Prostatic Smooth Muscle Cells Research Articles

Amphiregulin is coordinately expressed with heparin-binding epidermal growth factor-like growth factor in the interstitial smooth muscle of the human prostate.

Peptide growth factors have been proposed as mediators of smooth muscle-epithelial cell interactions in the human prostate; however, the identity of these molecules has not been established. In this study, we compared expression levels of messenger RNAs (mRNAs) encoding the epidermal growth factor (EGF) receptor-related receptor tyrosine kinases (ErbB1 through 4), the six EGF receptor ligands, EGF, transforming growth factor (TGF)-alpha, amphiregulin (ARG), HB-EGF, betacellulin, and epiregulin, and the related molecule heregulin-alpha, in a series of 10 prostate tissue specimens. Only EGF showed a disease-specific association, with increased mRNA levels in four of five PCa specimens in comparison to matched normal tissue from the same subject. In contrast, ARG and HB-EGF mRNAs showed a coordinate pattern of expression in 7/10 specimens that was distinct from all other growth factor or receptor genes examined and from mRNAs for prostate specific antigen, the androgen receptor and GAPDH, a house-keeping enzyme. Analysis of an additional series of benign prostatic hyperplasia and prostate cancer specimens from 60 individuals confirmed that ARG and HB-EGF mRNA levels varied in a highly coordinate manner (r = 0.93; P < 0.0001) but showed no association with disease. ARG was immunolocalized largely to interstitial smooth muscle cells (SMC), previously identified as the site of synthesis of HB-EGF in the prostate, while the cognate ARG and HB-EGF receptor, ErbB1, was localized exclusively to ductal epithelial cells and carcinoma cells. Although ARG was a relatively poor mitogen for Balb/c3T3 cells in comparison to HB-EGF, it was similar in potency to HB-EGF in stimulating human prostate epithelial cell growth, suggesting that prostate epithelia may be a physiologic target for ARG in vivo. Expression of both ARG and HB-EGF mRNAs was induced in cultured prostate SMC by fibroblast growth factor-2, a human prostate SMC mitogen linked to prostate disease. These findings indicate that ARG and HB-EGF are likely to be key mediators of directional signaling between SMC and epithelial cells in the human prostate and appear to be coordinately regulated.

Importance of Agonists in α-Adrenoceptor Classification and Localisation of α1-Adrenoceptors in Human Prostate

α-Adrenoceptor blocker drugs are commonly used in the clinical (non-surgical) treatment of BPH. α₁-adrenoceptors were originally sub-divided using agonists but, subsequently, were sub-divided using only antagonists in ligand-ligand interactions, which did not require agonists at all. Ultimately, proof that adrenoceptors are functional receptors for the natural ligands, noradrenaline and adrenaline, requires that agonists be used. The earlier excitement engendered by finding varying agonist potency series in different tissues has not been revisited to place it in the context of current concepts of α₁-adrenoceptor subtypes. This review will consider the advantages and limitations of different agonists for the study of α₁-adrenoceptor subtypes including ‘extreme’ examples where the archetypal α₁-adrenoceptor agonist phenylephrine activates α₂-adrenoceptors and others where UK14304, often the α₂-adrenoceptor agonist of choice, activates α₁-adrenoceptors. New work will also be presented showing the interaction between agonists and the fluorescent α₁-adrenoceptor antagonist QAPB. This introduces the novel point of view of studying the displacement of antagonists by agonists. Possible errors in antagonist classification arising from complexity in the actions of agonists and the recently developed method of fluorescent ligand binding on isolated living human prostatic smooth muscle cells will be discussed.

Alpha 1-ADRENOCEPTOR ANTAGONISTS TERAZOSIN AND DOXAZOSIN INDUCE PROSTATE APOPTOSIS WITHOUT AFFECTING CELL PROLIFERATION IN PATIENTS WITH BENIGN PROSTATIC HYPERPLASIA

Purpose Recent evidence indicated that an alpha 1 blocker, doxazosin, induces prostate apoptosis in patients with benign prostatic hyperplasia (BPH). [1] in this study, to determine whether this apoptotic response was mediated by alpha 1 adrenoceptor-dependent mechanism or was specific to doxazosin, we examined the effect of another alpha 1 blocker, terazosin, in addition to doxazosin, on the dynamics of prostate cell growth. Materials and Methods Cell proliferation and apoptosis were evaluated in BPH patients, an untreated (control) group (n = 31), and men treated with terazosin (n = 42) and doxazosin (n = 61) for the relief of the obstructive symptoms. Terazosin (1 to 10 mg./day) and doxazosin (2 to 8 mg./day) treatment varied from 1 week to 3 years. Ki-67 immunostaining and the TUNEL assay were used to evaluate the proliferative and apoptotic indices, respectively, in both the epithelial and stromal components of prostate (biopsy and prostatectomy) specimens. The smooth muscle cell content of the prostatic stroma was identified on the basis of smooth muscle alpha-actin immunoreactivity. Results A significant induction of apoptosis was observed in both the prostatic epithelial and stromal cells within the first month of terazosin and doxazosin therapy, as compared with untreated controls (p <0.05). Furthermore, the marked induction of prostatic stroma apoptosis in response to both alpha 1 adrenoceptor antagonists was paralleled by a significant decrease in the smooth muscle alpha-actin expression. This loss of prostatic smooth muscle cells correlated with morphological stromal regression (as detected by trichrome staining) and BPH symptom improvement. Neither terazosin nor doxazosin therapy resulted in significant changes in prostate cell proliferation. Conclusions These findings demonstrated that alpha-blockers as a class may regulate prostate growth by inducing apoptosis in both the epithelial and stromal cells, with little effect on cell proliferation. Apoptosis-mediated prostate stromal regression appears as a molecular mechanism underlying the therapeutic response to alpha 1 blockade in the treatment of BPH.

Modulation of the differentiation status of cultured prostatic smooth muscle cells by an alpha1-adrenergic receptor antagonist.

Prostatic stromal cells are believed to be a key factor in the pathogenesis of benign prostatic hyperplasia (BPH). The effect of phenylephrine, an alpha1-adrenergic receptor agonist, and doxazosin, an alpha1-adrenergic receptor-specific antagonist, on the expression of smooth muscle myosin-heavy-chain isotypes SM-1 and SM-2 was tested in an in vitro model of prostatic smooth muscle cells (SMC). Primary prostatic stromal cells, grown in SMC-specific medium, were treated with 10 microM of phenylephrine or 1 microM of doxazosin or a combination of both. SM-2 to SM-1 mRNA ratios and expression of alpha1-adrenergic receptor subtypes were determined by means of reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Cell growth was measured by a cell viability assay. SM-1 mRNA and only very low levels of SM-2 mRNA were detected in prostatic SMC cultures grown for 4 days in a serum-free base medium. After 6 days of treatment, SM-2 expression increased, highest in the doxazosin-treated cultures. In comparison to unstimulated cells, a statistically significant 10-fold increase of the SM-2:SM-1 ratio was measured in doxazosin-treated cultures. Analysis of alpha1-adrenergic receptor subtype expression revealed the presence of mRNAs of subtypes 1d and 1b mRNAs. Subtype 1a was not expressed. Phenylephrine and doxazosin showed no significant effect on cell proliferation and on alpha1d-adrenergic receptor expression. SMC can differentiate from a proliferative to a contractile phenotype, which is accompanied by increased expression of isotope 2 of smooth muscle myosin heavy chain. Our results suggest that doxazosin seems to have a long-term effect on the differentiation of prostatic stromal cells, indicating that alpha1-adrenergic receptor antagonists do not act solely on SMC contractility.

Cell proliferation in human prostatic smooth muscle cells involves the modulation of protein kinase C isozymes

We have examined the role of protein kinase C in the regulation of foetal-calf serum-stimulated cell proliferation in human prostatic smooth muscle cells. The data showed that the proliferative effect to foetal-calf serum (10%, v/v) was partially inhibited by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3,4-c)-carbazole (Go-6976), a selective Ca2+-dependent protein kinase C inhibitor, suggesting that Ca2+-dependent protein kinase C isozymes might play roles in this proliferative regulation. Additionally, foetal-calf serum caused a significant translocation of protein kinase C-βII and -ε from a cytosolic to a membrane distribution. These findings combined with the aforementioned functional experiments suggest that foetal-calf serum-stimulated cell proliferation might involve the activation of protein kinase C-βII in human prostatic smooth muscle cells; however, the role of protein kinase C-ε in mediating cellular functions other than cell proliferation remains further investigation in these cells.

Transforming growth factor-beta1 induces nuclear to cytoplasmic distribution of androgen receptor and inhibits androgen response in prostate smooth muscle cells.

Stromal-epithelial interactions in the prostate gland are dependent on androgen regulation of prostate stromal cells, yet little is known about androgen action in these cell types. Recent reports have demonstrated that androgen-regulated gene transcription can be stimulated or inhibited by certain growth factors, indicating cross-talk mechanisms. To address potential cross-talk in signaling pathways between androgen and transforming growth factor-beta1 (TGFbeta1) in prostate stromal cells, the PS-1 prostate smooth muscle cell line was examined. In the presence of physiological concentrations of androgen, PS-1 cell proliferation was stimulated, and androgen receptor (AR) exhibited a nuclear localization pattern. The addition of TGFbeta1 (25 pM) was capable of blocking androgen-induced proliferation, but had no direct effect in cultures without androgen. Immunocytochemistry to localize AR subcellular distribution showed that TGFbeta1 (5-100 pM) altered the distribution of AR from the nucleus to the cytoplasm. Other growth factors, including fibroblast growth factor-2, epidermal growth factor, and TGFbeta2 had no effect on AR distribution. The TGFbeta1-induced nuclear to cytoplasmic change in receptor localization was rapid (initiated within 30 min), was neutralized by TGFbeta1 antibodies, did not require new protein synthesis, and was complete by 6 h. Removal of TGFbeta1 from the culture medium resulted in a rapid redistribution of AR to the nucleus, indicating reversible mechanisms. Northern analysis of the ddp17 marker transcript for androgen action in PS-1 cells showed that androgen-stimulated ddp17 expression was inhibited in the presence of TGFbeta1 (25 pM). TGFbeta1 induced a similar nuclear to cytoplasmic distribution of AR in primary cultures of rat prostate stromal cells. TGFbeta1, however, had no effect on AR distribution in either the LNCaP prostatic carcinoma cell line or the DDT1MF-2 leiomyosarcoma cell line. Specific cross-talk between TGFbeta1 and AR signaling pathways in prostate stromal cells may play a significant role in prostate development and stromal cell response in carcinoma progression.

Transforming Growth Factor- 1 Induces Nuclear to Cytoplasmic Distribution of Androgen Receptor and Inhibits Androgen Response in Prostate Smooth Muscle Cells

Transforming Growth Factor- 1 Induces Nuclear to Cytoplasmic Distribution of Androgen Receptor and Inhibits Androgen Response in Prostate Smooth Muscle Cells

Biphasic Action of Phenylephrine on the Ca2+-Activated K+ Channel of Human Prostatic Smooth Muscle Cells

The elevation of cytosolic Ca²⁺ ([Ca²⁺]_i) is known to regulate smooth muscle contractility. A physiological concentration of phenylephrine induced the elevation in [Ca²⁺]_i of human prostatic smooth muscle cells; however, contraction of prostatic tissues in vitro needs a higher concentration of phenylephrine than the physiological level. To investigate this discrepancy, we investigated the functional importance of the Ca²⁺-activated K⁺ channel (K_Ca channel) of human prostatic smooth muscle cells in phenylephrine-induced contraction. Using the patch-clamp technique, the K_Ca channel of human prostatic smooth muscle cells was activated by phenylephrine at a physiological concentration (10^–7–10^–5 M) but was inhibited at a higher concentration (10^–4–10^–3 M). Phenylephrine (10^–3 M) also inhibited the K_Ca channel which was activated by 10 µM A23187, a calcium ionophore. Similar inhibition was obtained with 1 µM phorbol 12-myristate 13-acetate, an activator of protein kinase C (C-kinase). Both inhibitions were reversed by subsequent application of 1 nM staurosporine, a protein kinase inhibitor. These results suggested that C-kinase mediated the phenylephrine-induced inhibition of the K_Ca channel. In this study, a physiological concentration of phenylephrine induced activation of the K_Ca channel of human prostatic smooth muscle cells, which brought about membrane hyperpolarization and relaxation of human prostatic smooth muscle cells. The regulation of the K_Ca channel by phenylephrine may explain the need of a high concentration of phenylephrine for the contraction of prostatic tissue.

Mitogenic activity of endothelin on human cultured prostatic smooth muscle cells

The effects of endothelins on human prostatic smooth-muscle cell growth were examined. Endothelin-1 and endothelin-3 induced a concentration-dependent increase in DNA synthesis and also promoted cell growth. Use of subtype selective antagonists BQ-123 ((cyclo( d-Trp- d-Asp(ONa)-Pro- d-Val-Leu); endothelin ET A receptor selective) and BQ-788 (( N- cis-2,6-dimethylpiperidinocarbonyl- l- γ-methyl Leu- d-Trp-(COOMe)- d-Nle-ONa); endothelin ET B receptor selective), indicated that mitogenic effects of endothelin were mediated through activation of both endothelin ET A and ET B receptors. The mitogenic effects of endothelin-1 and endothelin-3 were significantly inhibited by pretreatment of the cells with pertussis toxin. However, mitogenesis due to basic fibroblast growth factor was not affected. In conclusion, endothelin has mitogenic effects on human prostatic smooth muscle cells through activation of both endothelin ET A and ET B receptors via different signalling pathways from basic fibroblast growth factor. This may contribute to smooth muscle hyperplasia associated with benign prostatic hyperplasia.

Antiproliferative effect in human prostatic smooth muscle cells by nitric oxide donor.

We obtained a primary culture of prostatic cells through explantation from patients with benign prostatic hyperplasia. Structural morphology, immunohistochemical staining, and growth characteristics of these cells demonstrate that they are consistent with the population of smooth muscle cells (SMCs). We examined the influence of a nitric oxide donor, sodium nitroprusside (SNP), on the regulation of human prostatic SMC proliferation. SNP exhibited a concentration-dependent (0.1-10 microM) inhibition of fetal calf serum-induced proliferation in human prostatic SMCs. In addition, growth-inhibitory responses to 8-bromo-cGMP (1-30 muM) were observed. However, the responses to SNP were significantly diminished by the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 microM; a selective guanylate cyclase inhibitor). Furthermore, SNP induced an increased concentration-dependent accumulation of intracellular cGMP in human prostatic SMCs. After 48-hr period of deprivation of serum, cells were restimulated with serum to permit cell cycle progression. The addition of SNP (10 microM) at various times after the addition of serum to serum-deprived cells showed maximal inhibition of cell proliferation even when added 6 hr after the serum. This blocking effect of cell cycle progression was lost gradually as the delay from serum to SNP application increased from 6 to 18 hr. The membrane-associated protein kinase C (PKC) activity was studied in human prostatic SMCs; results showed that fetal calf serum (10%, v/v) significantly increased membrane-associated PKC activity. SNP (10 muM), which had little effect on basal kinase activity, completely abolished serum-induced augmentation of PKC activity. Therefore, we suggest that SNP mediates its antiproliferative effect by the inhibition of PKC activity on human prostatic SMCs; furthermore, its antiproliferative effect occurs at the early G1 phase of the cell cycle.

Culture of Human Prostatic Smooth Muscle Cell and Their Response to Nitric Oxide

Culture of Human Prostatic Smooth Muscle Cell and Their Response to Nitric Oxide

Endothelin attenuates apoptosis in human smooth muscle cells.

An imbalance between proliferation and apoptosis is an important causal factor for disorders involving abnormal cell accumulation. Endothelin (ET)-1, a 21-amino-acid peptide with mitogenic and vasoconstricting activities, not only acts as a mitogen, but also attenuates paclitaxel-induced apoptosis in smooth muscle cells. In both human pericardial and prostatic smooth muscle cells, addition of ET-1 reduced paclitaxel-induced DNA fragmentation and phosphatidylserine on the cell surface, two characteristics of apoptosis. By comparison, angiotensin II, another vasoactive peptide, did not have a significant effect on apoptosis. The effect of ET-1 was dose-dependent with an EC50 of 1 nM. These results suggest that ET is a potential survival factor for smooth muscle cells, and that altered activity of the ET system in disease states has potential to contribute to aberrant cell growth.

Transurethral balloon laser thermotherapy induces long-lasting apoptosis of prostatic smooth muscle cells in human prostatic hyperplasia

Transurethral balloon laser thermotherapy (TUBAL-T) has been developed recently as a noninvasive treatment for benign prostatic hyperplasia (BPH), but the cellular events occurrmg after TUBAL-T were not yet fully understood. We examined prostate tissues by electron microscopy and the DNA nick end-labeling method at 2 and 4 weeks after a single TUBAL-T treatment in an attempt to elucidate the mechanism by which TUBAL-T progressively improves obstructive complications of patients with BPH. Condensation of chromatin at the nuclear margin was observed in prostatic smooth muscle cells (PSMCs) located in the area adjacent to periurethral coagulative necrosis. PSMCs with condensed chromatin were often accompanied by cytoplasmic vacuoles in perinuclear areas. Application of the nick end-labeling method at the light microscopic level revealed that most PSMC nuclei were positive for terminal TdT-mediated dUTP-dioxygenic nick end-labeling (TUNEL) staining. These results strongly suggested that long-standing apoptosis is induced in PSMCs by TUBAL-T. as well as massive coagulative necrosis of prostatic tissues. The role of apoptosis in PSMCs in the clinical improvement of outlet obstruction after TUBAL-T is discussed.

Endothelin receptors and their cellular signal transduction mechanism in human cultured prostatic smooth muscle cells.

1. Endothelin (ET) receptors, and their cellular signal transduction mechanism, were characterized in a primary culture of human prostatic smooth muscle cells (HP cell). 2. [125I]-ET-1 and [125I]-ET-3 binding studies revealed that both ETA and ETB receptors were present in the HP cells, and the ratio of ETA to ETB receptors was 1.4:1. 3. Analysis of ET receptor mRNA by reverse transcription-polymerase chain reaction also demonstrated that HP cells express both ETA and ETB receptors. 4. ET-1 and ET-3 increased intracellular free Ca2+ concentration ([Ca2+]i) in the HP cells in a concentration-dependent manner. Use of subtype selective antagonists BQ-123 and BQ-788, indicated that both ETA and ETB receptors were coupled to an increase in [Ca2+]i. 5. Pretreatment of the cells with pertussis toxin resulted in a significant but partial attenuation of the [Ca2+]i increase mediated through the ETA and ETB receptors. However, sensitivity to pertussis toxin (PTX) was significantly different between them. 6. In conclusion, HP cells possess ETA and ETB receptors. Further, these two endothelin receptor subtypes evoke an increase in [Ca2+]i possibly via the action of different GTP-binding proteins.

Morphological and pharmacological characterization of guinea-pig prostatic smooth muscle cells in vitro.

Prostatic smooth muscle is thought to play a major role in the pathogenesis of bladder outlet obstruction in patients with benign prostatic hypertrophy. However, the physiology of prostatic smooth muscle cells remains largely unknown, in part due to the lack of a suitable model system. We therefore sought to establish an in vitro culture of guinea pig prostatic smooth muscle cells. Immature guinea pig prostate was treated by enzymatic digestion and the cells obtained were used to initiate the primary culture. After 3 to 4 passages, cultured smooth muscle cells were examined morphologically by immunocytochemistry and electron microscopy. The contractile properties of cultured smooth muscle cells were also examined. The cultured prostatic cells demonstrated hill and valley morphology, which is a hallmark of smooth muscle cells in vitro, and stained positively for desmin. In addition, electron microscopic examination of ultrastructural morphology revealed myofilaments. Confluent cultures of prostatic smooth muscle cells showed a clear, dose-dependent contractile response to phenylephrine. Furthermore, contraction of the prostatic smooth muscle cells by 10(-6) mol/L phenylephrine was completely inhibited by pretreatment with 10(-6) mol/L terazosin. An in vitro culture of prostatic smooth muscle cells was established. This culture is likely to provide a powerful tool for elucidating the physiology and pathophysiology of prostatic smooth muscle.

Human prostatic smooth muscle cells in culture: estradiol enhances expression of smooth muscle cell-specific markers.

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 microM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle alpha-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs.

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell‐specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.

Androgen-regulated proliferation and gene transcription in a prostate smooth muscle cell line (PS-1)

Androgens play a key role in directing stromal-epithelial interactions in prostate gland development, in maintenance of adult phenotype, and in disease progression. To address the molecular mechanisms of androgen action in prostate stromal cells and the genes regulated by androgens in this cell type, a stromal cell line (PS-1) was developed from rat ventral prostate. The PS-1 cell line was adapted to chemically defined media and characterized as smooth muscle based on expression of desmin, smooth muscle alpha-actin, and nuclear androgen receptor, markers for prostate smooth muscle. To examine steroid hormone regulation, PS-1 cells were analyzed for response to a panel of steroid hormones in serum-free, chemically defined media. PS-1 cells were significantly growth stimulated by physiological concentrations of androgens (10nM) relative to other steroids tested. To ascertain whether this cell line could be used to examine androgen-regulated gene expression, differential display PCR was used to demonstrate the presence of androgen-regulated transcripts and provide the initial steps in cloning these genes. Replicate differential display PCR analyses showed consistent androgen stimulation of 16 messenger RNA species. Northern analysis confirmed androgen regulation of 6 species. Sequence analysis of each indicated no regions of significant homology or direct matches to existing sequences. Further study of the PS-1 stromal cell line and androgen-regulated genes identified here will provide a novel in vitro system for defining molecular mechanisms of androgen action in prostate stromal cells and the significance of stromal androgen-regulated genes to stromal-epithelial interactions.

Androgen-regulated proliferation and gene transcription in a prostate smooth muscle cell line (PS-1).

Androgens play a key role in directing stromal-epithelial interactions in prostate gland development, in maintenance of adult phenotype, and in disease progression. To address the molecular mechanisms of androgen action in prostate stromal cells and the genes regulated by androgens in this cell type, a stromal cell line (PS-1) was developed from rat ventral prostate. The PS-1 cell line was adapted to chemically defined media and characterized as smooth muscle based on expression of desmin, smooth muscle alpha-actin, and nuclear androgen receptor, markers for prostate smooth muscle. To examine steroid hormone regulation, PS-1 cells were analyzed for response to a panel of steroid hormones in serum-free, chemically defined media. PS-1 cells were significantly growth stimulated by physiological concentrations of androgens (10nM) relative to other steroids tested. To ascertain whether this cell line could be used to examine androgen-regulated gene expression, differential display PCR was used to demonstrate the presence of androgen-regulated transcripts and provide the initial steps in cloning these genes. Replicate differential display PCR analyses showed consistent androgen stimulation of 16 messenger RNA species. Northern analysis confirmed androgen regulation of 6 species. Sequence analysis of each indicated no regions of significant homology or direct matches to existing sequences. Further study of the PS-1 stromal cell line and androgen-regulated genes identified here will provide a novel in vitro system for defining molecular mechanisms of androgen action in prostate stromal cells and the significance of stromal androgen-regulated genes to stromal-epithelial interactions.

Stromal cells of the human prostate: Initial isolation and characterization

The present study was conducted to isolate and to characterize stromal cells from the human prostate and to study the effects of androgen and different growth factors in this model system. Benign prostatic hyperplasia (BPH) tissue samples were obtained from transurethral resection of the prostate (TURP). Tissue specimens were mechanically and enzymatically dissociated by treatment with DNAse and collagenase. Epithelial cells were separated from stromal cells by discontinuous Percoll gradient centrifugation. The stromal cells obtained were cultured in phenol red-free RPMI-1640 supplemented with 10% fetal bovine serum. Immunocytochemical analysis revealed that the stromal cell cultures were composed of both smooth muscle cells and fibroblasts. The short and broad, smooth muscle cells wee identified by using an antibody directed against alpha-smooth muscle actin. The thin and elongated fibroblasts stained positively for prolyl 4-hydroxylase. Smooth muscle cells were the predominant cell type in the present investigation. Typical cultures contained up to 99% of cells staining positively for alpha-smooth muscle actin. The prostate smooth muscle cultures were treated with dihydrotestosterone (DHT), bovine pituitary extract (BPE), basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). When cells were cultured in serum free RPMI-1640 supplemented with ITS+ (insulin, transferrin, and selenious acid) no significant (P > 0.05) mitogenic effect in medium supplemented with ITS+. In the presence of 10% charcoal-stripped fetal bovine serum (cFBS) DHT, at a concentration of 0.1 nM, was able to cause a slight but significant (P < 0.05) mitogenic effect on BPH smooth muscle cells growth. Basic FGF was able to stimulate BPH smooth muscle cells in a concentration-dependent fashion. The combination of DHT and 0.1 ng/ml bFGF was able to increase the proliferation of prostate smooth muscle cells above either agents alone. Addition of BPE to serum free RPMI-1640 caused a significant (P < 0.05) stimulation of cell proliferation in a concentration-dependent fashion. Addition to TGF-beta to serum or BPE containing RPMI-1640 caused a significant (P < 0.05) inhibition to cell proliferation in a concentration-dependent fashion. TGF-beta was cytostatic to the benign prostatic smooth muscle cells only in the presence of media containing growth stimulating factors found in charcoal-stripped serum or in bovine pituitary extract. These results demonstrated that stromal fraction isolated from BPH specimens was composed of both fibroblasts and smooth muscle cells. These cells could be cultured and were able to respond to various growth stimulatory and inhibitory agents.