Cultured human prostate-derived fibroblasts produce a factor that stimulates their growth with properties indistinguishable from basic fibroblast growth factor

Fibrostromal proliferation is believed to be important in the development of benign prostatic hyperplasia (BPH). We found that a mitogen for cultured mesodermal-derived cells was present in extracts of BPH tissue. The mitogen was identified as basic fibroblast growth factor (bFGF). Previous studies did not determine the cell population(s) responsible for bFGF production in the prostate. This information is important to the understanding of the role of bFGF in the etiology of BPH. Human prostate-derived fibroblasts (PF) were initiated in culture. Recombinant bFGF and PF lysates stimulated tritiated thymidine uptake by quiescent PF cells. Greater than 90% of the mitogen in PF lysates bound to heparin-Sepharose and had the same elution profile and apparent molecular weight as bFGF isolated from BPH tissue. The growth factor in PF lysates competed with recombinant iodinated bFGF for binding to antiserum to (1-24)bFGF. Cultured PF incorporated 35S-methionine into protein that was precipitated by antiserum to bFGF. The apparent molecular weight of the radiolabeled protein, about 17,000, was similar to authentic bFGF. The observations are consistent with the interpretation that cultured PF synthesize a growth factor that stimulates their growth with properties that are indistinguishable from bFGF.     

Transforming growth factor beta I expression in benign and malignant prostatic tumors

The expression of transforming growth factor beta 1 (TGF-β1) in prostate specimens obtained from patients with benign prostatic hyperplasia (BPH, n = 32) and prostate carcinoma (n = 66) was investigated using Northern blot analysis and immunohistochemistry. Northern blot analysis revealed TGF-β1 message (2.5 kb) in virtually all of the samples examined, reflecting the ubiquitous nature of this growth factor. No statistical difference was found between the levels of mRNA detected in benign and malignant tissues due, in part, to the inherent heterogeneity of prostate tissue. Immunohistochemical methods using an antibody to native TGF-β1 revealed a novel pattern of immunoreactivity. Staining observed only in certain epithelial cells of benign glands was associated with areas of infection rather than tumorigenesis. Interestingly, intense staining was also seen in polymorphonuclear leukocytes. No correlation was found with the mRNA results, suggesting that this antibody is binding to TGF-β1 activated in response to infection rather than detecting sites of synthesis of latent TGF-β. © 1994 Wiley-Liss, Inc.     

Effect of prostatic growth factor,basic fibroblast growth factor,epidermal growth factor,and steroids on the proliferation of human fetal prostatic fibroblasts

To study the relationship between androgen metabolism and the pathogenesis of benign prostatic hypertrophy, we purified a growth factor from benign hyperplastic tissue of human prostates and assayed the proliferative responses of human fetal prostatic fibroblasts to the purified growth factor (hPGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), dihydrotestosterone (DHT), and estradiol (E₂). Prostatic tissue extracts were fractionated using heparin-Sepharose chromatography. The fraction that eluted with 1.3–1.7 M NaCl contained the majority of mitogenic activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) of the lyophilyzed active fraction showed a band at 17,000 daltons. Human prostatic fibroblasts were isolated from fetal prostate and tested for their proliferative responses to hPGF, bFGF, EGF, DHT, and E₂. hPGF, as well as bFGF and EGF, did increase tritiated thymidine incorporation into the cultured fibroblasts. DHT(10⁻⁷ M) had a significant stimulatory effect on cell growth in serum-free media after 6 days of culture. E₂(10⁻⁷ M) had no effect on cell proliferation. The combination of DHT and E₂ showed no synergistic effect. We conclude that our purified hPGF, bFGF, and EGF promote cell growth directly, DHT indirectly, while E₂ does not. The effect of DHT appears to be mediated via the increased production and/or secretion of growth factor(s). Possibly, the bFGF-like hPGF purified from human benign hyperplastic prostatic tissue is such a mediator. © 1996 Wiley-Liss, Inc.     

Expression of basic fibroblast growth factor mRNA in benign prostatic hyperplasia and prostatic carcinoma

In our previous work we demonstrated that prostate-derived growth factor (PrGF) is homologous to basic fibroblast growth factor (bFGF), not acidic fibroblast growth factor (aFGF). Using Northern blot analysis we now show that the messenger RNA for bFGF but not aFGF is expressed in benign prostatic hyperplastic (BPH) tissue as well as in carcinoma of the prostate (CAP). This not only corroborates our previous results, but suggests that PrGF is produced locally and not merely stored in the prostate. The demonstration of local production of bFGF by prostate tissue may indicate that this growth factor plays a role, either alone or in conjunction with other factors, in the etiology of benign hyperplasia or prostatic cancer.     

In vivo and in vitro effects of androgen on fibroblast growth factor-2 concentrations in the human prostate

Prostatic growth is primarily regulated by dihydrotestosterone (DHT). Recent studies have demonstrated that a large number of growth factors are present in the human benign prostatic hyperplasia (BPH) prostate, including epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), transforming growth factor beta (TGF-β), insulin-like growth factor (IGF), and basic fibroblast growth factor (bFGF) (FGF-2). DHT may mediate its mitogenic effects in the prostate by regulating growth factors. To test this hypothesis, we have utilized a histoculture androgen sensitivity assay (HASA) in which ³H-thymidine incorporation is measured in aliquots of BPH tissue in histoculture with either added DHT or hydroxyflutamide (HF). The resulting DHT/HF ratio is an expression of the androgen sensitivity of the tissue. In this study, we have compared the DHT/HF ratio for ³H-thymidine incorporation to the DHT/HF ratio for FGF-2 measured in the histocultured prostates. The DHT/HF ratio for the HASA studies of ³H-thymidine incorporation averaged 2.68 compared to the DHT/HF ratio for FGF-2 in the same specimens of 1.01. These values were significantly different, therefore indicating no relationship between DHT stimulation and FGF-2 levels. In addition, FGF-2 levels were measured in human BPH prostates from patients medically castrated with megesterol acetate and estradiol 17-β prior to surgery. These values were not significantly different, and therefore do not suggest any effect of DHT on the concentration of prostatic FGF-2. Although these studies did not show any effect of DHT on the regulation of prostatic FGF-2, they do indicate that the HASA assay is feasible and appropriate to use in the study of relationships between DHT and various growth factors. © 1994 Wiley-Liss, Inc.     

Demonstration of fibroblast growth factor receptor-I in human prostate by polymerase chain reaction and immunohistochemistry

The expression and localization of fibroblast growth factor receptor-1 were investigated in human prostatic tissues with or without benign hyperplasia. Using a polymerase chain reaction method, we were able to demonstrate that prostatic tissues with benign hyperplasia expressed a significantly higher level of fibroblast growth factor receptor-1 mRNA than normal prostatic tissues (P < 0.01 by Anova). Western blot analysis using an antiserum against the receptor gave 2 bands with molecular weights of about 140 kDa and 80 kDa; these correspond to the expected sizes of the long and secreted forms of the fibroblast growth factor receptor-1, respectively. An immunohistochemical study using the same antiserum further demonstrated that the immunoreactive staining occurred mainly in the basal cells of the glandular epithelium and occasionally in the stromal cells. These results suggest that fibroblast growth factors may influence, at least in part, the proliferation of the epithelial cells seen in benign hyperplasia of human prostate. © 1995 Wiley-Liss, Inc.     

Secretion of transforming growth factor-β by the immature rat prostate

Immature rat ventral prostate was used to identify a potent cell growth inhibitory factor in normal prostate. Both conditioned media and peptide extracts derived from ventral prostates inhibit the cellular growth and DNA synthesis in metastatic androgen-independent human prostate carcinoma cell line (PC3). The prostatic inhibitory factor was partially purified using an hydrophobic HPLC column (C18). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (SDS-PAGE) of partially purified material revealed a major 25-kDa band. Immunoblot analysis showed similarity of the inhibitory factor to the transforming growth factor-β (TGF-β). Furthermore, the proliferation inhibiting activity of the prostatic factor was completely abolished by anti-TGF-β antibodies. These data indicate that normal prostate produces a TGF-β-like protein under non-physiological conditions. © 1994 Wiley-Liss, Inc.     

Androgen and prostatic stroma

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 TGFβ, bFGF, AR, and smooth muscle cell (SMC) specific proteins (myosin and/or sm     

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 were identified by using an antibody directed against α-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 α-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-β (TGF-β). When cells were cultured in serum free RPMI-1640 supplemented with ITS+ (insulin, transferrin, and selenious acid) no significant (P > 0.05) stimulative effect after treatment with DHT or TGF-β was detectable. Basic FGF had a slight but 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 of TGF-β to serum or BPE containing RPMI-1640 caused a significant (P < 0.05) inhibition of cell proliferation in a concentration-dependent fashion. TGF-β 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. © 1996 Wiley-Liss, Inc.     

Benign prostatic hyperplasia and growth factors.

A great deal of work has been accomplished in the attempt to determine the cause of benign prostatic hyperplasia (BPH). Early work by morphologists suggests that BPH starts as a stromal disease and that the hyperplastic stroma secretes a substance that stimulates the growth of epithelial cells. The quantitative morphometric data also suggest that BPH is primarily a stromal disease. Experimental embryology data have shown that the basic fibroblast growth factor, bFGF, is involved in early embryogenesis and is the primary inducer of mesodermal tissue. Work in mouse embryos has shown that a powerful inducer for prostatic epithelial growth is elaborated by the urogenital mesenchyme. Both of these findings fit the hypothesis that stromal hyperplasia may be initiated by a growth factor and that a second growth factor stimulates epithelial growth. Work in our laboratory has established that bFGF is the primary growth factor present in human BPH. We have also found that bFGF is synthesized by prostate fibroblasts and bFGF may be in higher concentration in the periurethral tissues of BPH. At this time, no definite link between growth factors and hyperplastic growth of the prostate has been established. However, circumstantial evidence has lead us to formulate several hypothesis regarding the role of growth factors in BPH. Hopefully, these hypothesis will be of some assistance in guiding future work on growth factors and BPH.     

前列腺增生症患者膀胱逼尿肌中转化生长因子-β1和结缔组织生长因子的表达及临床意义

目的 探讨良性前列腺增生症(BPH)患者逼尿肌中转化生长因子-β1(TGF-β1)和结缔组织生长因子(CTGF)的表达及临床意义.方法 应用免疫组织化学SP法检测29例BPH患者膀胱逼尿肌标本中TGF-β1和CTGF的表达并检测平均吸光度(MA),依据各临床参数参考值进行分组比较.结果 所有标本均有不同程度的TGF-β1和CTGF表达.前列腺体积(PV)＞50 ml组与PV≤50 ml组患者逼尿肌中TGF-β1的表达差异无统计学意义(P＞0.05);而CTGF在PV＞50 ml组的表达量显著高于PV≤50 ml组(P＜0.01).TGF-β1和CTGF的表达量在移行区指数(TZI)＞0.65、残余尿量＞50 ml组患者逼尿肌中分别高于TZI≤0.65、残余尿量≤50 ml(P＜0.01),而与IPSS、Qmax和病程大小无明显相关(P＞0.05).Pearson积差相关分析可见TGF-β1与CTGF的表达呈正相关(r=0.761,P＜0.01).结论 逼尿肌中TGF-β1和CTGF的高表达与前列腺体积、移行区指数及残余尿量相关,提示这些临床参数可作为评估BPH患者膀胱逼尿肌纤维化程度的指标.

Abstract：

Objective To investigate the correlation between the expression of transforming growth factor-β1 (TGF-β1 ) , connective tissue growth factor ( CTGF) in the detrusor of benign prostatic hyperplasia (BPH) patients and its clinical significance. Methods Immunohistochemical SP method was used to detect the expression of TGF-β1 and CTGF in 29 specimens of bladder wall of BPH patients. All patients were divided into 2 groups according to the meaningful value of each clinical parameter to compare the expression of TGF-β1 and CTGF. Results All the samples had different levels of TGF-β1 and CTGF expression. The TGF-β1 in BPH patients whose prostate volume (PV) was＞50 ml was not significantly different from that in patients whose PV was ≤50 ml, but CTGF in BPH patients with PV ＞50 ml was higher than that in the other team. The TGF-β1 and CTGF in groups with TZI ＞0. 65 and residual urine＞50 ml were higher than other groups with TZI ≤0. 65 and residual urine ≤50 ml, respectively. The expression levels of TGF-β1 and CTGF of detrusors were not related with IPSS, Qmax, disease duration (P＞0. 05).Pearson-analysis reavealed that there was a positive correlation between the expression of TGF-β1 and CTGF in detrusors (r = 0. 761 ,P ＜0. 01). Conclusion TGF-β1 and CTGF have a strong correlation with PV, TZI and residual urine, suggesting that these clinical parameters may be useful in assessing the degree of the detrusor fibrosis in BPH patients.     

Regulation of prostatic growth and function by peptide growth factors

Polypeptide growth factors are positive and negative regulators of prostatic growth and function. Expression and biological effects of epidermal growth factor (EGF), transforming growth factors (TGFs) α and β, fibroblast growth factors (FGFs), and insulin-like growth factors (IGFs) in the prostate have been extensively studied. EGF and TGFα, which share the same receptor, are strong mitogens for prostatic epithelial and stromal cells. Their paracrine mode of action in normal tissue and early-stage tumors is apparently altered towards an autocrine stimulation in hormone-independent tumors, which gain the ability to produce TGFα by themselves. TGFβ has a dual role in the regulation of prostatic growth. It inhibits growth of prostatic epithelial cells in culture and mediates programmed cell death after androgen withdrawal. However, advanced prostatic carcinomas become insensitive to the inhibitory effect of TGFβ. Several members of the FGF family have been identified in the prostate. They are mainly or exclusively expressed in the stromal cells, and stimulate the epithelial cells. In the rat Dunning tumor model, progression is accompanied by distinct changes in the expression of FGFs and their receptors. In the hyperplastic tissue, basic FGF (bFGF) is accumulated. This growth factor is also a potent angiogenic inducer, expression of which may determine the metastatic capability of a tumor. IGFs are paracrine growth stimulators in the normal and hyperplastic prostate. It is still under consideration whether prostatic cancer cells gain the ability to produce IGF-I by themselves and thus shift to an autocrine mode of IGF-I stimulation. Growth factors also interact with the androgen-signaling pathway. IGF-I in particular, other growth factors as well, can activate the androgen receptor. © 1996 Wiley-Liss, Inc.     

Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma

Objectives. Scatter factor (hepatocyte growth factor) (SF/HGF) is a multifunctional polypeptide growth factor that has been implicated in tumor proliferation, angiogenesis, invasiveness, and metastasis. Little is known of the expression of SF/HGF in human prostatic carcinoma. The aims of this investigation were to quantitate the level of SF/HGF expression in benign versus malignant human prostatic tissues and to assess regulation of SF/HGF expression by human prostatic stromal myofibroblasts.Methods. We determined the level of SF/HGF expression in 10 human prostatic tissue samples (5 benign, 5 carcinoma) by Western blot analysis. Five purified growth factors—basic fibroblast growth factor (bFGF), interleukin-1beta (IL-1β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and endothelial growth factor (EGF)—were tested for their capacity to induce SF/HGF expression by a human prostatic stromal myofibroblastic cell line, as assessed by enzyme-linked immunosorbent assay. Supernatant from the normal PrEC prostatic epithelial cell line and the DU 145 carcinoma cell line were assayed for SF/HGF-inducing activity.Results. SF/HGF exhibited a mean fourfold overexpression in carcinoma tissues compared with benign prostatic tissue. Significant stimulation of SF/HGF expression by prostatic stromal myofibroblasts was detected for IL-1β (8.1-fold), PDGF (6.2-fold), bFGF (4.0-fold), VEGF (3.7-fold), and EGF (2.9-fold). DU 145-conditioned media, but not the PrEC-conditioned media, contained SF/HGF-inducing activity, which was determined to include IL-1β, bFGF, and PDGF by antibody-blocking experiments.Conclusions. SF/HGF is overexpressed in human prostatic carcinoma tissues. Prostatic carcinoma cell stimulation of SF/HGF expression by adjacent benign myofibroblastic cells as a type of epithelial–stromal paracrine interaction could potentially influence prostatic carcinoma cell behaviors.