Relation of endocrine-paracrine cells to cell proliferation in normal, hyperplastic, and neoplastic human prostate.

The relative distribution pattern of the pan-endocrine marker Chromogranin A (Chr A) and the proliferation-associated Ki-67 antigen was investigated in 20 prospectively sampled prostatectomy specimens. In cryostat sections, the Chr A immunoreactivity showed evidence of endocrine differentiation in all 15 prostatic adenocarcinomas. Nine tumors displayed a weak, 5 a moderate, and 1 adenocarcinoma a strong endocrine differentiation. These findings highlight the importance of endocrine differentiation in prostate malignancy that histologically resembles ordinary adenocarcinomas. The simultaneous demonstration of Ki-67 and Chr A revealed that in normal, hyperplastic, and neoplastic prostate tissue Chr A-positive cells were preferentially situated in proximity to Ki-67-labeled cells. This relative distribution pattern of both markers may indicate that endocrine cells are involved in controlling cell proliferation through a paracrine hormonal mechanism. However, an obvious correlation was not found between the degree of endocrine differentiation and proliferative activity in prostatic adenocarcinomas. Furthermore, a coexpression of Ki-67 and Chr A in the same (tumor) cells was not observed suggesting that the endocrine phenotype is only expressed in the G0 phase of the cell cycle as well as in normal, hyperplastic, and neoplastic conditions.     

Neuroendocrine cells in the normal,hyperplastic and neoplastic prostate

Neuroendocrine cells can be demonstrated in normal, hyperplastic and neoplastic prostatic tissues. The products secreted by these cells can be used as tissue and/or serum markers but may also have biological effects. Neuroendocrine cells in prostate cancer most probably do not contain the androgen receptor and are therefore primarily androgen independent. Some of the neuropeptides secreted by the neuroendocrine cells may act as growth factor by activation of membrane receptors in an autocrine-paracrine fashion or by ligand-independent activation of the androgen receptor in neighboring non-neuroendocrine cells. Evidence is accumulating from experiments with tumor models that neuropeptides indeed can influence the growth of prostatic tumor cells. Future research on neuroendocrine differentiation may answer some questions concerning the biological behavior of clinical prostatic tumors.     

The proliferative function of basal cells in the normal and hyperplastic human prostate

To obtain more insight into the proliferative function of basal and secretory cell types in human prostate, we studied the immunoprofile of three well-characterized proliferation-associated antigens (Ki-67, PCNA, MIB 1) in normal and hyperplastic prostate tissue. Distinction between labeled basal and secretory cell types was made by simultaneous demonstration of the proliferation-associated antigens and basal cell-specific cytokeratins in identical sections. In normal and hyperplastic acini, approximately 70% of labeled cells were of the basal cell phenotype. These data clearly suggest that the proliferative compartment of the normal and hyperplastic epithelium is located in the basal cell layer. Compared to normal and hyperplastic conditions, severe proliferative abnormalities were detected in high-grade prostate intraepithelial neoplasias (PIN), as documented by the extension of the proliferative compartment up to the luminal border. Conversely, approximately 70% of proliferating cells detected in atypical hyperplasias that progressed in invasive carcinomas were localized in the remaining basal cell layer. These findings may indicate the proliferative role of basal cells in the epithelial renewal, and the development of hyperplastic and neoplastic disorders in the human prostate. © 1994 Wiley-Liss, Inc.     

Sex steroid receptors in normal and hyperplastic human prostate

The concentrations of sex steroid receptors (per unit DNA) were measured in normal periurethral and peripheral prostatic tissue samples from seven men (mean age 64 years; range 54-71 years) undergoing cystectomy for bladder cancer, and in hyperplastic nodules from 15 men with BPH (mean age 69 years; range 60-89). Occupied androgen (AR) and estrogen (ER) receptors were measured with an improved exchange procedure, where receptor-binding sites were stabilized by a combinatorial procedure involving careful washout of extracellular secretory products (including proteases) prior to homogenization, inclusion of 0.5 mM phenylmethyl sulfonylfluoride (PMSF) and 20 mM molybdate in the exchange medium, and long-term incubation at 0-4 degrees C. Bound radioligands were separated by a hydroxylapatite (HAP) batch adsorption procedure. Maximal specific exchange binding of 3H-R 1881 or 3H-estradiol in total homogenates of human prostate samples was achieved after incubation periods of about 72 h at 0-4 degrees C. In contrast, progestin receptors (PR) were readily available for binding 3H-R 5020; thus overnight binding at 0-4 degrees C was routinely used to measure PR. Binding specificities and equilibrium binding constants (calculated from 8-point Scatchard plots, correcting for nonsaturable binding) were found to be characteristic for AR, PR, and ER, respectively. The receptor results obtained in this study demonstrate that no significant differences existed in total AR per unit DNA between hyperplastic and either central or peripheral prostatic tissue samples; PR was present in both zones of normal prostatic tissue as often as in BPH samples, with PR concentrations significantly lower in hyperplastic samples; and ER was irregularly detected in both normal and hyperplastic tissue in low concentration relative to AR and PR; the frequency of ER detection was much lower in BPH than in normal prostate tissue. Studies of steroid receptor content relative to enzyme markers specific for epithelial and stromal cells in BPH samples showed a positive correlation between acid phosphatase activity (a specific marker for epithelial cells) and both AR and PR. No correlation was observed between AR or PR with either prolyl hydroxylase or myosin ATPase (specific markers for stromal cells). These observations suggest that PR, as well as AR, is primarily associated with the epithelial elements of prostate. Because of the relative infrequency of ER, similar correlation of ER with enzyme markers was not possible.     

Growth responses of normal, benign hyperplastic, and malignant human prostatic epithelial cells in vitro to cholera toxin, pituitary extract, and hydrocortisone

Normal, benign hyperplastic (BPH), and malignant prostatic epithelial cells from adult humans can be serially passaged in medium PFMR-4 supplemented with 20% whole serum, cholera toxin, and epidermal growth factor (EGF). Improved growth of these cells has been achieved by optimizing the concentrations of the components of the basal medium. In the new modified medium, PFMR-4A, the only supplements required are 5% dialyzed serum, cholera toxin, EGF, pituitary extract, and hydrocortisone. Comparative studies of the growth responses of prostatic cells derived from normal central zone, normal peripheral zone, BPH nodules, and adenocarcinomas did not reveal any qualitative differences; all respond positively to the addition of cholera toxin, pituitary extract, and hydrocortisone to the culture medium. Cells of one cancer strain, however, were much less density-dependent for growth than were the other strains of normal, BPH, or malignant cells.     

Differential expression of specific cytokeratin polypeptides in the basal and luminal epithelia of the human prostate.

The purpose of the present study was to identify cytokeratin polypeptides that are specifically associated with the basal and luminal epithelia of the human prostate. This aim was accomplished by immunohistochemical and immunoblot analysis of human prostate using cytokeratin-specific monoclonal antibodies. In immunohistochemical studies, monoclonal anticytokeratin 8.12 exhibited immunoreactivity with the basal, but not luminal, epithelial cells of fetal, juvenile, normal adult, and hyperplastic prostate. The 8.12 antibody did not stain prostate cancer tissues. Epithelia of 30 and 36 week fetal prostate contained only basal cells whereas both luminal and basal cells were noted in 7 month and 1 year old juvenile prostate. This finding suggests a stem cell function for the prostatic basal cells. Immunoblot analysis of proteins separated by two-dimensional electrophoresis showed that cytokeratins 5 and 15 were basal-cell-specific cytokeratins that were absent from prostatic carcinoma while cytokeratins 8 and 18 appear to be luminal-cell-specific. These results indicate that antibodies to specific cytokeratin polypeptides can be used not only to differentiate between prostatic basal and luminal cells but also to study the biological processes of prostatic organogenesis and carcinogenesis.     

E-cadherin expression and PSA secretion in human prostate epithelial cells

Prostate-specific antigen (PSA) is the most widely used marker for the diagnosis of prostate cancer and is an independent predictor of prostatic capsular invasion. A number of studies have identified E-cadherin, a cell adhesion protein, as a potential invasion suppressor which is decreased in prostate adenocarcinoma. Our goal in the present study was to evaluate E-cadherin expression in primary cultures and determine the relationship between E-cadherin expression and PSA secretion in both primary cultures and the prostate tumor cell line, LNCaP. Immunohistochemical studies and Western blot analysis confirmed greater expression of E-cadherin in normal epithelial cells than tumor-derived prostate cells. This is the first report that the incubation of normal prostate epithelial cells with E-cadherin antibody increases the amount of PSA detected in the media of normal cells as well as in LNCaP. Since E-cadherin may function as an invasion suppressor, an understanding of the decreased expression of this adhesion factor and the impact on PSA secretion may aid in understanding epithelial tumorigenesis.     

Regulation of cell-to-cell communication in non-tumorigenic and malignant human prostate epithelial cells

BACKGROUND: Gap-junction-mediated intercellular communication (GJIC) is required for normal development and tissue homeostasis. However, the role of GJIC in human prostate carcinogenesis and progression remains ill-defined. METHODS: The ability of hormones, anti-hormones, and the anti-hypertensive drug, forskolin, to restore GJIC in non-tumorigenic (RWPE-1 and PWR-1E) and malignant (RWPE-2, LNCaP, DU-145) human prostate epithelial cell lines, was examined by Scrape-Loading/Dye Transfer (SL/DT) and Fluorescence Recovery After Photobleaching (FRAP) methods using an Ultima laser cytometer. RESULTS: Results from both assays show that PWR-1E, RWPE-2, LNCaP, and DU-145 cells have weak or absent GJIC activity. However, the non-tumorigenic RWPE-1 cells showed restoration of some GJIC (nearly 10%) after 1 hr in the FRAP assay. Forskolin and estrone, which increase intracellular cAMP levels, induced a significant and consistent increase (2.8- and 4.4-fold, respectively) in cell-to-cell communication only in the non-tumorigenic RWPE-1 cells. Furthermore, estrone induced a two-fold increase in connexin 43 (Cx43) and a 30% decrease in Cx32 expression, while forskolin caused a 50% reduction in Cx32 with no effect on Cx43 expression in RWPE-1 cells. CONCLUSIONS: These data suggest that agents that increase Cx43:Cx32 ratio may be used to restore GJIC in junctionally-deficient, non-tumorigenic immortalized cells, thus providing insights into potential mechanisms responsible for the multistep carcinogenesis in the human prostate.     

Prostatic epithelial and luminal area in the transition zone acini: morphometric analysis in normal and hyperplastic human prostate

OBJECTIVE: To analyse quantitatively the acini and changes in the acinar epithelium and lumen in the transitional zone of normal and hyperplastic human prostates. PATIENTS AND METHODS: Tissue samples of the transitional zone were taken from prostates with benign prostatic hyperplasia (BPH) obtained from 20 patients with clinical symptoms of bladder outlet obstruction who underwent open prostatectomy. The control tissue comprised 20 transitional zones of prostates obtained during autopsy of adults aged < 30 years (killed in accidents). The following variables were measured; the number of acini, total acinar area, area of the lumen, epithelial area, and the median (range) epithelial height, using computerized histomorphometric techniques. RESULTS: The total area of the acini and the luminal area was statistically significantly greater in BPH. In normal and hyperplastic prostates, respectively, the total mean (sd) area (mm2) of the acini was 0.041 (0.007) and 0.056 (0.016), of the lumen was 0.016 (0.003) and 0.036 (0.013), and of the epithelium was 0.025 (0.004) and 0.019 (0.003) (all P < 0.001). There was no statistically significant difference in the number of acini between controls and BPH. The area and the height of the acinar epithelium was statistically significantly greater in BPH; for epithelial height ( micro m) in normal and BPH tissue, respectively the minimum height was 9.92 (1.67) and 6.45 (1.14), the maximum 54.38 (4.09) and 41.52 (4.51) and the median 27.89 (2.48) and 19.96 (2.20) (all P < 0.001). CONCLUSIONS: There was no significant difference in the number of acini between control and BPH tissue, but the area and the height of the acinar epithelium was significantly lower in BPH.     

Discrimination between normal, hyperplastic and malignant human prostatic tissues by enzymatic profiles

A relative enzymatic index has been developed which differentiates normal, hyperplastic (BPH) and malignant human prostatic tissues. Enzymatic activities have been calculated at Vmax conditions in 10 normal, 14 BPH and 11 carcinoma samples. Five enzymes have been assayed: 1) 5 alpha-reductase, 2) 3 alpha-hydroxysteroid oxidoreductase, 3) 3 beta-hydroxysteroid oxidoreductase, 4) 17 beta-hydroxysteroid oxidoreductase and 5) acid phosphatase. The following observations were made when comparing individual enzymatic activities between the 3 tissue groups: 1) mean 5 alpha-reductase activity was lower in carcinoma than in both normal prostate and BPH (p less than 0.05), 2) mean 3 alpha-hydroxysteroid oxidoreductase and 3 beta-hydroxysteroid oxidoreductase activities were greater in carcinoma than in BPH (p less than 0.05) and 3) mean acid phosphatase activity was higher in BPH than in both normal prostate and carcinoma (p less than 0.01). The absolute enzymatic activities were then expressed as relative activities by dividing each absolute value by the mean value for that enzyme in normal prostatic tissue. Relative enzymatic activities were used to derive the ratio: (Formula: see text) The mean value of this ratio was statistically different in normal, BPH and carcinoma tissue (p less than 0.01). The mean value was 3.6 times higher in BPH than in normal tissue, and was 3.8 times higher in normal tissue than in carcinoma. This suggests that BPH and carcinoma diverge in opposite directions biochemically from normal prostatic growth and supports histologic evidence that the 2 neoplastic conditions have a different pathogenesis rather than being part of the same disease spectrum.     

Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells

BACKGROUND: Cell-matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis. METHODS: 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression. RESULTS: RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in laminin-1. Anti-laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of alpha6 and beta1 integrin subunits. Blocking antibodies to alpha6 or beta1 reduced acinar formation to 9 and 6 percent of control, respectively. The beta1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38 percent of control, suggesting that beta1 integrin-mediated signal transduction may be regulated through a FAK pathway. CONCLUSIONS: While basal expression of alpha6beta1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of alpha6, and diffused beta1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that laminin-1 and a functional alpha6beta1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis.     

基于生物信息学探讨低剂量镉对人类正常前列腺上皮细胞基因表达谱的影响

目的:分析低剂量镉作用于人正常前列腺上皮细胞后其基因表达芯片数据的生物学网络调控及关键蛋白,为镉相关的前列腺癌发生机制提供新的理论依据。方法:从基因芯片公共数据库(GEO)中下载19份基因芯片数据,其中经过低剂量镉处理的前列腺上皮细胞样本9例,对照10例,利用基因云-基因大数据分析(GCBI)实验平台、GenClip 2.0、Sytoscape 3.5.1等分析软件,筛选差异表达基因,探讨差异基因的蛋白交互作用网络和生物学通路,从转录组角度阐述低剂量镉作用于正常人前列腺上皮细胞后其基因分子网络改变情况,及其可能涉及的分子生物学功能。结果:与正常前列腺上皮细胞相比,共发现差异表达基因1 050(1.92%)个,主要涉及细胞生理过程、MAPK调节、细胞内信号转导的调控、免疫效应等生物学功能。HSP90AB1、BUB3和PRKAR1A基因为蛋白网络核心节点,且表达差异有统计学意义,均与正常细胞的恶性转化有关。结论:低剂量镉作用于正常人前列腺上皮细胞后,能够导致基因改变,差异基因主要涉及细胞生理过程、MAPK调节、细胞内信号转导的调控、免疫效应等生物学过程。     

Pentagastrin stimulates in vitro growth of normal and malignant human colon epithelial cells

Five normal and four malignant human colon epithelial cultures initiated and maintained in our laboratories as well as the standardized in vitro human adenocarcinoma cell line HT-29 were plated in multiwell plates and incubated at 37 degrees C for 72 hours with either phosphate-buffered saline solution or pentagastrin (5 micrograms/ml). Pentagastrin stimulated normal cells to increase (p less than 0.05) in number by an average of 65 percent compared with saline control cells, whereas malignant cells increased an average of 59 percent compared with control cells. There was no difference in the magnitude of trophic effect between the normal and malignant cells. Further studies are indicated to elucidate the role of gastrin in either initiating, promoting, or both, the growth of carcinoma of the colon.     

Differential gene expression profiling of functionally and developmentally distinct human prostate epithelial populations

BACKGROUND

Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam⁺CD44^?CD49f^Hi basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule‐initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC.

METHODS

Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam⁺CD44^? with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam⁺CD44^?CD49f^Hi FC, adult Epcam⁺CD44^?CD49f^Hi TIC, Epcam⁺CD44⁺CD49f^Hi basal cells (BC), and Epcam⁺CD44^?CD49f^Lo luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2‐fold difference in expression and P < 5.00E‐2. Results were validated with RT‐PCR.

RESULTS

Grafts retrieved from Epcam⁺CD44^? fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam‐ fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC. Functional, network, and canonical pathway identification using Ingenuity Pathway Analysis Version 7.6 compiled genes with the highest differential expression (TIC relative to BC or LC). Many of these genes were found to be significantly associated with prostate tumorigenesis.

CONCLUSIONS

Our results demonstrate clustering gene expression profiles of FC and adult TIC. Pathways associated with TIC are known to be deregulated in cancer, suggesting a cell‐of‐origin role for TIC versus re‐emergence of pathways common to these cells in tumorigenesis. Prostate 75: 764–776, 2015. © The Authors. The Prostate, published by Wiley Periodicals, Inc.

     

The glycosaminoglycans of normal and hyperplastic prostate

A study has been made of the glycosaminoglycans (GAG) of the fetus, normal adult, and benign hyperplastic prostate. In the adult normal prostate dermatan sulphate (DS) is the predominant GAG. The central zone has a slightly higher chondroitin sulphate (Ch AC) content than the peripheral zone. Fetal prostates (22-27 weeks gestation) contain no heparan sulphate (HS). Benign prostatic hyperplasia (BPH) has an increased content of Ch AC. On separation of BPH stroma and epithelium, the epithelial fraction contains only Ch AC and DS. HS and hyaluronic acid (HA) are confined to the prostatic stroma.     

Immunohistochemical localization of laminin in the basement membranes of normal, hyperplastic, and neoplastic human prostate

We examined the effects of fixatives and antibody sources on the immunohistologic localization of laminin in normal and cancer-containing human prostates and studied the localization patterns in carcinomas of varying degrees of histologic differentiation. Two different polyclonal antibodies were localized in paraffin-embedded or cryostat sections of fixed (alcohol, formalin, and paraformaldehyde) or unfixed tissue, using the immunofluorescence (IF) or immunoperoxidase (IP) techniques, with positive and negative controls. We found that the IF reactions were more intense in unfixed or alcohol-fixed sections than in paraformaldehyde-fixed specimens. IP reactions were very weak or absent in fixed and paraffin-embedded sections, but pepsin treatment of these sections resulted in more intense and uniform IP reaction products, stronger than in unfixed or ethanol-fixed cryostat sections. With the IP technique, laminin localization was intense and uniform in the basement membranes (BM) of acini, blood vessels, smooth muscle, and nerve fibers in normal prostate, benign hyperplasia (BPH), and well-differentiated carcinomas. The BM of poorly differentiated carcinomas showed widespread absence of laminin reactivity. In normal BPH and well-differentiated tumors, occasional epithelial cells and their surface and acinar lumina had laminin reactivity. However, in higher grade tumors, numerous neoplastic cells had laminin reactivity in cytoplasm, their surface, and secretory material. Some macrophages and neutrophils also contained laminin reactivity, presumably of degraded laminin. In some moderately and poorly differentiated tumors, the BM of small capillaries did not contain laminin. The BM of larger vessels always had laminin reactivity, even in the higher grade tumors.