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Endocrinology Vol. 140, No. 7 3282-3291
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential Posttranscriptional Regulation of Androgen Receptor Gene Expression by Androgen in Prostate and Breast Cancer Cells1

Bu B. Yeap, Romano G. Krueger and Peter J. Leedman

Laboratory for Cancer Medicine and University Department of Medicine (B.B.Y., P.J.L.), University of Western Australia; Royal Perth Hospital, Perth, Western Australia 6000; Flow Cytometry Unit (R.G.K.), Royal Perth Hospital, Perth, Western Australia 6000

Address all correspondence and requests for reprints to: Dr. Peter J. Leedman, University Department of Medicine, 4th Floor, Medical Research Foundation Building, Rear, 50 Murray Street, Perth, Western Australia 6001. E-mail: peterl{at}cyllene.uwa.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens, via the androgen receptor (AR), modulate the growth and proliferation of prostate and breast cancer cells. However, the molecular mechanisms underlying the regulation of AR gene expression by androgen in these cells remain to be fully elucidated. To explore differences in AR gene expression between these hormone-responsive tumor cell types, we studied androgen-responsive LNCaP prostate cancer and AR positive MDA453 breast cancer cells. Dihydrotestosterone (DHT) 10 nM increased LNCaP cell proliferation and the proportion of LNCaP cells in S-phase of the cell cycle but inhibited MDA453 cell proliferation and reduced the proportion of MDA453 cells in S-phase of cell cycle. In both these cell lines, DHT decreased total AR messenger RNA (mRNA) but increased AR protein. In LNCaP cells, DHT down-regulated AR mRNA transcription but stabilized AR mRNA. In contrast, in MDA453 cells, DHT had no effect on AR mRNA transcription but destabilized AR mRNA. In summary, transcriptional down-regulation induced by androgens in LNCaP cells results in down-regulation of steady-state AR mRNA despite an androgen-induced increase in AR mRNA stability. However, in MDA453 cells, posttranscriptional destabilization of AR mRNA appears to be the predominant mechanism resulting in down-regulation of AR mRNA by androgen. These results demonstrate cell-specific and divergent regulation of AR mRNA turnover by androgen and identify a novel pathway of androgen-induced posttranscriptional destabilization and down-regulation of AR mRNA in human breast cancer cells. Furthermore, these data establish an important role for posttranscriptional pathways in the regulation of AR gene expression by androgen in human prostate and breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTATE CANCER is a leading cause of male cancer mortality and morbidity in Western societies (1, 2, 3). Prostate cancers are characterized by an initial stage of androgen dependency during which growth is enhanced by the presence of androgen and repressed by androgen deprivation or blockade, with even metastatic disease being readily responsive to hormonal manipulation (4, 5, 6). Unfortunately, androgen responsiveness is transient with ultimate progression of disease despite continued androgen deprivation (7). Androgens such as dihydrotestosterone (DHT) interact with the androgen receptor (AR), the ligand-receptor complex functioning as a nuclear transcription factor regulating the expression of androgen-dependent genes (8, 9). Recently, mutations in the AR gene have been reported in patients with prostate cancer (10, 11). Mutations such as the codon 877 Thr to Ala mutation in exon H have been identified in prostatic tissue from patients with prostate cancer and in the human LNCaP prostate cancer cell line (12, 13). This mutation alters the ligand binding specificity of the AR enhancing the binding of progestagens and estrogen, and increases transactivation by dehydroepiandrosterone (14, 15). Similarly, the Val to Met mutation at position 715 confers responsiveness to adrenal androgens as well as progesterone, and the Arg to Leu mutation at position 726 results in a mutant AR activated by estrogen (16, 17). The identification of these several AR mutations provide potential mechanisms by which prostate cancer cells may exhibit altered biological activity and escape the effect of antiandrogen therapy. An improved understanding of the biology of mutant AR gene expression may thus provide valuable insight into mechanisms of malignant progression in prostate cancer.

Breast cancer in women provides a complementary model of hormone-responsive cancer. Expression of estrogen receptors (ERs) in breast cancer tissue correlates with responsiveness to hormonal therapy and improved prognosis (18, 19). AR expression in breast cancer tissue samples has been associated with an improved response to hormone therapy and longer survival in patients with breast cancer (20, 21). The AR is detectable in the majority of tumor specimens from patients undergoing mastectomy for breast cancer, AR and ER tending to be coexpressed (22). Recent in vitro studies have demonstrated direct and indirect growth inhibitory effects of androgen on several AR positive (AR+) breast cancer cell lines (23, 24, 25, 26, 27), suggesting an independent effect of androgens, via the AR, on breast cancer progression. Consequently, it would be of interest to establish whether mechanisms underlying the regulation of AR gene expression differ between prostate compared with breast cancer cells.

The AR is autoregulated by androgen, which reduces AR messenger RNA (mRNA) in both rat tissues and LNCaP cells (28, 29, 30). Repression of AR mRNA expression in LNCaP cells has been attributed to reduced transcription with AR mRNA stability being paradoxically increased (31). However, different authors have found conflicting effects of androgen on AR protein expression, ranging from an increase in absolute AR protein content (30), to either posttranslational modification without alteration in AR protein level (31) or stabilization of the AR protein (32, 33). Androgens down-regulate AR mRNA in the T47D and MDA453 breast cancer cell lines (34, 35), but no data are available for the effect of androgens on AR mRNA transcription or stability in breast cancer cells. We used LNCaP and MDA453 cells to determine the relative importance of transcriptional and posttranscriptional mechanisms on androgen regulated AR gene expression in both prostate and breast cancer cells. Our work in LNCaP cells shows that androgens regulate AR mRNA transcription and mRNA turnover for a net decrease in AR mRNA, accompanied by increased AR protein expression and cellular proliferation. Remarkably, however, in MDA453 cells, despite producing a similar increase in AR protein expression, androgens have no effect on transcription and induce an opposite effect on AR mRNA turnover and cellular proliferation. These studies provide novel insight into the differential regulation of AR gene expression in each tumor cell type and establish a central role for posttranscriptional events in the process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
LNCaP and MDA453 cells were obtained from the American Type Culture Collection (Rockville, MD) and used for experiments within ten passages of the original stock. Cells were grown in phenol red-free RPMI medium 1640 supplemented with 10% FCS [dextran-charcoal stripped FCS (DCS FCS)], for experiments), 50 U/ml penicillin G and 50 µg/ml streptomycin (Gibco BRL Life Technologies, Grand Island, NY). DHT and actinomycin D (Act D) were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell proliferation and cell cycle analysis
For cell proliferation assays, cells were trypsinized and counted using an automated hematology analyzer (Technicon H1, Technicon Instruments Corp., Tarrytown, NY). For cell cycle analysis, DHT-treated and control cells were harvested by trypsinization, permeabilized, and stained for DNA overnight at 4 C in 0.2% Triton X-100 with 50 µg/ml propidium iodide. RNase A 100 µg/ml was added at room temperature 2 h before analysis by flow cytometry (Coulter EPICS XL-MCL, Beckman Coulter, Inc., Hialeah, FL). MultiPlus AV MultiParameter Data Analysis Software (Phoenix Flow Systems, San Diego, CA) was used to determine the proportion of cells in the various phases of cell cycle.

Northern analysis
For RNA extraction, cells were lyzed in a buffer containing 4 M guanidinium thioisocynate, 0.5% lauroylsarcosine, 25 mM Na citrate, pH 7.0, 0.2 M Na acetate, pH 4.1, and 4 mM 2-mercaptoethanol followed by phenol-chloroform extraction and isopropanol precipitation. Northern gel electrophoresis was performed with 10–12 µg total RNA per lane in a denaturing gel in 1 x MOPS buffer followed by overnight blot transfer in 10 x SSC to nylon membrane (Hybond N+, Amersham Life Sciences, Little Chalfont, Buckinghamshire, UK). A linearized Bluescript vector containing the 713 bp HindIII/EcoRI fragment (nucleotides 1850 to 2563) of the AR complementary DNA (cDNA) sequence (36) was used to synthesize a 32P-labeled complementary RNA (cRNA) probe using the T7 RNA polymerase promoter in a 10-µl reaction mix containing 100 µCi 32P UTP (Amersham Life Sciences), 1 µg linearized plasmid, 1 x transcription buffer, 0.5 mM ATP, CTP, GTP, 20 mM dithiothreitol (DTT), 40 U RNAsin, and 19 U T7 RNA polymerase (Promega Corp., Madison, WI) at 37 C for 45 min. Hybridization was performed overnight at 65 C in 50% formamide, 5 x SSC, 5 x Denhardt’s solution, 5% SDS, 50 mM NaPO4, pH 7.0, and 500 µg/ml yeast RNA followed by sequential washes in 1 x SSC/1% SDS, 0.5 x SSC/0.5%SDS and 0.1 x SSC/0.1% SDS. A predominant AR mRNA of 10.5 kb was detected, as described previously. Membranes were stripped and reprobed with a rat 18S ribosomal RNA cDNA probe as a loading control. Quantitation was performed using a PhosphorImager 445 SI with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Nuclear run-on assays
Nuclei were prepared by washing cells in ice-cold PBS followed by two rounds of lysis in a buffer containing 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2 and 0.5% Nonidet P-40 and centrifugation at 800 rpm for 5 min at 4 C. The final pellet of 5–10 x 106 nuclei was resuspended in a 100 µl volume of 50 mM Tris-HCl, pH 8.3, 40% glycerol, 0.1 mM EDTA, and 0.1 mM DTT and stored at -80 C. Transcription was performed in a 200 µl volume of nuclei in 5 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 150 mM KCl, 50 mM DTT, 0.5 mM each of ATP, CTP, GTP, and 100 µCi 32P UTP for 30 min at 30 C. Labeled RNA was phenol-chloroform extracted in GTC/Na acetate with 30 µg/ml yeast transfer RNA and precipitated with isopropanol before resuspension in 10 mM TES (N-Tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid), pH 7.4, 10 mM EDTA, 0.2% SDS and 0.3 M NaCl. Amersham Life Sciences Hybond N+ filters were slot blotted with an excess of detection cDNA probes (10 µg 3.1 kb AR cDNA sequence in linearized pBluescript vector, 2.5-µg, 1.1-kb r18S plasmid, and 10 µg 1.3-kb GAPDH plasmid in separate slots for normalizing controls). Hybridization was performed in 50% formamide, 5 x SSC, 5 x Denhardt’s solution and 1% SDS at 42 C for 48 h.

Western immunoblotting
Cells were lysed in a buffer containing 0.5% deoxycholic acid, 0.5% SDS, 0.5% Nonidet P40, 50 mM NaCl, 10 mM Tris, pH 7.6, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride before centrifugation at 13,000 rpm for 10 min at 4 C and storage in aliquots at -80 C. Protein concentration was quantitated in triplicate using the Bio-Rad Laboratories, Inc. (Hercules, CA) assay dye and Microplate Reader 450 at a wavelength of 595 nm. SDS-PAGE was performed using 20 µg of protein lysate per lane followed by blot transfer onto nitrocellulose membrane (Optitran BA-S 85, Schleicher & Schuell, Inc., Dassel, Germany) in Tris 20 mM, glycine 150 mM, methanol 20%, and SDS 0.02%. Membranes were blocked with 5% skim milk powder in TBS-T (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) before sequential incubations with rabbit polyclonal antibody to human AR in a 1:1000 dilution (Signet PG21, Dedham, MA) and HRP conjugated secondary antibody in a 1:2000 dilution (Sigma Chemical Co.). Enhanced chemiluminescence (ECL) was performed with Amersham Life Science ECL Western blotting detection reagents according to the manufacturer’s protocol. For a loading control, blots were washed in TBS-T and reprobed with a mouse polyclonal antibody to poly(A)-binding protein (PABP) (37) in a 1:600 dilution followed by HRP conjugated mouse secondary antibody in a 1:20,000 dilution (Sigma Chemical Co.) before ECL using the Amersham Life Science ECL Plus protocol.

35S-labeling and immunoprecipitation
Cells were prepared for experiments as above. After a 5-h incubation in the presence or absence of DHT 10 nM, the media were aspirated, cells were washed with PBS and replaced with methionine-free, phenol red-free RPMI medium 1640 with 10% DCS FCS and antibiotics ± DHT for 2 h. We added 200 µCi/ml 35S methionine (Promix, Amersham Life Sciences) harvested cells after 1 h of 35S incorporation by twice washing in cold PBS and lysis as described previously. A 2 x 107 cpm protein lysate was used for immunoprecipitation overnight at 4 C with AR antibody at a dilution of 3:500 followed by addition of protein A Sepharose beads (1:10 volume, Pharmacia Biotech, Uppsala, Sweden). Beads were washed three times in lysis buffer, once in cold PBS, and boiled for 4 min in 80 mM Tris, pH 6.8, 7.5% glycerol, 2% SDS, 0.2% mercaptoethanol, and 24 µg/ml bromophenol blue before SDS-PAGE. Additional experiments were performed using the rabbit polyclonal AR antibody AR52 (32).

Data analysis and statistics
Statistical analysis was performed using General Linear Models. ANOVA was performed to assess the significance of treatment (DHT) x time interactions. Where appropriate, means comparisons at specific time points were made using Student’s t test. A P value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgens exert opposing effects on cell proliferation and differentially modulate cell cycle progression in LNCaP and MDA453 cells
The human prostate cancer cell line LNCaP, which contains a mutant AR (877 Thr to Ala) (38, 39), has been extensively used as a model of androgen-dependent prostate cancer (40). The human breast cancer cell line MDA453 provides a suitable model of androgen action as it expresses a comparable level of AR in the absence of estrogen receptor-{alpha} (ER{alpha}) and progesterone receptor (35). Thus, these two cell lines provide an ideal comparative system to identify differences in the mechanisms involved in androgen-induced regulation of AR gene expression. We first sought to characterize the effect of DHT on LNCaP and MDA453 cell proliferation. LNCaP cells were seeded in equal density in identical tissue culture plates and cultured in 10% DCS FCS for 48 h before the addition of DHT with control cells maintained in androgen-depleted condition. Media were replaced at the time of hormone addition and then left for the duration of the experiment. DHT-treated and control cells were harvested and counted at progressive time points to determine the effect of DHT on cell proliferation. In LNCaP cells, DHT 10 nM significantly increased cell proliferation (ANOVA; DHT x time interaction 0–72 h: P = 0.002, means comparison after 72 h: P < 0.01), consistent with the androgen dependence of LNCaP cells (38, 40, 41) (Fig. 1AGo). A comparable proliferative effect of DHT was demonstrable in LNCaP cells at a 1 nM concentration (data not shown). A similar protocol was employed in MDA453 cells with a shorter 24 h period of growth in media with 10% DCS FCS before addition of DHT to prevent the faster growing MDA453 cells from becoming overconfluent during the course of experiments. In MDA453 cells, DHT 10 nM reduced cell proliferation (Fig. 1BGo). The effect of DHT appeared to be greatest after 24 h (means comparison at 24 h: P = 0.005) with a lesser effect thereafter (ANOVA; DHT x time interaction 0–72 h: P = 0.07). The more rapid proliferation of MDA453 cells both in the absence and presence of DHT compared with DHT-treated and control LNCaP cells reflects the faster growth of the MDA453 cell line. To extend these observations, we analyzed the effect of androgen on cell cycle progression. Androgen deprivation resulted in a low proportion of LNCaP cells in S-phase of the cell cycle. DHT 10 nM significantly increased the proportion of LNCaP cells in S-phase by greater than 2-fold (Fig. 2AGo). In marked contrast, in MDA453 cells DHT 10 nM significantly reduced the percentage of cells in S-phase of the cell cycle (Fig. 2BGo). The faster growth of the MDA453 cells in 10% DCS FCS was reflected by the greater proportion of these cells in S-phase of cell cycle both in the presence and absence of DHT. These observations indicate differential effects of androgens on cell proliferation and cell cycle modulation in these two cell types. DHT increases LNCaP cell proliferation by increasing the proportion of cells actively engaged in mitosis and inhibits MDA453 cell proliferation by reducing the proportion of cells entering S-phase of the cell cycle.



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  		Figure 1. Androgens increase proliferation of LNCaP cells but inhibit proliferation of MDA453 cells. Cells were cultured ± DHT and harvested at sequential time points. Cell numbers are shown relative to the cell count at time 0 h = 1.0. A, LNCaP cells ± 10 nM DHT from 0 to 72 h. B, MDA453 cells ± 10 nM DHT from 0 to 72 h. Data are mean ± SE from at least two experiments in quadruplicate for each cell line (ANOVA; **, P < 0.01; #, P = 0.07: DHT vs. control, means comparisons; (a), (b) P < 0.01: DHT vs. control).

 


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  		Figure 2. Androgens increase the proportion of cells in S-phase of cell cycle in LNCaP cells but have an opposite effect in MDA453 cells. Cells were cultured in the presence and absence of 10 nM DHT and harvested at specific time points. Permeabilization and DNA staining with propidium iodide was carried out before flow cytometric analysis. A, S-phase fraction of LNCaP cells following 24 h treatment with DHT 10 nM. B, S-phase fraction of MDA453 cells following 24 h treatment with DHT 10 nM. Data are mean ± SD from experiments in triplicate repeated twice for each cell line (means comparisons; (a) to (d) P < 0.01: DHT vs. control).

 
Androgens down-regulate steady-state AR mRNA in LNCaP and MDA453 cells
Having established that DHT at 10 nM concentration exerted significant and differing effects on LNCaP and MDA453 cellular proliferation and cell cycle modulation, we proceeded to examine the regulation of AR mRNA by androgen in these cells. Cells were cultured under identical experimental conditions as above and RNA extracted at sequential time points. Northern analysis identified AR mRNA bands at 10.5 and 8.0 kb, consistent with previous reports (29, 31, 34) (Fig. 3AGo). The major message was located at 10.5 kb, and this band was used for subsequent quantitation of AR mRNA. Incubation of the cells with DHT 10 nM significantly decreased steady-state AR mRNA (10.5 kb band) with AR mRNA levels at 2 and 8 h being 60 and 55% of levels found in control cells, respectively (Fig. 3BGo). This decrease was maintained at 24 and 48 h (70 and 45% of control, respectively) with some loss of effect apparent by 72 h (Fig. 3BGo) (ANOVA; DHT x time interaction 0–48 h: P = 0.001). When DHT 1 nM was used, down-regulation of AR mRNA to 75% of control was found with similar loss of effect occurring at 72 h (data not shown). Similar down-regulation of AR mRNA was found when the synthetic androgen mibolerone at doses of 3.3 and 10 nM was substituted for DHT, except that down-regulation persisted at 72 h (data not shown). The 10 nM concentration of DHT gave optimal responses for AR mRNA and was thus continued for subsequent experiments, its effects on cell proliferation and cell cycle modulation having previously been determined. MDA453 cells were cultured in identical conditions and left in androgen-free conditions for 24 h before the addition of DHT. A similar effect was noted in MDA453 cells with a decrease in AR mRNA to 60% of control levels after 8 and 24 h incubation with DHT 10 nM (ANOVA; DHT x time interaction 0–24 h: P = 0.04) (Fig. 3CGo).



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  		Figure 3. Androgens down-regulate AR mRNA levels in prostate and breast cancer cells. Cells were incubated ± 10 nM DHT and harvested at sequential time points. Total RNA was extracted and subjected to Northern analysis. A, AR mRNA was detected using an AR cRNA probe that identified a major message of 10.5 kb and a subsidiary message of 8.0 kb with a rat 18S rRNA cDNA probe for a loading control. RNA size markers are shown to the right of the figure. B, LNCaP cells ± DHT 10 nM for 0 to 72 h. C, MDA453 cells ± DHT 10 nM for 0 to 24 h. Values of AR mRNA for DHT treated cells are shown relative to the amount in control cells harvested at equivalent time points. Data are mean ± SD from representative experiments performed in triplicate at least twice in each cell line (ANOVA; DHT x time interactions: **, P < 0.01; *, P < 0.05).

 
Androgens reduce transcription of AR mRNA in LNCaP cells but not in MDA453 cells
To determine the effect of DHT on AR transcription in both cell lines, nuclear run-on assays were performed. LNCaP and MDA453 cells were treated with DHT 10 nM for between 8 and 24 h before extraction of nuclei for run-on assays. Transcription of AR mRNA was significantly reduced to 50% of control levels after 8 h pretreatment with DHT in LNCaP cells (means comparison at 8 h; P = 0.04 DHT < control) (Fig. 4AGo). A lesser effect of DHT was found after 24 h treatment with DHT (Fig. 4AGo). In contrast, there was no effect of DHT on AR mRNA transcription in MDA453 cells similarly treated for 8 h with comparable quantities of AR mRNA synthesized both in the presence and absence of DHT (Fig. 4BGo). These data confirm the contribution of DHT-induced transcriptional down-regulation in LNCaP cells. However, the data also suggests that in MDA453 cells, DHT was modulating AR mRNA predominantly via posttranscriptional changes in AR mRNA turnover.



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  		Figure 4. Androgens reduce transcription of AR mRNA in LNCaP but not in MDA453 cells. Nuclei were isolated from cells treated ± 10 nM DHT, nuclear run-on assays performed and transcribed labeled RNA hybridized to filters blotted with cDNA detection probes in individual slots. For AR mRNA, a 3.1-kb human AR cDNA was used as a probe and rat 18S ribosomal RNA cDNA used for a loading control. Transcribed AR mRNA is shown as the ratio of normalized AR mRNA detected in nuclei from DHT treated cells relative to that found in nuclei from control cells harvested at equivalent time points. A, LNCaP cells with 10 nM DHT for 8 and 24 h. (means comparisons; (a) P < 0.05: DHT < control). B, MDA453 cells with DHT for 8 h. Data are shown as means with error bars denoting the entire range of values obtained from at least two experiments performed in duplicate for each cell line.

 
Androgens induce divergent effects on AR mRNA stability in LNCaP and MDA453 cells
To assess the effect of DHT on AR mRNA stability, LNCaP and MDA453 cells were treated with DHT 10 nM before addition of the transcriptional inhibitor actinomycin D (Act D). Figure 5AGo shows that AR mRNA half-life in LNCaP cells was significantly increased in the presence of DHT by greater than 2-fold from 5.5 to >12 h (ANOVA; P < 0.001). The DHT induced approximately 2-fold stabilization of AR mRNA was observed with preincubation times for DHT 10 nM of 2 and 8 h before addition of Act D. In contrast to the stabilization of AR mRNA documented in LNCaP cells, in MDA453 cells, AR mRNA half-life was significantly reduced from 3 h in control cells to 2 h in cells treated with DHT 10 nM for 8 h before addition of Act D (ANOVA; P < 0.001) (Fig. 5BGo). Thus, androgens induced divergent effects on AR mRNA turnover in these two cell lines. In conjunction with the transcriptional data, these results suggest fundamental differences in the mechanisms underlying AR mRNA autoregulation by androgen in these two cell types. In LNCaP cells, the down-regulation of steady-state AR mRNA by DHT occurs at the transcriptional level. In MDA453 cells, the effect of DHT to down-regulate AR mRNA expression results primarily from posttranscriptional destabilization of AR mRNA.



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  		Figure 5. Androgens increase AR mRNA stability in LNCaP cells but decrease AR mRNA stability in MDA453 cells. Cells were incubated ± 10 nM DHT for 8 h followed by addition of actinomycin D (Act D) 5 µg/ml. Cells were harvested at sequential time points, Northern analysis performed and AR mRNA levels plotted on a semilogarithmic scale for estimation of AR mRNA half-life (T1/2). Values for AR mRNA are shown relative to the amount present at time 0 h = 1.0 for each set of cells (control or DHT treated). T1/2 was measured at the x-intercept of the decay curve from y = 0.5. A, Act D decay curve in LNCaP cells showing stabilization of AR mRNA in the presence of DHT (ANOVA; **, P < 0.01). B, Act D decay curve in MDA453 cells showing a decrease in AR mRNA half-life in DHT treated cells compared with control cells (ANOVA; **, P < 0.01). At least two actinomycin D decay experiments in triplicate were performed in each cell line.

 
Androgens increase steady-state AR protein expression in LNCaP and MDA453 cells
To assess steady-state AR protein expression, LNCaP and MDA453 cells were cultured and prepared for experiments as before. Cell lysates were prepared from cells treated with and without androgen, 20 µg aliquots resolved by SDS PAGE, transferred to nitrocellulose membranes and analyzed by ECL Western immunoblotting. A single AR protein band at the expected Mr of 110 kDa was found (Fig. 6AGo). Blots were washed and reprobed with an antibody to the 70-kDa PABP as a loading control (37). Treatment of LNCaP cells with androgen resulted in a 2- to 3-fold increase in AR protein, maximal after 8 h of DHT and reducing after 24 h (means comparisons; 8 h P < 0.001, 24 h P = 0.01: DHT > control) (Fig. 6BGo). A similar increase in AR protein levels was seen in DHT treated MDA453 cells (means comparisons; 8 h P = 0.047, 24 h P = 0.005: DHT > control) (Fig. 6CGo). The parameters of the SDS PAGE allowed quantitation of immunoreactive AR protein but did not provide sufficient size resolution to show protein mobility shifts from androgen induced AR phosphorylation (42, 43).



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  		Figure 6. Androgens increase steady-state expression of AR protein in LNCaP and MDA453 cells. Cell lysates were prepared from cells treated ± 10 nM DHT for Western immunoblots using 20 µg protein lysate per lane. A, Polyclonal AR antibody and a HRP conjugated secondary antibody for ECL assay detecting an AR protein of Mr 110 kDa. Blots were washed and reprobed with an antibody directed against the 70 kDa PABP as a loading control. Protein molecular weight markers are shown to the left of the figure. B, LNCaP cells ± 10 nM DHT for 0 to 72 h (means comparisons; (a) P < 0.001, (b) P < 0.01: DHT > control). C, MDA453 cells ± 10 nM DHT for 0 to 24 h (means comparisons; (c) P < 0.05, (d) P < 0.01: DHT > control). AR protein expression is shown as the amount of AR protein in DHT-treated cells relative to AR protein in control cells harvested at identical time points. Data are mean ± SE of three experiments in duplicate for each cell line.

 
The combination of decreased steady-state AR mRNA with increased total AR protein suggested that posttranscriptional regulation might also occur at the level of de novo AR protein synthesis. To determine the contribution of de novo AR protein synthesis in LNCaP and MDA453 cells, cells were labeled with 35S methionine and immunoprecipitated with an AR-specific antibody. In LNCaP cells, DHT 10 nM pretreatment for 8 h resulted in a marginal increase in labeled immunoprecipitated AR protein to 120% of the value for control (means comparison; P value not significant) (Fig. 7AGo). In MDA453 cells treated under identical conditions, DHT did not alter the quantity of labeled immunoprecipitated AR protein (Fig. 7BGo).



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  		Figure 7. Androgens do not significantly increase de novo synthesis of AR protein in LNCaP and MDA453 cells. Cells were incubated ± 10 nM DHT for 8 h, being methionine starved for 2 h before addition of 35S-labeled methionine in the final 1 h. Cell lysates were prepared, AR protein immunoprecipitated using a polyclonal AR antibody and protein A beads and resolved using 6% SDS-PAGE. A, Lane 1: parallel Western blot probed with polyclonal AR antibody and secondary antibody for ECL. Lanes 2 and 3: Immunoprecipitated 35S-labeled AR from LNCaP cells ± DHT 10 nM for 8 h. The increase in AR with DHT treatment was not statistically significant. Two experiments in duplicate were performed. B, Lanes 1 and 2: Immunoprecipitated 35S- labeled AR from MDA453 cells ± DHT 10 nM for 8 h. Data are shown as mean ± SE for three experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Negative autoregulation of AR mRNA by androgen has been described for prostate (28, 29, 30, 31) and breast cancer cell lines (34, 35), but the mechanisms linking reduced AR mRNA and increased AR protein expression in these cells have not been thoroughly explored. The results shown here identify these mechanisms, which, unexpectedly, are completely different in each cell line. Our findings indicate that androgen regulation of AR gene expression is complex, with cell-specific, differential androgen effects occurring at multiple levels of transcription and mRNA turnover. Thus, androgen regulation of AR gene expression resembles glucocorticoid mediated down-regulation of glucocorticoid receptor (GR) gene expression in that it is characterized by multiple level complexity and cell specificity (44). Further, our studies confirm that, despite androgens stabilizing AR mRNA in LNCaP cells, reduced transcription is the predominant mechanism accounting for androgen-induced down-regulation of AR mRNA (Fig. 8AGo). In LNCaP cells, the reduction in AR mRNA occurs in the presence of AR mRNA stabilization. Thus, the transcriptional down-regulation that occurs predominates over the reduction in AR mRNA turnover at that point in time (8 h). However, this mechanism is cell specific and is not the case for MDA453 cells. Instead, in MDA453 cells, posttranscriptional destabilization of AR mRNA provides an equally rapid and significant down-regulation of AR mRNA, in the absence of simultaneous transcriptional change (Fig. 8BGo). These results demonstrate the cell-specific, divergent nature of posttranscriptional regulation of AR mRNA by androgen and establishes that AR mRNA destabilization is the major determinant of steady-state AR mRNA expression in MDA453 cells under these conditions.



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  		Figure 8. Schematic illustrating mechanisms involved in androgen-induced regulation of AR gene expression in prostate (A, LNCaP) and breast (B, MDA453) cancer cells. Mib, Mibolerone; A, androgen.

 
As AR transcription is down-regulated by DHT in LNCaP but not MDA453 cells, androgen-responsive regulatory mechanisms for AR transcription appear to be differentially expressed in different cell types. The synthetic androgen R1881 down-regulates the expression of AR mRNA from COS 1 cells transfected with AR cDNA, suggestive of a regulatory mechanism contained within the coding sequence of the AR (45). However, transcriptional regulation of the AR mRNA appears to involve a potential cAMP response element (CRE) and other non-androgen response element (ARE) motifs within the 5' promoter region of the AR gene (46, 47, 48). AR mediated transcriptional down-regulation of AR gene expression in LNCaP cells may thus involve AR interactions with a second messenger such as cAMP rather than more proximal upstream AREs (49). In osteoblastic cells, however, DHT increases AR mRNA and up-regulates AR promoter activity (50). Thus, a number of potential regulatory elements affecting AR transcription exist that may be differentially expressed or modulated by androgen in LNCaP compared with MDA453 cells.

We found rapid down-regulation of AR mRNA by DHT with concomitant increases in AR protein expression occurring within 8 h of exposure to androgen in both LNCaP and MDA453 cells. These results differ from the slower time course of AR mRNA down-regulation by DHT or mibolerone previously reported in LNCaP cells (40- to 96-h incubations) (30, 31). With regard to AR protein expression, Krongard et al. (30) found a 2-fold increase in immunoreactive AR protein, an increase in ligand binding, and a protein gel mobility shift in androgen treated LNCaP cells. Wolf and colleagues (31) found no change in AR protein levels, noting instead an alteration in protein mobility, suggesting posttranslational modification or phosphorylation (43). However, we found a significant, rapid increase in AR protein expression occurring within 8 h of treatment with DHT. The earlier time course response may account for the lack of alteration in AR protein levels after 96 h incubation with mibolerone (31) when the androgen response may have diminished. Additionally, DHT 10 nM may represent an optimal concentration of ligand to use in tissue culture conditions to demonstrate regulation of both AR mRNA and AR protein. Given the rapid increase (in LNCaP cells) or decrease (in MDA453 cells) in the number of cells in S-phase of cell cycle occurring within 24 h of androgen exposure, short incubation times with androgens may be more physiologically relevant in these tumor cells.

The quantity of 35S-labeled, immunoprecipitated AR that we observed is determined by the rate of de novo synthesis and subsequent degradation of AR protein within the 60-min period of 35S-methionine incorporation. The slight enhancement of AR signal in DHT-treated LNCaP cells implies that no large increase in AR synthesis occurs as a result of DHT treatment (unless a concomitant more rapid degradation also takes place). Thus, we believe that the increased steady-state AR protein levels found in DHT treated LNCaP and MDA453 cells on Western immunoblotting is most likely a result of stabilization of the ligand receptor complex by ligand binding, as has been previously described (32, 33). As similar increases in AR protein expression with DHT were found in LNCaP and MDA453 cells despite different effects of DHT on cellular proliferation and cell cycle modulation, androgen-induced alterations in the number of ARs alone does not appear to mediate proliferative as opposed to inhibitory effects on growth. Rather, AR target genes or AR-related transcriptional coactivators and/or corepressors influencing growth and cell cycle progression might be differentially expressed in prostate compared with breast cancer cells.

In LNCaP cells, DHT from 1 to 100 nM exerts a proliferative effect lost at lower or higher concentrations of DHT (38), emphasizing the importance of selecting an appropriate ligand concentration for experiments. Previous work has utilized up to 10–20 nM DHT and between 1 to 10 nM mib for AR mRNA regulatory experiments in LNCaP cells (30, 31). We found that DHT 10 nM produced divergent proliferative and cell cycle responses in LNCaP compared with MDA453 cells. This concentration was used consistently in our experiments examining regulation of AR gene expression in the two cell types.

The divergent effect of androgen on AR mRNA turnover in these cell lines suggests that very different mechanisms are involved in maintaining AR mRNA stability. Previous work has shown stabilization of AR mRNA in LNCaP cells by mibolerone (31). In ventral and dorsal lobes of the rat prostate, up-regulation of AR mRNA on androgen withdrawal occurred without any alteration in AR gene transcription, suggesting posttranscriptional stabilization (51). Posttranscriptional destabilization of AR mRNA in the absence of transcriptional change by androgens in MDA453 cells thus appears to be a novel mechanism of AR mRNA autoregulation. Interactions between specific sequences within mRNA (cis-acting elements) and cellular RNA-binding proteins (trans-acting factors) as well as ribonuclease activity targeting the mRNA, are factors that determine the rate of mRNA turnover (52, 53, 54, 55). Thus, differentially expressed androgen-regulated RNA binding proteins might influence AR mRNA stability. The differences in androgen-regulated AR mRNA half-life in LNCaP compared with MDA453 cells are strongly suggestive of the existence and differential expression of such trans-acting factors. However, little is known of the identity of cis-acting elements and trans-acting factors for AR mRNA. Identification and cloning of such cellular trans-acting factors and determination of their functional role in relation to AR mRNA stability would be an important advance in further understanding the mechanism of androgen-regulated AR gene expression in these cancers. Furthermore, it would allow exploration of new therapeutic interventions based on targeted disruption of specific cellular cis-trans RNA-protein interactions (56).


   Acknowledgments
 
Bluescript vectors containing the full-length 3.1-kb and the 713-bp HindIII/EcoRI fragment of the AR cDNA and a 1.1-kb rat 18S ribosomal RNA cDNA were a kind gift from Dr. Wayne Tilley. A bluescript vector containing a 1.3-kb sequence GAPDH was kindly provided by Dr. S. Peter Klinken. The mouse polyclonal antibody to PABP was a kind gift from Dr. Matthias Görlach. The authors thank Dr. Elizabeth Wilson for kindly providing the rabbit polyclonal antibody to AR, AR52, and Dr. Valerie Burke for helpful advice regarding statistical analysis. We also thank the Medical Illustrations Department of Royal Perth Hospital for preparing the photographic prints for this manuscript.


   Footnotes
 
1 This work was supported in part by the Cancer Foundation of Western Australia, Merck Sharp & Dohme, the Sandoz Foundation for Gerontological Research, and the Medical Research Foundation of Royal Perth Hospital. Back

Received October 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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