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Glucocorticoid and Mineralocorticoid Cross-Talk with Progesterone Receptor to Induce Focal Adhesion and Growth Inhibition in Breast Cancer Cells

Department of Clinical Research (J.C.L.L., C.G., C.T.W., S.E.A.), Singapore General Hospital; and School of Biological Sciences (V.C.L.L.), Nanyang Technological University, Singapore, Singapore 637616

Address all correspondence and requests for reprints to: Valerie C.-L. Lin, School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Block 5 Level 3, Singapore, Singapore 637616. E-mail: cllin{at}ntu.edu.sg.

	Abstract

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Progesterone receptor (PR), glucocorticoid receptor, and mineralocorticoid receptor belong to a subfamily of nuclear receptor superfamily with similar sequence and structural characteristics. Many reports have documented glucocorticoid-like effects of progesterone in various tissues. This study addresses the issue of cross-talk between corticosteroids and PR using PR-transfected MDA-MB-231 cells ABC28 and vector-transfected control cells CTC15. At physiological concentrations, dexamethasone, cortisol, and aldosterone mimic the effects of progesterone by inducing significant growth inhibition, cell spreading, and focal adhesions in PR-positive ABC28 cells. These hormones also induce progesterone-like effects in increasing the expression of p21^CIP1/WAF1 protein and decreasing the level of phospho-p42/p44 MAPK. Two lines of evidence suggest that these effects are mediated by cross-talk with PR. First, these compounds do not exhibit the same progesterone-like effects in PR-negative CTC15 cells. Second, PR blocker ZK98299 abolishes their effect on cell spreading and focal adhesion in ABC28 cells. The cross-talk is corticosteroid specific because estradiol and thyroid hormone triiodothyronine have no effect on PR-transfected cells ABC28. It is also interesting to note that dexamethasone induces a small but detectable increase of focal adhesions and limited growth stimulation in vector-transfected cells CTC15. In contrast, progesterone exhibits no detectable effect on CTC15 cells. This study provides evidence that glucocorticoid and mineralocorticoid cross-talk with PR to produce progesterone-like effects in breast cancer cells. Glucocorticoid receptor and PR share some overlapping activity in mediating focal adhesion but not in regulating cell proliferation.

	Introduction

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GLUCOCORTICOID, MINERALOCORTICOID, AND progesterone are steroid hormones that are classically known to mediate distinct physiological functions. Glucocorticoids are mainly involved in the regulation of glucose metabolism, inhibition of inflammation, and immune functions (1). Mineralocorticoids regulate Na⁺ and K⁺ balance and water excretion. The major functions of progesterone are to establish and maintain pregnancy, promote the development of mammary lobule-alveolar tissues, and inhibit lactogenesis (2). More recent studies reveal that these three hormones also share overlapping biological activities. For example, mineralocorticoid receptor (MR) may compensate for the absence of glucocorticoid receptor (GR) at some stages of mammary development (3). Both glucocorticoid and progesterone have been shown to either stimulate or inhibit the growth of breast cancer cells (4, 5, 6, 7, 8, 9).

GR, progesterone receptor (PR), and MR belong to a subfamily of nuclear receptor superfamily with similar structural domains, cognate hormone response elements, and overlapping ligand binding specificities (10, 11, 12, 13, 14, 15, 16). It is known that GR and PR share substantial number of common target genes. In T47D/A1–2 breast cancer cells that express comparable levels of GR and PR, the number of common target genes for both GR and PR are comparable with the number of target genes for GR or PR alone (17). The effects could be either GR or PR mediated because RU486 was able to abolish both dexamethasone and progestin’s effect on the regulation of some selected target genes. Indeed, it is often difficult to differentiate between GR- and PR-mediated effects when the two receptors coexpress in the same tissue. Although the glucocorticoids have been demonstrated to compete with progestin for lower affinity binding sites in the cytosol preparation of mammary and uterine tissues (13, 14, 15), it is not known whether this cross-binding can elicit biological responses.

This study differentiates between GR- and PR-mediated effects of glucocorticoid, mineralocorticoid, and progesterone using cells that express either GR or both GR and PR. Estrogen receptor-- and PR-negative breast cancer cells MDA-MB-231 express both GR (7, 18) and MR. PR-transfected MDA-MB-231 cells ABC28 express PR (19). ABC28 cells and the vector-transfected cells CTC15 also express GR and MR because their parental cells do. Previous studies show that progesterone exhibits significant effect on growth inhibition and induction of focal adhesion in ABC28 cells (19, 20). This study provides evidence that glucocorticoid and mineralocorticoid cross-talk with PR to induce growth inhibition and focal adhesion in ABC28 cells. On the other hand, glucocorticoids induce a smaller degree of focal adhesion and limited growth stimulation in the PR-negative vector transfected control cells, CTC15. Progesterone exhibits no detectable effect in focal adhesion and growth regulation in CTC15 cells.

	Materials and Methods

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Cell line and reagents
MDA-MB-231 cells were obtained from American Tissue Culture Collection (Manassas, VA) in 1995 at passage 28. They were cloned using 96-well plates by the method of single cell dilution, and clone 2 (MDA-MB-231-C2) was transfected with PR expression vectors hPR1 and hPR2 that contain human PR cDNA coding for PR isoform B and isoform A, respectively, in pSG5 plasmid (21). Details of transfection and characterization were described previously (19). PR-transfected cells clone ABC28 was used in the present study. ABC28 cells expressed approximately 660 fmol PR per milligram protein as determined by enzyme immunoassay (Abbott Laboratories, Abbott Park, IL). The cells at passage numbers 5–30 were used. CTC15 cells are MDA-MB-231 cells stably transfected with empty vectors, and they were used as transfection controls.

Cells were routinely maintained in DMEM with phenol-red and supplemented with 7.5% fetal calf serum (FCS), 2 mM glutamine, and 40 mg/liter gentamicin. For all the experiments on the effects of steroid compounds, cells were grown in phenol-red-free DMEM containing 2 mM glutamine, 40 mg/liter gentamicin, and 5% dextran charcoal-treated FCS. FCS was treated with dextran-coated charcoal to remove the endogenous steroid hormones. Cells were treated with various steroid compounds from 1000-fold stock in ethanol. This gave a final concentration of ethanol of 0.1%. Treatment controls received 0.1% ethanol only. Triiodothyronine was added to the cell culture from 100x stock in PBS.

Chemicals
Progesterone, dexamethasone, cortisol, aldosterone, triiodothyronine, tamoxifen, estradiol-17ß, and fluorescein isothiocyanate (FITC)-labeled phalloidin were obtained from Sigma Chemical Co. (St. Louis, MO). Cy5-conjugated antimouse lgG were obtained from Amersham Biosciences (Buckinghamshire, UK). All tissue culture plastics and reagents were obtained from Gibco BRL, Life Technologies Inc (Gaithersburg, MD). Antibodies for cyclin p21^CIP1/WAF1 and MAPK were obtained from BD PharMingen (San Diego, CA). Phospho-p42/p44 MAPK was from Cell Signaling Technology (Beverly, MA). PR antibody used for Western bottling analysis of PR protein was from the PR enzyme immunoassay assay kit (Abbot Laboratories).

Real-time RT-PCR for GR and MR expression
Total RNA was extracted using TRIzol reagent (Life Technologies Inc.) according to the manufacturer’s instructions. Five micrograms of total RNA were reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Real-time PCR was performed with SYBR Green master mix on an ABI Prism 7700 sequence detection system according to the manufacturer’s protocol (PE Applied Biosystems, Foster City, CA). The primers for GR are 5'-CCT AAG GAC GGT CTG AAG AGC-3' (forward) and 5'-GCC AAG TCT TGG CCC TCT AT-3' (reverse). The primers for MR are 5'-AAC TTG CCT CTT GAG GAC CAA-3' (forward) and 5'-AGA ATT CCA GCA GGT CGC TC-3' (reverse). cDNAs were amplified by incubation for 10 min at 95 C to activate the Hot Start AmpliTaq Gold DNA polymerase, followed by 35 cycles of denaturation at 95 C for 30 sec, annealing at 60 C for 1 min, and extension at 72 C for 1 min. PCR for each gene fragment were performed in triplicate, and each primer set was repeated three times. To ensure PCR product specificity, melting curves were generated after amplification followed by agarose gel electrophoresis. Both melting curve and gel electrophoresis showed a single, specific product for GR and MR. The identity of the PCR product was further confirmed by restriction enzyme digestion. The changes in fluorescence of the SYBR Green I dye in each cycle were monitored by ABI 7700 system, and the threshold cycle, which is defined as the cycle number at which the amount of amplified target reaches a fixed threshold, was obtained for each gene. The relative amount of PCR products generated from each primer set was determined on the basis of the threshold cycle value. Primer sets for the 36B4 gene, which codes for human acidic ribosomal phosphoprotein P0, were included in each experiment because control for normalizing the quantity of cDNA used.

Immunofluorescence microscopy
Cells were grown on glass coverslips in six-well plates for 24 h before they were treated with progesterone, cortisol, dexamethasone, aldosterone, estradiol, RU486, and ZK 98299 alone or in combinations in 0.1% ethanol for 48 h. Control cells received 0.1% ethanol only. Triiodothyronine were added to the cell culture in 100-fold stock in PBS. After rinsing with PBS, the cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. This was followed by incubation with 2% normal FCS in PBS for 1 h to block nonspecific binding. Cells were incubated with antibody to paxillin in PBS containing 2% FCS overnight at 4 C, followed by incubation with Cy5-conjugated antimouse IgG together with 10 µg/ml FITC-phalloidin in PBS for 1 h at room temperature. After washing with PBS, the cover slips were mounted on slides with fluorescence mounting media from Dako (Carpinteria, CA). Stained cells were viewed and photographed using a confocal laser scanning microscope (model LSM 510, Carl Zeiss, Gottingen, Germany).

Light microscopy
Cells were grown in six-well plates and treated with progesterone, dexamethasone, aldosterone, and ZK 98299 alone or in combination in 0.1% ethanol respectively for 48 h before they were viewed and photographed under a AXIOVERT 35 phase contrast microscope (Carl Zeiss).

DNA synthesis
The 3 x 10³ cells were seeded onto 96-well plates and treated with test compounds after 48 h of plating. The effects of treatment on DNA synthesis after 48 h were measured by ELISA kit of bromodeoxyuridine incorporation (Roche, Mannheim, Germany) according to the manufacturer’s instructions.

Analysis of cell cycle by flow cytometry
1.5 x 10⁵ cells were grown in 6-well plates for 2 d before being treated with test compounds that were added from 1000-fold stock in ethanol. Treatment control received 0.1% ethanol only. At various time points after treatments, the cells were harvested and stained with propidium iodide (PI) in Vindelov’s cocktail (22) [10 mM Tris HCl (pH 8), 10 mM NaCl, 50 mg PI/liter, and 10 mg/liter ribonuclease A, and 0.1% Nonidet P-40] for 1 h at 4 C in the dark. The stained cells were analyzed using FACS Calibur flow cytometer (Becton Dickinson, Lincoln Park, NJ) with excitation wavelength of 488 nm. The resulting histograms were analyzed by MODFIT (Becton Dickinson) program for cell distribution in cell cycle phases. The average coefficient of variation was within 5%.

Western blotting analysis
For Western blotting analysis of MAPK and cyclin-dependent kinase (CDK) inhibitor 1 p21^CIP1/WAF1, 1 x 10⁶ cells were grown on 100-mm petri dishes for 48 h before they were treated with 0.1% ethanol, 0.1 µM progesterone, or 0.1 µM dexamethasone for various periods of time. The treated cells were lysed with 200 µl cold lysis buffer [50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 5 µg/ml pepstatin A, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, and 1 mM sodium vanadate (pH 7.5)] and left standing on ice for 20 min. Protein supernatants were then collected by centrifugation at 13,000 rpm for 20 min. Twenty micrograms of the protein were analyzed by Western blotting with antibodies against specific proteins.

Statistical analysis
Differences between treatments (Figs. 3–5) were tested by ANOVA of repeated measurements. Effects of a hormone at various concentrations on DNA synthesis (Figs. 3 and 5) were analyzed by the least significant difference test. For data in Fig. 4, one-way ANOVA was performed to compare treatments at different time points. This was followed by Tukey’s HSD post hoc test to provide all pair-wise comparisons among means at the same time point.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Analysis of the gene expression of GR and MR in various cell lines by real-time RT-PCR. The result is presented as the relative expression of GR and MR in MDA-MB-231-C2 cells, ABC28 cells, and vector-transfected cells CTC15 when the expression in MDA-MB-231 cells is given the value of 1. Primer sets for housekeeping gene 36B4 were included for normalizing the quantity of cDNA used. The results are the means ± SEM of three independent experiments, each of which has three replicates. The dotted line indicates the relative expression level of MR and GR in the parental cells MDA-MB-231.

View larger version (86K): [in this window] [in a new window]   FIG. 4. Effect of corticosteroids on cell spreading and formation of focal adhesion in PR-transfected cell ABC28. Cells grown on glass coverslips were treated with control vehicle (A), 0.1 µM progesterone (B), 0.1 µM cortisol (C), 0.1 µM dexamethasone (D), 0.1 µM aldosterone (E), 0.1 µM estradiol (F), 0.1 µM T3 (G), 1 µM ZK98299 (H), and 0.1 µM dexamethasone plus 1 µM ZK98299 (I) for 48 h. After fixation, the cells were costained for F-actin and focal adhesion protein paxillin. F-actin was probed with FITC-labeled (green) phalloidin. Paxillin was probed with paxillin antibody and detected by Cy5-labeled (red) antimouse lgG. Stained cells were viewed and photographed using the Zeiss confocal laser scanning microscope model LSM 510. Bar, 10 µm.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Effect of progesterone and corticosteroids (A) and nonglucocorticoid compounds (B) on DNA synthesis of PR-transfected cell ABC28. The 3 x 103 cells were seeded onto 96-well plates and treated with test compounds after 2 d. The effects on DNA synthesis after 48 h treatment were measured by bromodeoxyuridine incorporation ELISA kit (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Results are expressed as percentage of vehicle-treated controls (means ± SEM, n = 4).

	Results

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View larger version (61K): [in this window] [in a new window]   FIG. 1. Analysis of PR protein in MDA-MB-231 cells (lane 1), MDA-MB-231-C2 (lane 2), vector-transfected cells CTC15 (lane 3), and PR-transfected cells ABC28. Cells were gown to 70–80% confluency before the whole-cell lysate was collected. 25 µg protein was analyzed by Western blotting using specific PR antibody, which recognized both PR-A (90 kDa) and PR-B (120 kDa). After the detection of PR, the membrane was stripped and reprobed with antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to validate the consistency of sample loading.

View larger version (48K): [in this window] [in a new window]   FIG. 2. Analysis of the expression of GR and MR mRNA in MDA-MB-231 cells, MDA-MB-231-C2 cells, ABC28 cells, and vector-transfected cells CTC15 by RT-PCR. The specificity of the PCR gene products were confirmed by restriction enzyme digestion. M, DNA marker.

Glucocorticoids and mineralocorticoid exhibit progesterone-like effect in inducing cell spreading and focal adhesion in PR-transfected MDA-MB-231 cells ABC28
Our previous study showed that progesterone induces evident cell spreading and focal adhesion (20) in ABC28 cells (Fig. 4B), compared with vehicle-treated controls (Fig. 4A). Interestingly, the glucocorticoids cortisol (Fig. 4C) and dexamethasone at 10^-7 M (Fig. 4D) or higher also induced evident cell spreading and increases in F-actin (green) and focal adhesion protein paxillin (red) staining in ABC28 cells. The mineralocorticoid aldosterone also demonstrated similar effects as glucocorticoids (Fig. 4E). In contrast, estradiol (Fig. 4F) and thyroid hormone T₃ (Fig. 4G) did not exhibit these progesterone-like effects.

Whereas the antiprogestin ZK98299 did not induce progesterone-like effect on cell spreading and focal adhesion (Fig. 4H), it reversed the effect of dexamethasone on cell spreading and focal adhesion (Fig. 4I). ZK98299 also blocked the effect of aldosterone and cortisol on cell spreading and focal adhesion (data not shown).

Glucocorticoids and mineralocorticoid exhibit progesterone-like effect in inhibiting the cell proliferation of PR-transfected MDA-MB-231 cells ABC28
Progesterone markedly inhibits the proliferation of ABC28 cells by arresting them in the G₀/G₁ phase of the cycle (19). Dexamethasone, aldosterone, and cortisol also demonstrated significant (P < 0.01) growth-inhibitory effect on DNA synthesis of ABC28 cells (Fig. 5A). The effective concentration of aldosterone is from 10^-8 M, whereas that of dexamethasone and cortisol is from 10^-7 M and 10^-6 M, respectively. The potency of aldosterone is similar to that of progesterone at the concentrations of 10^-7 M and 10^-6 M. The potency of dexamethasone and cortisol is much lower than that of progesterone at the same concentrations. On the other hand, the noncorticosteroid compounds estradiol-17ß, tamoxifen, and thyroid hormone T₃ did not demonstrate any detectable effect on the DNA synthesis of ABC28 cells (Fig. 5B).

Flow cytometry analysis revealed that dexamethasone and aldosterone at 10^-7 M reduced the S-phase fraction of ABC28 cells by arresting them in the G₀/G₁ phase of the cell cycle (P < 0.005) (Fig. 6). The S-phase fractions of the cells treated with dexamethasone and aldosterone for 48 h were reduced to 76.9% and 65.5% of the vehicle-treated controls, respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Effect of progesterone, dexamethasone, and aldosterone on cell cycle progression in PR-transfected cell ABC28. The 1.5 x 105 cells were grown in six-well plates for 48 h before they were treated with the test compounds. At various time points after treatments, the cells were harvested and stained with PI in Vindelov’s cocktail for 1 h at 4 C in the dark. The stained cells were analyzed using FACS Calibur flow cytometer (Becton Dickinson) with excitation wavelength of 488 nm. The resulting histograms were analyzed by program MODFIT (Becton Dickinson) for cell distribution in cell cycle phases. The average coefficient of variation is within 5%. The results are expressed as the means ± SEM (n = 4). The SEMs are too small to be visible in most data points.

View larger version (20K): [in this window] [in a new window]   FIG. 7. Dexamethasone and progesterone induced up-regulation of p21WAF1/CIP1 protein and down-regulation of phospho-p42/p44 MAPK in ABC28 cells. Cells were treated with control vehicle, 0.1 µM progesterone, or 0.1 µM dexamethasone for the indicated periods of time, and cell lysate were collected. Twenty micrograms of protein were analyzed for p21WAF1/CIP1, p42/44 MAPK, and total MAPK by Western blotting. After detection of active p42/44 MAPK with anti-phospho-p42/p44 MAPK, the membrane was stripped and reprobed with anti-ERK antibody to determine total MAPK. The number below each lane indicates the relative density of the band signal when the same time point control is given the value of 1. The relative amount of activated p42/p44 MAPK was normalized against the total MAPK at each time point. C, Vehicle-treated control, P, progesterone and D, dexamethasone.

View larger version (22K): [in this window] [in a new window]   FIG. 8. Effects of various compounds on DNA synthesis (A) and cell cycle progression (B and C) in PR-negative vector-transfected control cells CTC15. Procedures of analysis are stated in Fig. 5 for DNA analysis and Fig. 6 for cell cycle analysis (means ± SEM, n = 4). The SEMs are too small to be visible in most data points of plate B and C.

View larger version (91K): [in this window] [in a new window]   FIG. 9. Effect of various hormones on cell spreading and focal adhesion in PR-negative vector-transfected control cells CTC15. Cells grown on glass coverslips were treated with control vehicle (A and B), 0.1 µM progesterone (C and D), dexamethasone (E and F), aldosterone (G and H), 1 µM ZK98299 (I and J), and 0.1 µM dexamethasone plus 1 µM ZK98299 (K and L) for 48 h. Cells were viewed and photographed under a Zeiss AXIOVERT 35 phase contrast microscope (bar, 100 µm). Procedures of analysis for immunostaining are stated in Fig. 1 (bar, 10 µm).

	Discussion

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The effective concentrations of dexamethasone, aldosterone, and cortisol in ABC28 cells were in the range between 10^-8 M and 10^-6 M. Serum cortisol concentrations in normal individuals range from 0.08–0.17 x 10^-6 M during the early night to 0.22–0.7 x 10^-6 M peak concentrations (25, 26, 27). It is reasonable to assume that concentrations of 10^-8 M to 10^-7 M used in this study are close to physiological levels for most individuals for at least 12 h a day. Therefore, the cross-talk between corticosteroids and PR demonstrated in this study is of physiological relevance.

It has long been recognized that progesterone cross-binds with GR in mammary gland and breast cancer cells (14, 28). It is a common practice to include cortisol in the PR binding assay to saturate GR site to minimize the nonspecific binding of progesterone (13, 29, 30). Numerous studies also document the cross-binding of glucocorticoids with PR (14, 31, 32). However, the cross-binding of glucocorticoids with PR exhibited lower affinity than that of progesterone, and hence the significance of this cross-binding has not been widely recognized. Judging from their effective concentrations in ABC28 cells, the lower affinity binding may indeed induce biological responses. Because the physiological concentrations of plasma glucocorticoids reach the range of 10^-7 to 10^-6 M, the low-affinity binding of glucocorticoid with PR is of physiological significance.

Another interesting finding of this study is that dexamethasone is able to induce an increase of focal adhesions with cobblestone-like morphology in vector-transfected control cells CTC15, which are GR positive but PR negative. The effect is likely GR-mediated because progesterone and aldosterone had no effect on CTC15 cells. Other studies have provided supporting evidence that dexamethasone plays a role in cell adhesion. Studies by Koukouritaki and Lianos (33) revealed that in human mesangial cells, dexamethasone increased the levels of tyrosine phosphorylation of focal adhesion protein paxillin and focal adhesion kinase. Hebert and Baker (34) reported that dexamethasone was able to reduce the number and intensity of the urokinase strands, which appear to focus urokinase at cell surface; hence, dexamethasone may participate in tissue matrix destruction and cell invasion.

Our finding that dexamethasone induces focal adhesion via both PR and GR suggests that GR and PR share common target genes responsible for formation of stress fibers and focal adhesion. In contrast, dexamethasone stimulates cell proliferation in vector-transfected control cells but inhibits it in PR-transfected cells, suggesting that GR and PR do not share common genes in regulating cell proliferation. In T47-D cells, however, both glucocorticoid and progesterone inhibited cell cycle progression (17). Surprisingly, although progesterone is well known to cross-bind with GR, it did not demonstrate a glucocorticoid-like effect on focal adhesion and growth stimulation in CTC15 cells.

It is interesting that GR, MR, and PR have evolved to share many similar features yet also distinct functional identities. Although homeostasis is maintained under physiological conditions by various regulatory mechanisms, the cross-reactivity of corticosteroids with PR is of significant relevance under pathological conditions. Glucocorticoids have been used widely in many clinical conditions such as in the primary combination chemotherapy of breast cancer, inflammation, and autoimmune disease (35, 36). Caution must be exercised to avoid undesirable progesterone-like effect of the corticosteroids, depending on the condition of the patient or the target organ of interest. This study also suggests that analogs of corticosteroids may be used in place of progestin for therapeutic application under some conditions. For instance, because both glucocorticoids and progestins have been used in the treatment of breast cancer, glucocorticoid therapy in PR-positive breast cancer may have combined effect of both progestin and glucocorticoids.

	Acknowledgments

 
The authors express their sincere thanks to Professor Pierre Chambon (Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France) for kindly providing the progesterone receptor expression vectors hPR1 and hPR2 and to Ms. Stephanie Fook-Chong for her assistance in statistical analysis.

	Footnotes

 
This work was supported by the National Medical Research Council of Singapore.

Abbreviations: CDK, Cyclin-dependent kinase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PI, propidium iodide; PR, progesterone receptor; PR-A and -B, PR isoform A and B.

Received June 11, 2003.

Accepted for publication November 4, 2003.

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