Clusterin as a possible predictor for biochemical recurrence of prostate cancer following radical prostatectomy with intermediate Gleason scores: a preliminary report.

Disease recurrence following radical prostatectomy is a major concern in prostate cancer patients. Gleason scores are useful in predicting recurrence. Low Gleason scores are usually associated with long disease-free intervals, while high Gleason scores are suggestive of early recurrence. However, prediction of recurrence has been difficult with intermediate Gleason scores. Clusterin is a ubiquitous secretory sulfated glycoprotein. It is also an antiapoptotic mediator in prostate cancer. The objective of the present study is to determine if clusterin can serve as a predictive biomarker for recurrence of prostate cancer with intermediate Gleason scores in patients following radical prostatectomy. Prostatic specimens with Gleason score of 6 (3+3) or 7 (3+4) were obtained from the archival bank. Three groups of specimens were investigated. The first group was from nine patients who developed recurrent disease according to a persistent rise of serum PSA within 3 years following radical prostatectomy. Those in the second group and the third group were from patients who showed no evidence of disease recurrence for at least 5 y (11 patients) and 10 y (eight patients), respectively following the surgery. Histological sections were subjected to immunohistochemical staining using a monoclonal antibody specific for clusterin. The staining intensity was scored as 0, 1, 2, and 3, with 0 being no staining, 1 showing less than 25% positive staining, 2 being 25-50% positive, and 3 showing greater than 75% positive staining. One-way ANOVA with Bonferroni correction was used for statistical analysis. Evaluation of the scores of clusterin staining was carried out according to four specific areas in each specimen. They were (a) benign epithelial cells, (b) malignant epithelial cells (cancer epithelia), (c) stromal cells surrounding benign cells, and (d) stromal cells surrounding malignant cells (cancer stroma). Staining score in prostatic epithelial cells, benign as well as malignant, showed no significant relationship among the three patient groups. However, when staining scores in stromal cells were compared, there was a significant difference between patients with recurrent disease and those showed no evidence of disease recurrence for at least 10 y. Results of this preliminary study support the important role of clusterin in the stromal component for prostate cancer progression. Clusterin immunostaining may be useful to aid the prediction of chance of disease recurrence in patients with Gleason score 6 or 7 prostate cancer following radical prostatectomy. Further studies with a large number of cases are warranted to verify this preliminary finding.

High fat diet increases the weight of rat ventral prostate.

BACKGROUND: Understanding the mechanisms by which diet influences the prostate may eventually lead to novel approaches for preventing prostate cancer. The objective of this research is to examine the impact of dietary fat, vitamin D, and genistein on prostate weight, serum and intraprostatic androgen levels, and the expression of several androgen-response genes. METHODS: Sprague-Dawley rats were fed, beginning at 21 days of age, for 1 or 3 months of experimental diets with high saturated fat (32.2% calories from fat), low saturated fat (3.6% calories from fat), genistein plus (20 mg/kg), genistein deficient, vitamin D surplus (4,000 U/kg), or vitamin D deficient. The body weight, food intake, the weights of the ventral prostate and dorsolateral prostate, and the levels of testosterone and dihydrotestosterone (DHT) in the serum and in the prostate were determined. The expression of androgen-response genes was characterized by Northern blot analysis. RESULTS: The pilot experiments showed that high dietary fat appeared to consistently increase the weight of the ventral prostate, while vitamin D or genistein did not have a consistent effect on prostate weight. Further analysis confirmed that the ventral prostate is 15% (P < 0.001) heavier in the rat on a high fat diet as compared to a low fat diet. Dietary fat had no significant influence on the levels of serum and intraprostatic androgens and the expression of androgen-response genes. CONCLUSIONS: Our results suggested that the ventral prostate weight of the rat is increased without affecting the androgen axis by feeding the animals with high fat diet beginning at 21 days of age. This observation is potentially important since epidemiological data suggest that saturated fat consumption is a major risk factor associated with prostate cancer incidence rate.

Apoptotic activity of doxazosin on prostate stroma in vitro is mediated through an autocrine expression of TGF-beta1.

BACKGROUND: Doxazosin, an alpha-adrenergic antagonist, has been shown to induce apoptosis in prostatic stromal cells. The mechanism of this apoptotic action by Doxazosin remains undefined. The present study was carried out to demonstrate that the effect of Doxazosin on apoptosis of prostate stromal cells is mediated through an autocrine action of TGF-beta1. METHODS: Primary cultures of human prostate cells were treated with varying concentrations of Doxazosin (0, 0.1, 1, 10, and 100 microM) for a period up to 3 days. At the end of the 3-day culture, cell numbers were counted. Apoptosis was assessed by a colorimetric terminal deoxyribonucleotide transferase labeling technique. TGF-beta1 was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to control cultures, cell numbers were significantly decreased as much as 68.4% in cultures treated with 10 microM of Doxazosin after 3 days incubation, while apoptosis increased by 64.7% in cultures treated with the same concentration of Doxazosin after 24 h. This decrease in cell number was reversed when antibody to TGF-beta1 was added to these cultures. Addition of TGF-beta1 (0, 1.0, and 10 ng/mL) to the cultures also decreased the cell numbers. Quantitation of TGF-beta1 in lysates of cells by ELISA revealed that the cells treated with Doxazosin (10 microM) produced as much as 62.5% more TGF-beta1 than in that of untreated cells. CONCLUSIONS: These results demonstrate that the apoptotic effect of Doxazosin on human prostatic stromal cells is mediated through an autocrine production of TGF-beta1.

Autocrine effect of DHT on FGF signaling and cell proliferation in LNCaP cells: role of heparin/heparan-degrading enzymes.

BACKGROUND: LNCaP cells are androgen-sensitive human prostate cancer cells. They are characterized by a bell-shaped growth curve in response to increasing doses of dihydrotestosterone (DHT) in culture. At a low concentration of DHT (0.1 nM), these cells show an increase in proliferation, but their growth is arrested at a high concentration (100 nM) of DHT. Results of our previous study demonstrated that the inhibitory effect of DHT at a high concentration was mediated through the action of TGF-beta1. The objective of the present study was to elucidate the mechanism of the proliferative effect of DHT in LNCaP cells. METHODS AND RESULTS DHT stimulated LNCaP proliferation only when cells were cultured in the presence of serum. In serum-free cultures, the characteristic DHT-induced proliferation was not observed. The addition of neutralizing antibody against FGF-2 (basic fibroblast growth factor) was able to inhibit this DHT-induced proliferation. These results suggest that the proliferative effect of DHT was mediated through the action of FGF-2. However, results of the reverse transcriptase polymerase chain reaction indicated that LNCaP cells did not express FGF-2 message. As a result, the source of FGF-2 in these cultures must be the serum supplemented in the culture media. FGF-2 can bind to heparin sulfate chains within the extracellular matrix (ECM). In cultures treated with exogenous heparin, the proliferative effect of DHT was abolished. These results led to the development of the hypothesis that DHT treatment mediates the release of FGF-2 entrapped in the ECM through increased heparinase activity. The addition of heparinase to cultures of LNCaP cells, in the absence of DHT, was able to stimulate cell proliferation. Moreover, 0.1 nM DHT caused a significant increase in heparinase activity. CONCLUSIONS: These results provide a possible mechanism for DHT action in LNCaP cells. In the absence of DHT, FGF-2 in culture was trapped in the extracellular matrix and was not available to interact with LNCaP cells. However, in the presence of 0.1 nM DHT, heparinase activity in the culture was elevated and, as a result, it liberated the trapped FGF-2 which, in turn, stimulated proliferation in LNCaP cells.

NCCN Practice Guidelines for Prostate Cancer.

Systemic therapies for prostate cancer are likely to improve, and as they do, they will have enormous impact on the treatment of high-risk and locally advanced cancers. Further technical improvements in radiotherapy and alternative local modalities, such as cryoablation, are also likely, and will bring even more options for local control. It is certain these guidelines will continue to evolve.

Cytotoxic sensitivity to tumor necrosis factor-alpha in PC3 and LNCaP prostatic cancer cells is regulated by extracellular levels of SGP-2 (clusterin).

BACKGROUND: SGP-2 is a ubiquitous secreted glycoprotein that prevents cellular apoptosis. This study was carried out to determine the extracellular action of SGP-2 in a model of tumor necrosis factor-alpha (TNF)-induced cytotoxicity using two human prostatic cancer lines, LNCaP and PC3. These two lines were selected because LNCaP cells are highly sensitive to the cytotoxic effect of TNF, while PC3 cells are resistant to TNF at 24 hr. METHODS: Cells were cultured in the presence or absence of TNF (10 ng/ml). LNCaP cells were treated with varying concentrations of exogenous SGP-2, while PC3 cells were treated with antisera to SGP-2 with and without exogenous SGP-2. Following a 24-hr treatment, cultures were assessed by counting of cell number and by the trypan blue exclusion assay. RESULTS: Western blot analysis of conditioned media revealed that PC3 secreted more SGP-2 than did LNCaP. The sensitivity to TNF in LNCaP cells was reduced by the addition of exogenous SGP-2. PC3 cells became sensitive to TNF when SGP-2 antibody was added to the culture. The effect of SGP-2 antibody on PC3 cells was reversed by the addition of exogenous SGP-2 to the culture. CONCLUSIONS: These results suggest that SGP-2 can act as an extracellular mediator of anti-TNF-induced cytotoxicity.

Differential growth rates in stromal cultures of human prostate derived from patients of varying ages.

BACKGROUND: This study was undertaken to attempt to characterize changes in in vitro growth rates and cellular phenotypes of human prostatic stroma associated with aging and/or development of benign prostatic hyperplasia (BPH). METHODS: Prostate stromal cell strains were established from 12 tissue donors of varying age. Culture growth rate was determined by cell counts over a 6-day period. Cell phenotype was assessed by immunocytochemical staining for smooth muscle alpha-actin, smooth muscle myosin, and prolyl-4-hydroxylase. RESULTS: Growth rates of prostate stromal strains in vitro varied. Stromal cells derived from aged males with BPH had significantly slower growth rates than cells from younger donors. A positive reaction for prolyl-4-hydroxylase, a mesenchymal cell marker, was present in all cell cultures regardless of donor age. Expression of smooth muscle-specific actin, a nonspecific smooth muscle cell marker, was present in 48-79% of prostate stromal cultures. Staining for smooth muscle myosin, a specific smooth muscle cell marker, was found to vary significantly with age. The percentage of smooth muscle myosin-positive cells derived from males aged 15, 45, 57, and 72 years were 0.70 +/- 0.14%, 2.12 +/- 0.30%, 4.20 +/- 0.89%, and 26.25 +/- 1.0%, respectively. The shape and size of actin- and/or myosin-positive stromal cells from a 72-year-old donor culture were also usually larger and polygonal in shape as compared to thin and elongated shapes in 15-year-old donor cultures. The shape of actin- and/or myosin-positive cells from a 45-year-old donor culture demonstrated both phenotypes. CONCLUSIONS: These results suggest that in human prostate stromal cells cultured as described, the growth rate decreases, the percent of smooth muscle cells increases, and the cellular shape changes with increasing donor age and/or development of BPH.

The pathogenesis of benign prostatic hyperplasia: a proposed hypothesis and critical evaluation.

PURPOSE: We used expanding observations regarding effects of testicular epididymal plasma and nonandrogenic testis factor(s) (NATF) on prostate growth to propose and evaluate a hypothesis regarding the development of benign prostatic hyperplasia (BPH) in man. MATERIALS AND METHODS: Current experimental data regarding the presence of NATF were reviewed. The potential for their exposure to the prostate by various routes was assessed. These observations were coupled with recognized anatomical, histological and epidemiological characteristics of BPH to construct a hypothesis regarding its pathogenesis. RESULTS: In vivo observations in man, rats and dogs supported the systemic secretion of NATF. These factors probably are, at least in part, spermatogenesis related. In vitro evaluation of the effect of spermatocele derived testicular epididymal plasma on human prostate stromal cells indicated the presence of androgen independent and androgen synergistic stromal growth promoters. These factors have potential local and systemic access to the prostate. The almost ubiquitous development of a regional, histologically variegated nodular growth occurring in the prostate in the androgen diminished environment of the aging man is compatible with local as well as systemic exposure to an age associated secretion of NATF. CONCLUSIONS: We propose that human BPH is an induced phenomenon that is usually initiated by local episodic exposure of periurethral prostate to mitogens secreted by the testis/epididymis. Once initiated, isolated or complex interacting proliferative stimuli from the testis/epididymis and a variety of other sources may achieve exposure to the prostate by several routes and simulate prostate growth.

Loss of expression of transforming growth factor-beta receptors is associated with poor prognosis in prostate cancer patients.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of proliferation in most cells and exerts its effects through an interaction with membrane receptors type I (TGF-betaRI) and type II (TGF-betaRII). Recently, we have demonstrated a correlation between the loss of expression of TGF-betaRI and TGF-betaRII and increasing Gleason score in archival human prostate cancer tissues. To evaluate the potential prognostic value of this observation, the present study investigated the expression of TGF-beta receptors in association with disease progression after the initial diagnosis in 52 archival human prostate cancer tissues. The expression of both TGF-betaRI and TGF-betaRII was correlated with the Gleason score, clinical tumor stage, 4-year survival rate, and serological recurrence rate after radical prostatectomy. Results revealed that there was a significant association between the Gleason score and the loss of expression of TGF-betaRI (P < 0.025) and TGF-betaRII (P < 0.01). However, only the loss of TGF-betaRI expression showed a statistically significant association with the clinical tumor stage (P < 0.05), 4-year survival rate (P < 0.05), and serological recurrence rate after radical prostatectomy (P < 0.025). Therefore, these data indicate that the loss of TGF-betaRI expression as measured by immunohistochemical staining may be a potential prognostic marker in prostate cancer patients.

Synergistic action of steroids and spermatocele fluid on in vitro proliferation of prostate stroma.

PURPOSE: Our goal is to understand human prostate growth phenomena potentially important to BPH development and growth. The objective of the present study is to characterize in vitro prostate stromal proliferative factors in testis epididymal secretions. MATERIALS AND METHODS: Human spermatocele fluids were used as a source of testicular epididymal plasma (STEP). Primary cultures of human prostate stromal cells were routinely grown in RPMI-1640 with 10% fetal bovine serum. During a 6-day experimental period, cells were cultured in RPMI-1640 in the absence of serum but supplemented with ITS. Whole STEP, ether stripped STEP, or heparin affinity column treated STEP was included in the culture medium with and without the addition of testosterone (T), dihydrotestosterone (DHT), or estradiol (E). Results of these treatments were assessed by cell counts. Antibodies against smooth muscle myosin heavy chain, smooth muscle alpha actin, and prolyl-4-hydroxylase were utilized in immunocytochemical characterization of cultured cells. RESULTS: Whole STEP stimulated prostatic stromal cells derived from prostates of 15, 45, 70 and 72-year-old men. Treatment of STEP by ether stripping or heparin affinity column exposure did not result in a significant reduction in cell counts. With the exception of the 15-year-old specimen, addition of T or DHT to ether stripped STEP resulted in a significant increase in cell counts over that of ether stripped STEP treatment alone. Preliminary immunocytochemical evaluation indicated the presence of variable mixture of fibroblasts, myofibroblasts, and smooth muscle cells in these cultures. CONCLUSIONS: These in vitro observations indicate that testis epididymal secretions contain androgen/STEP synergistic and androgen independent STEP factors promoting prostate stromal growth. These factors are not heparin binding. These observations are consistent with the concept that, in addition to the production of steroids, the testis produces non-androgenic factors that act in concert with, as well as independently of, androgen to stimulate prostatic growth.

Epidermal growth factor receptor activation in androgen-independent but not androgen-stimulated growth of human prostatic carcinoma cells.

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.

Acute adrenal insufficiency following unilateral radical nephrectomy: a case report.

Adrenal insufficiency following unilateral radical nephrectomy has not been previously described in medical literature. We present a 78-year-old male patient who experienced a difficult postoperative course with vague findings, and was ultimately diagnosed with acute adrenal insufficiency. Treatment with glucocorticoids and mineralocorticoids resulted in prompt control of the disease.

Intrinsic and extrinsic factors controlling benign prostatic growth.

This review will present a new concept on the etiology of the development of benign prostatic hyperplasia (BPH). Conventionally, two known etiological factors for the development of BPH have been aging and the presence of functional testes. Assignment of these two factors, although reasonable, has not been conducive to aid the research community to identify and isolate the patho-physiological agents that are directly responsible for the development of this disease. In the present review, we proposed a broadened concept of intrinsic and extrinsic factors for BPH. This concept offers identifiable research opportunities that will facilitate our quest in search for etiological agents for BPH. A brief description of various intrinsic and extrinsic factors and justifications for their selection will be discussed.

Comparative genomic hybridization and molecular cytogenetic characterization of two prostate cancer xenografts.

Conventional cytogenetic analysis of two prostate tumor xenografts, LuCaP 23.1 and RP22090, was unsatisfactory for comprehensive genetic evaluation of the cell lines. Fluorescence in situ hybridization (FISH) for chromosome enumeration and comparative genomic hybridization (CGH) for numerical imbalance detection were performed and resulted in a more complete molecular cytogenetic characterization of these lines. Both xenografts were hypertriploid and had significant numerical imbalances. For example, LuCaP 23.1 had gain of all or part of chromosomes 3, 5, 6, 7, 8, 11, and 12 and the X chromosome and loss of all or part of chromosomes 2, 3 6, 8, 9, 10, 17, and 18. In RP22090, gain of all or part of chromosomes 5, 7, 8, 9, 10, 12, 14, and 15 was seen, whereas loss was seen for all or part of chromosomes 4, 6, 8, 15, 16, 17, 19, 20, and 22. Both xenografts reflect the high frequency of chromosomal changes seen in some late-stage prostate cancers, including many novel changes and some changes such as the loss of 8p and gain of 8q, which have been reported previously in primary and metastatic prostate cancers. Consistent changes in both lines, such as loss of chromosomes 6 and chromosome arm 8p and gain of chromosome 7 and chromosome arm 8q, may represent genetic events specific for prostate cancer development, but imbalances on other chromosomes such as 3, 9, 19, and 20, not frequently reported in prostate cancers, may reflect potentially important changes that should also be examined.

Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer.

Our previous observations in LNCaP cells in vitro demonstrated an association between apoptotic cell death resistance and SGP-2 (Clusterin) overexpression. Accordingly, we hypothesized that high levels of cellular SGP-2 would aid in identifying biologically aggressive prostate cancer cells with unique survival advantages. To test this hypothesis, 40 archival radical prostatectomy and/or biopsy specimens of varying grades of prostate cancer were subjected to immunohistochemical SGP-2 staining. The resulting epithelial stains were quantified subjectively on a scale of 1-3 by four independent observers. Benign prostatic epithelial cells from young donors served as controls and showed a consistently weak staining intensity. In contrast, prostate cancer specimens showed varying degrees of staining intensity that correlated with a Gleason pattern (P = 0.006). This correlation supports the hypothesis that protection from apoptotic death may account, in part, for biologically aggressive tumor behavior.

The differentiating agent phenylacetate increases prostate-specific antigen production by prostate cancer cells.

The prostatic-specific antigen (PSA) is the tumor marker most widely relied upon for the monitoring of patients with prostate cancer. Recently, declines in the serum concentrations of PSA have been advocated as a surrogate marker of tumor response in clinical trials of investigational antitumor agents. We examined the hypothesis that this postulate may not apply to the evaluation of drugs such as phenylacetate, a differentiating agent endowed with mechanisms of action different from those of classic cytotoxic chemotherapy. Using human prostatic carcinoma LNCaP cells as a model, we show that phenylacetate induces PSA production despite inhibition of tumor cell proliferation. Incubation of LNCaP cultures with cytostatic doses of phenylacetate (3-10 mM) resulted in a three- to fourfold increase in PSA secretion per cell. This appears to result from upregulation of PSA gene expression, as indicated by elevated PSA mRNA steady-state levels in treated cells. The increase in PSA production per cell was confirmed in rats bearing subcutaneous LNCaP tumor implants that were treated systemically with phenylacetate. Further comparative studies indicate that upregulation of PSA is common to various differentiation inducers, including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and butyrate but is not induced by other antitumor agents of clinical interest such as suramin. We conclude that declines in PSA may be treatment specific and that the exclusive use of this criterion as a marker of disease response may mislead the proper evaluation of differentiating agents in prostate cancer patients.

Loss of expression of transforming growth factor beta type I and type II receptors correlates with tumor grade in human prostate cancer tissues.

Transforming growth factor beta1 (TGF-beta1) is a potential regulator of prostate cancer cell growth that signals through a heteromeric complex composed of type I and type II receptors. In the present study, an attempt was made to establish a correlation between expression of TGF-beta receptors and tumor grade in archival human prostate cancer tissues. To this end, immunohistochemical studies for TGF-beta receptors were carried out on 32 cases of human prostate cancer and 8 samples of benign human prostate. In both benign and malignant human prostate tissues, immunoreactivity for both type I and type II receptors was detected predominantly in epithelial cells. In addition, there was an inverse correlation between the loss of expression of TGF-beta1 type I and type II receptors and the tumor grade. Of the 32 prostate cancer cases screened, staining was completely absent in four samples for type II receptor (P < 0.05) and eight samples for type I receptor (P < 0.025). In contrast, all eight samples of benign prostate tissues investigated in this study showed strong staining for both type I and type II receptors. These results, taken together, indicate that human prostate cancer cells frequently have loss of expression of TGF-beta type I and/or type II receptors. Furthermore, these observations provide a potential mechanism for prostate cancer cells to escape the growth-inhibitory effect of TGF-beta.

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.

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

Etiology of benign prostatic hyperplasia.

Benign Prostatic Hyperplasia (BPH) is the most common neoplastic condition that afflicts men, and it constitutes a major factor impacting the health of the American male. This article reviews voiding dysfunction and the role of aging, the testis, and androgen in the development of BPH. Emphasis is placed on new concepts in the basic aspects of BPH etiology as a result of recent investigations.