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Endocrinology Vol. 143, No. 7 2708-2714
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Mechanism of Androgen Action on Cell Proliferation: AS3 Protein as a Mediator of Proliferative Arrest in the Rat Prostate

Maricel V. Maffini, Peter Geck, Charles E. Powell, Carlos Sonnenschein and Ana M. Soto

Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111-1800

Address all correspondence and requests for reprints to: Ana M. Soto, Department of Anatomy and Cell Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111. E-mail: . ana.soto{at}tufts.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens control the proliferation of their target cells first by increasing cell proliferation and later by inhibiting the proliferation of those same cells. Recently, we reported that the AS3 protein mediates the androgen-induced quiescence in androgen-target human cell lines. Our aims were to investigate the expression of the AS3 protein in the rat prostate in situ and in human cells in culture. Adult rats were separated into four groups (intact, castrated, castrated plus 3-d testosterone propionate replacement, and castrated plus 7-d testosterone propionate replacement). S9 cells expressing a tetracycline-regulated sense AS3 were also used. AS3 was expressed in the nuclei of over 90% of the epithelial cells and about 40% of the smooth muscle cells of the intact rat prostate. AS3 was not expressed in castrated rats or during the proliferative phase of androgen-induced regeneration. It was expressed in intact and castrated animals when the prostate has reached adult organ size. The AS3 protein was not expressed in cells that incorporate bromodeoxyuridine. These data suggest that AS3 is a mediator of the proliferative arrest in the normal rat prostate in situ and human prostate cell lines and that its expression is androgen-induced.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DEVELOPMENT AND maintenance of the morphology and functional activity of the prostate are androgen dependent. Plasma androgen levels are continuously high in the intact adult male rat; however, the prostate remains in a state of proliferative arrest. It is well established that after castration, extensive cell loss occurs rapidly in the prostate of adult rats. The steady administration of testosterone to these animals regenerates the gland within 7–10 d. Once maximal prostatic size is attained, further androgen treatment does not elicit a further increase in cell number, and the prostate shows the same state of proliferative arrest observed in the intact adult rat prostate (1, 2).

In previous studies, we have shown that in the human prostate LNCaP cell line variants, androgens control the proliferation of their target cells through a two-step mechanism (3, 4). In the first step, androgens mediate an increase in cell proliferation; whereas in the second step, androgens inhibit proliferation (proliferative shutoff) (1, 5). These two steps occur through discrete pathways (6). The AS3 gene, located in the human chromosome 13q12.3, has been shown to mediate the androgen-induced proliferative shutoff in the human prostate LNCaP-FGC cell line and the MCF-7-AR1 cell line, the human breast cancer MCF7 cell line transfected with the wild-type, full-length human androgen receptor (AR). During the androgen-induced proliferative arrest of both cell lines, there was an approximately 5-fold up-regulation in the expression of the AS3 protein (4). We used retroviral infection of MCF7-AR1 cells with virions containing tetracycline-regulated AS3 constructs to generate two cell lines; the S9 cell line contains the sense AS3 construct, whereas the A4 cell line contains the antisense AS3 construct. The tetracycline-regulated induction of the sense AS3 in S9 cells inhibited proliferation, as measured by bromodeoxyuridine (BrdU) incorporation. In addition, expression of antisense AS3 blocked the induction of the proliferative shutoff by androgens in A4 cells (7). These data suggest that AS3 plays a very important role in the androgen-induced proliferative arrest in cells in culture.

The aims of the present study were to investigate: 1) whether the AS3 gene is expressed in situ in the rat prostate; 2) which cell populations express the AS3 protein; and 3) how androgens regulate this expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
S9 and LNCaP-FGC cells were routinely maintained in DMEM containing 5% FBS. The medium for the S9 cells was supplemented with 1 µg/ml tetracycline. For the sense AS3 induction experiments, S9 cells were grown on coverslips using DMEM containing 5% charcoal-dextran stripped fetal bovine serum without tetracycline. The sense AS3 induction was carried out for 52 h. For the indigenous AS3 induction, LNCaP-FGC cells were exposed to the synthetic androgen R1881 at a 1-nM concentration for 52 h; these cells were used as positive control (7). Both cells were treated with 10 µg/ml BrdU (Roche Diagnostics, Mannheim, Germany) for 1 h before fixation in 50% methanol-50% acetone. Immunostaining for BrdU and AS3 was performed as described below.

Animals and tissue processing
All experimental procedures involving the use of animals were approved by the Tufts University-New England Medical Center Animal Research Committee. Three-month-old male Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were housed in an environmentally controlled room and provided with food and water ad libitum. The animals were separated into four groups: A) intact; B) chronically castrated (14 d after castration); C) castrated plus 3 d of testosterone propionate (TP) replacement; and D) castrated plus 7 d of TP replacement.

Seven days after castration, animals from groups C and D received a daily sc dose of 400 µg TP per 100 g body weight. Rats from group B received a daily dose of vehicle (sesame oil) for 7 d. All groups received a single ip injection of 0.2 ml 10-mM BrdU (Roche Diagnostics) 2 h before the ventral prostate lobes were removed. Tissues were fixed in 4% phosphate-buffered formalin and processed for paraffin embedding.

Northern blot analysis
Total RNA was prepared using the RNeasy kit (QIAGEN, Chatsworth, CA). RNA samples (20 µg) were separated on 1% agarose-formaldehyde gels and transferred to nylon membranes using the Turbo-Blot System (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). The PCR-amplified AS3 sequence was labeled by the Random Primed DNA Labeling Kit (Roche Molecular Biochemicals, Indianapolis, IN) and used as probe. Hybridization was performed using the human DNA fragment corresponding to nucleotide positions 1285–2471. The incubation was done overnight at 65 C in ExpressHyb solution (CLONTECH Laboratories, Inc., Palo Alto, CA). Membranes were analyzed by PhosphorImager (Molecular Dynamics, Inc., Stanton, CA) using the ImageQuant program.

Sequencing
Total RNA was prepared as described for Northern blot analysis. Random primed and oligo dT primed cDNA were synthesized using SuperScript II reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD), following the manufacturer’s instructions. PCR reactions were performed by the Expand High Fidelity thermostable enzyme combination (Roche Molecular Biochemicals). The PCR product was in the expected range (1300 bp); it was purified, sequenced, and analyzed using the GCG Wisconsin Package version 10.1 and SeqWeb 1.2 Best Fit programs.

Western blot analysis
Prostate ventral lobes were removed from intact rats; each lobe was cut into 3 small pieces, and each piece was immediately frozen in liquid nitrogen. Ice-cold lysis buffer [1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA, 50 mM sodium fluoride, 1 mM sodium vanadate, 0.2 mg/ml aprotinin (all from Sigma, St. Louis, MO) in PBS, pH 7] was added. The tissue was manually dounce-homogenized; 100 µg/ml phenylmethylsulfonyl fluoride was added, and the solution was incubated on ice for 30 min. The homogenate was centrifuged at 4 C for 20 min at 14,000 x g, and the supernatant was collected. The proteins were resolved by 4–20% SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-AS3 antibody at a 1:800 dilution; this rabbit antibody recognizes the antigenic epitope in positions 1369–1387 of the C-terminal region of the human AS3 protein (7). The peroxidase-conjugated secondary antibody (Zymed Laboratories, Inc., San Francisco, CA) was used at a 1:10,000 dilution, and the reaction was visualized with the Renaissance Chemiluminescence kit (NEN Life Science Products, Boston, MA). LNCaP-FGC cells treated with vehicle only (0.01% ethanol in DMEM) or with 1 nM R1881 for 48 h were harvested and lysed as described previously (7).

Immunohistochemistry
An antigen retrieval method based on microwave pretreatment and 0.01 M sodium citrate buffer (pH 6) was used. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide (Sigma) in methanol. Blocking was done with normal goat serum diluted 1:20 in PBG (PBS containing 0.5% BSA and 0.1% gelatin, pH 7.4). Rabbit anti-AS3 was used at a 1:100 dilution in PBG and was allowed to react overnight at 4 C. Biotin-conjugated goat antirabbit IgG (Zymed Laboratories, Inc.) was diluted 1:500 in PBG. The antigen-antibody reaction was visualized using the streptavidin-biotin-peroxidase complex method, with diaminobenzidine tetrahydrochloride (Sigma) as the chromogen. Counterstaining was performed with Harris’ hematoxylin, and the sections were mounted using Permount (Fisher Scientific, Fairlawn, NJ). The competition assay was performed using the same protocol; the anti-AS3 antibody was coincubated with 5-fold excess of the specific C-terminal oligopeptide for 2 h at 4 C. Rabbit antiandrogen receptor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 1:800 dilution.

For localization of AS3, AR, and cytokeratin 5 by double immunohistochemistry, sections were stained for AS3 or AR as described above. Before counterstaining, sections were incubated with double stain enhancer (Zymed Laboratories, Inc.) for 20 min. After rinsing with PBS, sections were incubated with blocking solution followed by the primary antibody for cytokeratin 5 (Chemicon International, Inc., Temecula, CA) at 1:100 dilution. The incubation was carried out for 14–16 h at 4 C, and the reaction was developed as described above. After color reaction with a peroxidase substrate kit (Vector VIP; Vector Laboratories, Inc., Burlingame, CA), sections were counterstained with Harris’ hematoxylin.

For the double staining immunofluorescence technique, BrdU incorporation was detected by using 5-bromo-2'-deoxyuridine Labeling and Detection Kit I (Roche Diagnostic) according to the manufacturer’s instructions. Anti-AS3 antibody, secondary antibody, and streptavidin-Alexa 594 conjugate (Molecular Probes, Inc., Eugene, OR) were used at a 1:100 dilution. The antibodies were diluted in a blocking solution of 4% BSA in PBS supplemented with 10% normal goat serum. Cell nuclei were counterstained with Hoechst 33258. Images were captured with a SPOT RT color digital camera (Diagnostic Instruments, Sterling Heights, MI) attached to an Axioscope fluorescence microscope.

Quantification of AS3 expression
Epithelial cells expressing AS3 were counted at a magnification of x400. A total of 1000 epithelial cells per animal were counted in randomly chosen fields. The smooth muscle cells in the peritubular stroma were also counted. Three animals per group were analyzed. Because the data gathered were not normally distributed, the nonparametric Mann-Whitney test was used to compare the expression of AS3 among groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of AS3 mRNA and protein in the adult rat prostate
Northern blot analysis was performed to evaluate the AS3 mRNA expression during the androgen-mediated proliferative shutoff in the prostate of intact rats. The mRNA presented two isoforms, of 5.5 and 8 kb (Fig. 1Go). The size of these isoforms concurs with our previous results obtained using human LNCaP cell lines grown in the presence of the synthetic androgen R1881 (8).



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  		Figure 1. Northern blot analysis of AS3 gene expression in the rat prostate. The AS3 gene is expressed in both dorsal and ventral lobes. Rat and human AS3 RNA are both present in two isoforms (5.5 and 8 kb).

 
When comparing the rat and human C-terminal regions of the AS3 amino acid sequences, we observed an 89.6% overall identity, whereas there was a 100% homology in the area coding for the antigenic epitope (Fig. 2AGo). Immunoblot analysis of protein extracts from the intact rat ventral prostate revealed a single band (Fig. 2BGo). The molecular mass of the rat AS3 protein (164–166 kDa) was in the same range as human AS3 (165–170 kDa). These findings suggest that AS3 is a highly conserved protein.



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  		Figure 2. Comparison between the human and rat AS3 protein. A, Comparison of the C-terminus 403 amino acids of the human and rat AS3 sequences. The letters in bold represent amino acid changes in the rat sequence. The bold and underlined ones represent the antigenic epitope; the homology between the rat and human sequence in this epitope is 100%. B, Immunoblot analysis of AS3 protein in cellular and tissue extracts. Lanes 1 and 2, Lysates from LNCaP-FGC cells, treated with vehicle only and with R1881, respectively. Lane 3, Whole-cell extract from rat prostate ventral lobe. Both human and rat AS3 are in the range of 165–170 kDa. The numbers at the left indicate the molecular markers.

 
In the intact animal prostate, the AS3 protein exhibited nuclear localization (Fig. 3AGo). It was expressed in the nuclei of about 90% of the epithelial cells, both luminal and basal, and in about 40% of the smooth muscle cells in the stroma (Table 1Go). Fig. 3BGo revealed the specificity of the antibody as the signal disappeared completely after coincubation with the specific oligopeptide.



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  		Figure 3. AS3 immunodetection in prostate sections from adult intact rats. A, The epithelial cells show strong nuclear intensity (arrow). Approximately 40% of the muscle cells present in the stroma are positive for AS3 (arrowhead). B, The specificity of the nuclear signal was assessed by way of a competition assay whereby the anti-AS3 antibody was preincubated with a 5-fold excess of the specific oligopeptide. The lack of any staining confirms the specificity of the AS3 antibody. Photomicrographs were taken at x630.

 

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  		Table 1. Quantitative analysis of AS3-positive cells in the prostate

 
AS3 protein expression is regulated by androgens
The expression of the AS3 protein occurred exclusively in the rat prostate inhibited from proliferating by long-term androgen exposure (Fig. 4Go, A and D). The pattern of AS3 expression was the same in the animals from groups A (intact) and D (7 d of TP treatment) (Table 1Go). The protein was expressed in about 90% of epithelial cells, showing a similar strong nuclear signal in both groups. However, the expression of AS3 was not present in group B (chronically castrated animals) (Fig. 4BGo) nor in group C during the peak of the androgen-mediated proliferative state (Fig. 4CGo). Rats from groups A, C, and D expressed high levels of nuclear AR (Fig. 5Go; A, C, and D), whereas group B showed cytoplasmic AR staining (Fig. 5BGo). We also observed that basal epithelial cells from groups A and D expressed AS3 as well as AR (Fig. 6Go). The basal cells were identified by their ability to express cytokeratin 5.



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  		Figure 4. AS3 expression in the rat prostate occurs only during androgen-mediated proliferative shutoff. A, Intact adult rat; B, chronically castrated rat; C, maximal cell proliferation induced by 3 d androgen replacement in a castrated rat; D, proliferative shutoff after 7 d of androgen treatment in a castrated rat. Photomicrographs were taken at x630.

 


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  		Figure 5. AR expression. The AR exhibits nuclear localization and strong intensity when the levels of androgens are high (A, C, and D). In castrates, the AR exhibits cytoplasmic localization (B). Photomicrographs were taken at x630.

 


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  		Figure 6. AS3 and AR are expressed in luminal and basal epithelial cells. The basal cells were identified by cytokeratin 5 immunostaining (purple). Double staining for AS3 and cytokeratin 5 (A) and AR and cytokeratin 5 (B). Photomicrographs were taken at x630.

 
The AS3 gene product inhibits cell proliferation
AS3 expression and BrdU incorporation were examined in the rat ventral prostate from all experimental groups and in the S9 cells. Animals from groups A and D showed a very low BrdU incorporation rate, whereas the AS3 protein was expressed in the majority of the luminal epithelial cells (Fig. 7Go, A and D). The double immunofluorescence staining showed that cells that incorporated BrdU did not express AS3, and those that expressed AS3 did not incorporate BrdU. In animals from group B, there was neither BrdU nor AS3 expression (Fig. 7BGo). Among animals from group C, no AS3 expression was immunodetected; only cells that incorporated BrdU were stained (Fig. 7CGo).



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  		Figure 7. Proliferative arrest in cells expressing AS3. The double staining immunofluorescence technique shows that those cells that express AS3 (red) do not incorporate BrdU (green). A, Intact adult rat: the proliferative shutoff is expressed. AS3 expression is observed in most cells; few BrdU-labeled cells are observed. B, Lack of AS3 expression and BrdU incorporation in chronically castrated animals. C, Maximal proliferation during androgen-induced regeneration; note high BrdU incorporation and absence of AS3 expression in the prostate of castrated animals treated for 3 d with TP. The red signal is attributable to autofluorescence of red blood cells inside blood vessels. D, Proliferative shutoff after 7 d of TP treatment. As in the intact adult animal, AS3 is expressed in most cells; very few BrdU-labeled cells are observed. Photomicrographs were taken at x630 using standard fluorescein isothiocyanate filters for BrdU detection and rhodamine filters for AS3 detection.

 
To assess whether tetracycline-controlled AS3 protein expression induced the proliferative shutoff, double immunofluorescence staining for BrdU and AS3 was carried out in S9 cells. In the presence of tetracycline, cells proliferated, and the sense AS3 construct was repressed. When tetracycline was withdrawn, the induction of the AS3 leads the cells to a proliferative arrest. As is shown in Fig. 8Go, cells that expressed AS3 did not incorporate BrdU. Similar results were obtained in S9 cells when the indigenous AS3 gene was induced by androgens (not shown).



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  		Figure 8. Analysis of AS3 expression and BrdU incorporation in S9 cells. Double labeling with antibody against BrdU (green) and AS3 (red) using immunofluorescence. The AS3 protein expression in S9 cells is repressed by tetracycline. A, Maximal cell proliferation (cells grown in media containing tetracycline). B, Proliferative shutoff (cells grown in media without tetracycline for 52 h); note high expression of AS3 with low BrdU incorporation. Magnification, x630. Photomicrographs were taken by using standard fluorescein isothiocyanate filters for BrdU detection and rhodamine filters for AS3 detection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In laboratory rodents, prostatic growth and ductal morphogenesis are essentially continuous processes that extend from late fetal life until sexual maturity (9). Once the animal reaches sexual maturity, most prostate epithelial cells are in a state of proliferative quiescence (10). Using human cell lines in a cell culture model, we have previously demonstrated that androgens regulate this effect by inducing cell cycle arrest in the G0/G1 phase (11) and that this proliferative shutoff is mediated by the induction of AS3, a newly identified gene (4, 7). Moreover, when the expression of the AS3 mRNA is induced from a tetracycline-regulated construct by tetracycline withdrawal, a decrease in the rate of BrdU incorporation is observed in the host cells (7). Further, the expression of AS3 antisense blocks the inhibitory effect of androgens (7). Using double immunofluorescence staining, we now show that the staining patterns of the AS3 protein and BrdU are mutually exclusive.

The present study shows that, in the adult rat prostate, AS3 mRNA is present in two isoforms, a feature comparable with results obtained using human prostate cancer cells (8). The recently available human genome sequence (accession no. NT-009984.7), GenBank expressed sequence tag (EST) (accession nos. BF343183.1, BI086359.1, BF692419, AI636348, BI621230, and T515431) and AS3 splicing variant data (accession nos. XM-033043, AL137201) indicate that the 8-kb mRNA has an alternative polyA signal about 3 kb downstream from the termination point of the 5.5-kb isoform. The open reading frames are identical, however, and they code for the same protein. Moreover, the molecular mass of the rat AS3 protein is similar to that of the human AS3. A comparison of the sequence coding for the 403 amino acids of the C-terminal in humans and rats revealed strong homology between the two. Such evolutionary conservation indicates a potential important role in maintaining normal cellular physiology in an androgen-dependent tissue such as the prostate.

In castrated rats, the proliferative pattern observed during androgen-induced prostatic regeneration consists of an initial lag period followed by a massive wave of cell proliferation, with a peak 3 d after initiation of androgen replacement. This is followed by a period of proliferative arrest in which cell numbers decline despite a steadily high level of androgens (1, 2). To better understand the correlation between AS3 expression and cell proliferation, we evaluated the pattern of expression of this protein during the stages of maximal cell proliferation and proliferative shutoff after complete organ weight restoration. The lack of AS3 expression during the peak of cell proliferation, after 3 d of hormonal replacement, and its expression when the prostate has reached its adult size strongly suggest that AS3 plays a key role in the control of androgen-induced proliferative arrest in the prostate. These results are consistent with the observation that induction of a tetracycline-regulated sense AS3 construct in S9 cells leads to cell proliferation arrest.

Analysis of the AS3 sequence has shown that the protein has a DNA-binding domain and a nuclear localization sequence in the C-terminal region (4) resembling that of the AR (12) and DNA polymerase-{alpha} (13). The nuclear localization of the AS3 protein was demonstrated experimentally in the present study. In addition to the DNA recognition domain, AS3 has protein-protein interaction motifs (4), suggesting that this protein could act as a transcription factor, inducing the shutoff effect by controlling the expression of downstream genes. Protein phosphorylation is an important regulatory mechanism of signaling, and the presence of a protein-kinase domain in the AS3 protein suggests that it may directly activate other proteins. Functional analysis of the protein kinase domain is now in progress using a GST-fusion construct. Pilot data suggest that the fusion construct can form a complex and phosphorylate substrate proteins in LNCaP-FGC extracts. It is not yet clear whether this effect is catalyzed directly by the AS3 fusion protein or whether AS3 is involved indirectly, through binding a protein kinase and its substrate. Kokontis et al. (14) suggest that the androgen-mediated arrest results in enhanced expression of p27kip. We hypothesize that AS3 may act by interacting with the cell cycle negative effectors p21 and p27.

Bruchovsky et al. (1) proposed that the prostate homeostasis seems to be achieved, in part, by the balanced function of two cellular constraint mechanisms, with one responsible for initiating DNA synthesis and cell proliferation and the other responsible for suppressing these processes. Later, De Klerk and Coffey (15) showed that castration elicits a disproportionate loss of epithelial vs. stromal cells, which changed the epithelial-stroma ratio. This change in the epithelial-stroma ratio may have important consequences in the regulation of prostatic epithelial proliferation, both during prostatic regeneration in castrates after androgen replacement therapy and during prepubertal prostatic development (16). It is also known that androgens affect epithelial and stromal cell types, which, in turn, interact in the prostate (17, 18). AS3 is induced by androgens, and it localizes in the nuclei of basal and luminal epithelial cells and smooth muscle stromal cells. These cell types also express AR. AS3 is associated with inhibition of epithelial cell proliferation, suggesting that it may be a mediator in the control of prostate growth.

In conclusion, the results from the present study strongly suggest that the androgen-induced proliferative arrest in normal rat prostate tissue in situ and that in human androgen-target cell lines occur through a similar mechanism involving the expression of the AS3 protein. In addition, our results support previous findings that the regulation of AS3 expression is dependent on the AR. Finally, these data demonstrate that the androgen-mediated proliferative shutoff can now be studied in the rat prostate in situ, using AS3 as a marker for this phenotype.


   Acknowledgments
 
The technical contributions of Ms. Cheryl Michaelson and Mr. Aaron Cook are appreciated. We thank Dr. Karina Meiri, Ms. Janine Calabro, and Carise Wieloch for their suggestions.


   Footnotes
 
This work was supported by NIH Grants PHS-CA-55574 and CA-13410 and a grant from the Massachusetts Department of Public Health. M.V.M. is a recipient of a postdoctoral fellowship supported, in part, from the World Bank Grant 815 and Universidad Nacional del Litoral, Santa Fe, Argentina.

Abbreviations: AR, Androgen receptor; BrdU, bromodeoxyuridine; TP, testosterone propionate.

Received January 7, 2002.

Accepted for publication March 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bruchovsky N, Lesser B, Van Doorn E, Craven S 1975 Hormonal effects on cell proliferation in rat prostate. Vitam Horm 33:61–102[Medline]
  2. English HF, Santen RJ, Isaacs JT 1987 Response of glandular versus basal rat ventral prostatic epithelial cells to androgen withdrawal and replacement. Prostate 11:229–242[Medline]
  3. Sonnenschein C, Olea N, Pasanen ME, Soto AM 1989 Negative controls of cell proliferation: human prostate cancer cells and androgens. Cancer Res 49:3474–3481[Abstract/Free Full Text]
  4. Geck P, Szelei J, Jimenez J, Sonnenschein C, Soto AM 1999 Early gene expression during androgen-induced inhibition of proliferation of prostate cancer cells: a new suppressor candidate on chromosome 13, in the BRCA2-Rb1 locus. J Steroid Biochem Mol Biol 68:41–50[CrossRef][Medline]
  5. Olea N, Sakabe K, Soto AM, Sonnenschein C 1990 The proliferative effect of "anti-androgens" on the androgen-sensitive human prostate tumor cell line LNCaP. Endocrinology 126:1457–1463[Abstract]
  6. Soto AM, Lin TM, Sakabe K, Olea N, Damassa DA, Sonnenschein C 1995 Variants of the human prostate LNCaP cell line as a tool to study discrete components of the androgen-mediated proliferative response. Oncol Res 7:545–558[Medline]
  7. Geck P, Maffini MV, Szelei J, Sonnenschein C, Soto AM 2000 Androgen-induced proliferative quiescence in prostate cancer: the role of AS3 as its mediator. Proc Natl Acad Sci USA 97:10185–10190[Abstract/Free Full Text]
  8. Geck P, Szelei J, Jimenez J, Lin TM, Sonnenschein C, Soto AM 1997 Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells. J Steroid Biochem Mol Biol 63:211–218[CrossRef][Medline]
  9. Janulis L, Lee C 1999 Prostate gland. In: Knobil E, Neill JD, eds. Encyclopedia of reproduction. San Diego: Academic Press; 77–85
  10. English HF, Drago JR, Santen RJ 1985 Cellular response to androgen depletion and repletion in the rat ventral prostate: autoradiography and morphometric analysis. Prostate 7:41–51[Medline]
  11. Szelei J, Jimenez J, Soto AM, Luizzi MF, Sonnenschein C 1997 Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells transfected with androgen receptor. Endocrinology 138:1406–1412[Abstract/Free Full Text]
  12. Hanks SK, Quinn AM 1991 Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol 200:38–62[Medline]
  13. Taylor SS, Knighton DR, Zheng J, Ten Eyck LF, Sowadsky JM 1992 Structural framework for the protein kinase family. Annu Rev Cell Biol 8:429–462[CrossRef]
  14. Kokontis JM, Hay N, Liao S 1998 Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27kip1 in androgen-induced cell cycle arrest. Mol Endocrinol 12:941–953[Abstract/Free Full Text]
  15. DeKlerk DP, Coffey DS 1978 Quantitative determination of prostatic epithelial and stromal hyperplasia by a new technique. Biomorphometrics. Invest Urol 16:240–245[Medline]
  16. Cunha GR, Donjacour AA, Cooke PS, Mee S, Bigsby RM, Higgins SJ, Sugimura Y 1987 The endocrinology and developmental biology of the prostate. Endocr Rev 8:338–362[Medline]
  17. Chang SM, Chung LWK 1989 Interaction between prostatic fibroblast and epithelial cells in culture: role of androgen. Endocrinology 125:2719–2727[Abstract]
  18. Hayward SW, Rosen MA, Cunha GR 1997 Stromal-epithelial interactions in the normal and neoplastic prostate. Br J Urol 79(Suppl 2):18–26



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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals