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Biomedical subjects

E M Wilson

Publications and source records attributed to E M Wilson.

At least 55 records · Page 3Linked to original sources
PubMed1997

Generation and characterization of an IGFBP-7 antibody: identification of 31kD IGFBP-7 in human biological fluids and Hs578T human breast cancer conditioned media.

The cDNA encoding mac25 (IGFBP-7) was firsr derived from mRNA isolated from leptomeningial and senescent human mammary epithelial cells (1,2). The open reading frame was shown to predict a protein with homology to the amino terminus of the IGF binding proteins, (IGFBP)1-6. Studies in our laboratory have shown that baculovirus generated mac25 binds IGF-I and-II in a specific manner, leading to the renaming of mac25 as IGFBP-7 (3). Further studies at the cellular level, to identify the involvement of IGFBP-7 in IGF regulation and cell growth, require a specific antibody against the protein, which has yet to be identified in either cultured cells or in vivo. We have now generated three polyclonal antibodies against the purified baculovirus peptide and, by western immunoblots and immunoprecipitation, demonstrated the existence of a specific 31,000 dalton protein. It is a secreted protein, and can be identified in the conditioned media of Hs578T breast cancer cells, as well as in normal human urine, cerebrospinal fluid and amniotic fluid. Subsequent studies with these antibodies should help elucidate the physiological role(s) of this protein.

Animals↗
PubMed1997

Transcriptional repression of the alpha-subunit gene by androgen receptor occurs independently of DNA binding but requires the DNA-binding and ligand-binding domains of the receptor.

The pituitary glycoprotein hormones LH and FSH regulate the reproductive cycle and are sensitive to feedback by gonadal steroids. The common alpha-subunit shared by these hormones is transcriptionally repressed by androgen receptor (AR) in the presence of its ligand dihydrotestosterone. This identifies at least one mechanism that contributes to AR-dependent suppression of gonadotropin synthesis. Repression of alpha-subunit transcription by AR requires only the sequences within the first 480 bp of the promoter. While this region contains a high-affinity binding site for AR, this element does not mediate the suppressive effects of androgens. Instead, two other elements within the promoter-regulatory region (alpha-basal element and cAMP-regulatory element), which are important for expression of the alpha-subunit gene in gonadotropes, mediate the effects of AR. This suggests that AR inhibits activity of the alpha-subunit promoter by interfering with the transcriptional properties of the proteins that bind to alpha-basal element and the cAMP-regulatory elements. Furthermore, transfection analysis of various mutant ARs identified both the DNA-binding and ligand-binding domains of the receptor as critical for repression. Comparisons with the MMTV promoter revealed distinct structural requirements that underlie the transactivation and transrepression properties of AR.

Animals↗
PubMed1997

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.

Androgen Antagonists↗
PubMed1996

Growth inhibition of human prostate cells in vitro by novel inhibitors of androgen synthesis.

The long-standing strategy for the treatment of metastatic prostate cancer has been to reduce androgenic stimulation of tumor growth by removal of the testes, the primary site of testosterone synthesis. However, a low level of androgenic stimulation may continue, even after castration, by the conversion of adrenal androgens to 5alpha-dihydrotestosterone (DHT) in the prostate tumor cells. Two important enzymes of the androgen biosynthetic pathway are 17alpha-hydroxylase/C17,20-lyase, which regulates an early step in the synthesis of testosterone and other androgens in both the testes and adrenal glands, and 5alpha-reductase, which converts testosterone to the more potent androgen, DHT, in the prostate. We have identified new inhibitors of these enzymes that may be of use in achieving a more complete ablation of androgens in the treatment of metastatic prostate cancer. Three derivatives of androstene were shown to inhibit 17alpha-hydroxylase/C17,20-lyase with potencies 2-20-fold greater than that of ketoconazole, a previously established inhibitor of this enzyme. Derivatives of pregnane and pregnene displayed activities against 5alpha-reductase that were comparable to that of N-(1,1-dimethyl-ethyl)-3-oxo-4-aza-5alpha-androst-1-ene-17beta-car boxamide. All of the 5alpha-reductase inhibitors were able to at least partially inhibit the mitogenic effect of testosterone in either histocultures of human benign prostatic hypertrophic tissue or in cultures of the LNCaP human prostatic tumor cell line. For these compounds, it appears that this inhibition can be attributed to a reduction of DHT synthesis in these cultures, because no inhibitory effect was observed in DHT-treated cultures, and none of the compounds had a cytotoxic effect. Surprisingly, one of the inhibitors of 17alpha-hydroxylase/C17,20-lyase, 17beta-(4-imidazolyl)-5-pregnen-3beta-ol, was also able to inhibit the mitogenic effect of testosterone in both the histoculture and cell culture assays and had an effect against DHT as well. In transcriptional activation assays, it was found that this compound is an antagonist of both the wild-type androgen receptor and the mutant androgen receptor, which is present in LNCaP cells. In conclusion, the abilities of these compounds to inhibit androgen synthesis and, in some cases, to exert antiandrogen activity, did in fact translate to an inhibitory effect on the growth of human prostatic tissue in vitro, suggesting their potential utility in the treatment of prostatic cancer.

Androgen Antagonists↗
PubMed1996

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.

Adolescent↗
PubMed1996

Reduced androgen receptor gene expression with first exon CAG repeat expansion.

The molecular basis for partial androgen insensitivity associated with adult onset spinal/bulbar muscular atrophy was investigated by transient transfection of human androgen receptor (AR) expression vectors containing increasing CAG repeat lengths in the first exon. An inverse relationship was observed between CAG repeat length and AR mRNA and protein levels. Trinucleotide repeat lengths of 43 and 65 associated with spinal/bulbar muscular atrophy decreased AR mRNA and protein levels but did not alter equilibrium binding affinity for [3H]R1881 or inherent transcriptional activity of AR, expressed as androgen-dependent fold induction of a mouse mammary tumor virus promoter-luciferase reporter vector. The findings indicate that glutamine expansion up to 66 residues in the NH2-terminal domain of AR does not alter AR functional activity. Rather, CAG repeat expansion in the region of the first exon reduces AR mRNA and protein expression. The study reveals a previously unrecognized effect of CAG repeat length on AR mRNA expression and a novel molecular mechanism for androgen resistance.

Androgens↗
PubMed1996

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.

Aged↗
PubMed1995

Evidence for an anti-parallel orientation of the ligand-activated human androgen receptor dimer.

Domain interactions of the human androgen receptor (AR) dimer were investigated using a protein-protein interaction assay in which the NH2- and carboxyl-terminal regions of human AR were fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and herpes simplex virus VP16 transactivation domain to produce chimeric proteins. Transcriptional activation of a GAL4 luciferase reporter vector up to 100-fold was greater than Fos/Jun leucine zipper binding, indicating stable AR interaction between AR NH2-terminal residues 1-503 and steroid-binding domain residues 624-919 that was specific for and dependent on androgen binding to the steroid-binding domain and was inhibited by anti-androgen binding. Deletion mutagenesis within the NH2-terminal region indicated transactivation domain residues 142-337 were not required for dimerization, whereas deletions near the NH2 terminus (delta 14-150) or NH2-terminal to the DNA-binding domain (delta 339-499) reduced or eliminated the AR interaction, respectively. An NH2-/NH2-terminal interaction was also observed, but no interaction was detected between ligand-free or bound steroid-binding domains. The results indicate that high affinity androgen binding promotes interactions between the NH2-terminal and steroid-binding domains of human AR, raising the possibility of an androgen-induced anti-parallel AR dimer.

Animals↗
PubMed1995

Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide.

The mechanism of antiandrogenic activity of vinclozolin (3-(3,5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione), a dicarboximide fungicide under investigation for its potential adverse effects on human male reproduction, was investigated using recombinant human androgen receptor (AR). The two primary metabolites of vinclozolin in plants and mammals are M1 (2-[[3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) and M2 (3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide). Both metabolites, in a dose-dependent manner, target AR to the nucleus and inhibit androgen-induced transactivation mediated by the mouse mammary tumor virus promoter. M2 is a 50-fold more potent inhibitor than M1 and only 2-fold less than hydroxyflutamide. In the presence of dihydrotestosterone (50 nM), M2 (0.2-10 microM) inhibits androgen-induced AR binding to androgen response element DNA. In the absence of dihydrotestosterone, concentrations of 10 microM M2 or hydroxyflutamide promote AR binding to androgen response element DNA and activation of transcription. Agonist activities of M2 and hydroxyflutamide occur at 10-fold lower concentrations with the mutant AR (Thr877 to Ala) endogenous to LNCaP human prostate cancer cells. The results indicate that androgen antagonists can act as agonists, depending on ligand binding affinity, concentration, and the presence of competing natural ligands.

Androgen Receptor Antagonists↗
PubMed1995

Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist.

The increase in the number of reports of abnormalities in male sex development in wildlife and humans coincided with the introduction of 'oestrogenic' chemicals such as DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) into the environment. Although these phenotypic alterations are thought to be mediated by the oestrogen receptor, they are also consistent with inhibition of androgen receptor-mediated events. Here we report that the major and persistent DDT metabolite, p,p'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), has little ability to bind the oestrogen receptor, but inhibits androgen binding to the androgen receptor, androgen-induced transcriptional activity, and androgen action in developing, pubertal and adult male rats. The results suggest that abnormalities in male sex development induced by p,p'-DDE and related environmental chemicals may be mediated at the level of the androgen receptor.

Androgen Antagonists↗
PubMed1995

Identification of sequences mediating guanylyl cyclase dimerization.

Deletion mutagenesis was used to identify sequences required for dimerization and enzymatic activity of the intracellular domain of the membrane guanylyl cyclase, GC-A. The intracellular domain of GC-A contains a protein kinase-like domain near its amino terminus, a guanylyl cyclase catalytic domain near its carboxyl terminus, and, between these domains, a region of unknown function predicted to form an amphipathic alpha-helix. Gel filtration analysis of deletion mutants of the GC-A intracellular domain suggested that a 43 amino acid sequence within the interdomain region was both necessary and sufficient for dimerization and was required for guanylyl cyclase catalytic activity. The ability of this sequence to mediate protein dimerization was confirmed in the yeast two-hybrid system, in which its fusion to the lexA DNA-binding domain and to the VP16 transcriptional activation domain led to their dimerization and consequent activation of a lexA-HIS3 gene. Thus, we have identified sequences responsible for dimerization of the intracellular domain of a guanylyl cyclase and shown that they are required for enzyme activity. Modulation of their interaction may be important in guanylyl cyclase activation.

Amino Acid Sequence↗
PubMed1995

Hormone-dependent transactivation by the human androgen receptor is regulated by a dnaJ protein.

Genetic studies were performed to examine the role of eukaryotic dnaJ protein, Ydj1p, in the regulated activation of human androgen receptor (hAR) after heterologous expression in Saccharomyces cerevisiae. Hormone-dependent activation of hAR was measured as a function of lacZ reporter gene expression, which was defective in ydj1-151 and ydj1-2 delta null mutant strains compared to the wild type. This defect was not due to receptor misfolding, since hAR in both wild type and mutant strains had a similar capacity to bind hormone. The target for Ydj1p action was determined to be the hAR hormone binding domain since an N-terminal fragment lacking this region was constitutively active in both wild type and ydj1-151 mutant strains. These data correlate hormone dependence of hAR activation with a requirement for Ydj1p function and are consistent with a role for dnaJ proteins in signal transduction by steroid hormone receptors.

Bacterial Proteins↗
PubMed1995

Stability of an expanded trinucleotide repeat in the androgen receptor gene in transgenic mice.

The expansion of trinucleotide repeat sequences underlies a number of hereditary neurological disorders. To study the stability of a trinucleotide repeat and to develop an animal model of one of these disorders, spinal and bulbar muscular atrophy (SBMA), we have generated transgenic mice carrying either the normal or expanded repeat human androgen receptor (AR) gene. Unlike the disease allele in humans, the AR cDNA containing the expanded repeat in transgenic mice showed no change in repeat length with transmission. Expression of the SBMA AR was found in transgenic mice, but at a lower level than normal endogenous expression. The lack of a physiological pattern of expression may explain why no phenotypic effects of the transgene were observed.

Animals↗