an adenoviral vector blocked c-Myc-mediated cell cycle progression of ICI-treated cells, suggesting that the decrease in p21 was important for this process. It has been reported that E2 treatment of MCF-7 cells leads to decreased p21 expression (18, 22). Using RNA interference to knock down c-Myc expression, we have determined that c-Myc is required for the E2-mediated decrease in p21, thus providing a link between E2, c-Myc, and the cell cycle machinery. A previous study demonstrated that decreasing p21 levels with antisense oligonucleotides is sufficient to cause cell cycle progression of ICI-treated cells (22). Based on this finding and our current results, we propose that a key mechanism by which c-Myc promotes proliferation of breast cancer cells in the presence of ICI is by decreasing p21 levels. To determine whether deregulation of c-Myc is associated with acquired antiestrogen resistance, we examined LCC9, an MCF-7 derivative selected for both E2 independence and ICI resistance (42). We observed that neither c-Myc nor p21 expression was affected by E2 or ICI treatment of LCC9 cells. However, p21 levels in LCC9 cells increased when c-Myc expression was suppressed using RNA interference. This indicated that the loss of p21 regulation in LCC9 cells is likely to be a consequence of constitutive c-Myc expression. These results provide further evidence that aberrant regulation of c-Myc and/or p21 could play a role in the progression of tumors to an antiestrogen-resistant phenotype.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—MCF-7 and LCC9 cells were obtained from the Lombardi Cancer Center. Cells were maintained in improved modified Eagle's medium (BioFluids, Inc.) supplemented with 5% fetal bovine serum (FBS) (HyClone), 100 units/ml penicillin (Invitrogen), and 100 units/ml streptomycin (Invitrogen) and cultured at 37 °C with 5% CO2. When studying the effects of E2 and ICI, cells were cultured in E2-free medium, which is phenol red-free improved modified Eagle's medium (BioFluids, Inc.) containing 5% charcoal-stripped fetal bovine serum (CSS) (HyClone) and penicillin/streptomycin. For most experiments, cells were plated in medium with FBS for 24 h and prearrested for 48 h in CSS-containing medium with 10 nM ICI (Zeneca Pharmaceuticals). Cells were then treated with CSS-containing medium with either 10 nM E2 (Sigma) or 10 nM ICI and harvested at various time points. For some experiments, cells were not prearrested with ICI in order to improve transfection efficiency or to facilitate comparisons between MCF-7 cells and ICI-resistant LCC9 cells. In these experiments, cycling cells were directly treated with medium containing either 100 nM ICI or 1 nM E2 and then harvested. The higher ICI concentration was used to observe rapid arrest in cycling cells, and 1 nM E2 was used, since cells were not being released from an ICI arrest.

Plasmids—The pCEN-F3p65/Z1F3/Neo and pLH-Z12-I-pL plasmids were obtained from Ariad Pharmaceuticals (43). The pJ5 plasmid, containing human c-myc cDNA (exons 2 and 3), was a gift from Dr. S. Mai (44). The c-myc fragment was excised from pJ5 using HindIII and BglII, subcloned into pLH-Z12-I-pL at the HindIII and EcoRI sites to generate pLH-Z12-I-pL-Myc, and the orientation of the insert was confirmed by restriction digestion and sequencing. The c-myc cDNA fragment obtained from pJ5 was also used as a probe for Northern blot hybridizations. The p21 cDNA probe used for Northern blotting was excised from the pCEP-WAF1-S plasmid and was obtained from Dr. L. K. Olson (45). The pSP271 vector and the pSP271-Myc expression plasmid were a gift from Dr. R. N. Eisenman (46). The –194 p21 promoter-luciferase plasmid was obtained from Dr. H. R. Kim with permission from Dr. X. F. Wang (47).

Construction of MCF-7-inducible Cell Lines—MCF-7/Myc-stable transfectants were generated in two steps from MCF-7 cells using the ArgentTM Regulated Transcription Plasmid Kit (Version 1) provided by Ariad Pharmaceuticals. The system is based on small molecule-regulated protein dimerization and consists of two plasmids and a small molecule dimerizer AP1510 (AP) (43). The pCEN-F3p65/Z1F3/Neo plasmid encodes two fusion proteins, one containing a DNA-binding domain and the other a transcriptional activation domain. Each domain is fused to the FKBP protein that interacts with AP. The two proteins interact only in the presence of AP to form an active transcription factor. The pLH-Z12-I-pL vector is used to clone the gene to be regulated and has a promoter that contains DNA elements that are recognized by the DNA binding domain encoded by pCEN-F3p65/Z1F3/Neo. MCF-7 cells were first stably transfected with pCEN-F3p65/Z1F3/Neo using Lipofectin reagent (Invitrogen) and then transfected with pLH-Z12-I-pL-Myc. Transfections with the pLH-Z12-I-pL vector alone were also carried out to obtain vector control cell lines. Stable derivatives were selected using hygromycin (Invitrogen) at 35 µg/ml and were subsequently maintained in 10 µg/ml hygromycin. Individual colonies were picked and cultured separately. The transfectants were screened by both Northern and Western blot analyses for induction of c-myc mRNA and protein upon treatment with AP. Selected clones were thereafter maintained in medium containing Geneticin (50 µg/ml) and hygromycin (10 µg/ml).

Western Blotting—Cells were lysed as described previously (48), and protein in the cell lysates was quantitated using the Bradford protein assay (Bio-Rad). Proteins (10–50 µg) were resolved by SDS-PAGE (7.5% for retinoblastoma protein (pRB) hyperphosphorylation and 12% for all other proteins analyzed), transferred to polyvinylidene difluoride membranes (PerkinElmer Life Sciences), and probed with primary antibodies for c-Myc (clone 9E10; ATCC) (49), cyclin A (clone BF683; Pharmingen), pRB (clone G3–245; Pharmingen), cyclin D1 (UBI06137 Upstate Biotechnology, Inc., Lake Placid, NY), cyclin E (clone HE12; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p21WAF1/CIP1 (p21-C-19; Santa Cruz Biotechnology), p27KIP1 (p27-C-19; Santa Cruz Biotechnology), CDK4 (clone H22; Santa Cruz Biotechnology), or {beta}-actin (clone AC-40; Sigma). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (Bio-Rad) or goat anti-mouse (American Qualex) secondary antibodies. Bands were visualized using Super Signal West Pico chemiluminescent substrate (Pierce).

Infections with Recombinant Adenoviruses—A recombinant adenovirus containing the cDNA encoding human p21 (Ad-p21) and a control virus encoding {beta}-galactosidase (Ad-{beta}-gal) were obtained from Dr. L. K. Olson (45). Cells were plated at 106 cells/100-mm dish in medium with FBS for 24 h and then treated with CSS-containing medium with 10 nM ICI. After 24 h, this medium was removed and saved. The Ad-p21 or Ad-{beta}-gal viruses were diluted in CSS containing medium, and cells were infected with 5 plaque-forming units/cell in a total volume of 1.0 ml. Infections were carried out at 37 °C for 2 h. Following infection, the medium was replaced with the original medium containing 10 nM ICI. The cells were prearrested for another 24 h and then treated with 10 nM ICI and 300 nM AP. After 24 h, the cells were harvested and subjected to cell cycle analysis and Western blotting.

Northern Blotting—Cells were lysed, and total RNA was purified using Trizol reagent (Invitrogen). Ten micrograms of RNA were electrophoresed on 1% formaldehyde-agarose gels, transferred to nitrocellulose membranes (Schleicher and Schuell), and UV-cross-linked. The blots were then hybridized with a 32P-labeled cDNA probe for c-myc or p21. The membranes were stripped and reprobed for GAPDH as a loading control. PhosphorImager scanning (Amersham Biosciences) was used to quantitate the bands obtained, and the c-myc or p21 mRNA levels in each sample were normalized to GAPDH.

Luciferase Assays—MCF-7 cells were plated at 5 x 105 cells/60-mm dish. After 24 h, cells were transfected using Lipofectin reagent (Invitrogen). One µg of pSP271-Myc or pSP271 vector was co-transfected with 0.25 µg of either –194 p21 promoter-luciferase plasmid or pGL2Basic vector. All transfections also included 0.1 µg of pCMV-{beta}-galactosidase, which served as a control for transfection efficiency. Transfections were carried out for 5 h in serum-free medium, followed by incubation overnight in complete medium containing FBS. Cells were then treated with CSS containing medium with 100 nM ICI and harvested after 24 h. Both luciferase and {beta}-galactosidase activities were measured using the protocol provided by the manufacturer (Promega, Clontech) on a Turner TD 20E luminometer (Turner Designs). Each transfection was done in triplicate, and the luciferase activity was normalized to {beta}-galactosidase activity in each sample.

Cell Cycle Analysis—Cells were fixed, stained with propidium iodide, and analyzed with a FACSVantage flow cytometer as previously described (51). Cell cycle distribution was determined using ModFit LT software.



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  		FIG. 1.
Induction of ectopic c-Myc and cell cycle distribution in ICI-treated MCF-7 cells. Stable MCF-7 derivatives in which ectopic c-Myc expression could be tightly regulated were established as described under "Experimental Procedures." The indicated stable transfectants were prearrested with medium containing CSS with ICI (10 nM) for 48 h and then treated with medium containing CSS with ICI (10 nM), ICI + AP (300 nM), or E2 (10 nM), and harvested after 24 h. A, cell lysates were subjected to Western blotting for c-Myc. Actin served as a loading control. B, cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The average percentage of cells in S phase for each treatment is shown ± S.D. Gray bars, ICI-treated cells. Black bars, ICI + AP-treated cells. White bars, E2-treated cells.

 
Gene Silencing with Small Interfering RNAs (siRNAs)—MCF-7 or LCC9 cells were cultured in 6-well plates containing 3 ml of medium with FBS. After 24 h, cells were transfected with 100 nM of c-myc or negative control siRNA duplexes (Ambion), using siPORTTM Lipid Transfection Agent (Ambion), according to the manufacturer's recommendations. Transfections were carried out for 4 h in serum-free medium, followed by treatment with CSS-containing medium with either 1 nM E2 or 100 nM ICI. Cells were harvested after 48 h, and the cell lysates were analyzed by Western blotting. Cells maintained in serum-free medium for 4 h and harvested after treatment with 100 nM ICI or 1 nM E2 for 48 h served as untransfected controls.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Cell Lines with Inducible c-Myc Expression—c-Myc expression induces cell cycle progression in the presence of ICI (19, 31). To examine the target(s) involved, stable MCF-7 derivatives, in which expression of ectopic c-Myc could be induced by the small molecule dimerizer AP, were established as described under "Experimental Procedures." Both c-Myc expression and cell cycle progression were characterized in several independently derived transfectants. Cells were pretreated with ICI for 48 h to block ER activity and cell proliferation. They were then treated with medium containing ICI, ICI + AP, or E2 for 24 h, harvested, and analyzed by flow cytometry and Western blotting. Northern blotting was also carried out to confirm induction of c-myc mRNA upon treatment with ICI + AP (data not shown).

c-Myc protein levels were low in the presence of ICI and increased upon E2 treatment in all transfectants (Fig. 1A), indicating that endogenous c-Myc was regulated as expected. Expression of ectopic c-Myc was tightly regulated and could be induced by AP in the presence of ICI to levels comparable with those seen in E2-treated cells. AP treatment did not induce c-Myc expression in a vector control cell line, demonstrating that treatment with AP does not induce endogenous c-Myc.

Cell cycle distribution was examined by flow cytometry, and the percentage of cells in S phase after 24 h of treatment is shown in Fig. 1B. In each transfectant, the percentage of cells in S phase was higher after E2 than ICI treatment. Upon treatment with ICI + AP, the percentage of cells in S phase was higher than in ICI but lower than in E2 treatments. This increase in the percentage of cells in S phase was specific to c-Myc expression, since it was not seen in vector control cells treated with ICI + AP. Thus, c-Myc expression alone is sufficient to promote cell cycle progression in ICI-treated transfectants but not as well as E2 in most cases. The cell cycle distribution differences between the various transfected cell lines might be due to the fact that only one time point was examined, and the clonal cell lines may have progressed through the cell cycle at somewhat different rates. The MCF-7/Myc33 and MCF-7/Myc31 cell lines were selected for further study, since they showed tight regulation of c-Myc and significant increases in S phase in response to AP treatment.

Induction of c-Myc in the Presence of ICI Decreases p21 Protein Levels and Causes Cell Cycle Progression with Kinetics Similar to Those Seen in E2-treated Cells—Time course experiments were carried out to allow a detailed comparison of the kinetics of cell cycle progression in E2 and ICI + AP-treated cells and to investigate whether, upon c-Myc induction, changes occur in the levels of key cell cycle regulators that might contribute to cell cycle progression. MCF-7/Myc33 cells were prearrested with ICI, treated with ICI, ICI + AP or E2, and harvested at 6-h intervals for analysis by flow cytometry and Western blotting.

The distribution of cells in different phases of the cell cycle was determined, and the data for S phase are shown in Fig. 2A. Fewer than 5% of ICI-treated cells were in S phase, and no significant changes were seen throughout the time course. Cell cycle phase distributions for ICI + AP- and E2-treated cells were similar over the duration of this experiment. Cells from both treatments began to enter S phase by 18 h, and substantial increases were seen between 18 and 24 h. The kinetics of S phase progression were somewhat different in that the percentage of E2-treated cells in S phase reached maximal levels at 30 h, whereas the ICI + AP-treated cells did so at 24 h. The percentage of ICI + AP- or E2-treated cells in G2/M increased between 24 and 30 h, with levels increasing until 36 h (data not shown).

As shown in Fig. 2B, ICI + AP treatment of prearrested MCF-7/Myc33 cells caused an increase in c-Myc protein within 6 h to levels similar to those seen in E2-treated cells, and its levels remained elevated throughout the time course. Thus, c-Myc expression at levels comparable with those seen in E2 is sufficient for cell cycle progression in the presence of ICI. Since cyclin A expression increases at the G1-S transition, it serves as a marker for cell cycle progression (52). In these experiments, there was a good correlation between cyclin A protein expression and S phase entry at 18 h.

To identify proteins that are regulated by c-Myc, the levels of key cell cycle regulatory proteins were compared in the three treatments (Fig. 2B). Cyclin D1 remained high in E2-treated cells and decreased upon ICI treatment. This decrease was not prevented by c-Myc induction in ICI + AP-treated cells, which is consistent with a previous report (19) that c-Myc does not induce proliferation by increasing cyclin D1. Although cyclin E and CDK4 are constitutively expressed in MCF-7 cells, their levels were examined, since they have been implicated as targets of c-Myc in other systems (32, 33). Ectopic c-Myc expression in the presence of ICI did not alter the levels of cyclin E or CDK4.

Next, the CDK inhibitors p27 and p21 were examined. In this experiment, p27 levels were decreased at 6–12 h in E2-treated cells as compared with ICI-treated cells. The expression pattern of p27 in ICI + AP-treated cells was similar to that in ICI-treated cells, and although subtle decreases in p27 levels occurred at 24–30 h in ICI + AP-treated cells, these decreases were not consistently observed over several experiments. Therefore, p27 is unlikely to be a major cause of c-Myc-mediated S phase entry. Expression of p21 was high in ICI-treated cells, and c-Myc induction caused a dramatic decrease to levels comparable with those seen in E2-treated cells. This decrease was consistently apparent by 12–18 h in several independent experiments and preceded major changes in the percentage of cells in S phase. To establish that this result was not unique to MCF-7/Myc33 cells, a second independently derived transfectant, MCF-7/Myc31, was examined. As shown in Fig. 2C, the decrease in p21 levels in MCF-7/Myc 31 cells was similar to that seen in the MCF-7/Myc33 cell line and occurred by 18 h of treatment with either E2 or ICI + AP. Cell cycle progression and both c-Myc and cyclin A protein levels were also examined in MCF-7/Myc31 cells, and the results were similar to those obtained with MCF-7/Myc33 cells (data not shown). In control cells stably transfected with vector alone, p21 levels decreased by 24 h of E2 treatment but remained high after ICI + AP treatment (Fig. 2D), establishing that the decrease in p21 was specific to c-Myc induction in the presence of ICI.



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  		FIG. 2.
Effects of c-Myc induction on cell cycle progression and protein expression in ICI-treated cells and in cells infected with an adenovirus expressing p21. MCF-7/Myc33 cells were prearrested with ICI and then treated with ICI, ICI + AP, or E2 and harvested at 6-h intervals. A, cell cycle analysis was performed as described in the legend to Fig. 1B. The average percentage of cells in S phase for each time point is shown ± S.D. {blacktriangleup}, E2-treated cells. {blacksquare}, ICI + AP-treated cells. •, ICI-treated cells. B, cell lysates were analyzed by Western blotting for c-Myc, cyclin A, cyclin D1, cyclin E, CDK4, p21, p27, and actin. These experiments were repeated once with similar results. C, the experiments described in A and B were also carried out with the MCF-7/Myc 31 cell line. Western blot results for p21 and actin are shown. D, control cells stably transfected with the vector were prearrested with ICI and then treated with ICI, ICI + AP, or E2 for 24 h. Cells were harvested, and the levels of c-Myc (data not shown), p21, and actin were determined by Western blotting. E, MCF-7/Myc 33 cells were prearrested with ICI for 24 h and then infected with adenoviruses encoding either p21 (Ad-p21) or {beta}-galactosidase (Ad-{beta}-gal) at 5 plaque-forming units/cell for 2 h. Arrest with ICI was continued for another 24 h, followed by treatment with ICI + AP. After 24 h, cells were harvested, and cell cycle analysis was performed as described in the legend to Fig. 1B. The average percentage of cells in S phase is shown ± S.D. F, the cells infected with Ad-p21 or Ad-{beta}-gal as described in E were harvested concurrently with those used for cell cycle analysis, and the levels of p21 and actin were determined by Western blotting.

 
The data presented above confirmed previous reports that c-Myc can promote entry into S phase in the presence of ICI (19, 31) and demonstrated that the kinetics are similar to those seen with E2. They also established that expression of c-Myc in the presence of ICI leads to significant changes in the levels of two proteins, p21 and cyclin A. Decreasing p21 expression using antisense oligonucleotides is sufficient to induce cell cycle progression in ICI-treated cells (22), whereas induction of cyclin A alone using the AP inducible system is not.2 We therefore chose to focus on p21 in further experiments by determining whether this decrease in p21 is necessary for c-Myc-mediated cell cycle progression in the presence of antiestrogens and by characterizing the mechanism by which c-Myc down-regulates p21 expression.

p21 Expression Can Block c-Myc-mediated Cell Cycle Progression in the Presence of ICI—To determine whether a decrease in p21 levels is required in order for c-Myc to promote cell cycle progression in the presence of ICI, prearrested MCF-7/Myc33 cells were infected with adenoviruses encoding either p21 (Ad-p21) or {beta}-galactosidase (Ad-{beta}-gal). The Ad-{beta}-gal virus was used as a control for possible effects of viral infection. Cells were then treated with ICI + AP to induce c-Myc expression. Western blot analyses confirmed that p21 was expressed in Ad-p21-infected but not in Ad-{beta}-gal-infected cells (Fig. 2F). The percentage of cells in S phase was ~2.5-fold lower in cells infected with Ad-p21 than Ad-{beta}-gal (Fig. 2E). This indicated that p21 expression blocks the ability of c-Myc to promote cell cycle progression in the presence of antiestrogen and suggested that the c-Myc-mediated decrease in p21 was necessary for this process. Our results complement previous studies, which have demonstrated that p21 is a critical regulator of CDK activity in human breast cancer cells and that promoting the formation of p21-free CDK complexes is sufficient for CDK activation and cell cycle progression (16, 17, 51, 53). Together, these results suggest that a key mechanism by which c-Myc promotes proliferation of breast cancer cells in the presence of ICI is by decreasing p21 levels.

c-Myc Expression Leads to Decreased p21 mRNA Levels and Promoter Activity in MCF-7 Cells—To characterize the mechanism(s) by which c-Myc and E2 down-regulate p21 in MCF-7 cells, p21 mRNA and protein were examined in tandem. MCF-7/Myc33 cells were prearrested with ICI and then treated with ICI, ICI + AP, or E2. At various times after treatment, cells were harvested, and the levels of p21 mRNA and protein were analyzed by Northern and Western blotting, respectively. Relative to the ICI-treated cells, a decrease in p21 mRNA levels was seen at 12 h in both ICI + AP- and E2-treated cells, and a decrease in p21 protein levels was apparent by 18 h (Fig. 3, A and B). Using PhosphorImager analysis, the decrease in p21 mRNA at 24 h was determined to be 2–3-fold relative to the 12-h ICI-treated sample (Fig. 3A; also see Fig. 6C).

To accurately quantitate the decrease in p21 protein levels, the 24-h ICI-treated sample was diluted as indicated, and the intensities of the p21 bands were compared with those of the undiluted 24-h ICI + AP- and E2-treated samples by Western blotting (Fig. 3C). Actin protein levels served as controls and demonstrated the accuracy of the dilutions. The p21 levels in the ICI + AP- and E2-treated samples were lower than the 2-fold but higher than the 4-fold diluted ICI samples. Thus, the decrease in p21 protein levels caused by ectopic c-Myc expression or E2 treatment is ~3-fold and is similar to the reduction in mRNA. These results concur with a prior report, which demonstrated that E2 decreases p21 mRNA levels (54). In addition, previous studies in other systems indicate that c-Myc can repress transcription from the p21 promoter (34–37, 39), and our Northern blot results suggest that similar mechanisms may be operative in MCF-7 cells.

To directly test whether c-Myc can repress p21 promoter activity in MCF-7 cells, we co-transfected cells with a c-Myc expression vector and a p21 promoter-luciferase reporter construct. Previous studies have shown that the p21 promoter region downstream of –119 bp is important for regulation by c-Myc (35), and footprinting experiments indicated that c-Myc binds to this region of the promoter (34). We therefore used a p21 promoter fragment beginning at –194 bp in our experiment. Expression from this promoter was decreased more than 2-fold in cells co-transfected with the c-Myc expression vector (Fig. 3D). The control promoterless luciferase plasmid showed very low levels of activity, and these levels were not regulated upon co-transfection with c-Myc. The level of repression observed in these luciferase assays is similar to that seen in other systems (34, 36, 37, 39) and is consistent with both our Northern and Western blot analyses.

E2 Mediates Down-regulation of p21 through c-Myc—Both c-Myc and p21 are established targets of E2 in breast cancer cells (22, 25, 26), and the results described above demonstrate that c-Myc expression is sufficient to decrease p21 levels in the presence of ICI. Together, these facts suggest that the decrease in p21 caused by E2 is mediated primarily through c-Myc. To test this hypothesis, we suppressed c-Myc expression using RNA interference and assayed whether E2 can decrease p21 in the absence of c-Myc.

MCF-7 cells were transfected with either c-myc or control siRNA and then incubated in the presence of E2. After 48 h, cells were harvested, and the levels of c-Myc and p21 were determined by Western blotting. As shown in Fig. 4, c-myc siRNA but not control siRNA decreased c-Myc protein in E2-treated cells to levels similar to those in ICI-treated cells. As a result, the levels of p21 in cells transfected with c-myc siRNA were similar to those in ICI-treated cells. This demonstrates that c-myc and p21 are not independent targets of E2 but rather that the decreased p21 expression caused by E2 is mediated primarily through c-Myc.



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  		FIG. 3.
Analysis of p21 protein and mRNA levels and p21 promoter-luciferase activity following c-Myc induction in ICI-treated cells. MCF-7/Myc33 cells were prearrested with ICI and then treated with ICI, ICI + AP, or E2 and harvested at the indicated times. A, RNA was isolated, and Northern blotting was carried out as described under "Experimental Procedures." Bands were quantitated by phosphorimaging. The p21 mRNA levels were normalized to the GAPDH levels in each sample and are represented as expression relative to the 12-h ICI treatment. B, cell lysates were prepared and analyzed for p21 and actin protein levels by Western blotting. C, the 24-h ICI sample was diluted as indicated, and the intensity of the p21 bands was compared with those seen in the undiluted 24-h ICI + AP and E2 samples. This experiment was repeated once with similar results. In both experiments, expression of c-myc mRNA and protein was confirmed (data not shown). D, MCF-7 cells were co-transfected with a pSP271-Myc expression plasmid or pSP271 vector and either –194 p21 promoter-luciferase plasmid or pGL2Basic vector. All transfections also included a cytomegalovirus promoter-driven {beta}-galactosidase gene as a control for transfection efficiency, and luciferase activity was normalized to {beta}-galactosidase activity in each sample. The results shown represent -fold change compared with the pSP271 vector control, and are the average ± S.D. of three independent experiments, each done in triplicate.

 



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  		FIG. 4.
Effect of c-myc siRNA on p21 expression in E2-treated MCF-7 cells. Cycling MCF-7 cells were transfected with 100 nM c-myc siRNA (msi) or control siRNA (csi) duplexes, as described under "Experimental Procedures," and then treated with E2. Cells were harvested after 48 h, and proteins were analyzed by Western blotting. Levels of c-Myc, p21, and actin were compared with those seen in untransfected cells treated with ICI or E2. This experiment was repeated twice with similar results.

 
c-Myc Expression Is Deregulated in the Antiestrogen-resistant LCC9 Cell Line—As shown by the above results (Figs. 1 and 2) and others (19, 31), aberrant c-Myc expression promotes proliferation of MCF-7 cells in the presence of ICI, suggesting that it could contribute to antiestrogen resistance. To determine whether the regulation of c-Myc expression is altered in cells with acquired antiestrogen resistance, we examined the LCC9 cell line, an ICI-resistant MCF-7 derivative (42).

MCF-7 and LCC9 cells were pretreated with ICI for 48 h and then incubated with medium containing either ICI or E2 and harvested every 24 h for 72 h. As shown in Fig. 5A, c-Myc levels were low in ICI-treated MCF-7 cells and were induced by E2 treatment. In contrast, c-Myc was expressed at similar levels in both E2- and ICI-treated LCC9 cells. pRB is a substrate for G1 CDKs, and hyperphosphorylation of pRB precedes S phase entry (55). Hence, both cyclin A and hyperphosphorylated pRB serve as markers for CDK activity and cell cycle progression. Deregulated c-Myc expression in LCC9 cells correlated with their ability to proliferate in the presence of ICI, as indicated by both pRB hyperphosphorylation and cyclin A expression.

To determine whether the deregulated c-Myc protein expression in LCC9 cells was a result of altered mRNA levels, MCF-7 and LCC9 cells were treated with ICI or E2 for 48 h, and c-myc mRNA levels were analyzed by Northern blotting. Expression of c-myc mRNA was high in E2 and low in ICI-treated MCF-7 cells, and this regulation was lost in LCC9 cells (Fig. 5B). Data from three independent experiments indicated that the levels of c-myc mRNA in ICI-treated MCF-7 cells were at least 2-fold lower than in all other samples (Fig. 5C). Together, these results established that c-Myc mRNA and protein are expressed at similar levels in E2- and ICI-treated LCC9 cells and that these levels are similar to those in E2-treated MCF-7 cells. This suggests that deregulation of c-Myc expression may contribute to or causes the acquired antiestrogen-resistant phenotype of LCC9 cells.

ICI Does Not Increase p21 mRNA Levels in LCC9 Cells—The results shown in Figs. 2, 3, 4 indicated that c-Myc decreases both p21 mRNA and protein levels in MCF-7 cells and that E2 treatment causes a decrease in p21 through c-Myc. We also showed that c-Myc expression is deregulated in LCC9 cells (Fig. 5). Previous experiments in our laboratory had indicated that CDK activity in LCC9 cells was resistant to ICI treatment and that this resistance was correlated with aberrant regulation of p21 protein levels.3 To determine whether altered regulation of p21 mRNA expression accounted for the changes in p21 protein levels in LCC9 cells, we conducted tandem Northern and Western blot analyses.

Cycling MCF-7 and LCC9 cells in FBS-containing medium were harvested before treatment (0 h) or treated with ICI or E2 for 48 h and then harvested. The levels of p21 protein and mRNA were examined by Western and Northern blotting, respectively (Fig. 6, A and B). In MCF-7 cells, p21 protein and mRNA levels were low at 0 h, since the FBS-containing medium has sufficient E2 to support proliferation. The levels of p21 were increased by 48 h of ICI treatment but remained low in the E2 treatment. In contrast, p21 protein and mRNA were expressed at similar levels in both E2- and ICI-treated LCC9 cells. The p21 mRNA levels were ~3-fold higher in ICI-treated MCF-7 cells than in all other samples (Fig. 6C), demonstrating that p21 mRNA expression increases in response to ICI treatment in MCF-7 cells, but not in LCC9 cells. These results indicate that the aberrant p21 protein levels observed in LCC9 cells are probably a result of altered regulation of p21 mRNA, which in turn may be the result of deregulated c-Myc expression.



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  		FIG. 5.
Analysis of c-Myc expression in an antiestrogen-resistant cell line. A, cycling MCF-7 and LCC9 cells were pretreated with ICI for 48 h and then treated with either ICI or E2 and harvested every 24 h for 72 h. c-Myc, cyclin A, pRB, and actin protein levels were analyzed by Western blotting. ppRB, the hyperphosphorylated form of pRB, which migrates more slowly than the hypophosphorylated form marked pRB. B, cycling MCF-7 and LCC9 cells were treated with either ICI or E2 for 48 h and harvested. RNA was prepared and analyzed for c-myc and GAPDH mRNA levels by Northern blotting. C, bands seen in B were quantitated by phosphorimaging. c-myc mRNA levels were normalized to GAPDH levels in the same sample and are represented as -fold increase over the ICI-treated MCF-7 sample. The graph represents the results as the mean ± S.D. of three independent experiments.

 
p21 Levels in LCC9 Cells Are Regulated by c-Myc—The results in Figs. 5 and 6 demonstrated that neither c-Myc nor p21 levels are regulated by E2 or ICI in LCC9 cells, and the data in Figs. 2 and 3 showed that p21 is a target of c-Myc in MCF-7 cells. This suggested that the loss of p21 regulation in LCC9 cells is a consequence of constitutive c-Myc expression. To test this hypothesis, RNA interference was used to knock down c-Myc expression in LCC9 cells.

LCC9 were transfected with c-myc siRNA and then incubated in the presence of ICI. MCF-7 cells served as controls and were transfected with either c-myc or control siRNA and incubated in the presence of E2. After 48 h, cells were harvested, and the levels of c-Myc and p21 were determined by Western blotting (Fig. 7). As expected, c-Myc expression was not decreased in ICI-treated relative to E2-treated LCC9 cells, and the levels were equivalent to those seen in E2-treated MCF-7 cells. Treatment with c-myc siRNA decreased c-Myc expression in both ICI-treated LCC9 cells and E2-treated MCF-7 cells, to levels similar to ICI-treated MCF-7 cells. This decrease was specific to c-myc siRNA, since it was not seen when cells were treated with control siRNA. In agreement with the results shown in Fig. 6, p21 levels did not increase in ICI-treated relative to E2-treated LCC9 cells and were comparable with those in E2-treated MCF-7 cells. However, in LCC9 cells transfected with c-myc siRNA in the presence of ICI, p21 expression increased and was similar to that seen in ICI-treated MCF-7 cells. This demonstrates that p21 can be regulated by c-Myc in LCC9 cells and suggests that the loss of p21 regulation by E2 and ICI in these cells is a consequence of constitutive c-Myc expression.



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  		FIG. 6.
Analysis of p21 protein and mRNA levels in LCC9 cells. Cycling MCF-7 and LCC9 cells in FBS-containing medium were harvested at 0 h before treatment or treated with ICI or E2 for 48 h and then harvested. A, p21 and actin protein levels were analyzed by Western blotting. B, p21 and GAPDH mRNA levels were determined by Northern blotting. C, bands seen in B were quantitated by phosphorimaging. p21 mRNA levels were normalized to GAPDH levels in the same sample and are represented as -fold increase over the E2-treated MCF-7 sample. The graph represents the results as the mean ± S.D. of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Various mechanisms have been proposed to account for acquired antiestrogen resistance in breast cancer cells, but since E2 and antiestrogens control cell cycle progression (56, 57), the development of resistance must ultimately be associated with changes in cell cycle regulation. Numerous studies support the idea that antiestrogen resistance can result from deregulated expression and/or activity of cell cycle regulatory proteins, including positive regulators, such as cyclin D1 and cyclin E, and negative regulators, such as p21, p27, and pRB (19–24). We chose to study the proto-oncogene product c-Myc, since it is a direct target of E2 that is required for E2-stimulated proliferation (25, 26, 30) and because c-Myc expression alone is sufficient to induce cell cycle progression in the presence of antiestrogens (19, 31). Although c-Myc has been extensively studied in many systems, the targets through which it mediates E2-stimulated proliferation and contributes to cell cycle progression in the presence of antiestrogens in breast cancer cells have not been identified.



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  		FIG. 7.
Effect of c-myc siRNA on p21 expression in LCC9 cells. Cycling LCC9 or MCF-7 cells were transfected with 100 nM c-myc siRNA (msi) or control siRNA (csi) duplexes as described under "Experimental Procedures" and then treated with E2 or ICI. Cells were harvested after 48 h, and proteins were analyzed by Western blotting. Levels of c-Myc, p21, and actin were compared with those seen in untransfected cells treated with ICI or E2. This experiment was repeated once with similar results.

 
To elucidate the role of c-Myc in E2 signaling, we established stable MCF-7 derivatives in which ectopic c-Myc expression could be tightly regulated. Using these derivatives, we confirmed previous reports (19, 31) that expression of c-Myc at levels comparable with those induced by E2 is sufficient to promote cell cycle progression in the presence of ICI (Figs. 1 and 2). However, inducing c-Myc expression alone in the presence of ICI is not sufficient to restore cell cycle progression to levels equivalent to those induced by E2. It is therefore likely that ICI affects multiple targets that together with c-Myc regulate proliferation. To identify the target(s) through which c-Myc mediates cell cycle progression in the presence of ICI, we examined the expression of key cell cycle regulators reported to be targets of c-Myc in other cell types (29). Among the proteins tested, c-Myc caused dramatic changes in the levels of two proteins, p21 and cyclin A (Fig. 2). Decreasing p21 expression using antisense oligonucleotides is sufficient to induce cell cycle progression in ICI-treated cells (22), whereas induction of cyclin A using the AP-inducible system is not.2 In addition, previous work has demonstrated that p21 is a critical regulator of CDK activity in human breast cancer cells and that promoting the formation of p21 free CDK complexes is sufficient for CDK activation and cell cycle progression (16, 17, 51, 53). This suggests that a key mechanism by which c-Myc promotes proliferation of breast cancer cells in the presence of ICI is by decreasing p21 levels. Further support for this viewpoint is provided by the fact that expression of p21 from an adenoviral vector blocks the ability of c-Myc to promote cell cycle progression in the presence of ICI (Fig. 2, E and F).

Our results with stable MCF-7 derivatives indicated that c-Myc can repress p21 expression but did not directly address whether the previously reported (18, 22, 51) decreases in p21 in response to E2 treatment are mediated via c-Myc. We therefore used RNA interference to demonstrate that c-Myc expression is required for the E2-mediated decrease in p21 in MCF-7 cells and thus identified c-Myc as the first direct link between E2 and p21 in breast cancer cells. Together, our studies suggest a model (Fig. 8) for antiestrogen-sensitive cells, in which E2 induces c-Myc, which in turn represses p21 and results in CDK activation and cell cycle progression. Antiestrogens such as ICI repress c-Myc expression in these cells, thereby leading to increased levels of p21, CDK inhibition, and cell cycle arrest. Antiestrogen resistance could arise if c-Myc expression is no longer inhibited by antiestrogens, since this would lead to decreased p21 levels, CDK activation, and cell cycle progression.



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  		FIG. 8.
Proposed model for the role of c-Myc in E2 signaling and antiestrogen resistance. A, in antiestrogen-sensitive cells, E2 induces c-Myc, which in turn represses p21 and results in CDK activation and cell cycle progression. B, antiestrogens such as ICI repress c-Myc expression in antiestrogen-sensitive cells, thereby leading to increased levels of p21, CDK inhibition, and cell cycle arrest. Antiestrogen resistance could arise if c-Myc expression is no longer inhibited by antiestrogens, since this would lead to decreased p21 levels, CDK activation, and cell cycle progression.

 
Our experiments suggest that regulation of p21 by c-Myc occurs at a transcriptional level, since p21 protein levels, mRNA levels, and promoter activity all decrease 2–3-fold in the presence of c-Myc. They are in agreement with several reports in other systems, which show that c-Myc can repress transcription from the p21 promoter (34–37, 39), and offer a potential explanation for the observation that under certain conditions only the trans-repression and not the trans-activation activity of c-Myc may be required for cell cycle progression (58, 59). However, they differ from an earlier study in which antisense oligonucleotides were used to decrease c-Myc expression in MCF-7 cells, and no increases in p21 levels were observed (60). One difference between the two studies is that the earlier report focused on early events (6–16 h) following transfection with c-Myc antisense, whereas we studied cells at 48 h after transfection with c-Myc siRNA. We chose this time point because our previous work and the work of others have shown that, upon ICI treatment, p21 accumulates over 48 h, leading to CDK inhibition and complete cell cycle arrest (51, 53).

Several interesting questions are raised by our findings, including whether additional targets of c-Myc are contributing to its ability to promote cell cycle progression in the presence of antiestrogens. Previous studies have shown that decreasing p21 levels using antisense oligonucleotides is sufficient to abrogate a cell cycle arrest caused by antiestrogens (22), indicating that additional targets need not be involved. However, another report showed that c-Myc induction in ICI-treated MCF-7 cells promoted the formation of p21-free CDK2 complexes without decreasing p21 protein levels (19), suggesting that other c-Myc targets might titrate p21 away from CDK2 complexes. In that report, early S phase entry was observed in the c-Myc-inducible cells, and the authors suggested that this was due to leaky ectopic c-Myc expression. Such leaky c-Myc expression might have led to lower p21 levels in the absence of c-Myc induction, and subtle changes in p21 levels that were undetectable on Western blots could have occurred upon further c-Myc induction. These changes could be sufficient to activate CDKs and promote cell cycle progression, since only a small percentage of all CDK2 complexes need to be free of p21 to mediate S phase entry (16).

A second question arising from our initial results was whether deregulation of c-Myc expression actually occurs during the acquisition of antiestrogen resistance. To address this question, we examined the LCC9 cell line, which is an ER-positive MCF-7 derivative that was selected in vivo for E2 independence and in vitro for ICI resistance (42). We found that c-Myc expression was not decreased in ICI-treated LCC9 cells, and its levels were comparable with E2-treated MCF-7 cells (Fig. 5A). Thus, c-Myc expression is deregulated in this in vitro model of antiestrogen resistance. Whether c-Myc deregulation contributes to the development of antiestrogen-resistant tumors or to unresponsiveness in primary tumors remains to be determined. There is evidence for aberrant c-Myc expression in breast tumors; a meta-analysis has shown that 15.5% of breast cancer biopsies bear a c-myc gene amplification, and in a recent study, 70% of high grade breast carcinomas showed elevated c-Myc expression (61, 62). However, more rigorous clinical studies designed specifically to compare c-Myc expression in tumors before and after recurrence on antiestrogens are required to directly correlate aberrant c-Myc expression with antiestrogen resistance.

Additional questions concern the mechanism by which the c-myc gene is activated in antiestrogen-resistant cells. Our results in LCC9 cells show that c-Myc deregulation occurs at the mRNA level (Fig. 5B). Altered expression or activity of factor(s) regulating c-myc transcription, mutations in the c-myc promoter, c-myc gene amplification, chromosomal rearrangements, or increased mRNA stability could all provide explanations for the deregulated c-Myc expression. However, since ER binds and activates the c-myc promoter (27, 28), deregulated c-Myc expression is likely to be the result of altered transcriptional control. NF-{kappa}B expression and activity is up-regulated in LCC9 cells (63); this could provide a mechanism for altered c-Myc expression, since NF-{kappa}B is a potent transcriptional activator of the c-myc promoter (64). Increased expression of the nucleolar phosphoprotein nucleophosmin has also been observed in LCC9 cells (63). Since nucleophosmin is a direct target of c-Myc (65), our results offer a potential explanation of this observation.

Previous experiments in our laboratory have established that the ability of ICI to increase p21 protein expression is lost or attenuated in LCC9 cells,3 and the current study showed that this is a result of altered p21 mRNA regulation (Fig. 6). We further demonstrated that suppressing c-Myc expression can increase p21 levels in LCC9 cells, indicating that the loss of p21 regulation is likely to be a result of constitutive c-Myc expression (Fig. 7). The fact that p21 is highly regulated by E2/ICI in MCF-7 cells and that disrupting this regulation by inhibiting p21 expression with antisense oligonucleotides promotes ICI resistance makes it very likely that altered p21 regulation contributes to the ICI-resistant phenotype of LCC9 cells. Several other mechanisms proposed to account for antiestrogen resistance also converge on p21. Cyclin D1 overexpression titrates p21 away from CDK2 complexes into cyclin D1-CDK4 complexes (19), and cyclin E overexpression decreases p21 protein, but not mRNA levels (21). In addition, antisense oligonucleotides to p21 or p27 abrogate an antiestrogen-mediated cell cycle arrest (22), and p27 deregulation contributes to antiestrogen resistance in LY2 cells (66). Together, our results and these reports strongly support a central role for CDK inhibitors and their direct upstream regulators in antiestrogen resistance. Levels of these CDK inhibitors are also suppressed in androgen-independent prostrate cancer cells (50, 67, 68), indicating that they may play a more general role in resistance to hormonal regulation.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grant CA76647, Susan G. Komen Breast Cancer Foundation Grant DISS02-1283, and the Schultz Oncology Research Award (to the Michigan State University College of Human Medicine). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, 2209 BPS, Michigan State University, East Lansing, MI 48824. Tel.: 517-353-5161; Fax: 517-353-8957; E-mail: conrad{at}msu.edu.

1 The abbreviations used are: ER, estrogen receptor; E2, 17{beta}-estradiol; CDK, cyclin-dependent kinase; p21, p21WAF1/CIP1; p27, p27KIP1; ICI, ICI 182,780; pRB, retinoblastoma protein, FBS, fetal bovine serum; CSS, charcoal-stripped fetal bovine serum; AP, AP1510; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

2 C. Zhang and S. E. Conrad, unpublished observations. Back

3 A. Skildum, H. Varma, S. Mukherjee, S. J. Santos, R. Clarke, V. C. Jordan, and S. E. Conrad, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank ARIAD Pharmaceuticals for the ArgentTM regulated transcription plasmid kit, Cunjie Zhang for constructing the initial stable transfectants that were used for generating theMCF-7 inducible cell line, and Dr. Robert Clarke for the LCC9 cell line. We also thank Drs. Richard Miksicek, Laura McCabe, Hemant Varma, Rami Saba, and Richard Schwartz for review of the manuscript and helpful discussions. Drs. Pam Fraker and Louis King at the Michigan State University Flow Cytometry Facility provided assistance with cell cycle analysis.



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 DISCUSSION
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