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Endocrinology Vol. 142, No. 10 4331-4338
Copyright © 2001 by The Endocrine Society

ARTICLES

Role of the Cyclin-Dependent Kinase Inhibitor p27Kip1 in Androgen-Induced Inhibition of CAMA-1 Breast Cancer Cell Proliferation

Jacques Lapointe and Claude Labrie

Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Laval University, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Claude Labrie, Oncology and Molecular Endocrinology Research Center, CHUL Research Center, 2705 Laurier Boulevard, Québec, Québec, Canada G1V 4G2. E-mail: claude.labrie{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens are known to inhibit the growth of breast cancer cells, but the molecular mechanism of androgen-induced growth inhibition remains unknown. To address this question, we examined functional and quantitative alterations in cell cycle regulators in the E-responsive CAMA-1 breast cancer cell line. We report here that the androgen 5{alpha}-dihydrotestosterone inhibits the proliferation of CAMA-1 breast cancer cells. This inhibition of cell proliferation was dose dependent, and maximal inhibition of E2-stimulated proliferation was observed at the concentration of 1 nM 5{alpha}-dihydrotestosterone. 5{alpha}-Dihydrotestosterone-induced growth arrest was accompanied by an increase in the proportion of cells in the G1 phase of the cell cycle. Compared with control cells, 5{alpha}-dihydrotestosterone-treated cells showed an increase in the relative proportion of hypophosphorylated retinoblastoma protein consistent with G1 arrest. In CAMA-1 cells, 5{alpha}-dihydrotestosterone caused an accumulation of the cyclin-dependent kinase inhibitor p27Kip1. Cyclin E-cyclin-dependent kinase-2-associated kinase activity was strongly inhibited in 5{alpha}-dihydrotestosterone-treated cells, and immunoprecipitation-Western blot analysis showed an increase in the amount of p27Kip1 associated with cyclin E-cyclin-dependent kinase-2 complexes. These results suggest that inhibition of breast cancer cell growth by androgens may be mediated at least in part by inactivation of the cyclin E-cyclin-dependent kinase-2 complexes by p27Kip1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS OR ANDROGENIC compounds can have beneficial effects on breast cancer growth in women (1, 2). Studies performed in vitro and in vivo have demonstrated a direct growth inhibitory effect of androgens in breast cancer cells (3, 4). This effect is mediated by activation of the AR, which is expressed in a large proportion of breast tumors as well as in normal mammary gland (3, 5). However, the role of androgens in breast physiology and the mechanisms of androgen-induced inhibition of breast cancer cell proliferation are poorly understood.

Despite the high prevalence of AR expression in breast tumors, there are relatively few in vitro models of androgen-responsive human breast cancer cells. In fact, a limited number of breast cancer cell lines express both ER and AR, and among these, only ZR-75-1 cells exhibit E-induced and androgen-inhibited proliferation that is characteristic of hormone-responsive tumor behavior in women (6). Of the other cell lines that express both ER and AR, MFM-223 cells are growth inhibited by androgen and are insensitive to E, whereas T47D cells are sensitive to E and are minimally affected by androgen (7, 8). Because androgens have proven efficacious, although not without side-effects, in the treatment of breast cancer, the identification of additional models of androgen-inhibited human breast cancer would be of value in understanding the mechanism of growth inhibition by androgen. Such knowledge could potentially lead to new therapeutic approaches in the management of breast cancer.

The control of cell number is determined by a balance between cell proliferation and cell death. Recently, we observed that androgens down-regulate the expression of the antiapoptotic protooncogene bcl-2 in ZR-75-1 breast cancer cells, suggesting that modulation of apoptosis may be involved in androgen-induced growth inhibition (9). However, to our knowledge, the effect of androgens on cell cycle regulatory molecules in breast cancer has not been previously reported. In fact, most studies of the effects of steroid hormones on breast cancer cell cycle dealt exclusively with E and progestins (10, 11, 12).

Progression through the cell cycle is regulated by a group of proteins called cyclin-dependent kinases (CDK). The major activating mechanism of CDKs is cyclin binding. The activity of cyclin-CDK complexes is modulated by phosphorylation and interactions with CDK inhibitors (13, 14). The CDK inhibitors are classified into two families: the INK4 family, p15INK4a, p16INK4b, p18INK4c, and p19INK4d, which specifically inhibits CDK4 and CDK6; and the Cip/Kip family, p21Cip1, p27Kip1, and p57Kip2, which inhibits a wide range of cyclin-CDK complexes. The members of the Cip/Kip family can also act as assembly factors for CDK4/6-cyclin complexes (15, 16, 17, 18).

To gain additional insight into the mechanisms by which androgens inhibit breast cancer cell growth, we studied the effect of the natural androgen 5{alpha}-dihydrotestosterone (DHT) on the proliferation of CAMA-1 human breast cancer cells (19). We demonstrate that CAMA-1 cells express AR, and that DHT exerts a potent inhibitory effect on CAMA-1 cell proliferation. Androgen treatment led to an increase in the proportion of cells in the G1 phase of the cell cycle, with an increase in the relative proportion of hypophosphorylated retinoblastoma protein. Analysis of cell cycle regulatory proteins revealed that the antiproliferative effect of DHT is associated with a decrease in cyclin E-CDK2 kinase activity and an increase in the amount of p27Kip1 associated with this complex, suggesting a role for p27Kip1 in this response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
E2 and DHT were purchased from Steraloids (Wilton, NH). The antiandrogen hydroxyflutamide was provided by Dr. R. Neri (Schering-Plough Corp., Kenilworth, NJ).

Cell culture
CAMA-1 cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were propagated in RPMI 1640 medium containing 10% (vol/vol) FBS supplemented with 2 mM L-glutamine, 100 IU penicillin/ml, 50 µg/ml streptomycin, and 1 nM E2. All experimental protocols were performed between passages 35 and 76. Before treatments, the cells were plated in phenol red-free RPMI 1640 supplemented with 2 mM L-glutamine, 100 IU penicillin/ml, 50 µg streptomycin/ml, and 10% (vol/vol) dextran-coated charcoal-treated FBS. The concentrations of E2 and DHT used in each experiment are indicated in the corresponding figure legends.

Cell proliferation assay
For the dose-response experiments shown in Fig. 2Go, the number of cells was determined by measurement of DNA content as described previously (20, 21).



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  		Figure 2. Regulation of human CAMA-1 breast cancer cell growth by E2 and DHT. Three days after plating in stripped medium, CAMA-1 cells were exposed for 10 d to the indicated concentrations of E2 alone (A) or 0.1 nM E2 and the indicated concentrations of DHT (B). At the end of the incubation period, cell number was determined by measurement of DNA content. Data are expressed as the mean ± SEM of triplicate dishes. When the SEM overlaps with the symbol used, only the symbol is illustrated. Similar results were obtained in three different experiments.

 
Flow cytometry
Cells were fixed with 70% ethanol in PBS for at least 20 min at -20 C. The cells were then centrifuged and resuspended in 0.5 ml PBS containing ribonuclease A (40 U/ml) and propidium iodide (50 µg/ml). The cells were incubated at 37 C for 30 min and then left on ice for 30 min. Analysis of DNA content in propidium iodide-stained cells was performed using an EPICS XL pulse cytometer (Coulter, Hialeah, FL). Cells were excited at 488 nm (15 mW), and red fluorescence (measure of total DNA) was recorded at 590 nm with a long-pass filter. The percentages of cells in the G1, S, and G2/M phases of the cell cycle were determined using the MultiCycle computer program provided by Phoenix Flow Systems, Inc. (San Diego, CA).

Treatment effects on the proportion of cells in phases G1, S, and G2/M were analyzed by ANOVA for randomized block design, where each block corresponds to an independent experiment. Identical conclusions were obtained using a nonparametric ANOVA (Kruskal-Wallis test) that is insensitive to the underlying assumptions of the data (22). A posteriori pairwise comparisons among the three treatment groups were performed to maintain an experiment-wise type I error of 5% using Bonferroni adjustment (23). All analyses were performed using SAS software (SAS Institute, Inc., Cary, NC).

Immunoblotting
Proteins were separated on 12.5% SDS-polyacrylamide gels, except that 7.5% gels were used for retinoblastoma protein immunoblots. The proteins were electroblotted to 0.2-µm nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH), and the resulting blots were blocked at room temperature for 1 h in 20 mM Tris and 137 mM NaCl containing 5% (wt/vol) dried milk and 0.1% (vol/vol) Tween 20. Incubations with the following primary antibodies were performed at room temperature for 3 h: anti-AR monoclonal antibody MS-443 (NeoMarkers, Union City, CA), anti-retinoblastoma protein (anti-Rb) monoclonal antibody 14001A (PharMingen, San Diego, CA), anticyclin E monoclonal antibody 14591 (PharMingen), anti-p27Kip1 monoclonal antibody K25020 (Transduction Laboratories, Inc., Lexington, KY), anti-CDK2 polyclonal antibody SC-163 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-{alpha} tubulin monoclonal antibody SC-8035 (Santa Cruz Biotechnology, Inc.). After a 1-h incubation with peroxidase-conjugated antirabbit (Amersham Pharmacia Biotech, Little Chalfont, UK) or antimouse (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) antibodies, proteins were detected using the SuperSignal Ultra chemiluminescent detection system (Pierce Chemical Co., Rockford, IL). The proteins of interest were quantitated by scanning densitometry using BioImage 110S (Millipore Corp., Bedford, MA). Equal loading of protein samples was confirmed by quantitating the amount of {alpha}-tubulin or by staining the nitrocellulose membranes with Ponceau S.

Northern blot analysis
Total RNA was isolated from CAMA-1 cells using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Northern blot hybridization was performed under stringent conditions essentially as previously described (24). The cDNA fragment used as probe to detect p27Kip1 mRNA consisted of coding nucleotides 1–596. The blot was probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a loading control. The cDNA fragment used as probe to detect GAPDH mRNA consisted of the HindIII-XbaI fragment (coding nucleotides 195–743) derived from the GAPDH cDNA. The cDNA fragments were labeled with [{alpha}-32P]deoxy-CTP (NEN Life Science Products, Boston, MA) using the DECAprime 2 labeling kit (Ambion, Inc., Austin, TX). The blots were hybridized to radiolabeled p27Kip1 and GAPDH cDNAs sequentially without stripping the blots, because the two mRNAs differ significantly in size. Northern blots were exposed to Hyperfilm MP (Amersham Pharmacia Biotech) at -80 C using intensifying screens. X-Ray films were quantitated by scanning densitometry using Bioimage 110 S (Millipore Corp.).

Immunoprecipitation
The cells were washed twice with ice-cold PBS and then harvested in PBS by scraping. After centrifugation to remove PBS, the cells were lysed on ice for 15 min in Nonidet P-40 lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.5), 0.5% (vol/vol) Nonidet P-40, 50 mM NaF, 1 mM sodium orthovanadate, and 1 x Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). Insoluble material was removed by ultracentrifugation at 100,000 x g for 30 min (rotor TL 100.2, Beckman Coulter, Inc., Palo Alto, CA). Protein concentrations were measured using the DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Cell lysates containing 500 µg protein were precleared with protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4 C. CDK2-containing protein complexes were recovered by incubation with 1 µg anti-CDK2 polyclonal antibody (Santa Cruz Biotechnology, Inc., SC-163), whereas cyclin E-containing complexes were recovered using 5 µl anti-cyclin E polyclonal antibody (NeoMarkers, RB-012) at 4 C for 1 h. Control immunoprecipitations were performed using nonimmune rabbit Igs. Immune complexes were precipitated with protein A-Sepharose for 30 min at 4 C and washed four times with Nonidet P-40 lysis buffer. Two additional washes were performed with 50 mM HEPES (pH 7.5) containing 1 mM dithiothreitol. Each immunoprecipitate was split in two: 75% of the immunoprecipitate was used for SDS-PAGE analysis, and the remaining 25% of each immunoprecipitate was designated for the in vitro histone kinase assay.

In vitro histone kinase assay
The immunoprecipitates were resuspended in kinase buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM dithiotheitol, 2.5 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM NaF, 20 µM ATP, 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol), and 1 µg calf thymus histone H1 (Roche Molecular Biochemicals). Incubations were performed at 30 C for 30 min. The reaction products were separated by SDS-PAGE. Phosphorylated histone H1 was visualized by autoradiography, and x-ray films were quantitated by scanning densitometry using Bioimage 110 S (Millipore Corp.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CAMA-1 cells express the AR
To determine whether human CAMA-1 breast cancer cells would be a suitable model for the study of androgen action in breast cancer, we performed an immunoblot analysis using a monoclonal antibody against the human AR. As illustrated in Fig. 1Go, a band corresponding to the expected size of AR protein (110 kDa) was detected in CAMA-1 cell protein extracts as well as in ZR-75-1 cells that are known to express AR mRNA (24). On the other hand, AR protein was not detected in BT-20 breast cancer cells, which do not express AR (25, 26), confirming the specificity of the anti-AR antibody.



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  		Figure 1. AR expression in CAMA-1 breast cancer cells. Twenty micrograms of total protein extract from CAMA-1 cells were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-AR antibody. ZR-75-1 and BT-20 cell extracts were used as positive and negative controls for AR expression, respectively. Equal sample loading was confirmed by Ponceau S staining of the nitrocellulose membrane.

 
Androgens inhibit CAMA-1 breast cancer cell growth
It has been reported that CAMA-1 cells depend on E for cell maintenance and growth in culture, but the effects of androgens have not been characterized in detail (19). As not all breast cancer cell lines that express AR are growth inhibited by androgen, we compared the effects of E2 and DHT on CAMA-1 cell proliferation.

CAMA-1 cells are exquisitely sensitive to E2. In the experiment presented in Fig. 2AGo, CAMA-1 cells were cultured in the presence of increasing concentrations of E2 (1 x 10-15 to 1 x 10-6 M) for 10 d. E2 caused a dose-related increase in cell number. A maximal 6-fold increase in cell number was observed at 1 x 10-10 M E2, whereas the EC50 was achieved at 2 x 10-11 M E2. These results are consistent with the data published by Leung et al. (19), who first showed that E stimulates CAMA-1 cell proliferation.

To assess the effect of androgens on CAMA-1 proliferation, CAMA-1 cells were incubated with a maximally stimulating dose of E2 (0.1 nM) and increasing concentrations of DHT (1 x 10-14 to 1 x 10-6 M) for 10 d. As shown in Fig. 2BGo, DHT exerted a potent inhibitory effect on CAMA-1 cell proliferation. A 50% inhibition of E2-induced proliferation (IC50) was achieved at a concentration of 0.1 nM DHT, whereas 1 nM DHT caused a maximal inhibition of E2-induced proliferation. DHT did not inhibit CAMA-1 cell proliferation in the absence of E2 (data not shown). However, under our experimental conditions, CAMA-1 cells do not proliferate in the absence of E2, which suggests that they are already in a quiescent or growth-arrested state in the absence of stimulation by E or other mitogens.

Androgens induce a G1 cell cycle arrest through activation of AR
Androgens could conceivably inhibit E2-induced CAMA-1 cell proliferation by inducing cell cycle arrest. We therefore used flow cytometry to examine the effect of DHT on the cell cycle distribution of CAMA-1 cells. CAMA-1 cells were cultured for 48 h in medium containing E2 (0.1 nM) alone, E2 in combination with DHT (1 nM), or E2 in combination with DHT and the antiandrogen hydroxyflutamide (1 µM). As shown in Table 1Go, DHT treatment increased the proportion of cells in the G1 phase from 69% to 82%. This shift in the number of cells in G1 appeared to result from a decrease in the proportion of cells in S phase (19% to 9%). The DHT-induced changes in G1 and S phase cell populations were statistically significant (P < 0.001). The proportion of cells in G2/M did not change significantly. The accumulation of CAMA-1 cells in G1 phase induced by DHT was completely prevented by the addition of hydroxyflutamide, indicating that DHT-induced G1 cell cycle arrest is mediated through activation of AR.


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  		Table 1. DHT increases the proportion of cells in the G1 phase of the cell cycle through activation of the AR

 
DHT inhibits Rb phosphorylation
As Rb is a regulator of cell cycle progression, we next sought to determine whether androgens affect the state of Rb phosphorylation. CAMA-1 cells were grown in medium containing E2 (10-10 M) alone or E2 in combination with DHT (10-9 M) and harvested at the beginning of the experiment as well as 1, 6, 24, and 48 h after the addition of steroid hormones. As shown in Fig. 3Go, both phosphorylated Rb (pRb) and hyperphosphorylated Rb (ppRb) were detected before the addition of steroids. E2 treatment did not affect the relative amounts of pRb and ppRb over the course of the experiment (compare the lanes labeled -DHT). On the other hand, DHT treatment caused a significant reduction in the amount of ppRb relative to that of pRb, effectively increasing the proportion of hypophosphorylated Rb. This effect was manifest 24 h after the addition of DHT and is consistent with G1 cell cycle arrest. In addition, total pRb levels were lower in DHT-treated cells at 24 and 48 h. This change is consistent with the earlier observation by Buchkovich et al. (27) that pRb levels are highest in S-G2/M and lowest in G1.



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  		Figure 3. DHT increases the relative proportion of pRb. Three days after plating in stripped medium containing 0.1 nM E2, CAMA-1 cells were grown for 0, 1, 6, 24, and 48 h in medium containing E2 (10-10 M) alone or supplemented with DHT (10-9 M). Twenty micrograms of total protein extract were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-Rb antibody. Upper band, ppRb; lower band, pRb.

 
DHT up-regulates p27Kip1 in CAMA-1 cells
Retinoblastoma protein phosphorylation is thought to be initiated by cyclin D-CDK4/6 complexes and completed by cyclin E-CDK2 complexes. Thus, a number of CDK inhibitors could be involved in the androgen-induced inhibition of Rb phosphorylation. We initially focused our attention on p27Kip1, because it has been implicated in G1 arrest induced by a number of antimitogenic hormones, including TGFß (28, 29, 30), progesterone (12), and vitamin D (31), in other cell types. The effect of DHT on p27Kip1 expression was evaluated by Western blot analysis. CAMA-1 cells were grown in medium containing E2 (10-10 M) alone or E2 in combination with DHT (10-9 M) and harvested 24 and 48 h after the addition of steroids. DHT treatment resulted in a reproducible increase in p27Kip1 protein. Based on densitometric quantification of p27Kip1 protein levels in at least four different experiments, the level of p27Kip1 protein detected in DHT-treated cells harvested 24 h after the addition of DHT was, on the average, 1.5-times that in control cells, whereas the level of p27Kip1 protein detected in DHT-treated cells harvested 48 h after the addition of DHT was, on the average, 2.8 times that in control cells (Fig. 4AGo). To determine whether the effect of DHT on p27Kip1 protein levels was attributable at least in part to an effect at the mRNA level, we examined the effect of DHT on p27Kip1 transcript levels. Northern blot analysis indicated that p27Kip1 mRNA levels were slightly higher (1.6-fold) than those in control cells after 48 h of DHT treatment (Fig. 4BGo). We did not detect any change in p21 protein levels or in the mRNA levels of the other cyclin-dependent kinase inhibitors in DHT-treated CAMA-1 cells (data not shown).



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  		Figure 4. DHT up-regulates p27Kip1 levels in CAMA-1 cells. A, Three days after plating in stripped medium containing 0.1 nM E2, CAMA-1 cells were grown for 24 and 48 h in medium containing E2 (10-10 M) alone or supplemented with DHT (10-9 M). Twenty micrograms of total protein extract were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-p27Kip1 antibody. The membrane was incubated with the anti-{alpha}-tubulin antibody as a loading control. B, Total RNA was isolated from CAMA-1 cells grown for 24 and 48 h in medium containing E2 (10-10 M) alone or supplemented with 10-9 M DHT. The complete coding p27Kip1 cDNA fragment was used as a probe for the Northern analysis. The blot was probed for GAPDH mRNA as a control.

 
DHT increases the amount of p27Kip1 associated with cyclin E-CDK2
The role of the CDK inhibitor p27Kip1 in cell cycle control is complex. p27Kip1 is known to contribute to the formation of catalytically active cyclin D/CDK complexes (15, 16, 17, 18), whereas p27Kip1-induced growth inhibition is linked to its ability to antagonize the activity of cyclin E-CDK2 that results from displacement of p27Kip1 from cyclin D/CDK complexes (29, 30). We therefore performed immunoprecipitation experiments to determine whether the increase in p27Kip1 protein observed in DHT-treated cells resulted in an increase in the amount of p27Kip1 associated with cyclin E-CDK2 complexes. CAMA-1 cells were cultured in medium containing E2 (10-10 M) alone or E2 in combination with DHT (10-9 M). Cells were harvested 24 and 48 h after the addition of steroids, and p27Kip1-containing cyclin E and CDK2 complexes were immunoprecipitated using antibodies against CDK2 or cyclin E. The immunoprecipitates were then processed for Western blot analysis.

As shown in Fig. 5Go, the anti-CDK2 immunoprecipitates of DHT-treated cells contained significantly more p27Kip1 protein than did immunoprecipitates of cells treated with E2 alone. Based on densitometry data averaged from at least three separate experiments, the anti-CDK2 immunoprecipitates of DHT-treated cells harvested 24 h after the addition of steroid hormones contained approximately 2.8 times more p27Kip1 than those of control cells, whereas the anti-CDK2 immunoprecipitates of DHT-treated cells harvested 48 h after the addition of steroid hormones contained approximately 10 times more p27Kip1 than those of control cells.



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  		Figure 5. DHT increases the amount of p27Kip1 associated with cyclin E-CDK2. Three days after plating in stripped medium containing 0.1 nM E2, CAMA-1 cells were grown for 24 and 48 h in medium containing E2 (10-10 M) alone or supplemented with DHT (10-9 M). Immunoprecipitates using anti-CDK2 or anti-cyclin E antibody were prepared from whole cell lysates of CAMA-1 cells grown for the indicated periods of time. Each immunoprecipitate was separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-p27Kip1, anti-cyclin E, and anti-CDK2 antibodies.

 
Similarly, the anticyclin E immunoprecipitates of DHT-treated cells contained significantly more p27Kip1 protein than those of cells treated with E2 alone. The anticyclin E immunoprecipitates of DHT-treated cells harvested 24 and 48 h after the addition of steroid hormones contained approximately 2.2 and 10.5, respectively, times more p27Kip1 than those of control cells.

DHT inhibits cyclin E-CDK2 kinase activity
To assess the functional consequences of the association between p27Kip1 and cyclin E-CDK2 complexes, we tested the kinase activity of cyclin E-CDK2 complexes isolated from DHT-treated CAMA-1 cells using the canonical CDK substrate histone H1 (32). Typical results are presented in Fig. 6Go. The histone H1 kinase activity of cyclin E-CDK2 complexes was markedly reduced in DHT-treated cells. In fact, the CDK2-associated histone kinase activity of cyclin E-CDK2 complexes isolated from DHT-treated cells was 83% and 94% lower, respectively, than that of control cells after 24 and 48 h of treatment. Similarly, the cyclin E-associated histone kinase activity of cyclin E-CDK2 complexes isolated from DHT-treated cells was 65% and 75% lower than that of control cells at 24 and 48 h, respectively. Taken together, these results suggest that inactivation of the cyclin E-CDK2 complex by p27Kip1 may contribute to androgen-induced growth inhibition of breast cancer cells.



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  		Figure 6. DHT inhibits cyclin E-CDK2-dependent histone kinase activity. The CDK2 and cyclin E immunoprecipitates were used for the kinase assay. Kinase activity was assayed using a histone H1 substrate. Phosphorylated histone H1 was visualized by autoradiography after SDS-PAGE. The amount of phosphorylated histone H1 was quantified by densitometry and compared with the level measured in immunoprecipitates from control cells, which was set at 100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A better understanding of the mechanism of androgen-induced growth inhibition in human breast cancer cells can lead to long-term benefits for the treatment and/or prevention of breast cancer. It is hoped that the identification of androgen-responsive genes in human breast cancer cells can potentially result in the development of new therapeutic agents that could mimic androgen action without the masculinizing effects currently associated with androgen therapy. In keeping with this objective we report two major findings. Firstly, we show that CAMA-1 cells can be used to study androgen action. Secondly, we provide experimental data suggesting that androgen-induced growth inhibition in CAMA-1 cells is mediated at least in part by the cyclin-dependent kinase inhibitor p27Kip1.

Until recently, the majority of studies aimed at elucidating the mechanism of androgen-induced growth inhibition in breast cancer cells were performed in ZR-75-1 cells, because this is the only cell line that displays the desired characteristics, i.e. ZR-75-1 cells express AR and ERs, they are stimulated to proliferate by E, and androgens inhibit both basal and E-induced proliferation in vitro as well as in vivo (6, 33). In contrast, T-47D cells, which also express both receptors, exhibit androgen-induced gene expression, but AR activation does not lead to strong growth inhibition (34, 35). MDA-MB-453 cells, on the other hand, express AR, but not ER (36). The first set of experiments reported here clearly demonstrates that androgens exert a potent inhibitory effect on CAMA-1 cell proliferation. CAMA-1 cells therefore represent a useful model in which to investigate the molecular mechanisms of AR action in breast cancer.

Previous data have shown that androgens and E exert antagonistic effects on several parameters in breast cancer cells. For example, androgens block E-induced cathepsin D and pS2 gene expression as well as cell proliferation in ZR-75-1 cells (6, 9, 37). Despite the identification of a number of androgen-responsive genes, the mechanism of androgen-induced growth inhibition remains undetermined. Androgens have been shown to down-regulate ER mRNA levels in ZR-75-1 cells (38). In addition, androgens down-regulate Bcl-2 expression in ZR-75-1 cells, which suggests that androgens may modulate apoptosis in breast cancer cells (9). Furthermore, the identification of UDP-glucose dehydrogenase as an androgen-responsive gene has raised the hypothesis that androgens could inhibit breast cancer cell proliferation by modulating sex steroid metabolism (24). However, these hypotheses remain to be tested experimentally.

In this paper we report data suggesting that p27Kip1 is implicated in the androgen-induced growth inhibition of breast cancer cells. Androgens induced G1 cell cycle arrest associated with Rb dephosphorylation in CAMA-1 cells. It is of interest to note that total Rb levels decreased in CAMA-1 cells after exposure to DHT. This suggests that other Rb-related pocket proteins may also play a role in DHT-induced growth arrest in CAMA-1 cells. In fact, Carroll et al. (39) found that Rb and p107 protein levels decreased by at least 75% 48 h after treatment of MCF-7 cells with the nonsteroidal ER antagonist ICI 182780. Conversely, p130 protein levels increased approximately 4-fold during the same period. In ICI 182780-treated MCF-7 cells, the increase in p130 levels coincided with the appearance of p130-E2F4 complexes that are characteristic or at least suggestive of a quiescent or differentiated state. Similar mechanisms may be involved in DHT-induced inhibition of CAMA-1 cell proliferation.

CAMA-1 cells treated with androgen exhibited an increase in the amount of p27Kip1 protein associated with cyclin E-CDK2-containing complexes and lower cyclin E-CDK2-dependent histone kinase activity. Although it can act as an assembly factor for active CDK4/6-cyclin complexes, p27Kip1 is a potent inhibitor of cyclin E-CDK2 kinase activity. Cyclin E-CDK2 can phosphorylate Rb and play a central role in the G1/S transition (14). Inhibition of cyclin E-CDK2 kinase activity by p27Kip1 may play an important role in the growth inhibitory effect of androgens in breast cancer cells. However, modulation of the activity of other cyclin/CDK complexes can also be implicated in the effect of androgens.

The present data indicate that androgens cause an up-regulation of p27Kip1 protein levels in CAMA-1 cells with a modest induction of the mRNA after 48 h of exposure to DHT. These results are in agreement with previous studies indicating that p27Kip1 levels are mainly regulated posttranscriptionally (40, 41, 42). Others have also observed regulation of p27Kip1 expression at the RNA level. In fact, Robson et al. (43) found that TGFß1 induced a marked up-regulation of p27Kip1 mRNA levels in primary prostatic epithelial cells. However, the results of the immunoprecipitation experiments argue strongly that the major effect of androgens on p27Kip1 protein function results from recruitment of p27Kip1 to cyclin-CDK complexes. In fact, the DHT-induced increase in the relative amount of cyclin E-CDK2-associated p27Kip1 was greater than the DHT-induced increase in total p27Kip1 levels. This result is in agreement with recent reports showing that p27Kip1 could inactivate cyclin E-CDK2 independently of p27Kip1 accumulation (12, 44).

The mechanisms of regulation of p27Kip1 localization within the cell by DHT remain unknown at the present time, but could involve D-type cyclins, c-Myc and other unknown p27Kip1-sequestering proteins (45, 46, 47, 48, 49). It is also possible that other cyclin-dependent kinase inhibitors could cooperate with p27 to induce G1 arrest in response to androgen in CAMA-1 cells. Reynisdottir et al. (50) previously reported that the cyclin-dependent kinase inhibitor p15INK4B displaces p27Kip1 from CDK4 to CDK2-containing complexes in TGFß-treated mink lung epithelial cells. Swarbrick et al. (12) demonstrated a similar role for p18INK4C in progestin-induced growth arrest in T-47D human breast cancer cells.

Deregulation of cyclin E-CDK2 kinase activity has been observed in breast tumor cells, and increased expression of cyclin E has been linked to more aggressive tumors and decreased patient survival (51, 52, 53, 54, 55, 56). Three studies conducted in women with primary breast cancer showed that lower p27Kip1 protein expression was associated with tumor progression and poor prognosis (56, 57, 58). Taken together, these data suggest that p27Kip1 may play a critical role in the control of breast cancer cell proliferation. The present data suggest that p27Kip1 may be an essential mediator of androgen-induced inhibition of breast cancer cell proliferation, at least in CAMA-1 cells. Additional experiments will be required to determine whether modulation of p27Kip1 is involved in the beneficial effects of androgens observed on breast cancer in experimental cell models (6, 8, 33) and in women treated with androgenic compounds (4).


   Acknowledgments
 
We thank A. Fournier and C. Fillion for their help, the members of our group for helpful discussions, and the members of the CHUL Research Center Illustration Service for artwork. We are also grateful to R. Faure for technical advice on the histone kinase assay, and B. Candas for statistical analyses.


   Footnotes
 
This work was supported in part by grants from the Canadian Breast Cancer Research Initiative (no. 6292 and 9430) and a fellowship from Le Fonds de Recherche en Santé du Québec (to C.L.), Endorecherche, and a scholarship from Le Fonds de Recherche en Santé du Québec (to J.L.).

Abbreviations: CDK, Cyclin-dependent kinase; DHT, 5{alpha}-dihydrotestosterone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pRb, phosphorylated retinoblastoma protein; ppRb, hyperphosphorylated retinoblastoma protein; Rb, retinoblastoma protein.

Received February 12, 2001.

Accepted for publication June 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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