SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer.

EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.

Relationship of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate to growth of dimethylbenz(a)anthracene-induced mammary tumors in rats.

Alteration of growth of dimethylbenz[a]anthracene-induced mammary tumors was caused by removal of estrogen (ovariectomy), or insulin (diabetes), or by inhibition of prolactin secretin (treatment with an ergoline derivative). The levels of cyclic AMP (cAMP) and cGMP were measured in carcinomas classified as growing, static, and regressing. The amount of cAMP, expressed as pmoles/mg tumor weight or pmoles/mg protein, was lowest in growing tumors, intermediate in static tumors, and highest in those regressing. No correlation was seen between tumor growth and cGMP levels. Cyclophosphamide-induced tumor stasis did not elevate cAMP levels. The data suggest a role of cAMP in arrest of hormone-induced tumor growth.

Use of the probasin promoter ARR2PB to express Bax in androgen receptor-positive prostate cancer cells.

BACKGROUND: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. METHODS: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. RESULTS: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001). CONCLUSIONS: Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.

Thymic origin of the prolactin-dependent Nb2 lymphoma cell line.

Blast cells in acute leukemia and lymphoma appear to be "frozen" at various stages of lymphoid cell differentiation. The enzymatic and antigenic phenotypes expressed by these cells often correspond to the gene products of their normal precursors. We have used various immunocytochemical and enzymatic techniques to identify membrane, nuclear, and cytoplasmic markers associated with the prolactin-dependent Nb2 lymphoma cell line. The Nb2 cells, whether stationary or in log-phase growth, did not express any surface immunoglobulin. However, 100% of the Nb2 cells bound both a monoclonal antibody raised to rat thymocyte W3/25-HLK, which specifically binds an antigenic determinant on rat T-helper cells, and second monoclonal antibody OX8-HL, which identifies rat nonhelper T-cells. Transmission electron microscopy showed no evidence of phagocytic vacuoles, and activity of the lysosomal enzyme muramidase was also absent. There was no evidence of the DNA polymerase enzyme terminal deoxynucleotidyl transferase. alpha-Naphthyl acetate esterase activity was indicated in about 50% of the Nb2 cells by a faint particulate cytoplasmic staining similar to that found in thymocytes. Rosette formation with guinea pig erythrocytes, a property of mature rat thymocytes, was not observed with Nb2 cells. The data suggest that the Nb2 tumor may have arisen from a thymocyte at an intermediate stage of differentiation. The presence of Thy-like alpha-naphthyl acetate esterase pattern and the binding of both W3/25-HLK and OX8-HL support the thymic origin and relative immaturity of these lymphoid cells. It is becoming increasingly apparent that a significant proportion of lymphomas and leukemias also originate in undifferentiated thymic cels.

Adenovirus-mediated tissue-targeted expression of a caspase-9-based artificial death switch for the treatment of prostate cancer.

Clinical experience with suicide gene therapy for prostate cancer using first-generation approaches has provided a basis for developing improved strategies. Given the low proliferation rate exhibited by prostate cancer, one improvement would be to develop suicide genes that effectively kill both dividing and nondividing cells. A second improvement would be to restrict cytotoxicity to prostate cancer cells, limiting injury of nondiseased tissue. Here we describe a novel approach to achieving both goals based on: (a) the use of a small, but potent, prostate-specific composite promoter, ARR(2)PB, based on the rat probasin gene; and (b) the use of a powerful artificial death switch, called inducible caspase-9 (iCaspase-9). ARR(2)PB includes two copies of the androgen response region (ARR), each containing two androgen receptor (AR)-binding sites, placed upstream of the probasin promoter elements necessary for basal transcription. Because iCaspase-9 contains two binding sites for the dimeric ligand, AP20187, administration of chemical inducers of dimerization leads to aggregation and caspase activation, followed by rapid apoptosis in both dividing and nondividing cells. Using both reagents, we constructed two novel adenoviruses (ADVs), ADV.ARR(2)PB-iCasp9 expressing iCaspase-9 and control ADV.ARR(2)PB-EGFP expressing enhanced green fluorescent protein (EGFP). We demonstrate that tissue specificity is not sacrificed in an ADV backbone because the marker protein, EGFP, is expressed in R1881-stimulated ADV.ARR(2)PB-EGFP-transduced LNCaP cells but not in AR(-) PC-3, 293, HuH-7, U-87, and MCF-7 cells. Similarly, Pro-iCaspase-9 is expressed in ADV.ARR(2)PB-iCasp9-infected LNCaP cells after R1881 administration and is activated after AP20187 administration. In vitro experiments revealed rapid and efficient iCaspase-9-induced apoptosis of LNCaP cells in both an R1881- and AP20187-dependent manner. Only 28, 8, and 0.5% survival of LNCaP cells was seen at multiplicities of infection of 2, 10, and 25, respectively. Furthermore, at a multiplicity of infection of 10, extraordinary sensitivity to AP20187 was seen (IC(50), approximately 3 pM). In vivo experiments showed that ADV.ARR(2)PB-iCasp9 induced apoptosis in LNCaP but not in HuH-7 xenograft tumors in an AP20187-dependent manner. Furthermore, a simple i.p. injection of AP20187 dramatically suppressed LNCaP tumor growth in nude mice and led to a significantly increased host survival. This study demonstrates the feasibility of using tissue-specific expression of cell cycle-independent iCaspases as a nonmutagenic alternative modality for prostate cancer suicide gene therapy.

Expression of the c-myc protooncogene in human prostatic carcinoma and benign prostatic hyperplasia.

We have examined the level of c-myc transcripts in prostate tissue obtained from patients with both benign prostatic hyperplasia and adenocarcinoma of the prostate. A significantly higher level of c-myc transcripts is observed in patients with adenocarcinoma (P less than 0.05). In addition, a subset of patients with adenocarcinoma had levels of c-myc transcripts 2-fold higher than the mean level for this group. These preliminary results indicate that the investigation of c-myc levels as a prognostic indicator in prostatic carcinoma is warranted.

A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential.

Neuroendocrine (NE) cells may be involved not only in growth and differentiation of the normal prostate but also in carcinogenesis and progression of prostate adenocarcinoma (Pca), including development of androgen resistance. However, the exact pathophysiology of NE cells in Pca remains poorly understood. Here we describe a transgenic model of Pca with progressive NE differentiation. Seven lines of transgenic mice with the rat prostate-specific large probasin promoter linked to the SV40-large T antigen (Tag) that develop prostatic neoplasia have been established. In this study, one of the seven lines (12T-10) was characterized by examination of 52 mice aged from 2-12 months. With advancing age, low-grade prostatic intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, microinvasion, invasive carcinoma, and poorly or undifferentiated carcinoma with NE differentiation appeared in the prostates in sequential order. Whereas Tag is expressed uniformly in prostate epithelium, only an increasing subset of cells in prostatic intraepithelial neoplasia showed NE differentiation by chromogranin immunostaining. Frankly invasive carcinoma developing subsequently showed occasional definitive glandular differentiation (adenocarcinoma) and particularly undifferentiated carcinoma with NE histological features similar to those observed in NE carcinomas in humans. The NE carcinomas occurred in the dorsolateral and ventral lobes and were generally androgen receptor negative. Twenty-one of 32 (66%) mice aged > or = 6 months and 15 of 17 (88%) mice aged > or = 9 months developed metastatic tumors, as confirmed by histology and/or Tag immunohistochemistry. Metastases occurred at the later time points, with metastasis to regional lymph nodes, liver, and lung being particularly common. Metastases showed histological features of NE differentiation, as confirmed by chromogranin immunostaining and electron microscopy. An athymic nude mouse that received a s.c. implant of a primary NE tumor developed Tag-positive metastatic tumors with similar NE differentiation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified identical protein profiles between the primary NE tumor and lesions in the extraprostatic organs. Hence, in the 12T-10 large probasin promoter-Tag mouse, high-grade prostatic intraepithelial neoplasia develops progressively greater NE differentiation and progresses to invasive adenocarcinoma and NE carcinoma, with a high percentage of metastases. The predictable progression through these stages will allow testing of therapeutic interventions as well as possible further delineation of the role of NE cells in Pca progression.

15S-Hydroxyeicosatetraenoic acid activates peroxisome proliferator-activated receptor gamma and inhibits proliferation in PC3 prostate carcinoma cells.

15-Lipoxygenase (15-LOX)-2 is expressed in benign prostate secretory cells and benign prostate produces 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA). In contrast, 15S-LOX-2 and 15S-HETE formation are reduced in prostate carcinoma (Pca). The mechanisms whereby reduced 15-LOX-2 may contribute to Pca development or progression are not known. We investigated the expression of peroxisome proliferator-activated receptor (PPAR) gamma in benign and malignant prostate tissues and the ability of 15S-HETE to activate PPARgamma-dependent transcription and modulate proliferation of the Pca cell line PC3. In contrast to benign prostate and similar to most Pca tissues, 15-LOX-2 mRNA was not detected in PC3 cells, and they did not produce detectable 15-HETE from [14C]AA. By reverse transcription-PCR, PPARgamma mRNA was present in 18 of 18 benign and 9 of 9 tumor specimens. The PPARgamma ligand BRL 49653 and 15S-HETE caused a dose-dependent inhibition of PC3 proliferation in a 14-day soft agar colony-forming assay (IC50 of 3 and 30 microM, respectively). 15S-HETE (10 microM) caused greater inhibition than 10 microM 15R-HETE. At 3 days, BRL 49653 and 15S-HETE caused a slight increase in cells in G0-G1 and a corresponding decrease in cells in S phase. In PC3 cells transiently transfected with a luciferase reporter linked to a PPAR response element, 1 microM BRL 49653 and 10 microM 15S-HETE caused approximately threefold and greater than twofold induction of PPAR-dependent transcription, respectively. By quantitative real-time reverse transcription-PCR and Northern analysis, 3-day treatment with BRL 49653 and 15S-HETE caused a reduction of PPARgamma expression but a marked up-regulation of the PPAR response element containing adipocyte type fatty acid binding protein. These results support the hypothesis that 15-LOX-2-derived 15S-HETE may constitute an endogenous ligand for PPARgamma in the prostate and that loss of this pathway by reduced expression of 15-LOX-2 may contribute to increased proliferation and reduced differentiation in prostate carcinoma.

Rat probasin: structure and function of an outlier lipocalin.

Probasin (PB) occurs both as a secreted and a nuclear protein that is abundantly expressed in the epithelial cells of the rat prostate. A genomic clone of 17.5 kb gene was isolated from a rat liver genomic library, determining that the probasin gene was comprised of seven exons where the splice donor/acceptor sites conformed to the GT/AG consensus sequence. The exon number and size are remarkably similar to those of aphrodisin, rat alpha(2)-urinary globulin and major urinary protein, outlier members of the lipocalin superfamily. In addition, alignment of the deduced amino acids determined that the probasin gene also contains the glycine-X-tryptophan (G-X-W) motif similar to that of human retinol serum binding protein which binds retinol, and the C-X-X-X-C motif also found in insect lipocalins that bind pheromones. The cysteine residues in exons 3 and 6 are conserved, predicting a secondary structure of eight beta-sheets and the alpha-helix commonly seen in the lipocalin superfamily. Unique PB characteristics include a large genomic fragment (17.5 kb compared to the 3-5 kb seen in other lipocalin genes) and an isoelectric point (pI) of 11.5 which is very basic compared to that of the other more acidic lipocalins. Functionally, PB gene expression is regulated by androgens and zinc in the epithelial cells of the rodent prostate. The 5'-flanking region of probasin contains two androgen receptor binding sites that allow androgen-specific gene expression as well as prostate-specific elements that target and maintain high levels of transgene expression in several PB transgenic mouse models.

Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.

To facilitate the elucidation of the genetic events that may play an important role in the development or tumorigenesis of the prostate gland, we have generated a transgenic mouse line with prostate-specific expression of Cre recombinase. This line, named PB-Cre4, carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter. Based on RT-PCR detection of Cre mRNA in PB-Cre4 mice or Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice, it is conclusively demonstrated that Cre expression is post-natal and prostatic epithelium-specific. Although the Cre recombination is detected in all lobes of the mouse prostate, there is a significant difference in expression levels between the lobes, being highest in the lateral lobe, followed by the ventral, and then the dorsal and anterior lobes. Besides the prostate gland, no other tissues of the adult PB-Cre4 mice demonstrate significant Cre expression, except for a few scattered areas in the gonads and the stroma of the seminal vesicle. By crossing the PB-Cre4 animals with floxed RXRalpha allelic mice, we demonstrate that mice, whose conventional knockout of this gene is lethal in embryogenesis, could be propagated with selective inactivation of RXRalpha in the prostate. Taken together, the results show that the PB-Cre4 mice have high levels of Cre expression and a high penetrance in the prostatic epithelium. The PB-Cre4 mice will be a useful resource for genetic-based studies on prostate development and prostatic disease.

Mutations at the boundary of the hinge and ligand binding domain of the androgen receptor confer increased transactivation function.

The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.

Inhibition of NF-kappa B signaling restores responsiveness of castrate-resistant prostate cancer cells to anti-androgen treatment by decreasing androgen receptor-variant expression.

Androgen receptor splicing variants (ARVs) that lack the ligand-binding domain (LBD) are associated with the development of castration-resistant prostate cancer (CRPC), including resistance to the new generation of high-affinity anti-androgens. However, the mechanism by which ARV expression is regulated is not fully understood. In this study, we show that the activation of classical nuclear factor-kappa B (NF-κB) signaling increases the expression of ARVs in prostate cancer (PCa) cells and converts androgen-sensitive PCa cells to become androgen-insensitive, whereas downregulation of NF-κB signaling inhibits ARV expression and restores responsiveness of CRPC to anti-androgen therapy. In addition, we demonstrated that combination of anti-androgen with NF-κB-targeted therapy inhibits efficiently tumor growth of human CRPC xenografts. These results indicate that induction of ARVs by activated NF-κB signaling in PCa cells is a critical mechanism by which the PCa progresses to CRPC. This has important implications as it can prolong the survival of CRPC patients by restoring the tumors to once again respond to conventional androgen-deprivation therapy (ADT).

A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo.

Transient transfection studies have shown that the probasin (PB) promoter confers androgen selectivity over other steroid hormones, and transgenic animal studies have demonstrated that the PB promoter will target androgen, but not glucocorticoid, regulation in a prostate-specific manner. Previous PB promoters either targeted low levels of transgene expression or became too large to be conveniently used. The goal was to design a PB promoter that would be small, yet target high levels of prostate-specific transgene expression. Thus, a composite probasin promoter (ARR2PB) coupled to the bacterial chloramphenicol acetyltransferase reporter (ARR2PBCAT) was generated and tested in prostatic and nonprostatic cell lines and in a transgenic mouse model. In PC-3, LNCaP, and DU145 prostate cancer cell lines, the ARR2PB promoter gave basal expression and was induced in response to androgen and glucocorticoid treatment after cotransfection with the respective steroid receptor. Basal expression of ARR2PBCAT in the nonprostatic COS-1, MCF-7, ZR-75-1, and PANC-1 cell lines was very low; however, CAT activity could be induced in response to androgens and glucocorticoids when cells were cotransfected with either the AR or GR. In contrast to the transfection studies, ARR2PBCAT transgene expression remained highly specific for prostatic epithelium in transgenic mice. CAT activity decreased after castration, and could be induced by androgens and, in addition, glucocorticoids. This demonstrates that the necessary sequences required to target prostate-specific epithelial expression are contained within the composite ARR2PB minimal promoter, and that high transgene expression can now be regulated by both androgens and glucocorticoids. The ARR2PB promoter represents a novel glucocorticoid inducible promoter that can be used for the generation of transgenic mouse models and in viral gene therapy vectors for the treatment of prostate cancer in humans.

Human growth hormone induces and maintains c-myc gene expression in Nb2 lymphoma cells.

The effect of human GH (hGH) on c-myc gene expression and cell proliferation in the Nb2 lymphoma cell line was examined. The addition of hormone to stationary cultures of Nb2 cells resulted in an increased accumulation of c-myc transcripts that was detectable within 15 min, reached a maximum induction of 25-fold in 3 h, and then gradually declined. Although maximal accumulation of c-myc transcripts occurred within 3 h after hGH addition, removal of the hormone even after 4 h of treatment resulted in a failure of the cells to proliferate. This loss of proliferative capacity following hormone removal was accompanied by a 20-fold reduction in the level of c-myc transcripts. These results indicate that hGH induced proliferation of Nb2 cells is associated with the induction and maintenance of c-myc gene expression.

Gene duplication gives rise to a new 17-kilodalton lipocalin that shows epididymal region-specific expression and testicular factor(s) regulation.

Using transgenic mice, we have recently shown that 5 kb of the 5'-flanking region of the mouse epididymal retinoic acid-binding protein (mE-RABP) gene contains all of the information required for spatial and temporal gene expression in the epididymis. To identify the important cis-DNA regulatory element(s) involved in the tissue-, region-, and cell-specific expression of the mE-RABP gene, the 5-kb DNA fragment was sequenced. A computer analysis of the nucleotide sequence showed the presence of a new gene located 1.7 kb upstream from the mE-RABP gene transcription initiation site. The analysis of the open reading frame showed that the new gene encoded a putative 17-kDa lipocalin (named mEP17) related to mE-RABP. A 600-bp complementary DNA encoding mEP17 was cloned by rapid amplification of 3'-cDNA ends from epididymal total RNA. Two mEP17 RNA species (1 and 3.1 kb in size) were detected by Northern blot in the epididymis, but not in other tissues tested. In situ hybridization analyses showed that, unlike mE-RABP messenger RNA (mRNA), which is expressed in the distal caput epididymidis, mEP17 mRNA was detected only in the principal cells of the initial segment. The spatial expression and homology with mE-RABP suggest that mEP17 may act as a retinoid carrier protein within the epididymis. mEP17 mRNA expression disappeared 5 days postcastration. Four days after unilateral castration, mEP17 mRNA had nearly disappeared in the epididymis from the castrated side, but not from the intact side. In addition, testosterone replacement to bilaterally castrated mice failed to restore gene expression. We conclude that mEP17 gene expression is dependent on testicular factors circulating in the luminal fluid. Together our results suggest that mE-RABP and mEP17 genes were generated by duplication and that evolution led to a different region-specific gene expression and regulation in the epididymis.

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid.

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.

Selective activation of the probasin androgen-responsive region by steroid hormones.

Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.

Effect of androgens on mRNA for a secretory protein of rat dorsolateral prostate and seminal vesicles.

The androgen dependence of a highly abundant mRNA found in the rat dorsolateral prostate and seminal vesicles has been investigated using a complementary DNA clone from a rat dorsal prostate library. The 1.5 kilobase (kb) mRNA codes for a 52 000 Da translation product which is processed to 49 000 Da in the presence of microsomal membranes. This product appears to correspond to the previously described SVS II protein secreted by rat seminal vesicles and can be immunoprecipitated with anti-SVS II antiserum. Dot hybridization assays indicated that the mRNA is abundant in the dorsal and lateral prostate glands and in seminal vesicles but not in the ventral prostate, coagulating gland or other non-accessory sex tissues. Castration of mature male rats reduces the 1.5 kb mRNA 10-fold in the seminal vesicles and 7-fold in the dorsolateral prostate in 9 days. Androgen administration to one-week castrates returned the mRNA level to normal in both tissues within 48 h. The levels of the 1.5 kb mRNA are very similar in the dorsolateral prostate and seminal vesicles at maturity but distinct patterns of developmental regulation of this gene exist in the two tissues. Between 3 and 6 weeks of age, the level of the 1.5 kb mRNA increases approximately 3-fold in the dorsolateral prostate while the increase in the seminal vesicles is more than 600-fold.

Induction of cell-free, in vitro transcription by recombinant androgen receptor peptides.

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 regulation of the human ornithine decarboxylase promoter in prostate cancer cells.

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.