Cationic peptide of the male reproductive tract, HE2alpha, displays antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis.

OBJECTIVES: To analyse the in vitro antimicrobial effects of synthetic HE2alpha peptide against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis. METHODS: The HE2alpha peptide was synthesized based on the C-terminal sequence of the HE2alpha protein. The bacterial strains tested included two antibiotic-susceptible strains of N. gonorrhoeae and four antibiotic-resistant clinical isolates, as well as S. aureus ATCC 29213 and E. faecalis ATCC 29212. Susceptibility determinations were carried out either in 0.7% casamino acids for N. gonorrhoeae isolates or in 10 mM phosphate buffer for S. aureus and E. faecalis strains. Antibacterial effects were measured in a dose- and time-dependent manner. After exposure to the peptide in solution, the number of viable cells was determined by counting colony forming units (cfu). RESULTS: The HE2alpha peptide exhibited time- and dose-dependent antibacterial effects on all N. gonorrhoeae isolates tested. S. aureus and E. faecalis strains were also susceptible to the peptide. All strains tested were susceptible to the peptide at high concentrations (50 or 100 mg/L) and some strains were susceptible to a peptide concentration of 25 mg/L. CONCLUSIONS: The peptide HE2alpha, which is derived from the male urogenital tract, exhibits antibacterial activity against both gram-positive and gram-negative pathogens in vitro. The peptide is active against both antibiotic-susceptible and -resistant N. gonorrhoeae isolates. Further investigation of the antimicrobial properties of the peptide is warranted.

Identification, characterization, and evolution of a primate beta-defensin gene cluster.

Defensins are members of a large diverse family of cationic antimicrobial peptides that share a signature pattern consisting of six conserved cysteine residues. Defensins have a wide variety of functions and their disruption has been implicated in various human diseases. Here we report the characterization of DEFB119-DEFB123, five genes in the human beta-defensin cluster locus on chromosome 20q11.1. The genomic structures of DEFB121 and DEFB122 were determined in silico. Sequences of the five macaque orthologs were obtained and expression patterns of the genes were analyzed in humans and macaque by semiquantitative reverse transcription polymerase chain reaction. Expression was restricted to the male reproductive tract. The genes in this cluster are differentially regulated by androgens. Evolutionary analyses suggest that this cluster originated by a series of duplication events and by positive selection. The evolutionary forces driving the proliferation and diversification of these defensins may be related to reproductive specialization and/or the host-parasite coevolutionary process.

Reversible immunocontraception in male monkeys immunized with eppin.

Various forms of birth control have been developed for women; however, there are currently few options for men. The development of male contraceptives that are effective, safe, and reversible is desired for family planning throughout the world. We now report contraception of male nonhuman primates (Macaca radiata) immunized with Eppin, a testis/epididymis-specific protein. Seven out of nine males (78%) developed high titers to Eppin, and all of these high-titer monkeys were infertile. Five out of seven (71%) high-anti-Eppin titer males recovered fertility when immunization was stopped. This study demonstrates that effective and reversible male immunocontraception is an attainable goal. This method of immunocontraception may be extended to humans.

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human.

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein.

Distinct effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells.

Androgen signaling influences the development and growth of prostate carcinoma. The transcriptional activity of androgen receptor (AR) is regulated by positive or negative transcriptional cofactors. We report here that PIAS1, PIAS3, and PIASy of the protein inhibitor of activated STAT (PIAS) family, which are expressed in human prostate, display distinct effects on AR-mediated gene activation in prostate cancer cells. While PIAS1 and PIAS3 enhance the transcriptional activity of AR, PIASy acts as a potent inhibitor of AR in prostate cancer cells. The effects of PIAS proteins on AR are competitive. PIASy binds to AR but does not affect the DNA binding activity of AR. An NH2-terminal LXXLL signature motif of PIASy, although not required for PIASy-AR interaction, is essential for the transrepression activity of PIASy. Our results identify PIASy as a transcriptional corepressor of AR and suggest that different PIAS proteins have distinct effects on AR signaling in prostate cancer cells.

Androgen receptor regulation of G1 cyclin and cyclin-dependent kinase function in the CWR22 human prostate cancer xenograft.

Human prostate cancer is initially dependent on androgens for growth, and androgen-dependent cells undergo apoptosis after castration. However, a subset of androgen-responsive cells survives and eventually proliferates in the absence of testicular androgen. The high levels of androgen receptor in both androgen-dependent and recurrent tumors led us to investigate androgen regulation of cell cycle proteins in human prostate cancer using the CWR22 xenograft. Cellular proliferation decreased dramatically in CWR22 tumors after castration. Testosterone propionate (TP) treatment of castrated mice restored cellular proliferation after 24-48 hours. Growth of CWR22 tumors in the absence of testicular androgen recurred several months after castration. CDK1 and CDK2, and cyclin A and cyclin B1 messenger RNAs were decreased 6 days after castration, increased 6-12 hours after TP treatment, and were expressed at high levels in recurrent CWR22 tumors. Coimmunoprecipitated cyclin B1/CDK1 and cyclin D1/CDK4 protein complexes decreased after castration and increased after TP treatment of castrated mice. In addition, CDK1 and CDK2 kinase activities were upregulated by androgen in parallel with hyperphosphorylation of retinoblastoma (Rb) protein. Despite the absence of testicular androgen in recurrent CWR22, the levels of these androgen-regulated cyclin/ CDK protein complexes and hyperphosphorylation of Rb were equal to or greater than in tumors from intact mice. The results indicate that androgen receptor regulates cellular proliferation by control of CDK and cyclins at the transcriptional level and by post-translational modifications that influence cell cycle protein activity.

A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy.

The development and growth of prostate cancer depends on the androgen receptor and its high-affinity binding of dihydrotestosterone, which derives from testosterone. Most prostate tumors regress after therapy to prevent testosterone production by the testes, but the tumors eventually recur and cause death. A critical question is whether the androgen receptor mediates recurrent tumor growth after androgen deprivation therapy. Here we report that a majority of recurrent prostate cancers express high levels of the androgen receptor and two nuclear receptor coactivators, transcriptional intermediary factor 2 and steroid receptor coactivator 1. Overexpression of these coactivators increases androgen receptor transactivation at physiological concentrations of adrenal androgen. Furthermore, we provide a molecular basis for this activation and suggest a general mechanism for recurrent prostate cancer growth.

Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis.

In this report we describe the discovery of Eppin (Epididymal protease inhibitor), a gene on human chromosome 20 expressing three mRNAs encoding two isoforms of a cystine-rich protein containing both Kunitz-type and WAP-type four disuffide core protease inhibitor consensus sequences. Analysis of Eppin's genomic sequence from chromosome 20q12-13.2 predicts the existence of all three splice variants of Eppin and that all the exons conform to the AG/GT splicing rule. The presence of single bands on a Southern blot of human genomic DNA suggests that Eppin is a single copy gene. TATA box transcription initiation sites are present for both of the different Eppin 5' UTRs and examination of the promoter region 1800 bp upstream of the start codon revealed a number of putative transcription enhancer binding sites typical of genes expressed in the epididymis or testis. Northern blot and tissue specific PCR data indicate Eppin-1 is expressed only in the testis and epididymis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the testis. Antiserum prepared against recombinant EPPIN recognizes several strong bands on Western blots of human epididymal extracts from the caput and corpus regions. Immunohistochemistry indicates a strong pattern of expression by the ciliated cells of the efferent ducts and strong staining of ejaculated spermatozoa. Eppin represents the first member of a family of protease inhibitors characterized by dual inhibitor consensus sequences, both WAP-type and Kunitz-type consensus sequences. A second family member is predicted to exist on chromosome 20 approximately 4 kb downstream from Eppin's exon I, which has two WAP-type sequences and one Kumtz-type consensus sequence.

Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen.

The androgen receptor (AR) is highly expressed in androgen-dependent and recurrent prostate cancer (CaP) suggesting it has a role in the growth and progression of CaP. Previously proposed mechanisms for AR reactivation in recurrent CaP include altered growth factor signaling leading to protein phosphorylation and AR mutations that broaden ligand specificity. To further establish a role for AR in recurrent CaP, we compared several properties of AR in relation to the growth response to low levels of androgens in model systems of androgen-dependent and recurrent CaP. AR from all of the tumors and cell lines bound [3H]R1881 with similar high affinity (mean Kd, 0.12 nM). In the absence of androgen, AR in androgen-dependent LNCaP cells was unstable with a degradation half-time (t(1/2)) of 3 h at 37 degrees C. In contrast, AR was 2-4 times more stable in recurrent CWR22 tumors (t(1/2), >12 h) and CWR-R1 or LNCaP-C4-2 cell lines (t(1/2), 6-7 h) derived from recurrent prostate tumors. In the recurrent CWR22 tumor and its CWR-R1 cell line grown in the absence of androgen, AR immunostaining was entirely nuclear, whereas under the same conditions AR in LNCaP-C4-2 and LNCaP cells was predominantly nuclear but was also detected in the cytoplasm. High level expression, increased stability, and nuclear localization of AR in recurrent tumor cells were associated with an increased sensitivity to the growth-promoting effects of dihydrotestosterone in the femtomolar range. The concentration of dihydrotestosterone required for growth stimulation in CWR-R1 and LNCaP-C4-2 cells was four orders of magnitude lower than that required for androgen-dependent LNCaP cells. The results suggest that AR is transcriptionally active in recurrent CaP and can increase cell proliferation at the low circulating levels of androgen reported in castrated men.

Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell complementary DNA library by two-hybrid screening in yeast using the AR DNA and ligand binding domains [amino acids (aa) 481-919] as bait. AR binding of the protein in yeast was dependent on the presence of testosterone or dihydrotestosterone (DHT). The isolated protein is identical to thyroid receptor activator molecule TRAM-1 but lacking aa 1-458. TRAM-1 is a steroid receptor coactivator-3 (SRC-3) subtype. In affinity matrix assays, 35S-labeled TRAM-1 bound the GST-AR ligand binding domain (aa 624-919) and GST-AR N-terminal and DNA binding domains (aa 1-660), but not the GST-AR DNA binding domain (aa 544-634) alone. Coexpression of TRAM-1 increased DHT-dependent AR transactivation 5-fold and constitutive activity of AR (aa 1-660) N-terminal and DNA-binding domains increased 9-fold. Full-length TRAM-1 (aa 1-1424) and the partial (aa 459-1424) were AR and GR coactivators as was SRC-1. In human testis, immunostaining of SRC-3 colocalized with AR in nuclei of Sertoli cells and peritubular myoid cells, indicating it could function as an AR coactivator in these cells. SRC-3 was also present in nuclei of spermatogenic cells where AR was not expressed, suggesting it might also be a coactivator with other nuclear receptors that regulate spermatogenesis.

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.

Cloning and characterization of an androgen-dependent acidic epididymal glycoprotein/CRISP1-like protein from the monkey.

A cDNA encoding an acidic epididymal glycoprotein (AEG)-like, CRISP1 (cysteine-rich secretory protein) protein from the monkey (Macaca mullata) epididymis has been cloned and sequenced. The monkey AEG (mAEG) has an open reading frame that encodes a protein containing 249 amino acids with a deduced molecular mass of 28 kDa. The mAEG protein sequence is 85% identical to human and 44% identical to mouse CRISP1, including all 16 conserved cysteine residues. mAEG also shows a significant amino acid homology with other CRISP proteins, rat AEG/DE, human TPX1/CRISP2, and guinea pig acrosomal autoantigen 1 (AA1). In addition, mAEG shows somewhat less homology to a toxin from the Mexican beaded lizard and to a human glioma pathogenesis-related protein. Northern blot analysis shows that the mRNA for mAEG is expressed in all the regions of the epididymis except the caput and was not detected in the testis, prostate, seminal vesicle, and brain. In castrated animals, mAEG gene expression in the epididymis is significantly diminished; however, testosterone enanthate replacement restored the normal level of expression, demonstrating that expression of mAEG is androgen dependent. Western blot analysis of monkey epididymal regions using mouse antirecombinant human AEG identified a 28-kDa protein only in the caudal region. Immunohistochemical analysis identified mAEG only in the principal cells of the cauda epididymal epithelium. Immunofluorescence analysis identified mAEG on the principal piece of the sperm tail and as small patches over the middle piece and head regions. The results described in the present study suggest that mAEG (CRISP1) is secreted in the monkey epididymis, regulated by androgens and present on epididymal spermatozoa.

Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important modulators of IGF action in many tissues including human prostate. IGFBPs and the androgen receptor (AR) are expressed in CWR22, an androgen-dependent epithelial cell human CaP xenograft that retains biological characteristics of human CaPs, including regression following androgen withdrawal and recurrent growth of AR-containing cells in the absence of testicular androgens beginning several months after castration. Northern blot and in situ hybridization analyses demonstrated that IGFBP-5 is androgen-regulated in CWR22. IGFBP-5 messenger RNA (mRNA) decreased by 90% following castration of tumor-bearing mice compared with noncastrate androgen-stimulated mice. Testosterone treatment of CWR22 tumor-bearing mice 6 or 12 days after castration increased IGFBP-5 mRNA 10- to 12-fold. Levels of other IGFBP mRNAs did not change following androgen withdrawal and replacement. IGFBP-5 protein in tumor extracts bound 125I-labeled IGF-I in ligand blot assays and the amounts of IGFBP-5 measured by immunoblotting paralleled the levels of IGFBP-5 mRNA. Androgen-induced expression of IGFBP-5 was at a maximum level within 24 h after testosterone replacement, whereas the major increase in cell proliferation as measured by Ki-67 immunostaining occurred between 24-48 h. This time course suggested IGFBP-5 may be a mediator of androgen-induced growth of CWR22. In tumors that recurred several months following castration, IGFBP-5 mRNA and protein increased to levels that approached those in androgen-stimulated CWR22 tumors from noncastrate mice. IGFBP-5 immunohistochemical staining of prostate tissue specimens from patients was stronger in androgen-dependent and androgen-independent CaP than in areas of intraepithelial neoplasia (PIN) or benign prostatic hyperplasia (BPH). IGFBP-5 mRNA in these specimens was localized predominantly to stromal cells and IGFBP-5 protein to epithelial cell membranes.

Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes.

The human prostate cancer (CaP) xenograft, CWR22, mimics human CaP. CWR22 grows in testosterone-stimulated nude mice, regresses after castration, and recurs after 5-6 months in the absence of testicular androgen. Like human CaP that recurs during androgen deprivation therapy, the recurrent CWR22 expresses high levels of androgen receptor (AR). Immunohistochemical, Western, and Northern blot analyses demonstrated that AR expression in the androgen-independent CWR22 is similar to AR expression in the androgen-dependent CWR22 prior to castration. Expression of prostate-specific antigen and human kallikrein-2 mRNAs, two well-characterized androgen-regulated genes in human CaP, was androgen dependent in CWR22. Despite the absence of testicular androgen, prostate-specific antigen and human kallikrein-2 mRNA levels in recurrent CWR22 were higher than the levels in regressing CWR22 tumors from 12-day castrate mice and similar to those in the androgen-stimulated CWR22. Other AR-regulated genes followed a similar pattern of expression. Differential expression screening identified androgen regulation of alpha-enolase and alpha-tubulin as well as other unknown mRNAs. Insulin-like growth factor binding protein-5, the homeobox gene Nkx 3.1, the AR coactivator ARA-70, and cell cycle genes Cdk1 and Cdk2 were androgen regulated in CWR22. In recurrent CWR22, the steady-state levels of all these AR-dependent mRNAs were similar to those in the androgen-stimulated CWR22, despite the absence of testicular androgen. Expression of AR and AR-regulated genes in the androgen-deprived recurrent CWR22 at levels similar to the androgen-stimulated CWR22 suggests that AR is transcriptionally active in recurrent CWR22. Induction of these AR-regulated genes may enhance cellular proliferation in relative androgen absence but through an AR-dependent mechanism. Alternatively, in androgen-independent tumors, induced expression of the AR-regulated gene network might result from a non-AR transcription control mechanism common to these genes.

Androgen-dependent protein interactions within an intron 1 regulatory region of the 20-kDa protein gene.

The 20-kDa protein gene is androgen regulated in rat ventral prostate. Intron 1 contains a 130-base pair complex response element (D2) that binds androgen (AR) and glucocorticoid receptor (GR) but transactivates only with AR in transient cotransfection assays in CV1 cells using the reporter vector D2-tkCAT. To better understand the function of this androgen-responsive unit, nuclear protein interactions with D2 were analyzed by DNase I footprinting in ventral prostate nuclei of intact or castrated rats and in vitro with ventral prostate nuclear protein extracts from intact, castrated, and testosterone-treated castrated rats. Multiple androgen-dependent protected regions and hypersensitive sites were identified in the D2 region with both methods. Mobility shift assays with 32P-labeled oligonucleotides spanning D2 revealed specific interactions with ventral prostate nuclear proteins. Four of the D2-protein complexes decreased in intensity within 24 h of castration. UV cross-linking of the androgen-dependent DNA binding proteins identified protein complexes of approximately 140 and 55 kDa. The results demonstrate androgen-dependent nuclear protein-DNA interactions within the complex androgen response element D2.

Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells.

An androgen receptor (AR) gene mutation identified in the androgen-dependent human prostate cancer xenograft, CWR22, changed codon 874 in the ligand-binding domain (exon H) from CAT for histidine to TAT for tyrosine and abolished a restriction site for the endonuclease SfaNI. SfaNI digestion of AR exon H DNA from normal but not from prostate cancer tissue indicated H874Y is a somatic mutation that occurred before the initial tumor transplant. CWR22, an epithelial cell tumor, expresses a 9.6-kb AR mRNA similar in size to the AR mRNA in human benign prostatic hyperplasia. AR protein is present in cell nuclei by immunostaining as in other androgen-responsive tissues. Transcriptional activity of recombinant H874Y transiently expressed in CV1 cells in the presence of testosterone or dihydrotestosterone was similar to that of wild type AR. With dihydrotestosterone at a near physiological concentration (0.01 nM), H874Y and wild type AR induced 2-fold greater luciferase activity than did the LNCaP mutant AR T877A. The adrenal androgen, dehydroepiandrosterone (10 and 100 nM) with H874Y stimulated a 3- to 8-fold greater response than with wild type AR and at 100 nM the response was similar with the LNCaP mutant. H874Y, like the LNCaP cell mutant, was more responsive to estradiol and progesterone than was wild type AR. The antiandrogen hydroxyflutamide (10 nM) had greater agonist activity (4- to 7-fold) with both mutant ARs than with wild type AR. AR mutations that alter ligand specificity may influence tumor progression subsequent to androgen withdrawal by making the AR more responsive to adrenal androgens or antiandrogens.

A novel missense mutation in the amino-terminal domain of the human androgen receptor gene in a family with partial androgen insensitivity syndrome causes reduced efficiency of protein translation.

The role of the androgen receptor (AR) in male sexual differentiation is revealed in part by the analysis of naturally occurring mutations in families with androgen insensitivity syndrome (AIS). We have investigated a family with partial AIS affecting three generations and have identified a G to A substitution in the AR gene at the fourth position 3' from the A of the ATG initiation codon changing the second amino acid residue from glutamic acid to lysine (EK2). Transient expression of the mutant EK2-pCMVhAR expression vector in COS cells revealed decreased translation with a 20-50% reduction in mutant protein relative to wild type AR by immunoblot analysis. The rate of dissociation of [3H]methyltrienolone from the EK2 mutant (half-time [t1/2] = 1.7 +/- 0.08 SE h) was increased compared with wild type AR (t1/2 = 2.4 +/- 0.11 h). Cotransfection studies using an androgen responsive luciferase reporter vector demonstrated a 50% reduction in transcriptional activation by EK2. These functional alterations are consistent with the partial AIS phenotype in affected males, corroborate the AR amino-terminal domain effect on kinetics of androgen binding, and provide physiological evidence for earlier translation experiments identifying the nucleotide sequence for optimal translation initiation.

Androgen and glucocorticoid receptors in the stroma and epithelium of prostatic hyperplasia and carcinoma.

Differences in stromal and epithelial cell staining for androgen and glucocorticoid receptors (ARs and GRs) were investigated in 20 patients with clinically localized prostatic carcinoma treated by radical prostatectomy. Sections of benign prostatic hyperplasia and prostatic carcinoma from each patient were stained with antibodies to AR and GR using an avidin-biotin peroxidase technique. The specificity of the GR immunoreactivity was established in benign prostatic hyperplasia and prostatic carcinoma by immunohistochemistry using the GR antibody absorbed with synthetic peptide and Western blotting. Nuclear staining intensity and percentage of nuclei stained were summed to obtain AR and GR immunostaining scores. AR staining of prostatic carcinoma epithelial [103 +/- 58 (SD)] and stromal (126 +/- 48) nuclei was less than in benign prostatic hyperplasia (142 +/- 47 and 169 +/- 56; paired Student's t tests, P = 0.02 and P = 0.01); however, no difference in staining intensity occurred between stroma and epithelium in either tissue type. GR stained intensely in stromal cells from benign prostatic hyperplasia (189 +/- 50) and prostatic carcinoma (163 +/- 60). However, prostatic carcinoma epithelial cells (34 +/- 57) had low levels of glucocorticoid receptor staining (P < 10(-7)), and benign prostatic hyperplasia epithelium (74 +/- 51) was intermediate. In most patients, GR could not be detected in nuclei of prostatic carcinoma epithelial cells but was undiminished in stromal cell nuclei. There was no relationship by multivariate regression analysis between AR or GR staining and age, serum prostate-specific antigen, Gleason grade, or pathological stage. In comparison with AR, the greater variability of GR staining in epithelium versus stroma of prostatic carcinoma warrants further study of GR, particularly in the area of stromal-epithelial interaction.