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A protein in rat ventral prostate cytosol that -binds estramustine [estradiol 3-bis(2-chloroethyl)carbamate] was purified to homogeneity by using chromatography on DEAEcellulose, Sephadex G-100 (superfine), octyl-Sepharose CL4B, and polyacrylamide gel electrophoresis. The [3H]estramustine, the drug is initially accumulated in the prostatic epithelium and subsequently secreted into the lumina of the prostatic lobuli (5). These findings might indicate that estramustine binds to a protein in the prostate cell and that the estramustine-protein complex is secreted into the prostatic fluid.To study this specific protein and the mechanism of action of estramustine further, the estramustine-binding protein has been purified from rat ventral prostate. Antibodies have been raised against this protein and its distribution in male and female rats has been studied by using radioimmunoassay. All of the following purification procedures were performed at 0-4°C unless stated otherwise. DEAE-Cellulose Chromatography. A column (1.5 X 22 cm) of DEAE-cellulose (Whatman DE-52) was prepared and equilibrated with TEKG buffer containing 0.25 mM dithiothreitol (TEKGD buffer). After application of sample to the column, elution was with 60 ml of TEKGD buffer and a 2 X 150 ml 0.01-0.30 M KCl linear gradient in TEKGD buffer. The flow rate was adjusted to 1 ml/min and absorbance was measured continuously '4 280 nm. Fractions (2 ml) were collected and analyzed for radioactivity and conductivity. Fractions containing radioactivity were pooled and concentrated to 4 ml in a collodion bag (Sartorius membrane filter).Gel Filtration on Sephadex G-100. Sephadex G-100 (superfine; Pharmacia) was packed in a 2.5 X 50 cm column. The column was equilibrated with TEKGD buffer and calibrated with eight proteins with known molecular weights ranging from 13,700 to 151,000. The concentrate from the DEAE-cellulose step was applied to the column and chromatography was carried out at a flow rate of 5 ml/hr. Fractions (2 ml) were collected and examined for radioactivity. Fractions containing radioactivity were pooled and a saturated ammonium sulfate solution (in TEKGD buffer at 0WC, pH 7.4) was added to give a final ammonium sulfate concentration of 20% saturation.Octyl-Sepharose Chromatography. A gel bed of 7-8-ml of octyl Sepharose CL-4B (Pharmacia) was layered on top of 1 ml of Sephadex G-25 (medium) in a 0.9 X 30 cm column. The Abbreviation: NaDodSO4, sodium dodecyl sulfate. 3149The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgeninduced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C ␣ and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C ␣ and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion. Prostate cancer (PCa)2 remains a leading cause of mortality worldwide. Despite important progress in the early diagnosis of prostate neoplasias through the measurement of prostate-specific antigen levels, ϳ10% of newly diagnosed patients have some evidence of locally advanced PCa and 5% already have distant metastasis at the time of diagnoses (1-3). Postmortem analysis of deaths attributed to PCa reveals that most subjects have evidence of metastatic disease (4). Some treatment alternatives exist with curative potential in the case of locally advanced PCa (5-7). In contrast, patients with evidence of distant metastasis have a very poor prognosis and no curative treatment exists (8).Androgen ablation therapy in its many modalities has been a mainstay in the treatment of prostatic adenocarcinoma. In general, androgen deprivation induces remission in 80 -90% of men with advanced PCa and results in a median progressionfree survival of 12-33 months. This period is usually followed by the emergence of an androgen-independent PCa resulting in a median overall survival of 23-37 months. In metastatic PCa disease, androgen ablation is also the first line of treatment. Androgen ablation in combination with external beam radiotherapy or prostatectomy delays disease progression and results in significant survival advantage when compared with radiation therapy or prostatectomy alone (5-7, 9).It is not entirely clear how hormone refractory cancer develops (10, 11). One hypothesis is that some of the mechanisms that are normally under androgen control become constitutively active in androgen-independent cells. This is supported by ...

Androgens are critical for prostate development, growth, and functions. In general, they support proliferation and prevent cell death of prostatic epithelial cells. Here, we studied changes of gene expression after castration and testosterone replacement therapy in the rat ventral prostate using cDNA microarrays analysis. We could identify 230 genes that were regulated in either experimental condition. Using hierarchical clustering analysis, different groups of genes could be detected according to their expression pattern. This enabled us to distinguish the putative androgen-responsive genes from the secondary-responsive ones. Among genes that altered during castration and testosterone replacement, a set of oxidative stress-related genes, including thioredoxin, peroxiredoxin 5, superoxide dismutase 2, glutathione peroxidase 1, selenoprotein 15 kDa, microsomal glutathione-S-transferase, glutathione reductase, and epoxide hydrolase, were changed by castration. We hypothesize that modulation of redox status can be a factor of relevance in androgen withdrawal-induced prostate apoptosis. In selective cases, quantitative RT-PCR was used to confirm changes in gene expression. Immunohistochemistry was performed to detect thioredoxin and ezrin. Both of these were detected in the prostate and seem to be regulated in a similar manner as shown by gene expression analysis. In conclusion, gene expression profiling provides a unique opportunity for understanding the molecular mechanisms of androgen actions in prostate gland.

The forced overexpression of c-Myc in mouse prostate and in normal human prostate epithelial cells results in tumor transformation with an invasive phenotype. How c-Myc regulates cell invasion is poorly understood. In this study, we have investigated the interplay of c-Myc and androgens in the regulation of prostate cancer cell invasion. We found that c-Myc induces cell invasion and anchorageindependent growth by regulating ezrin protein expression in the presence of androgens. The activity of the ezrin promoter is controlled by androgens through c-Myc, which binds to a phylogenetically conserved E-Box located in the proximal promoter region. Besides, we also show that ezrin is an important regulator of c-Myc protein levels. These effects are achieved through androgeninduced changes in ezrin phosphorylation, which results in the regulation of downstream signals. These downstream signals involve the modulation of Akt and GSK-3b activity resulting in increased c-Myc protein synthesis and inhibition of its degradation. In summary, we have shown a key role for ezrin as a mediator of c-Myc-induced tumorigenesis in prostate cancer cells.