Studies on the differentiation pathway and growth characteristics of epithelial culture cells of the human prostate.

We established explant primary cultures in order to study the growth and hormone responsiveness, and the differentiation process of prostatic epithelial cells. Cell outgrowth was achieved from explant tissue by using a new DU145-cell-conditioned medium and special plastic coverslips. To define the present model, proliferation assays were tested by [3H]thymidine assay and planimetric analysis. Cells were analyzed using immunocytochemistry, light, phase contrast and electron microscopy, polymerase chain reaction, telomerase ELISA and immunoassay (PSA). Morphology and electron microscopy revealed typical epithelial differentiation. Immunocytochemistry showed the content of basal and secretory epithelial cells, endocrine paracrine cells and a high level of proliferation. With increasing culture time, mature epithelial differentiation (PSA) increases and the initial increase of alpha-smooth muscle actin (alpha-SMA) decreases again. After further passaging, alpha-SMA expression is no longer detected and PSA expression decreases. Furthermore, epithelial cells showed both androgen responsiveness and androgen receptor expression. These findings show the presence of epithelial cells in a process of differentiation with endocrine paracrine cells and a high level of proliferation. This model may maintain the cellular and functional properties more closely related to the human prostate and may provide a valuable tool for studying stem cells and differentiation characteristics.

Cables enhances cdk2 tyrosine 15 phosphorylation by Wee1, inhibits cell growth, and is lost in many human colon and squamous cancers.

Cyclin-dependent kinase 2 (cdk2) is a small serine/threonine kinase that regulates cell cycle progression. Cdk2 activity is tightly controlled by several mechanisms, including phosphorylation and dephosphorylation events. Cables is a recently described novel cdk-interacting protein. In proliferating cells, Cables was predominantly localized in the nucleus by cell fractionation and immunostaining. Expression of Cables in HeLa cells inhibited cell growth and colony formation. Cables enhanced cdk2 tyrosine 15 phosphorylation by the Wee1 protein kinase, an inhibitory phosphorylation, which led to decreased cdk2 kinase activity. The gene encoding Cables is located on human chromosome 18q11-12, a site that is frequently lost in squamous, colon, and pancreas cancers. We found that Cables was strongly expressed in normal human epithelial cells including squamous and glandular mucosa. Breast and pancreatic cancers show strong Cables expression; however, loss of Cables expression was found in approximately 50-60% of primary colon and head and neck cancer specimens. Lack of Cables expression was associated with loss of heterozygosity on chromosome 18q11. The data provide evidence for a Cables-mediated interplay between cdk2 and Wee1 that leads to inhibition of cell growth. Conversely, loss of Cables may cause uncontrolled cell growth and enhance tumor formation.

Regulation of keratinocyte growth factor receptor and androgen receptor in epithelial cells of the human prostate.

PURPOSE: Stromal-epithelial interactions of growth factors and the androgen receptor may have implications for the pathophysiology of benign and neoplastic transformation of the human adult prostate. We investigated a possible interaction of keratinocyte growth factor with its receptor as well as with the androgen receptor signaling pathway in human prostatic epithelial cells. MATERIALS AND METHODS: Human prostatic epithelial cells were obtained from explant primary culture, established in DU145 cell conditioned medium and maintained in keratinocyte serum-free medium with supplements. Epithelial cells were characterized by light and electron microscopy, and immunocytochemical study using epithelial and mesenchymal markers. Androgen receptor, keratinocyte growth factor receptor and keratinocyte growth factor messenger RNA expression was measured by polymerase chain reaction (PCR). The response to 0.01 to 10 nM. dihydrotestosterone, 10 microM. flutamide and 1 to 1,000 ng./ml. keratinocyte growth factor was tested by [3H] thymidine assay. The difference in keratinocyte growth factor receptor and androgen receptor gene expression after treatment with and without keratinocyte growth factor and flutamide were determined by quantitative multiplex PCR and quantitated using densitometry analysis. RESULTS: Immunocytochemical and electron microscopy characterization revealed typical epithelial differentiation. PCR showed keratinocyte growth factor receptor and androgen receptor expression in epithelial cultured cells but no keratinocyte growth factor expression. Epithelial cells showed a significant time and dose dependent stimulation of cell proliferation with keratinocyte growth factor and dihydrotestosterone (p <0.05). When combined with the anti-androgen flutamide the effect of 100 ng./ml. keratinocyte growth factor was significantly decreased (p <0.05). At 100 ng./ml. keratinocyte growth factor quantitative multiplex PCR revealed stimulated keratinocyte growth factor receptor and androgen receptor messenger RNA expression. CONCLUSIONS: These results show that keratinocyte growth factor up-regulates the keratinocyte growth factor and androgen receptors in the absence of androgen. Thus, the androgen signaling pathway may be activated by growth factors such as keratinocyte growth factor in an androgen deficient environment.

Immunolocalization of the keratinocyte growth factor in benign and neoplastic human prostate and its relation to androgen receptor.

BACKGROUND: Growth and development of the prostate are androgen-dependent. Keratinocyte growth factor (KGF), widely expressed by mesenchymal cells, is thought to act like an andromedin between stroma and epithelium of the prostate. Since KGF has recently emerged as an autocrine mediator in prostate cancer, we investigated the role KGF plays in the human prostate and its relationship to androgen receptor (AR). METHODS: Normal (n = 13), benign hyperplastic (n = 5), and neoplastic (n = 14) human prostate tissues as well as cultured epithelial and stromal cells were analyzed using polymerase chain reaction (PCR), Western blot analysis, and immunohistochemistry. RESULTS: Reverse transcriptase polymerase chain reaction and Western blotting showed KGF expression in stromal cultured cells of the normal prostate but not in epithelial cells. Using immunohistochemistry, KGF was found to be localized in fibroblasts and smooth muscle cells, independent of prostate disease. There was KGF expression in epithelial cells of BPH and prostate cancer. Human androgen receptor was uniformly expressed in the same secretory glandular cells that were positive for KGF in BPH and prostate cancer. CONCLUSIONS: Our results provide evidence that KGF is a stromal-derived mediator, recently shown to act in a paracrine manner in normal prostate but now detected in epithelial cells in prostate cancer and BPH. These findings support the hypothesis that KGF might act as an autocrine factor in prostate cancer and BPH.

Characterization of a stromal cell model of the human benign and malignant prostate from explant culture.

PURPOSE: There is a lack of suitable in vitro models for the human prostate. To study stromal-epithelial interactions, we established stromal cells in cultures from benign and malignant prostate tissue that resemble more closely the in vivo conditions of the human prostate. MATERIALS AND METHODS: Stromal cells were obtained from explant primary culture, established in DU145 cell conditioned medium and maintained in RPMI-fetal bovine serum (FBS) supplemented with insulin, transferrin and selenium (ITS). Proliferation studies to compare different media were performed using a 3[H]thymidine assay. Stromal cells were characterized by immunocytochemistry using epithelial and mesenchymal markers. Morphology was evaluated by electron microscopy, light and phase-contrast microscopy. Androgen receptor (AR) mRNA expression was measured by polymerase-chain-reaction (PCR). The response to different concentrations of dihydrotestosterone (DHT) and the antihormones flutamide and hydroxyflutamide was tested by 3[H] thymidine assay. RESULTS: Microscopic evaluation revealed typical stromal morphology with elongated cell shapes, cilia, collagen and microfilaments. Immunocytochemical characterization revealed typical fibroblastic and smooth muscle differentiation. ITS supplemented in RPMI-FBS showed the best growth stimulation compared with other serum-free media (p <0.05) and became our basal medium. The presence of DU145 cell conditioned medium in this basal medium showed a significant increase in cell proliferation in stromal cells. Stromal cells maintained AR mRNA expression and significant DHT dose dependent growth stimulation in up to 10 passages. Both the antiandrogens flutamide and hydroxyflutamide counteracted the DHT effect (p <0.05). CONCLUSIONS: This stromal cell model maintains many cellular and functional properties of the human prostate, which may enable us to study growth factor modulation, drug and hormone metabolism in stromal-epithelial interaction with emphasis on the pathogenesis of BPH and prostate cancer.

Androgen responsiveness of stromal cells of the human prostate: regulation of cell proliferation and keratinocyte growth factor by androgen.

PURPOSE: Growth and development of the prostate are androgen dependent and mainly influenced by stromal-epithelial interaction. It is believed that indirect androgenic activation of paracrine factors like keratinocyte growth factor (KGF) in the prostatic stroma influences the growth of epithelial cells. In this study we investigated the role androgen plays in stromal cell growth and stimulation of KGF in the human prostate. MATERIALS AND METHODS: Stromal cells were derived from explant primary culture of human normal or benign prostatic tissue. The effect of different dihydrotestosterone (DHT) concentrations on cell proliferation was measured using 3[H]thymidine incorporation assay. The effect of DHT on levels of KGF protein was determined by Western blotting. The effect of DHT on levels of KGF gene expression was measured by various cycles of polymerase-chain-reaction (PCR) and multiplex PCR. RESULTS: Characterization of stromal cells showed epithelial cells less than 9.5% in all passages. DHT stimulated human prostate stromal cells in a dose dependent fashion over a concentration range of 0.001-10 nM. Immunocytochemical evaluation of KGF after DHT exposure showed a higher staining intensity. Relative quantitation of Western blotting showed a 1.93-fold increase in KGF protein in the androgen treated stromal cells. At 1 nM DHT conventional and multiplex PCR revealed a significant stimulation of the KGF mRNA expression. CONCLUSIONS: These data show for the first time that androgen stimulates cell proliferation as well as KGF protein and gene expression in human prostate stromal cells. This supports the hypothesis that androgen-induced stromal-derived KGF stimulates prostate epithelial cell growth.

Photodynamic destruction of lysosomes mediated by Nile blue photosensitizers.

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS-6I and sat-NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal. This is indicated by the light-and drug-dose-dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosome-containing subcellular fraction, and impairment of the lysosomes to take up and sequester acridine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat-NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat-NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells.

Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells.

Detection of exfoliated bladder cancer cells by monoclonal antibodies to tumor-associated cell surface antigens.

Monoclonal antibodies (Mabs) to human tumor antigens have potential for tumor detection and treatment. For bladder carcinoma, the detection of exfoliated tumor cells in urinary specimens may be accomplished with Mabs reacting to tumor cell-surface components. This method may be useful for screening and monitoring carcinogen-exposed workers. A Mab generated by our laboratory, 3G2-C6, reacts with high affinity to a cell-surface component expressed by bladder tumor cells. The potential utility of this Mab in detecting exfoliated tumor cells was evaluated in bladder wash specimens. The Mab method detected positive cells in 87% (56/64) of specimens from patients with bladder cancer, including a great majority with grade 1 tumor and carcinoma in situ, superior to the routine cytology done on the same specimens. Cells in specimens from patients with urinary calculi, chronic cystitis, and history of bladder cancer also reacted with the Mab, suggesting that other stimuli can induce antigen expression. The Mab method can also be performed on urine samples, thus allowing evaluation of the ability of the Mab to identify premalignant, malignant, and other abnormal exfoliated cells in urine. The Mab method represents a unique opportunity to develop noninvasive detection of bladder cancer and to monitor and screen bladder cancer high-risk groups.

Detection of tumor cells in bladder washings by a monoclonal antibody to human bladder tumor-associated antigen.

We have conducted two studies to evaluate the efficacy of using a specific monoclonal antibody (McAb) to detect exfoliated tumor cells in bladder washings. This is a preliminary step toward the development of immunological methods to improve the cytologic detection of bladder carcinoma. In this study, McAb 3G2-C6 was used. The McAb reacts to a bladder tumor-associated cell-surface antigen expressed in bladder tumors of various grades. Bladder washings from patients with and without carcinoma were stained with the McAb using two different indirect immunofluorescence methods (Methods A and B). The results of the immunological studies were compared with those obtained from the cytology laboratory and these in turn, were evaluated against the histopathological diagnosis of respective patients at the time the samples were taken. Immunofluorescence method A detected positive cells in 87% (56/64) of specimens from bladder cancer patients, including 18 of 19 from patients with grade 1 tumor. This method also had a low false-positive rate; only one of 17 specimens from patients with other urinary disorders had positively reacting cells. Immunofluorescence method B, evaluating a second group of specimens, detected positive cells in 68% (15/22) of specimens from patients with carcinoma, and in only one of 17 controls. However, it also identified positive cells in specimens from patients with chronic cystitis and urinary calculi. Overall the results of these studies indicate that the McAb method is superior to the routine cytology in detecting tumor cells in bladder washing specimens. More work must be done, however, to improve the specificity of the method before it can be used as an aid for routine tumor detection.

Visualization of urothelial blood group isoantigens A and B using direct biotin-labeled antibodies and avidin-biotin-peroxidase complex.

The loss of blood group isoantigens from the surface of bladder tumor cells has been correlated with the potential invasiveness of the tumor. Development of simple and reliable methods for detection of these isoantigens should facilitate the general clinical use of this test for predicting malignant potential in low grade, low stage cancer of the bladder. We now report a direct peroxidase technique for the detection of isoantigens A and B by utilizing the specific interaction between biotin and avidin, and the capability of labeling a single antibody with multiple biotin molecules. Antibodies specific to the isoantigens A and B were purified from human antisera by affinity chromatography using an immunoabsorbent containing chemically synthesized antigenic determinants. The purified antibodies were directly labeled with biotin. An avidin-biotin-peroxidase complex was used to bind the biotinylated antibody for the peroxidase staining reaction of the isoantigens on tissue section. Application of this technique to formalin-fixed, paraffin-embedded bladder tissue and tumor sections yielded specific and strong stainings of the isoantigens with low background staining. The potential clinical application of this method requires further evaluation.

Tumor and placental histaminase, I. affinity chromatography purification and characterization of the placental enzyme.

Histaminase (diamine oxidase) is an enzyme produced at very high levels by the decidua of the placenta and is found to be associated with a number of human cancers. A procedure for the affinity chromatography purification of this enzyme is described. In this procedure, cadaverine-AH-sepharose was used to bind the enzyme in the placental extract. After extensive washing of the column with 2.5% Triton X-100 in 1 M NaCl, the enzyme was released from the column by 0.1 N chromotropic acid. This purification, essentially a one step procedure, provided 1800-fold purification, and yielded mg quantities of histaminase, homogeneous by SDS-gel electrophoresis and immunodiffusion tests. The procedure usually recovered more than 40% of the enzyme applied and the specific activity of the final enzyme preparation was around 5000 units/mg protein. SDS-gel electrophoresis of the enzyme in different concentrations of acrylamide indicated that the subunit molecular weight of histaminase was about 90,000. Isoelectric focusing of the enzyme in polyacrylamide gel revealed 5 major enzyme components. Results of amino acid analyses indicated that the enzyme had a low content of sulfur-containing amino acids and a relatively high content of dicarboxylic amino acids. The availability of this purification will be useful for the development of immunological methods for detections and quantitation of this enzyme in specimens from cancer patients.