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Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.

Background: Proteins which regulate normal development may promote tumorigenesis, tumor progression, or metastasis through dysregulation of these functions. We postulate that proteins, which regulate prostate growth also promote prostate cancer (PCa) progression., Methods: Two Dimensional Gel Electrophoresis was utilized to compare patterns of protein expression in 12T-7f prostates (LPB-Tag mouse model for PCa) during tumor development and progression with those of normal developing and adult wild type CD-1 prostates. Stathmin expression and phosphorylation patterns were analyzed in mouse and human PCa cell lines as well as in human PCa tissue arrays., Results: Stathmin was identified by two-dimensional gel electrophoresis and mass spectrometry. Stathmin levels increase early during normal mouse prostate development and again during prostate tumor development and progression. In human prostate adenocarcinoma, stathmin increases in Gleason pattern 5. Further, stathmin is differentially phosphorylated in androgen-dependent LNCaP cells compared to androgen-independent PC-3 and DU145 cells. This differential phosphorylation is modulated by androgen and anti-androgen treatment., Conclusion: Stathmin expression is highest when the prostate is undergoing morphogenesis or tumorigenesis and these processes may be regulated through differential phosphorylation. Furthermore, modulation of stathmin phosphorylation may correlate with the development of androgen-independent PCa.

The aim of the present study was to determine whether a prostate-specific amplicon, containing a probasin-derived promoter (ARR(2)PB) upstream of an essential Herpes simplex virus-1 (HSV-1) viral gene, infected-cell polypeptide 4 (ICP4), could complement an HSV-1 helper virus with this gene deleted (ICP4-) and cause lytic replication specifically in prostate cancer cells. Two amplicon constructs, CMV-ICP4 and ARR(2)PB-ICP4, were packaged by a replication-deficient ICP4- helper virus. The amplicon viruses could complement ICP4- helper viruses to efficiently replicate and cause cell lysis in prostate cancer cells. Intratumoral injection of LNCaP human prostate cancer xenografts with either amplicon/helper virus resulted in >75% reduction in tumor volume and serum prostate specific antigen (PSA). Histological and Q-PCR (quantitative PCR) analyses indicated that the toxicity in nontumor tissues was much lower with ARR(2)PB-ICP4 than with CMV-ICP4 amplicon/helper virus. In conclusion, a replication-deficient HSV-1 virus could be complemented by an amplicon virus to restore its oncolytic activity in a tissue-specific and low toxicity fashion, illustrating that this approach could be a potentially useful strategy for developing an oncolytic viral therapy for prostate cancer.

Background: The androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis., Results: Comparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR., Conclusion: Taken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.

The oncogene DJ-1 has been associated with multiple cancers, including prostate cancer, where it can be stabilized by androgens and antiandrogens. However, little data exist on the expression pattern and function of DJ-1 in prostate cancer. To address the function of DJ-1 in prostate, a yeast two-hybrid screen was done to identify novel DJ-1 binding proteins. The androgen receptor (AR) was identified and confirmed as a DJ-1 binding partner. This is the first evidence that DJ-1 directly interacts with AR. We also show that modulation of DJ-1 expression regulated AR transcriptional activity. Importantly, both the subcellular localization of DJ-1 and the interaction with AR are regulated by androgens and antiandrogens. Additionally, immunohistochemical staining on two human prostate cancer tissue arrays was done providing the first large-scale expression analysis of DJ-1 in prostate. DJ-1 expression did not change with Gleason pattern but increased after androgen deprivation therapy, indicating that it may be involved in the development of androgen independence. These data provide a novel mechanism where DJ-1-mediated regulation of AR may promote the progression of prostate cancer to androgen independence.

Background & Aims: Lycopene has been credited with a number of health benefits including a decrease in prostate cancer risk. Our study investigates the molecular mechanism underlying anti-cancer activity of lycopene-based products in androgen-responsive (LNCaP) and androgen-independent (PC3) cells., Methods: The effect of lycopene-based agents on prostate cancer growth and survival were examined using proliferation assays, bromodeoxyuridine incorporation and flow cytometric analysis of cellular DNA content. Biochemical effects of lycopene treatment were investigated by immunoblotting for changes in the absolute levels and phosphorylation states of cell cycle regulatory and signalling proteins., Results: LNCaP and PC3 cells treated with the lycopene-based agents undergo mitotic arrest, accumulating in G0/G1 phase. Immunoblot screening indicated that lycopene's antiproliferative effects are likely achieved through a block in G1/S transition mediated by decreased levels of cyclins D1 and E and cyclin dependent kinase 4 and suppressed Retinoblastoma phosphorylation. These responses correlated with decreased insulin-like growth factor-I receptor expression and activation, increased insulin-like growth factor binding protein 2 expression and decreased AKT activation. Exposure to lycopene at doses as low as 10 nM for 48 h induced a profound apoptotic response in LNCaP cells. In contrast PC3 cells were resistant to apoptosis at doses up to 1 microM., Conclusions: Lycopene exposure can suppress phosphatidylinositol 3-kinase-dependent proliferative and survival signalling in androgen-responsive LNCaP and androgen-independent PC3 cells suggesting that the molecular mechanisms for the cytostatic and cytotoxic actions of lycopene involve induction of G0/G1 cell cycle arrest. This study supports further examination of lycopene as a potential agent for both the prevention and treatment of prostate cancer.

The promoter of the murine probasin (PB) gene exhibits strong androgen receptor (AR)-specific and tissue-specific regulation and is considered a promising candidate for gene therapy treatment of advanced prostate cancer. To characterize the determinants of chromatin specificity of the PB promoter with the AR we initially investigated the in vitro interactions of recombinant AR DNA binding domain (AR-DBD) with reconstituted nucleosomes incorporating the proximal PB promoter (nucleotides -268 to -76). We demonstrate that a DNA fragment of this promoter region exhibits strong nucleosome positioning. The phased DNA sequence protected by the histone octamer includes four androgen receptor response elements (AREs) which are arranged as two sets of class I and class II sites spaced approximately 90bp apart. Class I AREs form classical contacts with the AR, whereas class II AREs contain atypical binding sequences and have been shown to stabilize AR binding to adjacent class I sites, resulting in synergistic transcriptional activation and increased hormone sensitivity. We used DNase 1 footprinting and electrophoretic mobility shift assays (EMSA) to show that the AR-DBD binds to its cognate sequences independently of their nucleosomal organization. In addition, we show that the ability of the AR-DBD to interact with the nucleosomal PB promoter is not affected by histone acetylation. Thus the AR-DBD is able to bind to its cognate sequences within the PB promoter in a way that is indifferent to the presence or absence of histones and nucleosomal structure.

Background: We developed non-invasive, cell-based screening assays to rapidly and biologically assess factors that modulate prostate cancer growth and affect androgen receptor (AR) activity., Methods: LNCaP cells, which stably express enhanced green fluorescent protein (EGFP) either constitutively or upon AR activation, were treated with a variety of agents, and then monitored by fluorescence and MTS assays for dose-dependent changes in cell number and AR activity., Results: The assays were validated for rapid, fluorescence-based, quantitative measurement for the presence of growth and AR modulators. Using these assays, we found that osteoblast conditioned media (CM) enhanced prostate cancer cell growth, but not AR activity. After priming with androgen (<1 nM R1881), forskolin or the pesticide dichlorvos enhanced AR activation, whereas interleukin-6 (IL-6) inhibited it., Conclusion: These non-destructive, cell-based assays enable rapid systematic monitoring of the effects of drugs or complex mixtures on prostate cancer cell growth and/or AR activity.

Purpose: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses., Experimental Design: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment., Results: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2., Conclusion: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

Current hormone withdrawal therapies used for treatment of advanced prostate cancer lead to androgen-independent tumor growth. Increased prostatic neuroendocrine (NE) cell density has been implicated in promoting progression of prostate cancer, but the process by which this occurs remains unclear. The aim of this study was to determine whether there is an association of increased NE differentiation with neoadjuvant hormone therapy and Gleason grade. Using adjacently sectioned tissue microarrays, the expression profile of novel and known NE markers were monitored. L-Dopa decarboxylase (DDC), a catecholamine synthesis enzyme and androgen receptor (AR) coregulator protein, was identified as an additional NE marker of prostate cancer. Immunohistochemical analysis of DDC with the established NE markers, chromogranin A and bombesin, revealed a significant increase in NE differentiation after 6 months of hormone therapy and after progression to androgen independence but no apparent correlation with Gleason grade. In addition, dual immunofluorescence analysis revealed that approximately 55% of the mixed population of DDC- and chromogranin A-expressing NE cells continue to express AR. Taken together, these results suggest that the increase of NE differentiation in prostate cancers depends specifically on duration of hormone therapy. This increase may be due to the transdifferentiation of AR-expressing epithelial-derived adenocarcinoma cells into an NE cell phenotype.

Progression to androgen independence is the lethal end stage of prostate cancer. We used expression of androgen receptor (AR)-targeted short hairpin RNAs (shRNA) to directly test the requirement for AR in ligand-independent activation of androgen-regulated genes and hormone-independent tumor progression. Transient transfection of LNCaP human prostate cancer cells showed that AR shRNA decreased R1881 induction of the prostate-specific antigen (PSA)-luciferase reporter by 96%, whereas activation by forskolin, interleukin-6, or epidermal growth factor was inhibited 48% to 75%. Whereas the antiandrogen bicalutamide provided no further suppression, treatment with the mitogen-activated protein kinase (MAPK) inhibitor U0126 completely abrogated the residual activity, indicating a MAPK-dependent, AR-independent pathway for regulating the PSA promoter. Expression of doxycycline-inducible AR shRNA expression in LNCaP cells resulted in decreased levels of AR and PSA as well as reduced proliferation in vitro. When these cells were grown as xenografts in immunocompromised mice, induction of AR shRNA decreased serum PSA to below castration nadir levels and significantly retarded tumor growth over the entire 55-day experimental period. This is the first demonstration that, by inducibly suppressing AR expression in vivo, there is an extensive delay in progression to androgen independence as well as a dramatic inhibition of tumor growth and decrease in serum PSA, which exceeds that seen with castration alone. Based on these findings, we propose that suppressing AR expression may provide superior therapeutic benefit in reducing tumor growth rate than castration and may additionally be very effective in delaying progression to androgen independence.

The androgen receptor (AR), a steroid receptor family member, is a ligand-dependent transcription factor that has an integral role in normal prostate development. Alterations in AR-mediated activity can result in abnormal gene expression, dysregulated cell growth and prostate cancer. Coregulator proteins that interact with AR to influence activity and specificity of the AR-response may also have an important role in prostate cancer progression. Since the NH(2)-terminal domain (NTD) of AR encodes the ligand-independent activation function (AF)-1, this domain is incompatible with conventional yeast two-hybrid systems. Therefore, we have used the Tup1 repressed transactivator (RTA) system, which exploits the intrinsic transactivation properties of AR.NTD, for identification of novel AR-interacting proteins. Using this system, cyclin G-associated kinase (GAK) was identified as an AR interacting protein, and GST pull-down assays were used to confirm the interaction. GAK was shown to enhance the AF-1 function of AR activity in a ligand-dependent manner. Additionally, GAK enhanced the AR transcriptional response even at low concentrations of androgens, which is relevant to AR activity in androgen-independent prostate cancer. Finally, neo-adjuvant hormone therapy (NHT) tissue microarray analysis demonstrated that GAK expression increased significantly with prostate cancer progression to androgen independence, which suggests a prognostic role for GAK in advanced disease., ((c) 2005 Wiley-Liss, Inc.)

To develop a gene therapy that would selectively kill prostate cancer cells while sparing normal cells, we have constructed lentiviral vectors that contain a therapeutic gene with a short DNA sequence in the 5'-untranslated region (UTR) that is recognized by the translation initiation factor, eIF4E, which is often overexpressed in malignant cells. Infection of cancer (LNCaP, PC-3M, DU145, and MCF-7 cells) and noncancer cell lines (BPH-1, 267-B1, Plat-E, and Huvec-c cells) with lentivirus having a CMV-promoter and EGFP reporter resulted in high levels of EGFP expression in all cells, whereas, inclusion of the eIF4E UTR recognition sequence restricted high expression to cancer cells and Plat-E cells, which also express substantial levels of eIF4E. Infection of the cells with lentiviral vectors having this UTR in front of the HSV thymidine kinase suicide gene resulted in differential sensitivity to the killing effects of ganciclovir, with at least 100-fold more drug required to kill noncancer cells than cancer cells. Furthermore, in experiments where the CMV promoter was replaced by the prostate-specific ARR(2)PB promoter, the killing effects of ganciclovir were restricted to prostate cancer cells and not seen in nonprostate cancer cells. Our results indicate that combined translational regulation, by incorporation of an eIF4E-UTR recognition sequence into a therapeutic gene, together with transcriptional regulation with a prostate-specific promoter, may provide a means to selectively destroy prostate cancer cells while sparing normal prostate cells.

The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3beta, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3beta activity by LiCl augmented accumulation of beta-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. Beta-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of beta-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and beta-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of beta-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3beta pathway via the beta-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.

Background: Lentiviruses are retroviruses that can infect and stably integrate into the chromatin of non-dividing cells. The purpose of this study was to determine whether lentiviral vectors containing the probasin (PB) promoter displayed prostate-specific, androgen-regulated, and persistent gene expression., Methods: Three lentiviral-PB promoter/enhanced green fluorescent protein (EGFP)-reporter vectors together with a control lentiviral-CMV-EGFP, were tested by microscopy and flowcytometry for expression of EGFP after infection of human prostate cancer cells (LNCaP, PC-3, PC-3(hAR), and Du145 cells) and non-prostate cells (COS-1, HeLa, HeLa(hAR), and MCF-7 cells)., Results: All cells infected in vitro with lentiviral-CMV vectors expressed EGFP, whereas with lentiviral-PB vectors (the most potent being Lv-ARR(2)PB), reporter expression was only observed in LNCaP cells with a small amount seen in androgen-independent PC-3 cells. Stable or transient transfection of androgen receptor only raised EGFP expression in prostate-derived cell lines, but did not change tumor specificity. With Lv-ARR(2)PB infected LNCaP cells, androgens regulated EGFP both in vitro and in vivo. After intra-tumor injection of this vector, EGFP expression was observed in LNCaP tumors, but not in A-549 lung or CaKi-2 kidney tumors., Conclusions: Lv-ARR(2)PB may be an ideal vector for prostate-tumor targeting and for persistent, hormone-enhanced expression of a therapeutic gene to treat slow growing prostate tumors., (Copyright 2004 Wiley-Liss, Inc.)

The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.

Recent reports suggest that the beta-catenin-T-cell factor (Tcf) (BCT) signaling pathway is important in the progression of prostate cancer. Evidence suggests that the androgen receptor (AR) can repress BCT-mediated transcription both in prostate cancer and colon cancer cells (Chesire and Isaacs, 2002). In this study, we validate such findings and show that repression of BCT signaling is facilitated by competition between the AR and Tcf. Measurements of the Tcf transcriptional reporter (TOPFLASH) indicated that AR+DHT-mediated repression can inhibit BCT transcription in the presence of WT and exogenous activating beta-catenin (Delta1-130 bp). Transient transfections in SW480 cells (APC(mut/mut)) showed that this mode of repression is functionally independent of APC-mediated beta-catenin ubiquitination. Using a recently developed red flourescent protein (HcRed), we demonstrate novel observations about the nuclear distribution of Tcf. Furthermore, with the use of red (HcRed-AR and HcRed-Tcf) and green fusion proteins (beta-catenin-EGFP), we provide morphological evidence of a reciprocal balance of nuclear beta-catenin-EGFP (BC-EGFP). By cotransfecting in LNCaP prostate tumor cells and using quantitative imaging software, we demonstrated a 62.0% colocalization of HcRed-AR and BC-EGFP in the presence of DHT and 63.3% colocalization of HcRed-Tcf/BC-EGFP in the absence of DHT. Costaining for activated RNA Pol II (phosphoserine 2) and HcRed-Tcf suggested that Tcf foci contain transcriptional 'hotspots' validating that these sites have the capacity for transcriptional activity. Given this apparent androgen-dependent competition for nuclear BC-EGFP, we chose to assess our hypothesis by in vivo and in vitro binding assays. SW480 cells transiently transfected with an AR expression construct, treated with DHT and immunoprecipitated for Tcf showed less associated beta-catenin when compared to Tcf precipitates from untreated cells. Furthermore, by treating cells with DHT+Casodex, we were able to abrogate the androgen-sensitive AR/beta-catenin interaction, in addition to relieving transcriptional repression of the TOPFLASH reporter. In vitro binding assays, with increasing amounts of AR(S35), resulted in decreased Tcf(S35) association with immunoprecipitated recombinant beta-catenin-HIS. These data suggest that in steady-state conditions, AR has the ability to compete out Tcf binding for beta-catenin. Finally, using SW480 cells, we show that AR-mediated repression of the BCT pathway has implications for cell cycle progression and in vitro growth. Using FACs analysis, we observed a 26.1% increase in accumulation of cells in the G1 phase of the cell cycle, while in vitro growth assays showed a 35% reduction in viable cells transfected with AR+DHT treatment. Together, our data strongly suggest that a reciprocal balance of nuclear beta-catenin facilitates AR-mediated repression of BCT-driven transcription and cell growth.

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.

In this study we investigated the combination of transcription factors and proteins binding to the proximal part of the prostate-specific probasin (PB) promoter. Using DNaseI in vitro footprinting, several protected regions were identified on the proximal PB promoter (nucleotides -286 to +28 relative to the transcription start site) when nuclear extracts from LNCaP, a human prostate cancer cell line, were used. Four of the protected areas were observed only when LNCaP nuclear extracts treated with synthetic androgen (10 nM R1881) were used. Two other regions, referred to as FPI and FPII, showed protection regardless of the presence or absence of androgen. When DNaseI footprinting was done using other prostate and non-prostate nuclear extracts, protection of the FPII region was only seen in prostate cell lines. These androgen-independent regions were further tested for tissue and binding specificity using the electrophoretic mobility-shift assay. Eight complexes formed with the FPI probe while four complexes were observed with the FPII probe on incubation with the tested nuclear extracts. Methylation protection assays reveal that prostate cancer cell lines yield slightly different protection patterns for some of the protein complexes formed with non-prostate-derived cell lines, suggesting the presence of prostate-enriched or -exclusive proteins. Site-directed mutagenesis of the protected nucleotides within FPII resulted in a significant reduction in expression from the PB promoter. Identification of proteins binding to the FPII region revealed the participation of nuclear factor I (NF-I) or a closely related protein, although other unknown proteins are also involved. Defining the DNA and protein components that dictate prostate-specific expression of the PB promoter in an androgen-independent manner would provide a strong basis for the design and development of a gene therapy for systemic treatment of androgen-independent prostate cancer.

The androgen receptor (AR) is a ligand-dependent transcription factor that has an essential role in the normal growth, development, and maintenance of the prostate gland. The AR is part of a large family of steroid receptors that also includes the glucocorticoid, progesterone, and mineralocorticoid receptors. Steroid receptor family members share significant homology at their DNA and ligand-binding domains. However, these receptors exhibit a high degree of sequence variability at their NH(2)-terminal domain, which suggests the possibility of receptor-specific interactions with co-regulator proteins. Transcriptional co-regulators that interact with the AR may have a role in defining AR activity and may be involved in directing AR-specific responses. Here we have identified Ran-binding protein in the microtubule-organizing center (RanBPM) to be a novel AR-interacting protein by yeast two-hybrid assay and have confirmed this interaction by glutathione S-transferase- and His-tagged pull-down assays. In addition, transient overexpression of RanBPM in prostate cancer cell lines resulted in enhanced AR activity in a ligand-dependent fashion. Glucocorticoid receptor activity was also enhanced when RanBPM was overexpressed, whereas estrogen receptor activity remained unchanged. These data demonstrate that RanBPM interacts with steroid receptors to selectively modify their activity.

We provide evidence that the androgen receptor (AR) can promote nuclear translocation of beta-catenin in LNCaP and PC3 prostate cancer cells. Using AR-expressing cells (LNCaP) and non-AR-expressing cells (PC3) we showed by time course cell fractionation that the AR can shuttle beta-catenin into the nucleus when exposed to exogenous androgen. Cells exposed to the synthetic androgen, R1881, show distinct, punctate, nuclear co-localization of the AR and beta-catenin. We further showed that the AR does not interact with adenomatous polyposis coli or glycogen synthase kinase-3beta and, therefore, conclude that androgen-mediated transport of beta-catenin occurs through a distinct pathway. The minimal necessary components of the AR and beta-catenin required for binding nuclear accumulation of beta-catenin nuclear import appears to be the DNA/ligand binding regions and the Armadillo repeats of beta-catenin. We also employed a novel DNA binding assay to illustrate that beta-catenin has the capacity to bind to the probasin promoter in an AR-dependent manner. The physiological relevance of AR-mediated transport of beta-catenin and binding to an AR promoter appeared to be a substantial increase in AR transcriptional reporter activity. AR-mediated import represents a novel mode of nuclear accumulation of beta-catenin.

A common mutation in P53 protein occurs at amino acid residue 281 in the DNA binding domain (P53(gly(281))), which results in loss of transcriptional regulation of P53 target genes and has been reported to gain pro-oncogenic functions. In the present study, we investigated the activity of P53(gly(281)) in P53-null PC3 human prostate cancer cells and found that the P53(gly(281)) induced apoptosis as efficiently as the wild-type P53 (wtP53). However, in contrast to wtP53-induced apoptosis, the P53(gly(281))-induced apoptosis was insensitive to overexpression of bcl-2. Thus, our findings indicate that while a mutation in the DNA binding domain of p53 may result in a more oncogenic form of the protein, it may also paradoxically result in the 'gain' of a new, alternative pathway for apoptosis.

Polychlorinated biphenyls (PCBs) are ubiquitous highly persistent manufactured chemicals known to bioaccumulate in the food chain. Exposure to PCBs has been implicated in a wide range of human health effects, including altering normal endocrine processes and reproductive function. However, very little is understood regarding the specific mechanisms by which PCBs may exert their effects in biological systems. We have examined the ability of PCBs to interfere with transcriptional activation of the androgen receptor (AR) and glucocorticoid receptor (GR) in an in vitro transcription-based reporter assay system. Four Aroclor PCB mixtures were found to antagonize AR-mediated transcription in the presence of the natural AR ligand dihydrotestosterone (DHT). The antagonistic activity of Aroclor mixtures increased in the following order: 1260 < 1242 < 1254 < 1248. These Aroclor mixtures had no discernible effect on GR activity. Aroclor 1254 in the absence of DHT exhibited weak agonistic responses in a dose-dependent manner with AR. Within a series of individual congeners, congeners 42, 128, and 138 are shown to antagonize AR activity. These congeners all share a common core chlorine substitution pattern. Ligand-binding studies demonstrate that endocrine activities of PCB mixtures and congeners on AR are likely due to direct and specific binding to AR ligand-binding domain., (Copyright 2002 Elsevier Science (USA).)

The epididymis provides the optimal milieu for sperm maturation and storage. Epididymal secretory proteins are believed to be involved in that process. Androgens are the major endocrine and paracrine regulatory signals that regulate gene expression in the epididymis. We have previously identified an androgen-dependent retinoic acid-binding protein (mE-RABP) that is secreted into the luminal fluid from the mouse mid/distal caput epididymidis. The mE-RABP protein belongs to the lipocalin superfamily and may be involved in the trafficking of retinoic acid within the epididymis. We have recently demonstrated that 5 kilobases of the 5' flanking region of the mE-RABP gene contained all the information for the hormonal regulation and the tissue-, region-, and cell-specific expression of the mE-RABP gene. In this study, we have identified a complex androgen-specific response region (ARR) within the first 600 base pairs of the mE-RABP gene promoter. Androgen (DHT) but not glucocorticoid (DEX) activates the ARR in HeLa and PC-3 cells. Two androgen receptor binding sites have been located at positions -445/-459 and -102/-88 and were named ARBS-1 and ARBS-0, respectively. Point mutations of ARBS-0 resulted in a slight decrease of the androgen response. However, mutations of ARBS-1 led to a total loss of the androgen responsiveness, suggesting that it was a major cis-acting element. When ARBS-1 is isolated from its promoter context, it serves as a weak androgen-responsive element that was activated by both androgens and glucocorticoids. Also, the -543/-88 DNA promoter fragment behaved as a poor androgen-responsive region, suggesting that regulatory elements located within the proximal mE-RABP promoter were required for a full androgen response. In conclusion, the mE-RABP ARR is a good model for the study of molecular mechanisms that lead to an androgen-specific responsiveness in vivo.

Although insulin-like growth factor (IGF) binding protein-5 (IGFBP-5) is highly up-regulated in normal and malignant prostate tissues after androgen withdrawal, its functional role in castration-induced apoptosis and androgen-independent progression remains undefined. To analyze the functional significance of IGFBP-5 overexpression in IGF-I-mediated mitogenesis and progression to androgen-independence, IGFBP-5-overexpressing human androgen-dependent LNCaP prostate cancer cells were generated by stable transfection. The growth rates of IGFBP-5-transfected LNCaP cells were significantly faster, compared with either the parental or vector-only transfected LNCaP cells in both the presence and absence ofdihydrotestosterone. IGFBP-5-induced increases in LNCaP cell proliferation occurs through both IGF-I-dependent and -independent pathways, with corresponding increases in the cyclin D1 messenger RNA expression and the fraction of cells in S + G2/M phases of the cell cycle. Changes in Akt/protein kinase B, a downstream component of phosphatidylinositol 3'-kinase (PI3K) pathway, in the LNCaP sublines also paralleled changes in their growth rates. Although treatment with a PI3K inhibitor induced apoptosis in both control and IGFBP-5-overexpressing LNCaP cells, this PI3K inhibitor-induced apoptosis was prevented by exogenous IGF-I treatment only in IGFBP-5 transfectants, suggesting that IGFBP-5 overexpression can potentiate the antiapoptotic effects of IGF-I. Furthermore, tumor growth and serum prostate-specific antigen levels increased several fold faster in mice bearing IGFBP-5-transfected LNCaP tumors after castration, despite having similar tumor incidence and tumor growth rates with controls when grown in intact mice before castration. Collectively, these data suggest that IGFBP-5 overexpression in prostate cancer cells after castration is an adaptive cell survival mechanism that helps potentiate the antiapoptotic and mitogenic effects of IGF-I, thereby accelerating progression to androgen independence through activation of the PI3K-Akt/ protein kinase B signaling pathway.

Testosterone-repressed prostate message-2 (TRPM-2) expression is highly up-regulated in normal and malignant prostate cells after androgen withdrawal. Although recent studies have suggested a protective role of TRPM-2 expression against apoptosis in several experimental models, the functional role of TRPM-2 in chemotherapy-induced apoptosis remains undefined. Here, we demonstrated that overexpression of TRPM-2 in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to paclitaxel treatment than control LNCaP cells, with a 20-fold higher IC50 through the inhibition of apoptotic cell death. In mice bearing TRPM-2-overexpressing LNCaP tumors, tumor volume and serum prostate-specific antigen increased two to three times faster after castration and paclitaxel treatment compared with mice bearing control tumors. We then tested the efficacy of combined treatment with antisense TRPM-2 oligodeoxynucleotide (ODN) and paclitaxel in the mouse androgen-dependent Shionogi tumor model. Antisense TRPM-2 ODN treatment significantly enhanced paclitaxel chemosensitivity of Shionogi tumor cells in a dose-dependent manner, reducing the IC50 by 75%. Combined treatment of Shionogi cells with 500 nM antisense TRPM-2 ODN and 10 nM paclitaxel-induced apoptosis, either agent alone did not. Adjuvant administration of antisense TRPM-2 ODN and polymeric micellar paclitaxel after castration resulted in reduced TRPM-2 levels in vivo and a significant delay of emergence of androgen-independent recurrent Shionogi tumors compared with administration of either agent alone. Furthermore, combined treatment of mice bearing androgen-independent recurrent Shionogi tumors with antisense TRPM-2 ODN and micellar paclitaxel inhibited tumor growth compared with treatment with either agent alone. Collectively, these findings demonstrate that TRPM-2 overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis, and that antisense TRPM-2 ODN may be useful in enhancing the effects of cytotoxic chemotherapy in hormone-refractory prostate cancer.

Although initially reported as an androgen-repressed gene in the rat prostate, the functional role of testosterone-repressed prostate message-2 (TRPM-2) in apoptosis remains undefined. Inhibition of castration-induced apoptosis by calcium channel blocker treatment in androgen-dependent Shionogi tumors resulted in the prevention of TRPM-2 gene up-regulation, suggesting that TRPM-2 is not directly androgen-repressed, but is regulated by apoptotic stimuli. The overexpression of the TRPM-2 gene in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to androgen ablation in vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucleotide (ODN) therapy in the Shionogi tumor model and demonstrated that the systemic administration of antisense TRPM-2 ODNs in mice bearing Shionogi tumors after castration resulted in a more rapid onset of apoptosis and time to complete regression, as well as a significant delay of emergence of androgen-independent recurrent tumors compared to control ODN treatment. Collectively, these findings illustrate that TRPM-2 is an antiapoptotic rather than an androgen-repressed gene that confers resistance to androgen ablation and thereby helps accelerate the progression to androgen independence.

While androgen, progesterone, and glucocorticoid receptors perform distinct physiological functions by regulating unique sets of genes, in vitro they can transactivate a common high-affinity DNA-binding target. Naturally occurring steroid response elements display nucleotide divergence that lowers binding affinity in comparison to the optimal binding element, but enhances receptor-type specificity. We investigated the role of nucleotide deviations within the DNA-binding site for contribution to steroid receptor specificity. We hypothesized that receptor specificity drives the evolution of binding site sequence, rather than strictly receptor-binding affinity. Receptor-selective targets can evolve by some nucleotides selected on the basis of additional bond energy, and others may be selected by differential tolerance to discourage binding from inappropriate receptors. To identify receptor-specific binding sites, we mimicked these dual selection pressures in a receptor-competitive environment in which DNA binding sites for the androgen or progesterone receptors were selected in the presence of the glucocorticoid receptor. These analyses also demonstrated that steroid receptors strongly select nucleotides in the spacer and flanking regions of the half-site and do so in an asymmetric fashion, indicating that steroid receptors interact with DNA in an allosteric manner that affects the transcriptional activation potential.

Background: The purpose of this study was to determine the contribution of different transactivating regions of the androgen receptor (AR) to the induction of androgen-regulated promoters in poorly (PC3 cells) and well-differentiated (LNCaP cells) prostate cancer cell lines., Methods: PC3 and LNCaP cells were co-transfected with plasmids expressing full-length AR or deletion mutants together with luciferase reporters linked to the probasin (PB) and PSA promoters; as well as to ARR3tk, a PB-derived recombinant promoter., Results: Androgen induction of the ARR3tk promoter in the presence of AR was 8- to 10-fold higher than that seen with the PB promoter. Activation of ARR3tk was greatest with an androgen-independent construct in which the first 231 amino acids and the ligand binding domain had been removed, indicating that this promoter is more responsive to activating functions in the N-terminal domain than in the ligand binding domain. By comparison, induction of the PB promoter was greatest with the full-length AR, which suggests that the ligand binding domain also makes a major contribution to the activation of this promoter. In similar analyses with the PSA promoter, AR regions required for promoter induction was dependent on the host cell type. In PC3 cells, the predominant AR transactivation function was androgen-independent and resided in the N-terminal domain, whereas in LNCaP cells, the highest level of induction was androgen dependent and also required participation of the ligand binding domain., Conclusions: Our results indicate that the relative utilization of transactivating functions in N-terminal and ligand binding domains of the AR is promoter and cell specific.

We studied the response of the human ornithine decarboxylase (ODC) promoter to androgen in human prostate cancer cell lines. In the well-differentiated, androgen-sensitive human prostate cancer line LNCaP, a genomic ODC promoter fragment that includes putative androgen response elements was suppressed by androgen. In contrast, the androgen-regulated probasin promoter was induced by androgens. The ODC promoter was also induced by cotransfected androgen receptor in the poorly differentiated, androgen-insensitive human prostate cancer cell line PPC-1. We examined the effects of cotransfected mutant androgen receptors containing the LNCaP mutation or DNA-binding mutations. All cotransfected androgen receptors switched the ODC androgen response from suppression to induction in LNCaP cells. Gel-shift and DNA footprint assays demonstrated androgen receptor binding to an ODC sequence that does not contain a consensus androgen response element. Deletion of the sequence abolished androgen suppression of the ODC promoter. We propose a model of pleiotropic gene regulation by androgen that requires a regulatory balance between androgen receptor and a transcription factor binding to the nonconsensus androgen response element.

Epigenetic mechanisms may be the main driving force for critical changes in gene expression that are responsible for progression of prostate cancers. The three most extensively characterized mechanisms for epigenetic gene-regulation are (i) changing patterns of DNA methylation, (ii) histone acetylations/deacetylations, and (iii) alterations in regulatory feedback loops for growth factors. Several studies have indicated that DNA hypermethylation is an important mechanism in prostate cancer for inactivation of key regulatory genes such as E-cadherin, pi-class glutathione S-transferase, the tumor suppressors CDKN2 and PTEN, and IGF-II. Similarly, histone acetylations and deacetylations are frequently associated respectively with transcriptional activation (e.g. IGFBP-2 and p21) and repression (e.g. Mad:Max dimers) of genes linked to prostate cancer progression. Recently, histone acetyltransferase and deacetylase activities have been shown to be intrinsic with transcriptional coregulator proteins that bind to steroid receptors (e.g. SRC-1 and PCAF). Changes in regulatory feedback loops for growth factors with prostate cancer progression tend toward shifts from paracrine to autocrine control where the receptor and ligand are produced by the same cell. While there are several examples of this progression pattern in prostate tumors such as with IGF, FGF, TGF-alpha and their respective receptors, the precise mechanism (i.e. epigenetic or mutational) is less certain. In the context of treatment options, the contribution of mutational versus epigenetic events to prostate cancer progression is an important consideration. Irreversible genetic changes are likely to be less amenable to therapeutic control than are epigenetic ones.

In exploring the possible mechanisms of androgen independence of prostate-specific antigen (PSA) gene expression, we investigated the effect of elevating AP-1 by both 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment and transfection of the c-Jun expression vector in LNCaP cells. Transcription of PSA is initiated when ligand-activated androgen receptor (AR) binds to a region in the PSA promoter that contains an androgen-responsive element (ARE). It was found that TPA inhibited androgen-induced PSA gene expression by a mechanism that did not alter nuclear levels of AR protein. Overexpression of AP-1 (jun and fos proteins) also inhibited androgen-induced PSA promoter activity. These observations were apparently related to the disruption of AR.ARE complexes as demonstrated by the results of electrophoretic mobility shift assays. Specifically, c-Jun inhibited the formation of AR.ARE complexes and conversely that AR-glutathione S-transferase proteins inhibited the formation of c-Jun.TPA-responsive element (TRE) complexes. Consistent with the inhibitory effect of both proteins, anti-c-Jun antibody blocked the inhibition of AR.ARE complex formation by c-Jun. A similar, but less marked, effect was obtained when anti-AR antibody was used to prevent AR inhibition of c-Jun.TRE complex formation. These findings together with results obtained from co-immunoprecipitation experiments strongly suggest that mutual repression of DNA binding activity is due to direct interaction between the two proteins and that the degree of repression may be determined by the ratio of AR to c-Jun. The mechanism of repression studied in mutant analysis experiments yielded evidence of an interaction between the DNA- and ligand-binding domains of AR and the leucine zipper region of c-Jun. Thus, the AR is similar to other nuclear receptors in its ability to interact with AP-1. This association provides a link between AP-1 and AR signal transduction pathways and may play a role in the regulation of the androgen-responsive PSA gene.

Several metastasizing murine and human animal models for prostate cancer are available. However, these models are androgen-independent and lack differentiated features such as androgen receptor and androgen-regulated gene expression like prostate-specific antigen (PSA). The objective of this study was to develop a metastasizing prostate cancer model with differentiated features using the human LNCaP cell line. Athymic and SCID mice were injected either s.c. or intraprostatically with 1 x 10(6) LNCaP cells. Changes in serum and tumor PSA mRNA levels were determined before and after castration to assess time to androgen-independent progression. Local tumor and metastatic growth was assessed at sacrifice after 12 weeks. Reverse transcription-PCR (RT-PCR) was used to detect circulating LNCaP cells. LNCaP tumor incidence after s.c. injection was 100% (65 of 65) in SCID mice and 80% in athymic mice. No lymph node or distant metastases were observed with s.c. tumors, and RT-PCR for PSA transcripts was negative. Primary tumor incidence after intraprostatic injection was 89% (39 of 44) in SCID mice and 60% in athymic mice. In 10 SCID mice with primary tumors followed for 12 weeks, retroperitoneal or mediastinal lymph node metastases were found in 100%, and microscopic pulmonary metastases were identified in 40%. RT-PCR for PSA transcripts was positive in 3 of 10 mice tested. Serum PSA levels in mice with s.c. and intraprostatic tumors decreased by 65% to nadir levels at 7 and 4 days after castration, respectively. Serum PSA and LNCaP tumor PSA mRNA levels increased to precastration levels earlier in SCID mice with intraprostatic tumors compared to those with s.c. tumors. Intraprostatic injection of LNCaP cells in SCID mice provides a useful animal model to investigate mechanisms of metastasis and to evaluate therapies targeted toward inhibiting the metastatic cascade.

The proportion of tumorigenic stem cells and the expression of the apoptosis-related gene, TRPM-2 (clusterin), were studied in populations of Shionogi carcinoma cells subjected to multiple cycles of androgen withdrawal and replacement (intermittent androgen suppression). The parent androgen-dependent cell line was initially transplanted into a male mouse which was castrated when the estimated weight of the resultant tumour became approximately 3 g. After the tumour had regressed to 40% or less of the original weight, it was transplanted into the next non-castrated male. This was repeated for four cycles of transplantation and castration-induced apoptosis before the tumour progressed to an androgen-independent state. The proportion of total stem cells in the tumour, as determined by in vivo limiting dilution assays in male mice, was constant during the first three cycles but increased 15-fold between the third and fourth cycles. In the parent androgen-dependent tumour before androgen ablation, the androgen-independent stem cell population formed 0.8% of the total stem cell compartment. After the fourth cycle this population increased to 47%; a population of similar size (33%, P = 0.8) was found in the androgen-independent recurrent form of the tumour induced by one-time castration. Whether androgen withdrawal therapy was intermittent or continuous, conversion to androgen independence thus occurred when one-third to one-half of the total stem cell compartment was populated by androgen-independent stem cells. The androgen-repressed TRPM-2 (clusterin) gene was actively expressed in regressing tumours after androgen ablation, and also became constitutively expressed in non-regressing tumours after the first and subsequent cycles of androgen withdrawal. Staining of cytoplasm and nuclei with anti-clusterin antibody was observed in androgen-dependent tumour cells after each cycle of intermittent androgen suppression; the nuclear staining was more intense in recurrent androgen-independent cells. The anomalous nuclear localization of clusterin, an anti-cytolytic TRPM-2 encoded protein, may serve to inhibit early events in the apoptotic process and thereby foster the generation and outgrowth of androgen-independent stem cells in an androgen-depleted environment.

The expression of 5alpha-reductase type 1 and type 2 isoenzymes in hyperplastic human prostate tissue and several human prostate cell lines was investigated by Northern blot analyses, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme activity. Separation of stroma and epithelium was confirmed histologically and only preparations with no apparent contamination were employed in the subsequent studies. Poly(A)+ RNA was isolated from stromal and epithelial fractions and analysed by Northern blot and RT-PCR. Inhibition of epithelial and stromal 5alpha-reductase activities by 17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstan- 3-one (4MA) was assessed using a range of concentrations between 10(-13) and 10(-5) M. Results from Northern blot analyses and RT-PCR showed that the prostate stroma expressed 5alpha-reductase type 1 and type 2 isoenzymes, whereas the prostate epithelium only expressed 5alpha-reductase type 1. This was consistent with biphasic inhibition of 5alpha-reductase activity by 4MA in stroma and monophasic inhibition in epithelium. Cultured epithelial cells derived from human prostate only expressed 5alpha-reductase type 1 and had Vmax and Km values that approximated the lower end of the range reported for surgically removed prostate epithelium. The foregoing data explains the disparate activities of 5alpha-reductase, previously reported, in stroma and epithelium. The differential localization of these isoenzymes in the prostate suggests that future therapy of androgen-sensitive disease may be more successful through the use of selective inhibitors of the different 5alpha-reductase isoenzymes.

An in vitro, cell-free transcription system, based on prostate-derived transcriptional machinery and very powerful androgen response elements (AREs), has been developed. Multiple (p(ARR3)LovTATA) AREs from the androgen-regulated probasin gene were linked to G-free cassettes and used in nuclear extracts prepared from prostate carcinoma cell lines (PC3 and LNCaP cells) to test specific induction of transcription by full-length AR and by glutathione-S-transferase (GST)-fusion peptides in which the androgen receptor (AR) DNA-binding domain alone (AR524-649), or together with the ligand-binding domain (AR524-902), or a portion of the NH2-terminal domain (AR232-649) were incorporated. In the presence of AR, nuclear extracts from PC3 cells had greater activity in supporting transcription than those from LNCaP cells; and lower background activity than those from HeLa cells. All of the AR forms correctly initiated in vitro transcription of ARE-templates in an androgen-independent manner. The amount of specific, inducible transcript was dependent on the concentration of AR peptide present. AR524-902 was the most potent transactivator tested, with the maximal level of specific transcript over 900-fold higher than the minimal level. At all concentrations this peptide was three to four times more active than either AR524-649 or AR232-649. In conclusion, we have developed a very specific and sensitive cell-free transcription system for delineating trans-activational regions of the AR.

Androgen-independent progression invariably occurs following castration of patients with prostate cancer. Animal model data suggest that androgen resistance may result from adaptive cell survival mechanisms activated by androgen withdrawal. If this is true, then re-exposure to, or low levels of, androgens may suppress or downregulate these mechanisms. The objective of this study is to determine whether intermittent androgen suppression (IAS) delays the onset of nonandrogen-regulated PSA production in the LNCaP prostate tumour model, when compared to continuous androgen suppression (CAS). Serum PSA levels correlate highly with LNCaP tumour volume and decrease rapidly following castration; beginning 3-4 weeks post-castration LN CaP tumours become androgen-independent with respect to PSA gene expression and produce PSA in amounts similar to precastrate state. In this study, IAS-treated mice were implanted with testosterone pellets beginning two weeks post-castration; cycles of testosterone replacement for 1 week and withdrawal for 2 weeks were repeated until serum PSA levels no longer returned to baseline. IAS therapy prolonged time to androgen-independent PSA production 3-fold, from an average of 26 days in the CAS group to 77 days in the IAS group. Serum and LNCaP tumour mRNA PSA levels remained below precastrate levels by 60 days post-castration in 75% of the IAS group, while serum PSA in all mice in the CAS group exceeded precastrate PSA by 28 days post-castration. Following castration, serum PSA levels increased 9-fold faster with CAS compared to IAS therapy. Observations using IAS in the LNCaP tumour model suggest that the onset of androgen-independent PSA gene regulation is prolonged 3-fold, perhaps due to androgen-induced differentiation and/or downregulation of androgen-suppressed gene expression.

In most patients with prostate cancer, continuous androgen suppression (CAS) therapy causes tumour regression and an accompanying decrease in serum prostate specific antigen (PSA). However, with tumour progression, regulation of both tumour growth and PSA gene expression becomes androgen-independent. Because androgen resistance develops, in part, from adaptive cell survival mechanisms activated by androgen withdrawal, we hypothesize that intermittent re-exposure to androgens may prolong time to androgen-independent progression. The objective of this study was to determine whether intermittent androgen suppression (IAS) could delay the onset of androgen-independent PSA gene regulation in LNCaP prostate tumour model when compared to CAS. Five or six cycles of IAS were possible before progression developed. IAS prolonged time to androgen-independent PSA gene regulation from an average of 26 days in CAS to 77 days in IAS. Serum PSA increased above pre-castrate levels in all mice treated with CAS by 28 days post-castration, but remained below pre-castrate levels in 75% of IAS-treated mice by 60 days post-castration. By 15 weeks post-castration, serum PSA levels increased 7-fold above pre-castrate levels in CAS-treated mice compared to 1.9-fold increase in IAS-treated mice. PSA mRNA expression levels highly correlated with serum PSA levels in both groups. Maintenance of androgen dependency through IAS may be due to androgen-induced differentiation and/or down-regulation of androgen-suppressed gene expression.

The ability to induce multiple apoptotic regressions of an androgen-dependent tumor cell population by repeated cycles of androgen withdrawal and replacement may be advantageous in therapeutic strategies aimed at delaying or preventing tumor progression. With greater insight into factors that either initiate or limit apoptosis, more efficient application of intermittent therapy might be achieved, especially if methods could be devised to increase the length or number of treatment cycles. Both calreticulin and clusterin represent proteins with a potential role in the regulation of apoptosis. Calreticulin may inhibit target gene transcription by interacting with steroid hormone receptors, thereby masking their DNA-binding sites and triggering the onset of the apoptotic process. Clusterin, on the other hand, is a membrane-stabilizing protein that appears to be involved in limiting the autophagic lysis of epithelial cells during apoptosis. Also, the increasing tendency for nuclear localization of clusterin after androgen withdrawal may preserve the nuclear environment, limiting the lethal effect of treatment. Thus, tumor progression, characterized by the loss of apoptotic potential, appears to be linked in part to the inappropriate activation of TRPM-2 gene, which accounts for the constitutive expression of clusterin.

Androgen suppression is the routine approach to the treatment of advanced prostate cancer. Using intermittent androgen suppression by taking the advantage of the reversible action of medical castration results in the maintenance of apoptotic potential. The experiments in the androgen-dependent androgen-dependent Shionogi carcinoma tumor model as well as clinical experience in a group of men with prostate malignancy are presented in this report. These consecutive cycles of androgen withdrawal and replacement afford an improved quality of life when the patient is off therapy. It is possible to reduce toxicity, cost of treatment and to delay tumor progression. Whether survival is affected in a beneficial or adverse way still remains to be studied.

We have demonstrated the binding of the recombinant DNA binding domain of the rat androgen receptor to a DNA sequence of the canine prostate arginine esterase gene and have determined the functional significance of this sequence in transient transfection experiments. One of the binding sites was localized to a region (-172 to -148 bp) containing the sequence AGGACAACAGGTGTT that has 73% homology with the prostate-specific antigen (PSA) androgen response element (ARE) found at a similar position in the PSA promoter. Competition experiments showed that the androgen receptor had an approximately 100-fold more affinity for the PSA ARE than for the arginine sequence at -172 to -148. Transient cotransfection of 5'-deletion mutants of the arginine esterase promoter and 5'-flanking sequences driving the activity of the reporter gene along with the rat androgen receptor expression vector yielded only negligible inductions of chloramphenicol acetyl transferase (CAT) activity when dihydrotestosterone (DHT) was added to the culture medium. The introduction of one to four repeats of the -172 to -148 sequence of the arginine esterase gene upstream of the basal promoter of the mouse p12 gene in p12.108 also resulted in a minimal induction of CAT activity compared with a 10-fold induction of PSA AREs under similar conditions. These results suggest that the regulation of the canine arginine esterase gene by androgens is most probably achieved by mechanisms that differ from the ones prevailing with the human PSA and kallikrein-2 (hKLK2) genes.

The functional and structural interactions of two androgen receptor-binding sites in the 5'-flanking DNA of the rat probasin gene were determined. Deletion mapping and DNase I footprinting analysis had previously identified two androgen receptor-binding sites (ARBS) necessary for androgen induction of the probasin gene: ARBS-1, which resembled a glucocorticoid-responsive element, and ARBS-2, which had a unique sequence. In this study, maximal androgen induction in transient transfection studies only occurred when both sites were present. Neither binding site functioned independently, and deletion of the DNA sequence between the sites resulted in a 60% loss of androgen inducibility. Moreover, point mutations in either ARBS-1 or ARBS-2 led to > 90% loss in activity. Scatchard analysis indicated that ARBS-1 and ARBS-2 bound a synthetic androgen receptor, AR2, with Kd values of 20.0 and 6.7 nM, respectively. Consistent with the higher affinity, ARBS-2 bound AR2 at half the threshold concentration (200 ng) of that required in reciprocal DNase I footprinting experiments with ARBS-1. By comparison, protection occurred at a much lower threshold concentration of AR2 (60 ng) and to the same extent over each site when both sites were present, suggesting a cooperative interaction between the two sites. The cooperative effect was further substantiated when a point mutation in ARBS-1 blocked AR2 binding not only to ARBS-1, but also to ARBS-2. Similarly, a point mutation in ARBS-2 also prevented receptor binding to both sites. Androgen-specific regulation of probasin gene transcription therefore required an androgen-responsive region (positions -286 and +28) containing two androgen receptor-binding sites, where the binding of the androgen receptor to both sites occurred in a cooperative, mutually dependent manner.

Progression of an androgen-dependent tumour to an androgen-independent state is characterized by the loss of apoptotic potential, a property of cells which have differentiated under the influence of androgens. In an attempt to relate progression to mechanisms of apoptotic failure, we compared the relative levels of expression of androgen receptor and TRPM-2 (clusterin) genes in androgen-dependent and -independent tumours derived from the Shionogi carcinoma. The amount of 10 kb androgen receptor mRNA in androgen-dependent and -independent cells was similar thus showing no relationship to progression. Owing to cross-hybridization of androgen receptor cDNA with non-receptor transcripts, two new androgen-repressed mRNAs (ADS31 and ADS39) were cloned. Each was found to have a 20/21 bp GC-rich region of sequence homology with the androgen receptor, implying selective conservation of a domain whose function is unknown. Sequencing results also revealed that ADS31 cDNA encodes a polypeptide identical to mouse YPT1, a ras-related GTP-binding protein. Expression of the ADS31/YPT1, ADS39 and TRPM-2 genes was sensitive to androgen withdrawal and replacement both in the parent androgen-dependent and the recurrent androgen-independent carcinomas. The uncoupling of TRPM-2 expression and apoptosis observed in androgen-independent tumour cells implies that the function of androgen receptor becomes more restricted with tumour progression. Furthermore, the fact that the expression of ADS31/YPT1 transcript becomes dominant in the advanced stages of androgen-independent growth, suggests that the mechanism of progression is subserved by duplication and possibly redundancy of alternative (signal transduction) pathways mediating tumour cell survival and growth.

We have shown that a polypeptide of M(r) 60,000 (60K) that shares N-terminal homology with a calcium-binding protein, calreticulin, can bind to an amino-acid sequence motif, KXGFFKR, found in the cytoplasmic domains of all integrin alpha-subunits. The homologous amino-acid sequence, KXFFKR (where X is either G, A or V), is also present in the DNA-binding domain of all known members of the steroid hormone receptor family; amino acids in this sequence make direct contact with nucleotides in their DNA-responsive elements and are crucial for DNA binding. Here we show that both the 60K protein (p60), purified on a KLGFFKR-Sepharose affinity matrix, and recombinant calreticulin can inhibit the binding of androgen receptor to its hormone-responsive DNA element in a KXFFKR-sequence-specific manner. Calreticulin can also inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation. Our results indicate that calreticulin can act as an important modulator of the regulation of gene transcription by nuclear hormone receptors.

Background: Although smaller variant forms of estrogen receptor (ER) messenger RNA (mRNA) have been detected in breast tumors, neither their prevalence nor their prognostic significance have been evaluated. Similarly, TRPM-2 mRNA, the product of a gene induced principally during the onset of apoptosis, is present in mouse and human breast cancer cell lines, but whether it also occurs in primary breast tumors and is related to disease outcome is unknown., Methods: The relative expression and transcript size of ER mRNA and TRPM-2 mRNA in 126 primary breast tumors were measured by Northern analysis and compared with tumor grade, hormone receptor status, extent of tumor necrosis, and survival., Results: In ER-positive tumors, 64% of the tumors had only the normal 6.5 kb ER mRNA, an additional 9% had the normal plus smaller ER mRNA, and 2% had variant forms. Only 8% of ER-negative tumors had ER mRNA transcripts. There were significant relationships between the occurrence of ER mRNA and low tumor grade, ER-positive receptor status, and better survival. In contrast, TRPM-2 mRNA was found in only 17% of breast tumors, none of which could be grouped with respect to grade, hormone receptor status, or survival., Conclusions: The presence of smaller variant forms of ER mRNA either alone or in association with the normal ER transcript is not indicative of an unfavorable prognosis, whereas TRPM-2 mRNA occurs in many primary breast tumors, but has no apparent relationship to survival.

Background: In response to the first administration of a luteinizing hormone-releasing hormone (LHRH) agonist, the secretion of pituitary gonadotropin increases sharply and gives rise to a transient surge in the concentration of serum testosterone. This effect reaches a peak 4 to 7 days after the start of therapy and results in the onset of clinical symptoms and signs of tumor flare in 5% to 10% of patients., Methods: To determine whether the effects of the LHRH-induced flare reaction are preventable, cyproterone acetate (100 mg) and low-dose diethylstilbestrol (0.1 mg) were administered daily for 4 weeks to inhibit the pituitary before the initiation of therapy with a depot LHRH agonist, goserelin acetate (3.6 mg every 4 weeks). Diethylstilbestrol was stopped after 8 weeks to eliminate associated minor toxicity while administration of cyproterone acetate was continued to suppress vasomotor symptoms. Twenty-four men with histologically confirmed prostate cancer were enrolled in the study: 6 with Stage C, 2 with Stage D1, and 16 with Stage D2 disease., Results: Lead-in therapy reduced the concentration of serum testosterone into the castrate range within 1 week, and no significant change was observed in the mean level after administration of goserelin acetate. Neither was there an effect on the initial rate of normalization of serum prostate specific antigen (PSA); normal PSA values were obtained in 50% of patients after 10 weeks and in 70% after 32 weeks. In the subgroup of patients with Stage D2 disease, longer median survival was predicted by a normal serum PSA, either stable or decreasing, after 32 weeks of treatment. The regimen was well tolerated with a low incidence of hot flushes., Conclusions: These results imply that in the absence of LHRH-induced tumor flare, prognosis is related to the ability of therapy to maintain a PSA nadir in the normal range.

Background: Since postcastration progression of tumors to an androgen-independent state appears to be linked to the cessation of androgen-induced differentiation of tumorigenic stem cells, the authors hypothesized that the replacement of androgens at the end of a period of apoptotic regression might result in the regeneration of differentiated tumor cells with further apoptotic potential., Methods and Results: To determine the effect of intermittent exposure of androgens on the androgen-dependent Shionogi carcinoma, the tumor was transplanted into a succession of male mice, each of which was castrated when the estimated tumor weight became about 3 g. After the tumor had regressed to 30% of the original weight, it was transplanted into the next noncastrated male. This cycle of transplantation and castration-induced apoptosis was repeated successfully four times before growth became androgen-independent during the fifth cycle. In four of Stage C and three of Stage D patients with prostate cancer, androgen withdrawal was initiated with cyproterone acetate (100 mg/d) and diethylstilbestrol (0.1 mg/d) and then maintained with cyproterone acetate in combination with the luteinizing hormone-releasing hormone agonist, goserelin acetate (3.6 mg/month). After 6 or more months of suppression of serum prostate-specific antigen (PSA) into the normal range, treatment was interrupted for 2 to 11 months. After recovery of testicular function, androgen-withdrawal therapy was resumed when serum PSA increased to a level of about 20 micrograms/l. This cycle was repeated sequentially to a total of two to four times over treatment periods of 21 to 47 months with no loss of androgen dependence., Conclusions: These results demonstrate that intermittent androgen suppression can be used to induce multiple apoptotic regressions of a tumor; they also suggest that the cyclic effects of such treatment on prostate cancer can be followed by the sequential measurement of serum PSA levels.

The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.