Androgen and prostatic stroma*

Department of Prostatic Disease, Tianjin Institute of Urologial Surgery, Tianjin Medical University, Tianjin 300211, China

Asian J Androl 2003 Mar; 5: 19-26             

Keywords: androgen; castration; androgen receptor; prostatic stroma; smooth muscle cells

Abstract

Aim: To investigate the effect of androgen on the proliferation, differentiation and regression of canine prostatic stromal cells in vivo and human stromal cells in vitro. Methods: Twenty-two dogs, including 15 normal prostate dogs and 7 prostatic hyperplasia dogs, had their serum concentration of testosterone and estrodiol determined by radioimmunoassay before and after castration. The expression of androgen receptor (AR) and estrogen receptor (ER) in the prostate were analysed by immunohistochemistry and semi-quantitative RT-PCR before and after castration. Light microscopy, transmission electron microscopy and TUNEL assay were carried out successively before and after castration to evaluate the prostatic histomorphology. In vitro serum-free cell cultures from human prostatic stroma were established and exposed to dihydrotestosterone (DHT). The proliferation of the cell culture was detected by MTT assay. The expression of TGFb, bFGF, AR, and smooth muscle cell (SMC) specific proteins (myosin and/or smoothelin) were detected using immunohistochemistry and RT-PCR. The differentiation from fibroblasts to smooth muscle cells was deduced by measuring the expression of SMC specific proteins. Results: Before castration, the serum concentrations of testosterone and estrodiol were not statistically different between normal and hyperplasia groups. Following castration, the serum concentration of testosterone decreased rapidly in 2 days, and the concentration of estrodiol had no significant change compared with the pre-castration data. In the prostate, AR was presented in both the epithelial and stromal cells and the AR mRNA level was higher in hyperplasia than in normal prostate tissues (P<0.05). While ER predominantly existed in the prostate stromal cells and the ER mRNA had no difference between the hyperplasia and the normal group. Within the early phase of castration (<d7), the expression of AR was increased rapidly. Then it gradually dropped to a lower level than that of the pre-castration by the end of d90. The expression of ER remained unchanged in the whole course. The prostatic stromal cells, including SMCs and fibroblasts, diminished and underwent serial pathological changes of atrophy and apoptosis after castration. The atrophic cells were filled with huge intracellular lipofuscin. The expression of SMC myosin declined after castration, coincident with the increase in TGFb mRNA level and decline in bFGF mRNA level. In vitro, DHT caused a weak increase in the proliferation and expression of SMC-specific proteins (P<0.05). However, DHT and bFGF together stimulated the proliferation of stromal cells significantly more than either agent alone (P<0.01). The combination of DHT and TGFb greatly enhanced the expression of SMC-specific proteins (P<0.01) more strongly than either alone (P<0.01). Conclusions: The whole prostate gland is an androgen-sensitive organ with both the epithelium and stroma under the control of androgen. Androgen may direct the proliferation, differentiation and regression of stromal cells by regulating the expression of TGFb, bFGF, AR and smooth muscle cell specific proteins.

1 Introduction

Androgen is the most important male sex hormone and the most critical molecule in controlling prostate growth. The human embryonic prostate differentiates in response to androgens secreted by the foetal testis after 8 weeks of gestation. Exogenous testosterone accelerates the growth of an immature prostate, castration prior to puberty prevents prostatic development and testosterone administration to castrated adult causes the gland growing to the normal adult size [1].

Benign prostatic hyperplasia (BPH) is the most common benign tumor in aged men. The etiology of this disease remains poorly understood. Although both the stromal and epithelial components are involved in the processes of BPH, the stroma plays important roles in the development of BPH. McNeal [2] indicated that the reversion of the stroma to an "embryonic" phenotype and the formation of hyperplastic stromal nodules were the earliest events in BPH. The de novo epithelial glandular formation that occurs in BPH is a later event that is induced by paracrine factors from the stromal nodules. In the stromal-epithelial interaction mechanism of BPH formation, the stroma is the key factor.

Androgen may also be involved in the stromal-epithelial interaction. In the mature prostate, androgens are known to elicit various alterations on prostatic epithelium via the androgen receptors in the "stroma" Immunocytochemical studies have shown that prostatic smooth muscle cells (SMCs) are uniformly androgen receptor-positive, whereas many fibroblasts are androgen receptor-negative [3, 4]. These findings indicated that prostatic SMCs might be important targets for androgen action and were capable to regulate the expression of prostatic growth factors. Specific growth factors derived from the stroma, e.g. the transforming growth factor (TGF)-b and the fibroblastic growth factor families, accumulate and contribute to the progression of BPH [5]. The concentration of bFGF and TGFb₂ was increased in BPH tissue [6, 7]. The overproduction and/or malfunction in the growth factor response pathways may be related to the pathogenic mechanism of BPH.

It is not clear whether androgen and growth factors directly regulate SMCs proliferation and differentiation in a sympathetic and/or antogonistic manner. In this study, the effects of androgen, TGFb and bFGF on the stromal cell proliferation and differentiation were investigated in vivo and in vitro. It was indicated that the homeostasis of prostatic stromal cells was under the control of androgen and growth factors.

2 Materials and methods

2.1.1 Animals
Twenty-two adult male dogs (2~6 years old and 20~30 kg body weight) were obtained from the Department of Experimental Animal of the University. Bilateral orchiectomy was performed under pentobarbital sodium anesthesia. The serum and prostate samples were collected before and at d3, 7, 14, 30 and 90 after castration.

2.1.2 Serum testosterone and estradiol and prostatic expression of their receptors
The serum testosterone and estradiol concentrations were determined with radioimmunoassay (RIA) kits bought from the Larwin Bio-technological Co. (Shenzhen, China). Immunohistochemistry method was carried out to determine the expression of androgen receptor (AR) and estrogen receptor (ER) in the formalin-fixed prostatic tissue slide. The AR and ER immunostain kits were bought from the Zhongshan Bio-tech Ltd. (Beijing, China).

2.1.3 AR ,ER, TGFb1 and bFGF expression
The expression of AR, ER, TGFb1 and bFGF were determined in the prostate tissue by the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).

The total RNA was isolated from the frozen tissue or cells using a guanidinium thiocyanate-acid phenol-chloroform (pH 4.0) extraction method. Samples were precipitated with isopropanol and were treated with protease K (200 µg/mL, Sigma) at 55 for 15 min. Then the phenol-chloroform extraction procedure was repeated. The amount of RNA was determined by measuring the absorbance at 260 nm.

Reverse transcription was performed for 2 h at 37 on 500 ng of RNA in 20 µL final mixture containing 0.5 mmol/L of dNTP, 5 µL of N9-primers, 10 nmol/L of DTT, 20 units of ribonuclease inhibitor (Promega) and 200 units of Mo-MLV reverse transcriptase (Gibco).

PCR was performed with 1 µL of cDNA in a final volume of 20 µL containing 200 µmol/L of dNTP, 0.5 unit of Tag polymerase and 0.25 µmol/L of each primer. b-actin was used as an internal control. The sequence of primer was shown in Table 1. Synthesis was performed with 20 s at 94 , 15 s at 96 , 1 min at 55 and 30 s at 73 (32~35 cycles). The PCR product was quantitated using CCD methods.

Table 1. Nucleotide sequences used for PCR.

2.1.4 Histomorphologic evaluation

The prostatic samples were fixed and stored in 10 % neutral buffered formalin, embedded in paraffin blocks, cut at 3 µm thickness and stained with HE. The slides were examined under a light microscope.

Transmission electron microscopy was carried out. Samples were fixed in 2.5 % (v/v) glutaraldehyde. After postfixation with OsO₄ (1 %, w/v in water), they were embedded in Epon-812 (Japan). Ultrathin sections (60 nm) were stained at 25 with aqueous uranyl acetate (0.5 %, w/v) for 40 min followed by lead citrate solution for 5 min. The samples were examined with an electron microscope at 60 and 80 kV (JEM-100CX, Japan).

2.1.5 Apoptosis

Tissue sections were analyzed for in situ apoptosis using the terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end labeling (TUNEL) method. The DNA fragments in apoptotic cells were labeled at free 3'-OH DNA ends and DNA strand breaks. Incorporated fluorescin was detected by anti-fluorescin antibody conjugated with alkaline phosphatase. After substrate reaction stained cells were analyzed with light microscopy. Apoptotic staining of prostatic cell death induced by castration was performed in paraffin-embedded sections using the in situ Cell Death Detection Kit (Boehringer, Germany) according to the manufacturer's instructions.

2.2 In vitro experiments

Stromal cell cultures were established according to the method previously described [8]. Surgically obtained tissue specimens from histologically proven human BPH were cut into pieces (1 mm³) and digested for 4~5 h at 37 in collagenase (200 u/mL, Yakult Pharmaceutical Industries Ltd., Japan) associated with elastase (0.1 mg/mL, Sigma, USA) and deoxyribonuclease I (40 u/mL, Sigma) in MCDB131 medium. After three washes in HEPES -buffered saline, the cell suspension was decanted through a 100 µm mesh and cultured in MCDB131 medium (Sigma) supplied with 10 % fetal bovine serum, 10 mmol/L of HEPES (pH 7.2), 2 % penicillin-streptomycin solution, insulin (5 µg/mL, Boehringer), transferrin (10 µg/mL, Boehringer), sodium selenite (5 µg/mL, Sigma), estradiol (0.1 µmol/L, Sigma) and dexamethasone (0.1µmol/L, Sigma). Cell cultures were incubated in a humidified atmosphere at 37 with 95 % air and 5 % CO₂. Both the stromal and epithelial cells were attached for nearly 1 week and then the medium was changed every 2~3 days. After reaching confluence, the stromal cells significantly outgrew the epithelial cells. The cells were passed into new flasks at a ratio of 1:2 using trypsin/EDTA solution (Boehringer). The remaining epithelial cells were lost at the first passage. From passage 1 to passage 3, the serum in the medium withdrew gradually from 10 %, 5 % to 3 %. The cells were trypsinized as described above and resuspended at 410⁵ cells/mL in the serum-free medium and inoculated into collagen S coated flasks. The serum-free medium contains MCDB131 medium, 10 mmol/L of HEPES (pH 7.2), 1 % penicillin-streptomycin solution, ITS premix at 1:1000, 0.1 µmol/L of estradiol, 0.1 µmol/L of dexamethasone, 10 mg/L ATP and 50 µg/mL of trypsin inhibitor (from soy bean, Sigma). After one week, the medium was changed and the cells incubated with the experimental agents for 48 h. Eight experimental groups were divided with different combinations of the active agents as shown in Table 2.

Table 2. Treatment (TGFb1 and bFGF in ng/mL, DHT in µmol/L).

2.2.2 Cell growth analysis

MTT test was used to determine the effects of DHT, TGFb and bFGF on the growth of prostatic stromal cells. Mitochondrial enzymes in metabolically active cells could decompose a tetrazollium salt, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tertazolium bromide (MTT), to a colored formazan product with a maximum absorption at 560 nm. Cells were harvested by trypsinization, resuspended in fresh culture medium and plated in a volume of 0.1 mL/well (4000/well) in 96-well microtitor plates (costar). On d3, 30 µL of a 5 mg/mL solution of MTT in PBS was added to each well. After 4h incubation at 37 in 5 % CO₂/air, the medium was carefully removed and the purple dye dissolved in 0.1 mL DMSO. The plates were shaken for 5 min and the absorbance at 560 nm measured by spectrophotometry. Eight replicate wells were used for each sample. Wells containing no cells served as the blanks and wells with untreated cells as the normal control.

2.2.3 Immunohistochemical Staining

Cytoskeletal markers for the SMCs were assessed in cultured stromal cells, which were grown on chamber slides. Cytoskeletal proteins were stained with the anti-myosin (smooth muscle, Sigma)monoclonal mouse antibody used at a 1:200 dilution in blank solution. The slides were covered with antibody solutions at 4 overnight and then further developed using an anti-mouse IgG streptavidin kit, POD (Vector Labs., Burlinghame). Cells with their cytoplasm stained brownish-black were considered positive.

2.2.4 RT-PCR detection of TGFb1, bFGF, myosin and smoothelin expression

The expression of smoothelin was detected in the cultured stromal cells by semi-quantitative RT-PCR. b-Microglobulin was used as an internal control. The method was shown in 2.1.3 and the primer sequences in Table 1.

2.3 Statistical analysis

The serum hormone data were analyzed using a two-sample t-test and the results of immunohistochemistry using rank sum test and anova for the MTT assay and RT-PCR. All P values were two-sided and were deemed significant at P<0.05.

3 Results

Castration resulted in a significant decrease in circulating testosterone, but not the estradiol levels. The serum testosterone levels dropped 50 times from about 515 ng/L pre-castration to about 10 ng/L 3 days post-castration (P<0.01). The changes of estradiol serum level following castration were not statistically significant (P> 0.05). In the prostate, AR was presented in both the epithelial and stromal cells, while ER predominantly in the stromal cells. The AR mRNA level was higher in the hyperplasia than in the normal prostate tissue (P<0.05), while the ER mRNA did not have a significant difference. Following castration, the expression of AR was increased rapidly on d7 and then gradually dropped to a lower level than that of the pre-castration by d90. The variation in the expression of AR was coincident with the changes in the number of apoptotic cells in the tissue slide. There were not significant changes in ER mRNA levels before and after castration.

From d3 to d90 following castration, the morphological changes became gradually serious in both the epithelium and stroma. Under the light microscope, the glandular epithelial cells changed gradually form the high-columelliform pre-castration to the low-columelliform or cuboid form on d7 post-castration and finally turn to flatcytes and "small dark cells" on d90. The gland lumen shrank and was diminished in size after castration. Light microscope also showed that the volume of stromal cells shrank and the components of the stroma were decreased. By TUNEL assay, less than 1 % of the glandular cells died via apoptosis in intact dogs. Following castration, this percentage increased (P<0.01) drama-tically. From d7 to d14 about 40 % of the prostatic cells were apoptotic. Before d14, the stained positive cells were detected mainly in the prostatic epithelium with less stromal cells involved, leading to an elevated stroma/epithelium ratio. However, from d14, the stained apoptotic cells could be observed in the stroma. Interestedly, the endothelial cells and vascular wall SMCs were also positively stained, indicating the involvement of blood vessels. The basal epithelial cells did not fall into apoptotic process. Following castration, the ratio of the basal cells to the glandular cells was elevated.

With electron microscopy, the prostatic stromal fibroblasts, SMCs and glandular epithelial cells were involved in the morphological changes induced by castration. Apoptosis, atrophy and oncosis could be observed in both the epithelium and stroma. The morphological changes in the epithelium appeared early at d2 to d14 and were predominated by apoptosis. The apoptotic epithelial cells showed typical nuclear chromatin condensation into crescent-shaped caps adjacent to the nuclear membrane and "apoptotic bodies". However, stromal cells died in a process of so-called "para-apoptosis" with non-typical nuclear chromatin condensation (Figure 1). Characteristically, many stromal cells atrophied after d14. The cellular atrophy was characterized by numerous huge intracellular lipofuscin filled in the plasma that decreased the cell volume (Figure 2). Atrophied cells appeared after d14 and became gradually serious in the stroma and epithelium. Oncosis could also be found at the early phase following castration, but it was not the main pathological changes induced by castration.

Figure 1A. Electron micrograph (9900), apoptotic prostatic epithelial cells showing typical nuclear chromatin condensation at d3 following castration.
Figure 1B. Electron micrograph (9900) d30 after castration, para-apoptotic stromal SMCs with non-typical nuclear chromatin condensation.

Figure 2A. Electron micrograph (7500) atrophic cells in prostatic stroma at d14 after castration, showing decreased cell volume and characteristic huge intracellular lipofuscin.
Figure 2B. Electron micrograph (9900) d30 after castration, plasma of atrophic stromal fibroblasts filled with huge lipofuscin, so that nuclei could not be seen.
Figure 2C. Electron micrograph (9900) d30 after castration, plasma of atrophic glandular epithelial cell filled with huge lipofuscin, nuclear changes relatively insignificant.

Following castration, TGFb₁ mRNA levels in the prostate were elevated, while bFGF mRNA levels dropped. Within 14 days after castration, the expression of the TGFb₁ increased gradually to 2 times higher at d14 than pre-castration (P<0.01), while the expression of bFGF decreased dramatically to 4 times lower on the first 14 days (P<0.01). So the TGFb₁/bFGF ratio was up regulated (Table 3). The up-regulation of the ratio was correlated with the decrease of serum testosterone levels (P<0.01) and corresponding morphological changes in the stroma.

Table 3. Expression of TGFb₁ and bFGF induced by castration (meanSD).  ^cP<0.01. 

3.4 Cellular growth regulated by DHT, TGFb₁ and bFGF

For the prostatic stromal cells in the exponential growth period, TGFb₁ inhibited the cellular growth in a concentration-dependent manner (P<0.01), while bFGF stimulated the growth (P<0.01). DHT had a slightly stimulating effect (P<0.05). However, the combination of DHT and bFGF (10 ng/mL) significantly increased the proliferation of the stromal cells above either agent alone (P<0.01)(Figure 3).

Figure 3. Effect of DHT, TGFb and bFGF on growth rate of prostatic stromal cells in exponential growth phase. 10 ng/mL TGFb (T10) decreased growth rate by 77 %, 10 ng/mL bFGF (bF) increased growth rate by 168 % (P<0.01), 1 µmol/L DHT (D) increased growth rate by 124 % (P>0.05). DHT + bFGF increased growth rate by 240 %.

For the confluently cultured prostatic stromal cells, TGFb₁ stimulated the myosin staining (P<0.01) (Figure 4) and the expression of smoothelin (P<0.01) (Figure 5). The higher the TGFb₁ concentration is the stronger the expression of myosin and smoothelin would be. bFGF inhibited the expression of smoothelin (P<0.01) and had no significant effect on the expression of myosin (P> 0.05). DHT alone had little effect on the expression of smoothelin (P>0.05), nevertheless, the effect of combined DHT and TGFb₁ was much stronger than that of TGFb₁ by a rate of 433.3 % (P<0.01).

Figure 4A. (Immunostain of SMC myosin, 40) Human normal prostatic stromal cells cultured in serum-free medium, showing only a few smooth muscle cells (brown).
Figure 4B. (Immunostain of SMC myosin, 40) After 48 h exposure to 10 ng/mL TGFb₁, with increased smooth muscle cells.
Figure 4C. (Immunostain of SMC myosin, 200) Smooth muscle cells exposed to 10 ng/mL TGFb₁ for 48 h.
Figure 4D. (Immunostain of SMC myosin, 40) After 48 h exposure to 10 ng/mL bFGF, with fewer stained smooth muscle cells.
Figure 4E. (Immunostain of SMC myosin, 40) Smooth muscle cells increased by combination of DHT (10-6 mol/L) and TGFb₁ (10 ng/mL).

Figure 5. Effect of DHT, TGFb and bFGF on expression of smoothelin in confluently cultured prostatic stromal cells. 10 ng/mL TGFb significantly increased smoothelin expression. 10 mg/mL bFGF decreased smoothelin expression. DHT alone did not significantly increase expression of smoothelin (P>0.05). Combination of DHT and TGFb had a greater effect on expression than TGFb alone (P<0.01).

SMCs are the major cellular components of prostatic stroma. It was believed that the prostatic stromal fibroblasts could differentiate to SMCs with myofibro-blasts as a transition form. The differentiation could be investigated by measuring the expression of smooth muscle-associated proteins [9, 10]. Myosin may be one of the early markers [9] and smoothelin, a terminal marker for differentiation [11].

In vivo, we initially reported that SMCs were fallen into atrophy and apoptosis processes following castration [12]. The atrophic SMCs appeared later than apoptotic cells and were characterized by huge intracellular lipofuscin. In gross, the morphological changes of stroma following castration were slighter and more prolonged than that in epithelium. The atrophy of SMCs and epithelial cells may be due to the decreasing expression of some crucial enzymes for cell living.

The post-castration changes of the canine prostate indicated that the whole prostate gland must be an androgen-sensitive organ. Age-related changes in the density of SMCs have been observed in the fetal, childhood and pubertal prostate and these changes appear to parallel the serum testosterone surge, while no apparent changes were seen in the epithelium or glandular lumen during these periods [5]. Androgen receptors have been identified in the fibroblasts and the SMCs [13]. In this study, the expression of myosin was down-regulated following castration and DHT had some direct effects on the proliferation and differentiation of SMCs in culture (especially combined with bFGF or TGFb₁). Through quantitative stereologic evaluation of the morphological changes in the rat prostate treated with 5a-reductase inhibitor, it was found that castration led to a decrease in the number of epithelial and stromal cells in both the ventral and dorsolateral lobes of the prostate[14]. We also reported that the stromal cells in the whole prostate gland were decreased following castration [15]. In the present study the TUNEL positive cells were seen in the prostatic stroma following castration.

It is proposed that prostatic SMCs, under the influence of androgens, signal to epithelium to maintain epithelial differentiation and to suppress epithelial prolifera-tion, while prostatic epithelium signals to prostatic SMC to maintain smooth muscle differentiation. The peptide growth factors in the prostate play the role of mediators in the stromal-epithelium interaction. bFGF is a mitogen for cultured stromal cells and is also synthesized by the latter, suggesting that their growth is potentially under the autocrine control [16]. TGFb expressed in the stromal cells and could induce SMC phenotype in cultured human prostatic stromal cells [17]. Our experiments confirmed these facts.

Androgens negatively regulate TGFb expression in the prostate. Both TGFb₁ and bFGF are major regulators for SMCs. In this study, the expression of TGFb₁ was up-regulated and bFGF down-reglated after castration. The rate of TGFb₁/bFGF may determine the proliferation and differentiation of stromal cells. For stromal cells in the exponential growth period, TGFb₁ inhibits the proliferation and bFGF stimulates the growth rate. In confluent cultures, TGFb₁ induces the differentiation of fibroblast to SMC, but bFGF block it. We conclude that the opposing effects of TGFb₁ and bFGF may play important roles in maintaining stromal cell homeostasis.

If the prostatic stromal cells are exposed to high levels of androgen or the cells have a high sensitivity to androgen (e.g. the expression of androgen receptor elevated), a significant decrease in TGFb₁ levels and increase in bFGF levels may result in stromal proliferation. On the other hand, when BPH develops, the androgenic role in regulating stromal cells homeostasis may be in a state of chaos. The growth and differentiation of stromal cells may largely depend on local factors, such as TGFb₁/bFGF ratio and the cell density. TGFb₁ may not inhibit the growth of confluently cultured stromal cells in vitro, but stimulate their differentiation from fibroblast to SMC. Different local TGFb₁/bFGF ratios may lead to various components of BPH nodules. It is an interesting finding that the combination of DHT and bFGF or TGFb₁ will dramatically increase the effects on proliferation or differentiation, which may be important for the formation of BPH.

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Correspondence to: Dr Yuan-Jie NIU, Department of Prostatic Disease, Tianjin Institute of Urologial Surgery, 23 Pingjiang Road, Hexi District, Tianjin 300211, China.
E-mail: niuyj@public.tpt.tj.cn
Received 2002-11-26       Accepted 2002-12-12
* Presented at the First Asia-Pacific Forum on Andrology, 17-21 Shanghai, China.