metribolone and Adenocarcinoma

Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

Topics: Adenocarcinoma; ADP-Ribosylation; Antineoplastic Agents; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Metribolone; Neoplasm Proteins; Phthalazines; Piperazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Isoforms; Protein Processing, Post-Translational; Receptors, Androgen; Signal Transduction; Survival Analysis

Retinoid acid induced 16 (RAI16) was reported to enhance tumorigenesis in hepatocellular carcinoma (HCC). The androgen receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in several cancer progressions. However, whether RAI16 is a candidate AR target gene that may involve in prostate cancer progression was unclear.. RAI16 expression was detected in prostate cancer cells with or without the AR agonist R1881 treatment by quantitative RT-PCR and Western blot. Direct AR binding to the RAI16 promoter was tested using AR chromatin immunoprecipitation (ChIP) and luciferase assay. Cell viability and colony formation assays in response to R1881 were analyzed in cells with RAI16 knockdown by specific siRNA.. The expression of RAI16 was high in LNCaP(AI), LNCaP(AD), C4-2 expressing AR, but low in Du145 and Pc-3 cells without AR expressing. In addition, the expression of RAI16 could be induced by 10 nM R1881 treatment LNCaP(AD) and C4-2 cells, but inhibited by AR specific siRNA treatment. Furthermore, AR binds directly to ARE3 (-2003~-1982bp) of RAI16 promoter region by ChIP and luciferase assay. RAI16 knockdown inhibited the enhancement of cell viability and colony formation of AR stimulation.. We demonstrate for the first time that RAI16 is a direct target gene of AR. RAI16 may involved in cell growth of prostate cancer cells in response to AR signaling.

Topics: Adenocarcinoma; Androgens; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Metribolone; PC-3 Cells; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; Proteins; Receptors, Androgen

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.

Topics: Adenocarcinoma; Androgens; Cell Death; Cell Line, Tumor; Dihydrotestosterone; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Metribolone; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Prostate; Prostatic Neoplasms; Protein Kinase C; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate

Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa.. Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

Topics: Adenocarcinoma; Aged; Androgens; Animals; Cell Line, Tumor; Cell Proliferation; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Male; Metribolone; Mice, Inbred C57BL; Mice, Mutant Strains; Middle Aged; Predictive Value of Tests; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Receptors, Androgen; Signal Transduction; STAT5 Transcription Factor; Suppressor of Cytokine Signaling Proteins

Ultraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA: RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors.. Using a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2'deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString®-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA: RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs.. We observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363 + A, uc.454 + A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate < 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 (P < 0.05) whereas uc.283 + A was up-regulated following treatment with combined 5-Aza 2'deoxycytidine and trichostatin A (P < 0.05). Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs.. This first study of ucRNA expression in human prostate cancer indicates an altered transcript expression in the disease.

Topics: Adenocarcinoma; Aged; Azacitidine; Case-Control Studies; Cell Line, Tumor; Conserved Sequence; Decitabine; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Genome, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Metribolone; Middle Aged; Oligonucleotide Array Sequence Analysis; Prostate; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; RNA, Untranslated; Testosterone Congeners; Transcriptome

Prostate cancer (PrCa) risk is positively associated with levels of insulin-like growth factor I (IGF-I) and prostate specific antigen (PSA), both androgen receptor (AR) signaling target genes in PrCa cells. Although activated AR is required for androgen-induction of expression of both genes, effects of the IGF-I signaling pathways on the androgen-induction of PSA have not been studied.. Human prostate stromal and epithelial cancer cells were treated alone or in coculture with steroid hormone and/or inhibitors. Gene or protein expression was analyzed by real time RT-PCR or Western blotting of lysates, nuclear extracts, or immunoprecipitated products.. In PrCa epithelial cells, endogenous IGF-I, significantly induced by R1881, was required for R1881-induction of PSA. Increased IGF-I correlated with accumulation of cytoplasmic dephospho β-catenin (CPDP β-catenin), a co-activator of AR signaling. Exogenous IGF-I enhanced R1881-induced PSA and accumulation of CPDP β-catenin in LAPC-4 cells. Functional depletion of IGF-I or IGF-I receptor diminished PSA induction. Induction of IGF-I reached a plateau while PSA consecutively increased. Inhibiting PI3K abolished R1881-induced Akt phosphorylation, CPDP and nuclear β-catenin and nuclear association of AR/β-catenin, consequently abrogating R1881-induced expression of IGF-I and/or PSA.. By integrating androgen, IGF-I and β-catenin signaling pathways, these data reveal that androgen-induced PSA expression requires activation of AR and endogenous IGF-I or IGF-I/PI3K/Akt signaling, suggesting a positive feedback cycle for increased production of PSA associated with PrCa.

Topics: Adenocarcinoma; beta Catenin; Biomarkers, Tumor; Cell Line, Tumor; Epithelial Cells; Humans; Insulin-Like Growth Factor I; Male; Metribolone; Neoplasm Proteins; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoration of testosterone levels could suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets were implanted. Of 33 tumors, 24 adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen. We then studied the molecular mechanism underlying the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G(1) cell cycle arrest through reduction of S-phase kinase-associated protein 2 (Skp2) and c-Myc, and induction of p27(Kip1). 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with a slower growth rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27(Kip1). Nuclear AR and prostate-specific antigen expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype; knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells through AR, Skp2, and c-Myc.

Topics: Adenocarcinoma; Androgen Antagonists; Anilides; Animals; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Drug Implants; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Male; Metribolone; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Nitriles; Orchiectomy; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Receptors, Androgen; S-Phase Kinase-Associated Proteins; Testosterone; Tosyl Compounds; Xenograft Model Antitumor Assays

The androgen-signaling pathway with the androgen receptor (AR) as its key molecule is widely understood to influence prostate tumor growth significantly even after androgen ablation. Under androgen-deprived conditions, the AR may be activated inappropriately through interaction with other molecules, including cyclic AMP-dependent protein kinase A (PKA). In a previous study, we have shown that knocking down the AR significantly inhibits prostate tumor growth. In this study, we show that combined inhibition of the AR and the regulatory subunit I alpha of PKA (RIalpha) with small interference RNAs significantly increased the growth-inhibitory and proapoptotic effects of AR knockdown. This treatment strategy was effective in androgen-sensitive and in androgen ablation-resistant prostate cancer cells. In addition, we report that downregulating PKA RIalpha was sufficient to inhibit PKA signaling and interestingly also impaired AR expression and activation. Vice versa, AR knockdown induced a decline in PKA RIalpha, associated with reduced PKA activity. This mutual influence on expression level was specific, because siRNAs against the AR did not affect expression of PKA RIalpha in AR negative DU-145 cells and a siRNA control did not affect protein expression. Another important finding of our study was that depletion of PKA RIalpha also potentiated the antiproliferative effect of the antiandrogen bicalutamide in androgen-sensitive LNCaP. We therefore concluded that combined inhibition of PKA RIalpha and AR may be a promising new therapeutic option for prostate cancer patients and might be superior to solely preventing AR expression.

Topics: Adenocarcinoma; Androgen Antagonists; Androgen Receptor Antagonists; Androgens; Anilides; Animals; Apoptosis; Bucladesine; Cell Division; Colforsin; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Enzyme Induction; Gene Knockdown Techniques; Humans; Isoquinolines; Male; Metribolone; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Nitriles; Prostatic Neoplasms; Receptors, Androgen; RNA, Small Interfering; Signal Transduction; Sulfonamides; Tosyl Compounds

We determined protein levels and subcellular distribution of thioredoxin 1 (Trx1) in human prostate tissues using tissue microarrays and analyzed redox changes in Trx1 in the nucleus and cytoplasm in cell culture models with a redox Western blot technique. We demonstrated increased nuclear Trx1 levels in high- versus low-grade human prostate cancers. Despite increased protein levels, the oxidized forms of nuclear Trx1 were higher in prostate cancer cell lines compared to their benign counterparts, suggesting that nuclear redox imbalance occurred selectively in cancer cells. A growth-stimulating dose of androgen caused transient oxidation of Trx1 in androgen-responsive prostate cancer cells only, suggesting a loss of both androgen- and redox-signaling mechanisms during cancer progression. Androgen-independent PC3 cells showed a significant increase in nuclear and cytoplasmic Trx1 protein levels, but a significant decrease in total Trx activity. Trx1 redox state and activity correlated with the sensitivity of prostate cancer cells to pro-oxidant agents, and downregulation of Trx1 sensitized cancer cells to these agents. Our findings suggest that loss of Trx function because of oxidation and corresponding redox imbalance may play important roles in prostate cancer progression and response to therapies; and Trx1 may serve as a biomarker of subcellular redox imbalance in prostate cancer.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Death; Cell Line; Cell Nucleus; Cytoplasm; Humans; Lymphatic Metastasis; Male; Metribolone; Oxidation-Reduction; Prostate; Prostatic Neoplasms; Reactive Oxygen Species; RNA Interference; Thioredoxin-Disulfide Reductase; Thioredoxins; Up-Regulation

Human cytomegalovirus (HCMV) major immediate early (MIE) promoter, which is involved in viral reactivation, is specifically activated by androgen in LNCaP prostate cancer cells.. Androgen-dependent transcriptional activities of the HCMV MIE promoter were measured by transient transfection assay. Expression levels of genes related to protein kinase A (PKA)-signaling pathway was measured by RT-PCR.. Activation of the HCMV MIE promoter by androgen results from an increase in PKA activities. The expression level of PKA catalytic (C) subunit beta transcript variant 2 (PKA-C beta 2) was decreased by serum deprivation and was increased by R1881 treatment, in a pattern similar to changes of the HCMV MIE promoter activities. Furthermore the exogenous expression of PKA-C beta 2 strongly activated the HCMV MIE promoter, indicating that regulation of the PKA-C beta 2 expression might be a direct upstream factor in the HCMV MIE promoter regulation by androgen in LNCaP cells.. The data elucidated the mechanisms by which androgen activates PKA in androgen-dependent prostate cancer cells, resulting in activation of the HCMV MIE promoter. These results support the possibility that activation of the HCMV MIE promoter by androgen in the prostate could cause the productive infection, which might contribute to oncomodulation in prostate cancers.

Topics: Adenocarcinoma; Androgens; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cytomegalovirus; Cytomegalovirus Infections; Dinoprostone; Humans; Male; Metribolone; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Kinase C; Protein Kinase C beta; Receptors, Prostaglandin E; Transfection

MT1-MMP is a metalloproteinase involved in prostate cancer metastasis. The IGF-1R is a tyrosine kinase receptor involved with tumor progression and metastasis. The purpose of this investigation was to examine MT1-MMP and IGF-1R expression and localization in prostate cancer tissues and explore the role of IGF-1R in regulating MT1-MMP in prostate cancer cell lines.. Immunohistochemistry was utilized to study MT1-MMP and IGF-1R expression in human prostate tissues. IGF-1R regulation of MT1-MMP expression was determined by gene promoter analysis, quantitative RT-PCR and Western blot analysis following pharmacological inhibition of the receptor in PC-3N cells and treatment of LNCaP cells with androgen and IGF-1.. MT1-MMP expression was high in the apical regions of the luminal cells in PIN and prostate cancer and less intense in the basalateral regions of benign tissues. IGF-1R was expressed primarily in the basal cells of normal glands and highly expressed in prostate cancer. Inhibition of IGF-1R in PC-3N cells decreased MT1-MMP expression and treatment of LNCaP cells with a synthetic androgen and IGF-1 increased MT1-MMP expression.. These data demonstrate that MT1-MMP is highly expressed in the apical cytoplasmic regions of the luminal cells in PIN and prostate cancer when compared to basalateral cytoplasmic membrane staining in benign glands. Additionally, we demonstrate that IGF-1R is highly expressed in human prostate carcinoma. These findings suggest that MT1-MMP localization and IGF-1R expression in prostate carcinoma could be predictive biomarkers for aggressive disease and support IGF-1R as a promising therapeutic target to decrease processes of prostate cancer metastasis.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Line, Tumor; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Male; Matrix Metalloproteinase 14; Metribolone; Prostate; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Receptor, IGF Type 1; Testosterone Congeners

Androgens have key roles in normal physiology and in male sexual differentiation as well as in pathological conditions such as prostate cancer. Androgens act through the androgen receptor (AR), which is a ligand-modulated transcription factor. Antiandrogens block AR function and are widely used in disease states, but little is known about their mechanism of action in vivo. Here, we describe a rapid differential interaction of AR with target genomic sites in living cells in the presence of agonists which coincides with the recruitment of BRM ATPase complex and chromatin remodeling, resulting in transcriptional activation. In contrast, the interaction of antagonist-bound or mutant AR with its target was found to be kinetically different: it was dramatically faster, occurred without chromatin remodeling, and resulted in the lack of transcriptional inhibition. Fluorescent resonance energy transfer analysis of wild-type AR and a transcriptionally compromised mutant at the hormone response element showed that intramolecular interactions between the N and C termini of AR play a key functional role in vivo compared to intermolecular interactions between two neighboring ARs. These data provide a kinetic and mechanistic basis for regulation of gene expression by androgens and antiandrogens in living cells.

Topics: Adenocarcinoma; Androgen Antagonists; Androgens; Anilides; Animals; Cell Line, Tumor; Chromatin Assembly and Disassembly; Cyproterone Acetate; Dihydrotestosterone; Female; Fluorescence Recovery After Photobleaching; Flutamide; Genes, Reporter; Green Fluorescent Proteins; In Situ Hybridization, Fluorescence; Ligands; Luciferases; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Metribolone; Mice; Microscopy, Video; Mifepristone; Models, Biological; Nitriles; Plasmids; Promoter Regions, Genetic; Receptors, Androgen; Response Elements; Testosterone; Tosyl Compounds; Transcription, Genetic

Epidemiological and experimental studies suggest that both fatty acids and androgens have a role in the development and progression of prostate cancer (PC). Plasma membrane fatty acid binding protein (FABP(pm)) is a transporter of medium and long chain fatty acids (MCFA and LCFA) across the plasma membrane, and is identical to the mitochondrial protein aspartate aminotransferase (mAAT) that is regulated by testosterone only in prostate epithelial cells, a site where PC initially develops. We therefore hypothesized that FABP(pm) is also regulated by androgens.. We examined the effect of a synthetic androgen, R1881, and that of androgen receptor (AR) blocker, bicalutamide, on the expression of FABP(pm) and mAAT and on the uptake of fatty acids in the androgen-sensitive LNCaP, androgen responsive 22rv1 and androgen-independent CL1 human PC cells. This was done using immunofluorescence and confocal microscopy, Western blot, flow cytometry, and (3)H-oleate uptake studies.. Androgen supplementation increased the cellular and surface expression of FABP(pm) and mAAT and increased the uptake of fluorescently labeled MCFA and LCFA and that of (3)H-oleate only in PC cells that express the AR. Bicalutamide inhibited this phenomenon.. The uptake of MCFA and LCFA into PC cells is androgen regulated as well as the expression of FABP(pm) and mAAT.

Topics: Adenocarcinoma; Androgen Antagonists; Androgens; Anilides; Aspartate Aminotransferase, Mitochondrial; Blotting, Western; Cell Line, Tumor; Fatty Acid-Binding Proteins; Fatty Acids; Flow Cytometry; Humans; Male; Metribolone; Microscopy, Confocal; Neoplasms, Hormone-Dependent; Nitriles; Oleic Acid; Prostatic Neoplasms; Tosyl Compounds; Up-Regulation

High throughput sequencing-by-synthesis is an emerging technology that allows the rapid production of millions of bases of data. Although the sequence reads are short, they can readily be used for re-sequencing. By re-sequencing the mRNA products of a cell, one may rapidly discover polymorphisms and splice variants particular to that cell.. We present the utility of massively parallel sequencing by synthesis for profiling the transcriptome of a human prostate cancer cell-line, LNCaP, that has been treated with the synthetic androgen, R1881. Through the generation of approximately 20 megabases (MB) of EST data, we detect transcription from over 10,000 gene loci, 25 previously undescribed alternative splicing events involving known exons, and over 1,500 high quality single nucleotide discrepancies with the reference human sequence. Further, we map nearly 10,000 ESTs to positions on the genome where no transcription is currently predicted to occur. We also characterize various obstacles with using sequencing by synthesis for transcriptome analysis and propose solutions to these problems.. The use of high-throughput sequencing-by-synthesis methods for transcript profiling allows the specific and sensitive detection of many of a cell's transcripts, and also allows the discovery of high quality base discrepancies, and alternative splice variants. Thus, this technology may provide an effective means of understanding various disease states, discovering novel targets for disease treatment, and discovery of novel transcripts.

Topics: Adenocarcinoma; Alternative Splicing; Androgens; Cell Line, Tumor; Chromosome Mapping; Chromosomes, Human; DNA, Complementary; Exons; Expressed Sequence Tags; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Metribolone; Neoplasms, Hormone-Dependent; Polymorphism, Single Nucleotide; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Transcription, Genetic

AGR2, the human homologue of Xenopus anterior gradient 2 (XAG2), was identified by a suppression subtractive hybridization-based technique as an androgen-inducible gene. There are two AGR2 transcripts, which encode the same secretory protein of 175 amino acids. The androgen induction was time- and dose-dependent, with more than a 10-fold increase in the level of AGR2 mRNA after 48 hr of treatment with 10(-9) M R1881. Expression of AGR2 mRNA was specifically detected in limited human tissue rich in epithelial cells, including the prostate gland. Analysis of 46 microdissected primary prostate adenocarcinoma samples showed that AGR2 mRNA expression was markedly elevated in the majority of tumors as compared to matched adjacent benign tissues. Androgen-induced AGR2 protein expression was demonstrated in LNCaP cells by Western blot analysis with an anti-AGR2 antibody. Immunohistochemistry analysis indicated that AGR2 protein expression was highly restricted to the secretory epithelial cells in the prostate gland. In tissue sections from radical prostatectomy specimens, immunohistochemical staining of AGR2 showed markedly increased expression in high-grade prostatic intraepithelial neoplasia and Gleason pattern 3-4 prostatic adenocarcinoma. Therefore, the androgen-induced secretory protein AGR2 may serve as a potential therapeutic target and/or molecular marker for prostate cancer.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Metribolone; Molecular Sequence Data; Mucoproteins; Oncogene Proteins; Prostate; Prostatic Neoplasms; Protein Biosynthesis; Protein Disulfide-Isomerases; Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Testosterone Congeners; Transcription, Genetic; Xenopus; Xenopus Proteins

3,3'-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a potential anticancer component of cruciferous vegetables. Our results indicate that DIM exhibits potent antiproliferative and antiandrogenic properties in androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA synthesis. These activities were not produced in androgen-independent PC-3 cells. Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM inhibited, in a concentration-dependent manner, the DHT-induced expression of a prostate-specific antigen promoter-regulated reporter gene construct in transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3 cells only when these cells were co-transfected with a wild-type androgen receptor expression plasmid. Using fluorescence imaging with green fluorescent protein androgen receptor and Western blot analysis, we demonstrated that DIM inhibited androgen-induced androgen receptor (AR) translocation into the nucleus. Results of receptor binding assays indicated further that DIM is a strong competitive inhibitor of DHT binding to the AR. Results of structural modeling studies showed that DIM is remarkably similar in conformational geometry and surface charge distribution to an established synthetic AR antagonist, although the atomic compositions of the two substances are quite different. Taken together with our published reports of the estrogen agonist activities of DIM, the present results establish DIM as a unique bifunctional hormone disrupter. To our knowledge, DIM is the first example of a pure androgen receptor antagonist from plants.

Topics: Adenocarcinoma; Androgen Antagonists; Anilides; Animals; Anticarcinogenic Agents; Binding, Competitive; Cell Division; Cell Nucleus; Cytoplasm; Dihydrotestosterone; DNA; Gene Expression Regulation, Neoplastic; Indoles; Male; Metribolone; Nitriles; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; RNA, Messenger; Signal Transduction; Testosterone Congeners; Tosyl Compounds; Transcription, Genetic; Tumor Cells, Cultured; Vegetables

Poly(ADP-ribose) polymerase (PARP) has strong affinity for DNA strand breaks and cycles on and off the DNA ends to allow DNA repair. A DNA-binding domain of PARP (PARP-DBD) acts as a dominant-negative mutant by binding to DNA strand breaks irreversibly and sensitizing mammalian cells to DNA-damaging agents. Therefore, expression of PARP-DBD in prostate carcinoma cells offers a strategy to achieve sensitization to genotoxic treatments. Toward this end, we developed recombinant plasmids expressing the PARP-DBD under the control of the 5'-flanking sequences of the human prostate-specific antigen (PSA) gene. Tissue specificity of PARP-DBD expression in human tumor cells was confirmed using the PSA-producing (LNCaP) and PSA-negative (PC-3) prostate cancer cells, as well as cells of nonprostate origin, Ewing's sarcoma (A4573 cells). LNCaP cells stably transfected with the PSA-regulated cDNA for PARP-DBD exhibit an androgen-dependent induction of PARP-DBD expression as determined by Western blotting, reverse transcription-PCR, and in situ immunofluorescence. Furthermore, we found that PARP-DBD sensitized LNCaP cells to DNA-damaging agents, such as ionizing radiation and etoposide. Androgen (R1881) -dependent stimulation of PARP-DBD expression resulted in a 2-fold growth inhibition in LNCaP cells as compared with controls, and an augmented apoptotic cell death in response to ionizing radiation or etoposide. Taken together, the plasmid vector developed in this study permits the expression of the human PARP-DBD in an androgen-inducible and PSA-dependent fashion, and sensitizes prostatic adenocarcinoma cells to DNA-damaging treatments. These results provide proof-of-principle for a novel therapeutic strategy for the treatment of prostate cancer.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Combined Modality Therapy; DNA Damage; DNA-Binding Proteins; DNA, Neoplasm; Etoposide; Genetic Therapy; Humans; Male; Metribolone; Neoplasms, Hormone-Dependent; Plasmids; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Structure, Tertiary; Testosterone Congeners; Transfection; Tumor Cells, Cultured

In the prostate, androgens negatively regulate the expression of transforming growth factor-beta (TGF-beta) ligands and receptors and Smad activation through unknown mechanisms. We show that androgens (dihydrotestosterone and R1881) down-regulate TGF-beta1-induced expression of TGF-beta1, c-Fos, and Egr-1 in the human prostate adenocarcinoma cell line, LNCaP. Moreover, 5alpha-dihydrotestosterone (DHT) inhibits TGF-beta1 activation of three TGF-beta1-responsive promoter constructs, 3TP-luciferase, AP-1-luciferase, and SBE4(BV)-luciferase, in LNCaP cells either with or without enforced expression of TGF-beta receptors (TbetaRI and TbetaRII). Similarly, DHT inhibits the activation of Smad-binding element (SBE)4(BV)-luciferase by either constitutively activated TbetaRI (T204D) or constitutively activated Smad3 (S3*). Activation of SBE4(BV)-luciferase by S3* in the NRP-154 prostatic cell line, which is androgen receptor (AR)-negative but highly responsive to TGF-beta1, is blocked by co-transfection with either full-length AR or AR missing the DNA binding domain. Immunoprecipitation and GST pull-down assays show that AR directly associates with Smad3 but not Smad2 or Smad4. Electrophoretic mobility shift assays indicate that the AR ligand binding domain directly inhibits the association of Smad3 to the Smad-binding element. In conclusion, our data demonstrate for the first time that ligand-bound AR inhibits TGF-beta transcriptional responses through selectively repressing the binding of Smad3 to SBE.

Topics: Adenocarcinoma; Cycloheximide; Dactinomycin; Dihydrotestosterone; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Regulation; Genes, Reporter; Humans; Immediate-Early Proteins; Ligands; Male; Metribolone; Prostatic Neoplasms; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-fos; Receptors, Androgen; Signal Transduction; Smad3 Protein; Testosterone Congeners; Trans-Activators; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous studies have shown that androgen up-regulates expression of the p21 (WAF1, CIP1, SDI1, CAP20) gene, which contains a canonical androgen response element (ARE) in its proximal promoter region. We undertook the current studies to determine whether elements in the p21 promoter other than the ARE mediate androgen action. We found that deletion of the ARE did not completely abolish the promoter responsiveness to androgen, suggesting that additional cis-regulatory elements within the p21 core promoter may also be involved in androgen responsiveness. The p21 core promoter is GC-rich and contains six binding sites for transcription factor Sp1. We determined whether one or more of these Sp1 sites mediate androgen responsiveness of the p21 promoter. To do so, we used a transient transfection assay with p21 promoter-luciferase reporter constructs. The reporter activity of a construct lacking the ARE but containing all six Sp1 sites was induced approximately 3-fold by androgen. Mutation of Sp1-3 nearly eliminated basal promoter activity as well as androgen responsiveness, whereas deletion of Sp1-1 and Sp1-2 sites and mutation of Sp1-4, Sp1-5, and Sp1-6 sites had relatively little effect. We also used the mammalian one-hybrid assay and coimmunoprecipitation assay to show that androgen receptor (AR) and transcription factor Sp1 interact with one another. The current studies suggest a model in which AR and transcription factor Sp1 not only bind to their respective consensus sites within the p21 promoter, but also complex with one another, thereby recruiting coactivators and general transcription factors and inducing p21 transcription.

Topics: Adenocarcinoma; Androgens; Base Sequence; Consensus Sequence; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Gene Expression Regulation; Genes, Reporter; Humans; Luciferases; Macromolecular Substances; Male; Metribolone; Models, Genetic; Molecular Sequence Data; Neoplasms, Hormone-Dependent; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Sp1 Transcription Factor; Stimulation, Chemical; Testosterone Congeners; Transfection; Tumor Cells, Cultured

Androgen receptors (AR) have been identified in the human endometrium, but their role in endometrial function and development towards endometrial receptivity remains poorly understood. In an effort to study the regulation and possible function in endometrial epithelium, we utilized the well-differentiated endometrial adenocarcinoma cell line, Ishikawa, as a model system. This cell line has proven to be stable, hormonally responsive, contains both estrogen and progesterone receptors, and has been shown to express endometrial proteins in a hormone responsive manner. In the present study, we demonstrate that Ishikawa cells also express AR, based on immunohistochemical staining, radioactive binding studies, RT-PCR and Northern blot analysis. The expression of AR is induced in Ishikawa cells by estrogens, similar to that reported for normal endometrium. Further, using an estrogen-responsive gene that has been characterized in this cell line, alkaline phosphatase, we show that androgens act as antiestrogens in diethylstilbestrol (DES) treated cells, inhibiting enzymatic activity in a dose-dependent manner. These data support a physiologic role for AR in the endometrium. Elevations in endometrial AR in certain clinical situations such as polycystic ovarian syndrome (PCOS) may amplify the effects of androgens on the endometrium leading to suspected defects in uterine receptivity, higher than expected infertility and high miscarriage rates observed in patients with this disorder.

Topics: Adenocarcinoma; Alkaline Phosphatase; Androgens; Cell Differentiation; Cell Nucleus; Diethylstilbestrol; Dihydrotestosterone; Endometrial Neoplasms; Endometrium; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Flutamide; Fulvestrant; Humans; Metribolone; Progesterone; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Topics: Adenocarcinoma; Calcitriol; Cell Division; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Male; Metribolone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Retinoids; Testosterone Congeners; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

mRNA differential display polymerase chain reaction analysis was used to screen systematically for novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 232 bp PCR fragment was found to be consistently down-regulated by the synthetic androgen R1881. Sequencing revealed complete identity with the human homologue of mouse Paternally expressed gene 3 (Peg3), an imprinted gene that plays an important role as a downstream mediator of the effects of tumor necrosis factor (TNF). The down-regulation of Peg3 mRNA by androgens was confirmed by Northern blot hybridization. The effect proved time and dose dependent with maximal repression (3.5-fold) after 24 h of treatment with 10(-8) M R1881. The steroid specificity of Peg3 mRNA regulation reflected the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the repression process. Basal expression of Peg3 mRNA was almost completely abolished by the protein synthesis inhibitor cycloheximide. Experiments with Actinomycin D suggested that androgens act at a transcriptional level rather than by changing the stability of Peg3 mRNA. Comparison of the expression of Peg3 mRNA in 50 different human tissues revealed ubiquitous expression, but low levels in the prostate. The highest levels were observed in endocrine tissues such as ovary, placenta, adrenal and pituitary. High levels were also noted in various parts of the brain. No detectable levels of Peg3 mRNA were observed in two other androgen receptor-positive prostate tumor cell lines (MDA PCa-2a and -2b), and in the poorly differentiated and androgen receptor-negative prostate tumor lines PC-3 and DU-145. It is concluded that both androgens and loss of differentiation may affect the expression of Peg3, a mediator of the effects of TNF. Further experiments will be required to explore whether these changes affect the responsiveness of prostate tumor cells to TNF.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Kruppel-Like Transcription Factors; Male; Metribolone; Mice; Polymerase Chain Reaction; Prostatic Neoplasms; Protein Kinases; Proteins; Receptors, Androgen; RNA, Messenger; Testosterone Congeners; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Zinc Fingers

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.

Topics: Adenocarcinoma; Androgen Antagonists; Androgens; Anilides; Dihydrotestosterone; Dose-Response Relationship, Drug; Enzyme Induction; Fatty Acid Synthases; Gene Expression Regulation, Neoplastic; Humans; Lipids; Male; Metribolone; Nandrolone; Neoplasm Proteins; Nitriles; Prostatic Neoplasms; Receptors, Androgen; Testosterone; Testosterone Congeners; Tosyl Compounds; Tumor Cells, Cultured

Growth of prostatic epithelial cells is androgen-dependent; however, the mechanism of androgen action on cell growth is not well defined. We investigated whether androgen-dependent prostatic epithelial cell growth is mediated by androgen regulation of expression of genes controlling cell cycle progression. For this purpose, we used an androgen-dependent prostatic cancer cell line, LNCaP-FGC, as an in vitro model. We found that expression of CDK2 and CDK4 genes were up-regulated within hours of androgen treatment as detected in Northern and Western blot analyses. Kinase assay also confirmed that there was increased CDK2 kinase activity upon androgen stimulation. Moreover, androgen down-regulated expression of the cyclin-dependent kinase inhibitor p16 (MTS1, CDKN2) gene. The overall effects of these androgen actions result in an increased cyclin-dependent kinase activity and stimulation of the cell to enter S phase of the cell cycle, thereby enhancing cell proliferation. In contrast, an androgen-independent PC-3 cell line lost its response to androgen stimulation, and higher basal levels of CDK2, CDK4, and p16 genes were constitutively expressed in PC-3 cells. Collectively, these data suggest a possible signaling pathway of androgen in stimulating cell growth. These results also imply that in androgen-dependent prostate cancer, increased androgen receptor (AR) activity, resulting from AR gain-of-function mutations, AR gene amplification, or AR gene overexpression, malignantly stimulates proliferation of prostatic epithelial cells and constitutes one possible mechanism of androgen-dependent tumorigenesis.

Topics: Adenocarcinoma; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA Probes; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Genes, p16; Humans; Kinetics; Male; Metribolone; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Testosterone Congeners; Tumor Cells, Cultured

In a previous report we demonstrated that androgens markedly stimulate accumulation of lipid droplets in LNCaP cells. The effects were already evident at low concentrations of androgens optimal for proliferation but became much more pronounced at high concentrations optimal for differentiation. In the present report we explored whether other agonists acting by nuclear receptors and modulating LNCaP growth and differentiation also affect lipid accumulation. The agonists investigated were 1alpha,25-dihydroxycholecalciferol (VD3), all-trans-retinoic acid (atRA), and triiodothyronine (T3). Lipid accumulation was evaluated by Oil Red O staining followed by image analysis of Oil Red O-stained cells or by extraction and measurement of absorbency. Only marginal effects were noted for VD3 and T3. The atRA, on the contrary, increased lipid staining 5-12-fold. This effect required high concentrations of retinoids (10[-6] M) and was accompanied by growth stimulation. Lipid accumulation was less pronounced than that observed with maximally effective concentrations of androgens (10[-3] M R1881). Thin layer chromatography (TLC) and enzymatic determination of the various lipid fractions demonstrated that retinoids increase triacylglycerides and an unidentified lipid fraction with a slightly higher mobility. In contrast with androgens, however, they did not stimulate the accumulation of cholesterol esters. Incorporation studies with [2-14C]acetate revealed that the increased accumulation of the mentioned lipids is related both to increased synthesis and to decreased secretion. Retinoid-induced lipid accumulation is accompanied by increased steady-state levels of the mRNA encoding fatty acid synthase (FAS), a key enzyme involved in lipid synthesis, while the expression of HMG-CoA-reductase, an enzyme controlling cholesterol synthesis is only marginally affected. It is concluded that retinoids share the ability of androgens to increase lipid accumulation in LNCaP cells. The nature of the lipids affected by both agonists, however, differs at least in part suggesting that the underlying mechanisms may also be different. For the studied compounds (androgens, VD3, atRA, and T3) no simple and consistent relationship could be observed between their ability to decrease proliferation and increase differentiation on the one hand and their ability to promote lipid accumulation on the other hand.

Topics: Acetates; Adenocarcinoma; Azo Compounds; Calcitriol; Coloring Agents; Fatty Acid Synthases; Humans; Hydroxymethylglutaryl CoA Reductases; Lipids; Male; Metribolone; Prostatic Neoplasms; RNA, Messenger; Testosterone Congeners; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Microscopic evaluation of LNCaP cells stained with the lipophilic dye Oil red O revealed that androgens induce a marked stimulation of lipid droplet accumulation. As determined by quantitative analysis of the Oil red O extracted from the stained cells, stimulatory effects of the synthetic androgen R1881 became apparent at concentrations as low as 10(-11) M. Maximal induction (15-fold) was reached at 10(-8) M. Increases were observed 2 days after hormone addition and were maximal 1 day later. Accumulation of lipid droplets was also induced by mibolerone (another synthetic androgen) and by the natural androgens testosterone and dihydrotestosterone. In agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, stimulation of lipid accumulation was also apparent after treatment with progesterone and estradiol. Cortisol and the synthetic glucocorticoid dexamethasone were ineffective. The androgen antagonist Casodex (bicalutamide) abolished the stimulatory effect of R1881, further supporting the involvement of the androgen receptor. In agreement with this conclusion, no changes in lipid accumulation were observed after androgen treatment of the androgen receptor-negative prostate tumor lines PC-3 and DU-145. To investigate the nature of the lipids affected by androgens, lipid extracts were analyzed by TLC, complemented with enzymatic lipid analyses. Androgens were shown to have major effects on the content of triglycerides and cholesterol esters (33- and 7-fold stimulation, respectively), the two main classes of lipids stained by Oil red O. Phospholipid and cholesterol contents were increased by a factor of 2. Incorporation studies with [2-14C]acetate revealed that androgens caused a major stimulation of 2-14C incorporation into triglycerides and cholesterol esters (11- and 13-fold, respectively), suggesting that androgens act at least in part at the level of lipid synthesis. Taken together, these findings indicate that androgens, besides affecting proliferation and protein secretion, also markedly stimulate the production and accumulation of neutral lipids, revealing a novel interesting aspect of androgen regulation of LNCaP cells.

Topics: Adenocarcinoma; Androgens; Azo Compounds; Coloring Agents; Humans; Lipid Metabolism; Male; Metribolone; Prostatic Neoplasms; Testosterone Congeners; Tumor Cells, Cultured

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Topics: Adenocarcinoma; Androgens; Blotting, Northern; Carrier Proteins; Cloning, Molecular; Cycloheximide; Diazepam Binding Inhibitor; Dihydrotestosterone; DNA, Complementary; Humans; Immune Sera; Male; Metribolone; Nandrolone; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; Testosterone; Tumor Cells, Cultured

1. Prostate has kininogenase activity and expresses members of the tissue kallikrein gene family. The present study examined the effect of exogenous and endogenous kinins on growth of LNCaP prostate adenocarcinoma cells. 2. Rate of DNA synthesis was measured by incorporation over 4 h of [3H]-thymidine into a TCA insoluble fraction of LNCaP cells that had been cultured for 24 h. 3. Increased [3H]-thymidine incorporation was seen in response to 10 nmol/L testosterone (+103 +/- 5 s.e.%), dihydrotestosterone (+113 +/- 14%) and R1881 (+64 +/- 10%) (P < or = 0.001; n = 4). 4. In contrast 0.05, 5 and 1000 nmol/L lysyl-bradykinin had no effect (15 +/- 4, 10 +/- 9 and 5 +/- 3 s.e.%, respectively; n = 7). Des-Arg9[Leu8]-bradykinin (a B1 receptor antagonist) and/or D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (a B2 receptor antagonist), 1 nmol/L, and indomethacin, 5 mumol/L, also had little or no effect. 5. In conclusion, kallidin and endogenous kinins and prostaglandins have little or no effect on DNA synthesis and therefore on the growth of LNCaP cells in comparison to the two-fold stimulation produced by androgens.

Topics: Adenocarcinoma; Bradykinin Receptor Antagonists; Dihydrotestosterone; DNA, Neoplasm; Drug Interactions; Humans; Indomethacin; Kallidin; Male; Metribolone; Prostatic Neoplasms; Testosterone; Tumor Cells, Cultured

LNCaP cells contain androgen receptors with a mutation in the steroid binding domain (Thr 868 changed to Ala) resulting in a changed hormone specificity. Both the wild-type and mutated androgen receptors were transfected into COS cells. Transcription activation was studied in cells co-transfected with an androgen sensitive reporter (CAT) gene. The wild-type androgen receptor was activated by the agonist R1881, but the antiandrogens did not enhance transcription apart from a partial agonistic effect at high concentrations of cyproterone acetate. The mutated androgen receptor was fully activated by R1881, cyproterone acetate and hydroxyflutamide, but not by ICI 176,334. Receptor transformation to a tight nuclear binding state was studied by preparation of detergent washed nuclei and Western blotting with a specific antibody against the androgen receptor. Nuclei of COS cells transfected with wild-type receptor retained the receptor when the cells had been treated with the agonist R1881, partially retained receptors when treated with antiandrogen cyproterone acetate, but did not retain receptor when treated with hydroxyflutamide or ICI 176,334. The cells transfected with the mutated receptor additionally retained nuclear receptors after treatment with hydroxyflutamide. We conclude that each one of the three antiandrogens tested displayed different characteristics with respect to its effect on transformation and transcription activation.

Topics: Adenocarcinoma; Alanine; Amino Acid Sequence; Androgen Antagonists; Anilides; Animals; Blotting, Western; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cyproterone Acetate; Dose-Response Relationship, Drug; Flutamide; Humans; Male; Metribolone; Mutagenesis, Site-Directed; Nitriles; Point Mutation; Prostatic Neoplasms; Receptors, Androgen; Recombinant Proteins; Threonine; Tosyl Compounds; Transcription, Genetic; Transfection; Transformation, Genetic; Tumor Cells, Cultured

The transplantable human prostatic adenocarcinoma, PC-82, has been shown to be a suitable model for the study of several aspects of androgen-regulated tumor growth. This tumor contains an androgen receptor, and the purpose of the present investigation was to characterize this androgen receptor with respect to hormone specificity, sedimentation coefficient, dissociation constant, Stokes radius, ionic properties, and molecular mass. Cytosol was prepared from tumor tissues grown in athymic nude mice, which were castrated 10 days before harvesting the tumor. Scatchard plot analysis revealed a binding protein with a Kd of 0.1 nM for R1881 (methyltrienolone) and binding capacity of 120 fmol/mg protein. The receptor showed a high affinity for R1881, testosterone, and 5 alpha-dihydrotestosterone, respectively, whereas no or little affinity was found for progesterone and estradiol. In the presence of 10 mM molybdate the androgen receptor in PC-82 cytosol eluted from an FPLC anion exchange column (Mono Q) at 0.32 M NaCl, which is identical to what has been found for androgen receptors from rat prostate and calf uterine cytosol. Photoaffinity labeling of the [3H]R1881-androgen receptor complex and subsequent analysis on SDS-polyacrylamide gels resulted in a covalently labeled protein with a molecular mass of approximately 50 kD. The androgen receptor of the PC-82 tumor had a sedimentation coefficient of 4S and a Stokes radius of 3.3 nm at high ionic strength (0.4 M NaCl). It is concluded that the PC-82 tumor contains a binding protein with the properties described for androgen receptors present in prostate tissue.

Topics: Adenocarcinoma; Cell Line; Cyproterone; Cyproterone Acetate; Dihydrotestosterone; Estradiol; Estrenes; Humans; Male; Metribolone; Molecular Weight; Neoplasm Transplantation; Progesterone; Prostatic Neoplasms; Receptors, Androgen; Testosterone; Triamcinolone Acetonide

For basic studies of receptor dynamics in androgen-responsive tissues and cells, the autoradiographic and cytochemical procedures were applied to cultured tumor cells (DU-145 and PC-3). Uptake and retention of 3H-R1881, a potent synthetic androgen, were observed in DU-145 cells. The radioactive labelling was intense, and solely confined to the nuclei of DU-145 cells. Radioactivity over PC-3 cells was minimal. For assessing binding specificity, DU-145 cells were incubated with 3H-R1881 in the presence or absence of either unlabelled R1881, testosterone, progesterone, estradiol-17 beta, or corticosterone. The displacement of 3H-R1881 with R1881 and testosterone was significant, while no displacement was observed with other steroids. Nuclear localization of cytochemical staining of the dihydrotestosterone-peroxidase conjugate was evident in DU-145 cells. Our results indicate that androgen receptor may reside primarily in target cell nuclei of androgen-responsive tissues and tumors.

Topics: Adenocarcinoma; Autoradiography; Binding, Competitive; Cell Line; Estrenes; Histocytochemistry; Humans; Male; Metribolone; Prostatic Neoplasms; Receptors, Androgen

High affinity binding of the synthetic steroids methyltrienolone (R1881) and promegestone (R5020) to cytosol protein from the Dunning (R3327) experimental prostatic carcinoma of the rat was investigated. Animals bearing tumours of approx 1.5 cm mean diameter were either left untreated, or were administered diethylstilbestrol diphosphate (DESP) in the drinking water in doses close to those used clinically for the treatment of human prostatic carcinoma. Tumours were excised after 10-40 days, and binding of [3H]R1881 and [3H]R5020 to tumour cytosol was characterized using Scatchard analysis, sucrose density gradient centrifugation, and steroid competition, under conditions optimal for the conservation and assay of progesterone receptor. Both ligands were bound in much higher concentrations by cytosol from DESP-treated tumours than from untreated tumours. Binding was of high affinity (Kd congruent to 1 nM), was specific for progestins, and sedimented in peaks at approximately 8S and approximately 4S in sucrose density gradients. We conclude the DESP treatment of rats bearing the R3327 prostatic carcinoma induces synthesis of progesterone receptor in this tumour.

Topics: Adenocarcinoma; Animals; Binding, Competitive; Centrifugation, Density Gradient; Cytosol; Diethylstilbestrol; Estrenes; Male; Metribolone; Norpregnadienes; Promegestone; Prostatic Neoplasms; Rats; Receptors, Progesterone

The R3327H-G8-A1 cell line derived from the Dunning rat prostate adenocarcinoma contains both androgen and glucocorticoid receptors. Following steroid deprivation, androgens specifically increase the concentration of their receptors in these cells by approximately 2-fold within 6 h and 3-4-fold in 24 h. In the presence of potent glucocorticoids, androgen receptor augmentation is reduced by 40-50% in the first 6 h and completely inhibited during the subsequent 24 h. This event, which is specific for glucocorticoids, appears to be due to an inhibition of androgen receptor synthesis. Furthermore, glucocorticoids inhibit proliferation of these cells by inhibiting the release of growth factors and arresting them in the G0 or A state of the cell cycle. This inhibition can be overcome by addition of low concentrations of either epidermal growth factor or platelet-derived growth factor; however, the inhibitory effect of the glucocorticoid on androgen receptor augmentation is not released. These results suggest that glucocorticoids arrest cellular proliferation by altering the autoregulation of growth and that this event is not dependent upon inhibition of androgen receptor augmentation.

Topics: Adenocarcinoma; Androgens; Animals; Blood; Cell Division; Cell Line; Cell Nucleus; Cycloheximide; Dihydrotestosterone; Epidermal Growth Factor; Estrenes; Glucocorticoids; Interphase; Kinetics; Male; Metribolone; Platelet-Derived Growth Factor; Prostatic Neoplasms; Rats; Receptors, Androgen; Receptors, Glucocorticoid; Triamcinolone Acetonide

This paper describes the dose-response inhibitory effects of a synthetic androgen, methyltrienolone, on the growth of a grade II endometrial adenocarcinoma in vitro. Derived from a nude mouse heterotransplant, this cell line has maintained the morphological characteristics of the original human tumor, remains tumorigenic in the nude mouse, forms colonies on soft agar, and exhibits low levels of estrogen receptors. Data reported in this paper indicate that the cells contain androgen binding sites. At low doses of 0.25 micrograms/ml methyltrienolone had no effect on these cells, whereas at 1.0 and 10.0 micrograms/ml there was significant dose dependent inhibition of cell growth and an increase in the cell doubling time. Both testosterone and dihydrotestosterone were not inhibitory to the cells except at high doses where inhibition was slight. Methyltrienolone was not inhibitory to normal human foreskin fibroblast cultures tested as a control for cytotoxicity of the synthetic androgen. These data suggest that androgens may play a role in the treatment of tumors not responsive to conventional forms of hormonal therapy.

Topics: Adenocarcinoma; Cell Division; Cell Line; Dihydrotestosterone; Dose-Response Relationship, Drug; Estradiol; Estrenes; Female; Humans; Metribolone; Testosterone; Testosterone Congeners; Uterine Neoplasms

During the course of another investigation, three dogs had been castrated 3 months previously. Upon completion of the experiment, it was discovered that one dog presented a spontaneous prostatic adenocarcinoma of intraalveolar proliferative type at histology. Prostate weight of this dog before castration was estimated to be 22 g by tridimensional measurement at laparotomy and remained relatively constant (19 g) 3 months after castration. These results indicate that if regression had occurred in some cell populations (androgen-dependent) it was only partial and masked by growth of androgen-independent cells. Analysis of 12 individual steroids in peripheral blood and in prostatic tissue attested of a normal adrenal secretory activity. A series of 15 hydrolytic enzymes along with receptors for androgen, estrogen, and progesterone, were determined in prostatic tissue obtained at sacrifice. Enzymatic activities were those of typical epithelial cells, and most of them remained relatively high despite low levels of circulating testosterone. However, two markers of androgen action in dog prostate, acid phosphatase and arginine esterase, were significantly reduced. Receptor levels were similar to those of castrated animals. Thus, cancer cells had probably retained some androgen sensitivity.

Topics: Adenocarcinoma; Animals; Castration; Cell Nucleus; Cytosol; Disease Models, Animal; Dogs; Estradiol; Estrenes; Male; Metribolone; Promegestone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Steroid; Testosterone Congeners; Time Factors

Analyses of hormone receptors in cytosols from 41 renal cell carcinoma specimens were performed by the dextran-coated charcoal technique, using estradiol, synthetic progestin R5020 and synthetic androgen R1881. Binding data were calculated according to the method of Scatchard. Of 41 renal cell carcinomas estradiol receptor was detected in 11, R5020 receptor in 11 and R1881 receptor in 13. No significant correlation between histopathological findings and hormone receptors was observed. Patients were classified into those positive and negative for receptors. The clinical response of endocrine therapy for 17 with advanced residual or metastatic lesions after nephrectomy was studied in regard to the survival rates. Although there was no complete or partial regression in tumor size, the survival rate of patients with 1 or more receptors was significantly higher than that of patients negative for receptors (p less than 0.01). In conclusion, hormonal manipulation in patients with renal cell carcinoma cannot induce an antitumor effect but seems to increase survival in patients with receptors.

Topics: Adenocarcinoma; Aged; Estradiol; Estrenes; Female; Humans; Kidney Neoplasms; Male; Medroxyprogesterone; Medroxyprogesterone Acetate; Metribolone; Middle Aged; Promegestone; Receptors, Androgen; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Progesterone; Testosterone

A transplantable tumor CUB-II, a subline derived from the Dunning R 3327 rat prostatic adenocarcinoma, contains a unique sex steroid-binding protein. The protein possesses binding sites for androgens as well as for estrogens, and the binding affinity to androgen is higher than that to estrogen. The sedimentation coefficient of the protein is 10S. Sodium thiocyanate inhibits the binding to both sex steroids. This type of binding is not present in the 0.4M KC1 extract of nuclei. These results suggest that the binding protein is not the receptor for steroid hormones in spite of its high affinity binding to androgens and estrogens. Since the original tumor does not contain such protein, production of this binding protein seems to take place during culture in vitro and/or serial transplantations of the tumor.

Topics: Adenocarcinoma; Animals; Binding Sites; Binding, Competitive; Cell Line; Cytosol; Estradiol; Estrenes; Male; Metribolone; Prostatic Neoplasms; Rats; Sex Hormone-Binding Globulin; Thiocyanates

The incidence of prostatic cancer is highly correlated with advanced age, and it has been suggested that changes in androgen binding may be important in age-associated alterations in growth regulatory mechanisms of prostatic epithelial cells. In this study the effects of age on androgen binding characteristics in the dorsolateral prostate glands of young and aged Copenhagen rats were determined and the binding properties in the Dunning R3327/130 subline of rat prostatic adenocarcinoma were characterized. Tritium-labeled and nonlabeled methyltrienolone analogs (R1881) were used to study the binding properties of 5 alpha-dihydrotestosterone receptor in the cytosol of tumors and prostate glands. Binding of R1881 was low but specific for the androgen receptor as shown by competition studies in which nonlabeled R1881, 5 alpha-dihydrotestosterone, and testosterone competed successfully with 3H-R1881 for binding sites, but 17 beta-estradiol and low levels of progesterone did not. In Copenhagen dorsolateral prostate, Scatchard analysis suggested a single class of binding sites. In young animals (three to five months) the average binding capacity was 10.36 fmol/mg cytosol protein with a dissociation constant (Kd) of 2.28 nmol/L. The dorsolateral prostate of aged rats (11-16 months) showed no significant difference in specific binding characteristics as compared to the younger age group. Specific binding of 3H-R1881 in R3327/130 tumor was saturable with a single class of high-affinity binding sites having an average binding capacity of 64.77 fmol/mg cytosol protein and a Kd of 2.76 nmol/L. These data show that the tumor had approximately 6.5 times the number of binding sites as did the normal Copenhagen rat dorsolateral prostate gland. However, no age-related changes were detected through 11-16 months of age in the androgen binding characteristics of normal rat dorsolateral prostate gland that could be correlated with the higher concentration of androgen binding sites in the R3327/130 tumor subline.

Topics: Adenocarcinoma; Aging; Animals; Binding, Competitive; Cytosol; Dihydrotestosterone; Estrenes; Male; Metribolone; Prostate; Prostatic Neoplasms; Rats; Rats, Inbred Strains; Receptors, Androgen; Receptors, Steroid; Testosterone; Testosterone Congeners

Topics: Adenocarcinoma; Carrier Proteins; Estrenes; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescence; Humans; Male; Metribolone; Prostatic Neoplasms; Receptors, Androgen; Serum Albumin, Bovine; Thiocyanates

Topics: Adenocarcinoma; Binding, Competitive; Cell Nucleus; Cytosol; Estrenes; Humans; Kinetics; Lymph Nodes; Male; Metribolone; Neoplasm Metastasis; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Steroid

The binding of methyltrienolone (R 1881, 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one), a highly active synthetic androgen, by cytosol preparations from human renal cell carcinoma was investigated. High-affinity, low-capacity binding components for R 1881 were detected in 3 out of 8 tumours analysed. The apparent dissociation constant of the R 1881-binder complexes was found to be in the range of 1.1-2.3 x 10(-9) mol/l. The number of binding sites in the positive tumours varied from 2.1 to 9.7 fmol/mg cytosol protein. Studies of binding specificity indicated a requirement for androgens. It is concluded that these binding components may be hormone receptors. If in consecutive studies this hypothesis holds true, progestins, most widely used in hormonal therapy of metastatic renal cancer, may possibly act on the tumour tissue by inhibiting the secretion of gonadotropins, thus lowering the blood levels of eventually growth-promoting.

Topics: Adenocarcinoma; Aged; Binding Sites; Cytosol; Estrenes; Female; Humans; In Vitro Techniques; Kidney Neoplasms; Male; Metribolone; Middle Aged; Testosterone Congeners