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Endocrinology Vol. 140, No. 3 1205-1212
Copyright © 1999 by The Endocrine Society

ARTICLES

Induction of Androgen Receptor by 1{alpha},25-Dihydroxyvitamin D3 and 9-cis Retinoic Acid in LNCaP Human Prostate Cancer Cells1

Xiao-Yan Zhao, Lan H. Ly, Donna M. Peehl and David Feldman

Departments of Medicine (X.-Y.Z., L.H.L., D.F.) and Urology (D.M.P.), Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: David Feldman, M.D., Division of Endocrinology, Stanford University Medical Center, Room S-005, Stanford, California 94305-5103.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown that 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
1{alpha},25-DIHYDROXYVITAMIN D3 [1,25-(OH)2D3], the active metabolite of vitamin D, regulates calcium homeostasis in the body by actions in the intestine, bone, kidney, and parathyroid glands (1, 2). Recently, 1,25-(OH)2D3 has also been shown to have nonclassical actions. For example, the hormone exerts antiproliferative and prodifferentiating effects on many cell types, including cells derived from myeloid, breast, colon, and prostate tissues (3, 4, 5, 6). Biologic responses of target cells to 1,25-(OH)2D3 are mediated by its nuclear receptor, the vitamin D receptor (VDR) (7). The VDR belongs to the steroid/thyroid/retinoid receptor superfamily (1, 2). Numerous studies indicate that VDR controls target gene transcription by forming a heterodimeric complex with the retinoid X receptor (RXR), the receptor for 9-cis retinoic acid (RA), and binding to the vitamin D response element (VDRE) present in the promoter region of target genes.

Our group (8, 9), as well as others (10), have shown that VDRs are present in established human prostate cancer cell lines, as well as primary cultures of normal prostate and cancer cells (11). Moreover, 1,25-(OH)2D3 and its analogs significantly inhibit cellular proliferation of prostate cancer cells, including LNCaP (8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20). LNCaP cells express both the VDR and the androgen receptor (AR). Our recent studies (21) and those of others (15, 22) have demonstrated that cross-talk between 1,25-(OH)2D3 and androgens exists and that the antiproliferative actions of 1,25-(OH)2D3 in LNCaP cells are androgen-dependent. Blutt et al. (17) have shown that 9-cis RA acts synergistically with 1,25-(OH)2D3 to inhibit LNCaP cell growth.

Because cellular responsiveness to androgen depends on AR abundance, in the present study, we have analyzed the ability of 1,25-(OH)2D3 and 9-cis RA to regulate the level of AR gene expression in these cells. We found that 1,25-(OH)2D3 increased the levels of AR messenger RNA (mRNA) and AR protein in a concentration- and time-dependent manner. Such regulatory effects of 1,25-(OH)2D3 on AR gene expression required de novo protein synthesis. Furthermore, the stimulatory effect of 1,25-(OH)2D3 on AR mRNA was also enhanced by 9-cis RA. Because it has been reported that the antiproliferative effects of 1,25-(OH)2D3 on LNCaP cells can be synergistically enhanced by the addition of 9-cis RA (17), we examined the involvement of AR in the antiproliferative action of 9-cis RA, as well as 1,25-(OH)2D3. Using the pure AR antagonist, Casodex, we demonstrated that AR blockade prevented the growth inhibitory activity of both 1,25-(OH)2D3 and 9-cis RA. In contrast, Casodex did not affect the antiproliferative activity of dibutyrl cAMP, a well-known up-regulator of AR in LNCaP cells (23). Our studies demonstrate that both 1,25-(OH)2D3 and 9-cis RA up-regulate AR mRNA levels in LNCaP cells and that growth inhibition mediated by 1,25-(OH)2D3 and 9-cis RA requires the action of AR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
1,25-(OH)2D3 was the generous gift of Dr. M. Uskokovic (Hoffmann-LaRoche, Inc., Nutley, NJ). Bicalutamide (Casodex or ICI 17,334) was a gift from Zeneca Pharmaceuticals (Macclesfield, Cheshire, UK). Aprotinin, pepstatin, and soybean trypsin inhibitor were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Tissue culture media were purchased from Mediatech (Herndon, VA). All other reagents, except where indicated, were purchased from Sigma Chemical Co. (St. Louis, MO). The anti-AR monoclonal antibody F39.4 and the human AR complementary DNA (cDNA) were generous gifts from Dr. TH Van der Kwast (Erasmus University, Rotterdam, Netherlands) and Dr. M. McPhaul (University of Texas Southwestern Medical Center, Dallas, TX), respectively. FBS was obtained from Gibco BRL (Gaithersburg, MD). Charcoal-stripped FBS (CSS) was purchased from Sigma Chemical Co.

Cell culture and hormone treatment
The LNCaP human prostate carcinoma cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were routinely cultured in RPMI-1640 medium supplemented with 5% FBS and antibiotics (FBS medium), at 37 C in a humidified atmosphere of 5% CO2. For experiments, LNCaP cells were trypsinized and seeded at an appropriate density, and hormonal treatments were initiated, the next day, in FBS medium or in RPMI-1640 medium supplemented with 5% CSS and antibiotics (CSS medium).

Hormone stocks [1,25-(OH)2D3, 9-cis RA, and Casodex] were prepared in 100% ethanol, at a concentration 1000-fold higher than the working concentrations. Fresh culture media were premixed with hormone stock and then added to triplicate wells. Media and hormone were replenished every 2 days. Controls received ethanol vehicle at a concentration equal to that in hormone-treated cells.

Assay of cell proliferation
Cell proliferation was assessed by measurement of attained cell mass using an assay of DNA content. As previously described (21), LNCaP cells were seeded in six-well tissue culture plates (Becton Dickinson and Co., Lincoln Park, NJ), at a density of 50,000 cells per well, in 3 ml RPMI-1640 containing 5% FBS. After incubation for 24 h, the medium was replaced with fresh medium containing 5% FBS (FBS medium). Cells were treated with vehicle (ethanol, final concentration 0.1%), 1,25-(OH)2D3, 9-cis RA, dibutyrl cAMP, or Casodex. On the sixth day, cell monolayers were processed for DNA assay using the method of Burton (24). DNA content of each treatment was derived from the mean value of triplicate wells in an experiment. Each experiment was repeated three times.

Western blot analysis
Cells were treated with ethanol or 1,25-(OH)2D3 (10 nM) in RPMI-1640 medium containing 5% CSS (CSS medium) for 2 days. They were harvested, and sonicated extracts were prepared as described. Aliquots of 100 µg protein were heated in SDS sample buffer at 95 C, for 5 min, before electrophoresis in an 8% SDS-polyacrylamide gel. After electrophoresis, the gels were transferred and processed as previously described (25). After transfer, the blots were incubated with anti-AR monoclonal antibody F39.4 (1:100 dilution) for 1 h, at room temperature, with gentle shaking. The blots were washed and then incubated with a horseradish peroxidase-conjugated rabbit antimouse IgG (1:1000 dilution) for 1 h at room temperature. Blots were rewashed and developed with the Enhanced Chemiluminesence (ECL) System system, according to the manufacturer’s instructions (Amersham Chemical Co.).

Steroid receptor ligand-binding assay
LNCaP cells were seeded at a density of 150,000 cells per 100-mm dish in 10-ml medium containing 5% FBS or 5% CSS. At the end of the 6-day incubation with hormone (at concentrations of 0, 1, and 10 nM), cell monolayers were harvested, and high-salt nuclear extracts were made as previously described (21). Protein concentration of the extract was determined (26). In a typical binding experiment, 200 µl soluble extract (1–2 mg protein/ml) were incubated with 10 nM concentration of [3H]-5{alpha}-dihydrotestosterone (DHT) for 16–20 h at 4 C. Bound and free hormone were separated by hydroxylapatite (21). Specific binding was calculated by subtracting nonspecific binding (obtained in the presence of a 250-fold excess of radioinert DHT) from the total binding (measured in the absence of radioinert steroid). Data were expressed as femtomoles [3H]-DHT bound per milligram protein.

Northern blot analysis
Northern blot analysis was performed as previously described (8, 11). Briefly, semiconfluent LNCaP cells were treated with graded concentrations of 1,25-(OH)2D3, or 5 mM dibutyrl cAMP, or 9-cis RA in FBS medium and in CSS medium for 24 h before isolation of total RNA. Ten micrograms of total RNA were denatured, fractionated by electrophoresis, and transferred to Hybond-N nylon membrane (Amersham), as previously described (8, 11). The bound RNA was immobilized by UV cross-linking and then hybridized with a random primed [32P]-labeled 0.8-kb HindIII-BamHI fragment of the human AR cDNA at 60 C. To control for RNA sample loading and transfer, Northern blots were also hybridized with a [32P]-labeled 0.9-kb EcoRI fragment of the human cDNA for the ribosomal protein gene L7 (8, 11). The silver grain pixel intensity of each AR and L7 band was scanned by a densitometer, and the data were integrated by scanner software and indexed to the corresponding levels of L7 mRNA.

AR promoter-luciferase reporter gene assay
LNCaP cells were seeded at 3 x 106 cells/dish in 60-mm tissue culture dishes (Corning, Inc., Corning, NY) in RPMI-1640 medium containing 5% FCS and antibiotics. A 6-kb promoter-luciferase reporter was transfected using a calcium-phosphate method (23). Each transfection contained 1 µg pAR-LUC DNA (Drs. G. Mora and D. Tindall, personal communication) and 0.1 µg pSV-Renilla DNA. The control plasmid pSV-Renilla was used to monitor transfection efficiency. Cells were harvested after 32 h of incubation with tested compounds at 37 C. Luciferase activity was employed to measure induction using Promega Corp. (Madison, WI) dual luciferase assay system on luminometer TD-20 (Turner Design, Sunnyvale, CA). The results were expressed as the ratio of luciferase activity to Renilla activity.

Statistical analysis
ANOVA was used to assess the statistical significance of the difference. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have recently demonstrated that the antiproliferative action of 1,25-(OH)2D3 in LNCaP cells is androgen-dependent (21). Here, we investigate further the interaction between 1,25-(OH)2D3 and androgen signaling pathways by exploring the mechanism of 1,25-(OH)2D3 regulation of AR gene expression in these cells. We also examine the possible involvement of AR in the synergistic antiproliferative actions of 1,25-(OH)2D3 and 9-cis RA on LNCaP cells.

Dose response effect of 1,25-(OH)2D3 on AR mRNA
The effect of 1,25-(OH)2D3 on steady-state AR mRNA levels was assessed by Northern blot analysis. We have used two culture conditions (FBS medium and CSS medium) in this set of experiments and have observed similar results. As shown in Fig. 1Go, LNCaP cells express a major transcript of AR at 11 kb. In Fig. 1AGo, the cells were treated in CSS medium for 24 h with increasing concentrations of 1,25-(OH)2D3 (0–100 nM), and AR mRNA transcripts increased in a dose-dependent manner. The increased AR mRNA levels became evident with a concentration of 1,25-(OH)2D3 at 1 nM (lane 3). Increasing the 1,25-(OH)2D3 concentration caused further induction of AR mRNA (lanes 4–5). The levels of AR mRNA were quantitatively determined by densitometric scanning of the autoradiographs, with correction for the L7 mRNA signal (Fig. 1BGo). At 100 nM of 1,25-(OH)2D3, more than 5-fold up-regulation of AR mRNA was detected (lane 5). When we carried out the experiment using FBS medium (Figs. 1Go, C and D), we also detected a significant up-regulation of AR mRNA in LNCaP cells in response to 1,25-(OH)2D3 treatment for 24 h. Hence, 1,25-(OH)2D3 increased AR mRNA expression in LNCaP cells in a dose-dependent manner in either CSS medium or FBS medium.



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  		Figure 1. Dose-dependent effect of 1,25-(OH)2D3 on AR mRNA levels in LNCaP cells. A, Northern blot analysis in CSS medium. LNCaP cells were treated with 1,25-(OH)2D3, at the indicated concentrations, for 24 h in RPMI medium containing 5% charcoal stripped serum. Total RNA was isolated, and the RNA blot was hybridized with a 32P-labeled 712-bp HindIII-EcoRI fragment of the human AR cDNA at 60 C. The blot was simultaneously probed for expression of the L7 ribosomal protein gene as a control for sample loading and transfer. B, The pixel intensity of each AR band in panel A was scanned by computing densitometer, and the data were integrated by scanner software and indexed to the corresponding levels of L7 mRNA. C, Northern blot analysis in FBS medium. LNCaP cells were treated with 1,25-(OH)2D3, at the indicated concentrations, for 24 h in RPMI medium containing 5% FBS. Total RNA was isolated, and the RNA blot was hybridized with a 32P-labeled human AR cDNA and the L7 gene. D, The pixel intensity of each AR band indexed to L7 in panel C.

 
Time-course of AR mRNA expression in response to 1,25-(OH)2D3
In Fig. 2Go, time-course experiments using CSS medium revealed that addition of 10 nM 1,25-(OH)2D3 to LNCaP cells increased AR mRNA levels by 8 h. The AR mRNA levels peaked at 48 h in the treated cells, with a 10-fold higher level, compared with the untreated cells at the concurrent time point, and this inductive effect of 1,25-(OH)2D3 was sustained at 72 h. No change in AR mRNA could be detected at 4 h, suggesting a delayed primary response of AR gene expression to 1,25-(OH)2D3.



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  		Figure 2. Time-course of AR mRNA expression in LNCaP cells, in response to 1,25-(OH)2D3. A, Northern blot analysis. LNCaP cells were treated with 1,25-(OH)2D3, at 10 nM, for the indicated time period. Total RNA was isolated and analyzed by Northern blot using the human AR cDNA and L7 cDNA as probes. B, The pixel intensity of each AR band in panel A was scanned by computing densitometer, and the data were integrated by scanner software and indexed to the corresponding levels of L7 mRNA.

 
Up-regulation of the AR protein by 1,25-(OH)2D3
1,25-(OH)2D3 also caused a concentration-dependent stimulation of AR protein expression in LNCaP cells cultured in CSS medium, as measured by Western blot analysis (Fig. 3Go). The major species of AR in LNCaP cells was detected as a single band, at 108 kDa, by monoclonal antibody F39.4. There was no detectable increase in AR protein levels after 24 h treatment (data not shown), but levels rose approximately 4-fold in cells treated with 10 nM 1,25-(OH)2D3 for 48 h. As seen for AR mRNA levels (Fig. 1Go), a detectable increase in AR protein was evident with 1 nM 1,25-(OH)2D3.



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  		Figure 3. Western blot analysis of AR protein in LNCaP cells. LNCaP cells were incubated, in RPMI medium containing 5% charcoal stripped serum, with the indicated dose of 1,25-(OH)2D3 for 2 days. High-salt protein extracts were electrophoresed in an 8% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose and probed with anti-AR monoclonal antibody F39.4. Immunoreactive bands were detected by incubation of blots with a secondary antibody (rabbit antimouse IgG), followed by ECL. Molecular weight standards are indicated. hAR is indicated by an arrow. The experiment was repeated twice, with similar results.

 
[3H]DHT-binding analyses demonstrated that 1,25-(OH)2D3 increased the AR content in LNCaP cells when they were cultured in either CSS medium or FBS medium. As we have reported (21), cells treated with 1 nM 1,25-(OH)2D3 in CSS medium showed a more than 2-fold increase in DHT-binding (from 197 ± 17.4 to 430 ± 9.6 fmol/mg protein, n = 3). Addition of 10 nM of 1,25-(OH)2D3 further up-regulated the AR content (from 197 ± 17.4 to 532 ± 60 fmol/mg, n = 3). Meanwhile, cells cultured in FBS medium exhibited a higher baseline DHT-binding than in CSS medium (378 ± 33.9 fmol/mg vs. 197 ± 17.4 fmol/mg, n = 3). 1,25-(OH)2D3 at 1 nM in FBS medium also increased the AR content (from 378 ± 33.9 to 451 ± 54 fmol/mg protein, n = 3). Therefore, 1,25-(OH)2D3 up-regulates the AR content in LNCaP cells in either growth condition. Interestingly, CSS medium allowed us to detect a clear up-regulation of AR because charcoal treatment removes endogenous steroids in serum that may interfere with [3H]DHT-binding.

Requirement of new protein synthesis for 1,25-(OH)2D3 regulation of AR
To determine whether 1,25-(OH)2D3 affected AR mRNA levels via a direct mechanism, LNCaP cells in CSS medium were treated for 24 h with 1,25-(OH)2D3 in the presence of the protein synthesis inhibitor cycloheximide (CHX) at various doses (0, 2, 5, and 10 µg/ml). As shown in Fig. 4Go, CHX blocked the 1,25-(OH)2D3-induced increase in AR mRNA levels, such that in the presence of CHX and 1,25-(OH)2D3 (lane 3), AR mRNA levels were no higher than those in untreated cells (lane 1). The extent of blockade depended upon the concentration of CHX included in the media (lanes 3–5). Moreover, the effect of CHX could be detected at either 16 h (lane 6) or 24 h (lane 4).



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  		Figure 4. Effect of CHX on the 1,25-(OH)2D3 induction of AR mRNA in LNCaP cells. Cells were treated with ethanol (lane 1) or 10 nM 1,25-(OH)2D3 (lanes 2–6), in the presence of CHX, at the indicated concentrations, for 24 h (lanes 1–5) or 16 h (lane 6). Total RNA was isolated and analyzed by Northern blot using the human AR cDNA and L7 cDNA as probes.

 
In other studies, using a 6-kb AR promoter-luciferase reporter transfected into LNCaP cells, we attempted to directly induce expression of AR with 1,25-(OH)2D3. No increase in luciferase could be detected with 1,25-(OH)2D3, whereas dibutyrl cAMP induced a 5-fold rise in luciferase (data not shown). Taken together, these findings indicate that 1,25-(OH)2D3 regulates AR mRNA expression via an indirect mechanism requiring new protein synthesis.

Enhancement of 1,25-(OH)2D3-mediated up-regulation of AR by 9-cis RA
It recently has been reported that 1,25-(OH)2D3 acts synergistically with 9-cis RA to inhibit LNCaP cell proliferation (17). We therefore investigated the effect of 9-cis RA on 1,25-(OH)2D3 regulation of AR mRNA. Both culture conditions (FBS medium and CSS medium) gave similar results. As shown in Fig. 5Go, 1,25-(OH)2D3, at a dose of 10 nM, induced a 5-fold increase in AR mRNA levels (lane 1) over the control at 24 h (lane 2). LNCaP cells, treated with 100 nM 9-cis RA for 24 h, expressed 3-fold more AR mRNA (lane 3) than the untreated cells (lane 2). Combination treatment of 1,25-(OH)2D3 and 9-cis RA gave a more than 8-fold induction of AR mRNA (lane 4). Thus, although 1,25-(OH)2D3 was more effective than 9-cis RA in up-regulating AR mRNA, both hormones acted additively to increase AR gene expression in LNCaP cells.



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  		Figure 5. Enhancement of 1,25-(OH)2D3 action by 9-cis RA in the induction of AR mRNA in LNCaP cells. A, Northern blot analysis. LNCaP cells were treated with 1,25-(OH)2D3 at 10 nM, or 9-cis RA at 100 nM, individually or in combination for 24 h. Total RNA was extracted and analyzed by Northern blot. B, The pixel intensity of each AR band in panel A was scanned by computing densitometer, and the data were integrated by scanner software and indexed to the corresponding levels of L7 mRNA.

 
Involvement of AR action in the antiproliferative response of 1,25-(OH)2D3 and 9-cis RA
We further tested the possible involvement of AR action in the antiproliferative effect of 1,25-(OH)2D3 and its synergism with 9-cis RA. As shown in Fig. 6AGo, 10 nM 1,25-(OH)2D3 inhibited LNCaP cell growth 50%, whereas 100 nM 9-cis RA only reduced proliferation by 10%. However, combination treatment with 1,25-(OH)2D3 and 9-cis RA caused 80% growth inhibition. Casodex was used to determine whether the 9-cis RA action was also AR-dependent. In the presence of Casodex, neither 1,25-(OH)2D3 nor 9-cis RA inhibited cell growth individually or in combination (Fig. 6AGo). On the other hand, dibutyrl cAMP, a known up-regulator of AR in LNCaP cells, inhibited cell proliferation in a dose-dependent manner (Fig. 6BGo). With maximal growth inhibition of 90% occurring at a dose of 5 mM dibutyrl cAMP, addition of Casodex, however, did not block this effect (Fig. 6BGo). These data imply that the actions of 1,25-(OH)2D3 and 9-cis RA on LNCaP cell growth are both AR-dependent. In contrast, cAMP, although an inducer of AR (23), inhibits LNCaP cell proliferation by an AR-independent mechanism.



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  		Figure 6. Effect of Casodex on 1,25-(OH)2D3 and 9-cis RA-induced growth inhibition on LNCaP cells. A, LNCaP cells were treated with ethanol, 1,25-(OH)2D3 at 10 nM, or 9-cis RA at 100 nM, individually or both in the presence or absence of Casodex at 1 µM, for 6 days. Cellular DNA contents were determined by Burton’s method. The data are expressed as percent of control, a mean of three triplicate samples ± SEM. *, P < 0.01, compared with the untreated control group; **, P < 0.05, compared with the single-treatment group. B, LNCaP cells were treated with dibutyrl cAMP [Bu2cAMP], from 0–5 mM, in the presence or absence of Casodex at 1 µM, for 6 days.

 
Model of 1,25-(OH)2D3 and 9-cis RA action in LNCaP
The hormonal action of 1,25-(OH)2D3 is mediated by the VDR present in LNCaP cells. Figure 7Go depicts the possible events in the 1,25-(OH)2D3 signaling pathway in these cells. 1,25-(OH)2D3 binds to the VDR and activates the receptor. The activated VDR controls target gene transcription by forming a heterodimer with the partner RXR and binding to the VDRE in the promoter region of a target gene. A 1,25-(OH)2D3 target gene (or genes) encodes protein(s) X, mediators of the up-regulation of AR mRNA in response to 1,25-(OH)2D3. The production of protein(s) X is CHX-sensitive. Both 1,25-(OH)2D3 and 9-cis RA, individually or in combination, induce AR mRNA expression. The increased AR mRNA causes an increase in AR protein levels, which mediates the action of androgens on LNCaP cell growth. As we reported previously (23), either 1,25-(OH)2D3 or DHT was growth inhibitory on LNCaP when cells were cultured in FBS-containing medium. The androgen action is blocked by the AR antagonist, Casodex. Although both the AR mRNA and the AR protein are also induced in CSS medium, in the absence of androgens, 1,25-(OH)2D3 does not exhibit an antiproliferative effect on LNCaP cells in this culture system (21).



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  		Figure 7. A tentative model of 1,25-(OH)2D3 and 9-cis RA action on LNCaP cells. Both 1,25-(OH)2D3 and 9-cis RA induce AR mRNA expression. The increased AR mRNA leads to an increase in AR protein levels. AR protein mediates androgen action in cell proliferation. The pure AR antagonist, Casodex, blocks AR action; in turn, it blocks the growth-inhibitory action of 1,25-(OH)2D3 and 9-cis RA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The AR is the key element in the androgen signal transduction cascade, and it plays a critical role in the regulation of growth and differentiation of the prostate. The data presented here demonstrate that 1,25-(OH)2D3 up-regulates AR gene expression at both mRNA and protein levels in LNCaP cells, an androgen-responsive human prostate cancer cell line. This inductive action of 1,25-(OH)2D3 was enhanced by 9-cis RA, which by itself also up-regulates AR expression in LNCaP cells. Our data show that growth inhibition induced by 1,25-(OH)2D3 alone or in combination with 9-cis RA was accompanied by increased AR expression. Moreover, the antiproliferative actions of 1,25-(OH)2D3 and 9-cis RA were AR-dependent and could be blocked by the AR antagonist, Casodex. Although androgens are not added in these experiments, androgens are present in the serum supplement to the culture medium (FBS medium), and we hypothesize that the amplitude of the androgen response is augmented by the increased levels of AR expressed in these cells after treatment with 1,25-(OH)2D3 and/or 9-cis RA. Support for the effect of androgens, in FBS medium, on cell growth was presented in our earlier studies (21). In the absence of androgens (for example, in CSS medium), 1,25-(OH)2D3 does not exhibit an antiproliferative action on LNCaP cells.

Nonetheless, our finding that androgen mediates the antiproliferative activity of 1,25-(OH)2D3 in LNCaP cells is not the situation in all prostate cancer cells. 1,25-(OH)2D3 inhibits the growth of AR-negative prostate cancer cell line PC-3, as well as primary cultures of human prostate cells. In contrast to LNCaP cells, mechanisms other than androgen signaling are responsible for the growth inhibitory effect of 1,25-(OH)2D3 on these cells.

It is of interest to consider whether increasing the abundance of a steroid receptor, such as the AR, will cause an increased amplitude of response, i.e. antiproliferation. Although it has been well demonstrated that the level of receptors in LNCaP does not necessarily predict the ligand potency of a hormonal response (8, 14, 19, 27), it is clear that the presence of a receptor is essential for a response (1, 2, 13, 14, 28). In a given cell, in the presence of a constant level of hormone, up-regulation of the receptor does cause an enhanced response, whereas down-regulation of the receptor diminishes the response (13, 14, 28, 29, 30). Therefore, we believe that up-regulation of AR, in these studies, is the mechanism of the enhanced antiproliferative effect.

The expression of the AR gene has been found to be induced by a number of agents in several systems, such as the rat ventral prostate (31), and in LNCaP human prostate cancer cells (32, 33). Growth factors such as FSH, EGF, and TGF-ß regulate AR gene expression (34, 35). Activators of protein kinase A, such as forskolin and dibutyrl cAMP, are the known up-regulators of AR in LNCaP cells (23). The induction of AR by these reagents was not detected in the two other commonly studied prostate cancer cell lines, PC-3 and DU 145, which do not express basal levels of AR mRNA (36).

1,25-(OH)2D3 is the most potent inducer of AR in LNCaP cells, among the three agents that we tested. Consistent with the reported data (23), we found that treatment of LNCaP cells with dibutyrl cAMP for 24 h caused a 2-fold increase in AR mRNA levels (data not shown). In the same experiment, we observed an increase of 5-fold in AR mRNA, with 1,25-(OH)2D3 at 10 nM. 9-cis RA induced 3-fold induction of AR mRNA. It has been reported that dibutyrl cAMP increases AR gene transcription via the cAMP-response elements present in the 2.3-kb promoter region of the human AR gene (23). In contrast, the same promoter region of the AR gene seems to lack a VDRE and an RXRE. Computer searching of the 2.3-kb promoter region failed to identify a consensus sequence for these regulatory elements. Moreover, the luciferase reporter construct, driven by a 6-kb promoter region of the human AR gene, did not respond to 1,25-(OH)2D3 or 9-cis RA but did respond to dibutyrl cAMP.

An indirect mechanism for 1,25-(OH)2D3 action to induce AR was supported by several findings taken together: the delayed time of AR mRNA rise in time-course experiments (Fig. 2Go), the CHX studies (Fig. 3Go), and the failure of the promoter to respond to 1,25-(OH)2D3 (data not shown). We refer to the indirect, CHX-inhibited mediator(s) of 1,25-(OH)2D3 action to up-regulate AR as protein(s) X. We surmise that, in the presence of CHX, 1,25-(OH)2D3 was unable to induce protein(s) X, and as a consequence, 1,25-(OH)2D3 failed to up-regulate AR mRNA (Fig. 4Go). It is interesting to speculate on the nature of protein(s) X. Protein(s) X may be related to the chaperon proteins, given the fact that several chaperons have been identified in the regulation of steroid receptors (37). Further studies are needed to elucidate this mechanism.

We did not detect AR up-regulation by 1,25-(OH)2D3 in two human breast cancer cells, either MCF-7 or T47D (unpublished data). Both MCF-7 and T47D cells express the VDR, as well as the AR. However, the levels of AR protein did not change in both cell lines when treated with 1,25-(OH)2D3. Therefore, induction of protein(s) X by 1,25-(OH)2D3 may be tissue-specific. At present, it is difficult to examine this point because of the limited number of human prostate cancer cells that exhibit the AR. We have, thus far, been unable to induce AR in cells that lack the AR, including primary cultures of prostate cancer cells and established cell lines PC-3 or DU 145. To determine whether AR induction by 1,25-(OH)2D3 is LNCaP cell-specific, we hope to study other AR-positive human prostate cancer cell lines as they become available.

The action of androgens to inhibit proliferation of cultured prostate cancer cells is an interesting finding. We and others (21, 38) showed that LNCaP cells exhibit a biphasic growth response to DHT in charcoal-stripped serum-containing medium, with a growth stimulatory effect at a low concentration (less than 1 nM) and an inhibitory effect at a high concentration (greater than 1 nM). The levels of AR protein in LNCaP cells determine the concentration of DHT at which the stimulatory effect crosses over to an inhibitory effect. In other words, the stimulatory effect is favored at low abundance of AR, and an inhibitory effect at high abundance of AR (21). Liao and co-workers (39, 40, 41) found that high-passage LNCaP cells in an androgen-depleted medium express 10- to 20-fold higher AR levels and are growth inhibited by androgens in vitro and in an in vivo mouse model. Moreover, they demonstrated that G1 arrest of the high AR-expressing cells by androgen is caused by the induction of p27kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation (41). There are two additional examples to document the role of AR in the inhibition of growth of prostate cancer cells. Yuan et al. (42) have reported that PC-3 cells, stably transfected with the human AR cDNA, were growth inhibited by androgen. Recently, Zhau et al. (43) have established an androgen-repressed human prostate cancer cell line (ARCaP) derived from the ascites fluid of a patient with advanced metastatic disease, which is growth inhibited by androgens. Cumulatively, these findings support the hypothesis that higher levels of AR in cultured prostate cancer cells cause increased sensitivity to growth inhibition.

In summary, we have shown that the hormonally active forms of vitamin D and vitamin A are potent inducers of AR in LNCaP cells. Both 1,25-(OH)2D3 and 9-cis RA act in synergy to inhibit cell proliferation; moreover, their antiproliferative actions can be blocked by the AR antagonist, Casodex. In conclusion, our study provides direct evidence for an important role of the AR in mediating the growth inhibitory actions of 1,25-(OH)2D3 and 9-cis RA in LNCaP cells. More importantly, the newly discovered AR-inducing property of both vitamins A and D suggests a possible application of these potential chemo-preventive agents in increasing androgen sensitivity of prostate cancer cells. An understanding of the mechanisms of AR gene regulation may be of great importance in efforts to restore androgen responsiveness to the patients with androgen-independent prostate cancer, because this type of cancer is commonly unresponsive to most conventional therapies.


   Acknowledgments
 
We thank Dr. TH Van der Kwast (Erasmus University) for the anti-AR monoclonal antibody F39.4, Dr. D. Tindall (Mayo Clinic, Rochester, MN) for the AR-LUC reporter construct, and Dr. M. McPhaul (Univ. of Texas Southwestern Medical Center) for the human AR cDNA probe. We are also grateful to Dr. M. Uskokovic (Hoffmann La-Roche Co.) for providing 1,25-(OH)2D3 and 9-cis RA.


   Footnotes
 
1 Portions of this work were presented at the 19th American Society for Bone and Mineral Research meeting, Cincinnati, Ohio, September 1997. Supported by NIH Grant DK-42482, an American Institute for Cancer Research Grant 97-A-072, and U.S. Army Medical Research and Materiel Command Grant DAMD 17–98-8556 (to D.F.). Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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