BACKGROUND The aim of this study was to determine the effect of the phytotherapeutic agent, Permixon®, on a novel coculture model of benign prostatic hyperplasia (BPH) in an effort to better understand the mode of action of the drug in vivo. METHODS The effect of Permixon®, at the calculated therapeutic concentration, on the activity of 5α‐reductase isoenzymes was evaluated utilizing a pH‐specific assay. Prostate‐specific antigen (PSA) secretions into the medium were measured in the presence and absence of Permixon® and quantified by an ELISA assay. The morphological patterns before and following Permixon® treatment were also examined by electron microscopy. All results were compared to controls. RESULTS Permixon® at a concentration of 10 μg/ml (calculated plasma concentration in patient receiving recommended therapeutic dosage) was shown to be an effective inhibitor of both 5α‐reductase types I and II isoenzymes without influencing the secretion of PSA by the epithelial cells, even after stimulation with testosterone. The morphology of Permixon®‐treated cells was found to be markedly different from that of untreated controls. Cells which had been treated with the drug demonstrated extensive accumulation of lipids in the cytoplasm and widespread damage of intracellular membranes, including mitochondrial and nuclear membranes. CONCLUSIONS Permixon® is an effective dual inhibitor of 5α‐reductase isoenzyme activities in the prostate. Unlike other 5α‐reductase inhibitors, Permixon® induces this effect without interfering with the cells' capacity to secrete PSA, thus permitting the continued use of PSA measurements for prostate cancer screening. Prostate 40:232–241, 1999. © 1999 Wiley‐Liss, Inc.

Oestrogen action is mediated via specific receptors that act as ligand-activated transcription factors. A monoclonal antibody specific to the C-terminus of human oestrogen receptor beta has been characterized and the prevalence of expression of oestrogen receptor beta protein investigated in a well defined set of breast cancers. Reverse transcription-polymerase chain reaction analysis of RNA from tissue biopsies detected oestrogen receptor beta in all samples examined. The anti-oestrogen receptor beta antibody cross reacted specifically with both long (*59 Kd) and short (*53 Kd) forms of recombinant oestrogen receptor beta. Western blot analysis of breast tumours contained both forms of oestrogen receptor beta protein although in some samples lower molecular weight species (32 -45 Kd) were identified. Fifty-one breast cancer biopsies were examined using immunohistochemistry; 41 (80%) were immunopositive for oestrogen receptor alpha, 48 (94%) were immunopositive for oestrogen receptor beta and 38 (74.5%) co-expressed both receptors. Expression of oestrogen receptor beta was exclusively nuclear and occurred in multiple cell types. There was no quantitative relationship between staining for the two ERs although in tumours in which both receptors were present immunoexpression of oestrogen receptor alpha was invariably more intense. The significance of oestrogen receptor beta protein expression in breast cancers to therapy remains to be determined but the availability of a well characterized antibody capable of detecting oestrogen receptor beta in archive material will facilitate the process.

We have developed a coculture system for primary fibroblast and epithelial cells derived from benign prostatic hyperplasia (BPH) that retained many of the characteristics of the intact human prostate. In contrast to separately cultured prostate fibroblast and epithelial cells, cocultures of fibroblasts and epithelial cells maintained messenger ribonucleic acid expression and functional activity for both isoenzymes of 5 alpha-reductase (type I and type II) as well as maintained expression of androgen receptors and prostate-specific antigen. Furthermore, levels of prostate-specific antigen secreted by cocultured epithelial cells were increased by treatment with androgens, mimicking the situation in the human gland. This contrasted with conventionally cultured fibroblasts or epithelial cells, which failed to express 50 alpha-reductase type II and rapidly lost expression of androgen receptors and androgen sensitivity upon being placed into culture. Electron microscopy demonstrated intracellular structures indicative of the differentiated state of the cocultured cell types, including round nuclei, tonofibrils, and microvilli in epithelial cells and elongated nuclei; large amounts of Golgi and cilia; along with immature collagen fibers in fibroblasts. The present study demonstrates that the coculture model reflects more closely the in vivo system for human BPH and is thus a far more suitable model for investigating the molecular and cellular events that underlie BPH than current in vitro systems.

The expression and localisation of mRNAs for 5 reductase Type I (5 R-I) and Type II (5 R-II) isoenzymes in human benign prostatic hyperplasia (BPH) were investigated by RT-PCR and by in situ hybridisation (ISH) using digoxigenin labelled riboprobes. In addition, we also examined the isoenzymes mRNA expression in primary BPH cultures of separated stroma/fibroblast and epithelial cells to determine whether primary cultures are appropriate models in which to investigate 5 R activity and regulation. The results demonstrated conclusively the presence of mRNA encoding both isoenzymes in all specimens so far examined. Additionally, the presence of a functional 5 R-I and -II activity in BPH was confirmed by enzyme assays. ISH studies localised the mRNA expression to both the fibroblast/stromal component as well as the epithelial cells of the hyperplastic tissue. In the glandular regions the expression for both isoenzymes was particularly strong in the basal layers of the epithelium whereas mRNA expression in the secretory cells was less pronounced. Expression of 5 R-I and -II mRNAs in fibroblast was on the other hand variable with high expression in some areas and little in others. These findings were supported by our primary culture experiments which demonstrated that both the fibroblast and epithelial cells maintain a capacity to express both isoenzymes in vitro. In the case of the fibroblast, the capacity to express the isoenzymes was maintained following the sequential passaging of the cells up to passage 6, after which the cells no longer expressed either isoenzyme.

OBJECTIVE To detect the expression and transcription pattern of MUC1 in benign and malignant disease, and in two widely studied cell lines, and to investigate the glycosylation of MUC‐1 in bone metastasis of prostate cancer, as mucins have been implicated in the progression and behaviour of several cancers. MATERIALS AND METHODS RNA extracted from cell lines (DU145 and PC3), five samples of BPH and five samples of prostate cancer was reverse transcribed before amplification of MUC1‐specific sequences by polymerase chain reaction. Paraffin‐embedded sections were stained for glycosylated MUC1 and MUC1 core epitopes by HMFG1 and B27.29 antibodies, respectively. Steroid‐treated cell lines werre analysed by fluorescence‐activated cell sorting, using the same antibodies. RESULTS MUC1, in an under‐glycosylated form, was widely expressed in the prostate and in metastatic lesions. MUC1/Z and MUC1/Y RNA were differentially expressed in BPH and prostate cancer, with no detectable expression of splice variant mRNA. This is in contrast to prostate cancer cell line cells (PC3 and DU145), which express splice variant mRNA. CONCLUSIONS BPH, prostate cancer and metastatic prostate cancer all express high levels of under‐glycosylated MUC1. This may explain the inability of previous studies to detect MUC1 in prostate tissue, as the antibody used was specific for a carbohydrate epitope which is not expressed on the under‐glycosylated MUC1.