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E R Barrack

Publications and source records attributed to E R Barrack.

At least 37 records · Page 2Linked to original sources
PubMed1987

A new method for labeling and autoradiographic localization of androgen receptors.

We have used a novel receptor labeling and autoradiographic technique to localize androgen receptors in the intact rat ventral prostate at the morphological level. Frozen slide-mounted prostate tissue sections (10 micron thick) were incubated with increasing concentrations of [3H]-R1881 in the absence and presence of excess unlabeled R1881. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. After incubation and washing to remove free [3H]-steroid, some of the sections were wiped from the slides for scintillation counting in order to characterize and quantitate [3H]-R1881 binding. Androgen receptors could indeed be labeled in slide-mounted tissue sections, and specific [3H]-R1881 binding to these receptors was high-affinity (Kd = 1 nM), saturable, and androgen-specific. All cellular androgen receptors appear to be retained, because receptor content in sections was comparable to the sum of receptors in subcellular fractions of homogenized tissue. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We discuss here the unique features and advantages of labeling androgen receptors in slide-mounted frozen tissue sections for autoradiographic localization.

Androgens↗
PubMed1987

Estrogen receptors in the nuclear matrix: direct demonstration using monoclonal antireceptor antibody.

Estradiol-binding sites, as assayed by exchange with radiolabeled steroid, become associated with the nuclear matrix of estrogen-responsive tissues after treatment with estrogen in vivo. Using monoclonal estrogen receptor antibodies, we have now obtained direct evidence that these matrix-associated estradiol-binding sites are estrogen receptor proteins similar to those found in the cytosol before estrogen treatment. Proteins of the liver nuclear matrix from untreated or ethinyl estradiol-treated female rats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and probed with the monoclonal estrogen receptor antibody H222Sp gamma. A single prominent immunoreactive 67,000 mol wt band, indicating the presence of estrogen receptors, was found in the liver nuclear matrix of estrogen-treated animals. This band was detectable, but of much lower intensity, in the liver nuclear matrix of untreated animals. Liver cytosol estrogen receptor from untreated rats also migrated as a 67,000 mol wt band. These immunoreactivity data corroborated data obtained by [3H]estradiol-binding assays. Scatchard analysis of specific high affinity [3H]estradiol-binding sites showed high levels of these sites in the liver nuclear matrix of estrogen-treated rats and low levels in untreated rats. Therefore, both direct and indirect methods of receptor identification demonstrate the specific association of estrogen receptors with the nuclear matrix after estrogen treatment in vivo.

Animals↗
PubMed1987

Immunocytochemical localization of estrogen receptors in the normal male and female canine urinary tract and prostate.

We have used the monoclonal estrogen receptor (ER) antibody H222Sp gamma to localize ER by immunocytochemistry in frozen sections of the normal canine urinary tract of both sexes and of the normal prostate of the male. Striking regional heterogeneity of ER location was observed. In the urinary tract, specific ER staining was confined to nuclei of the transitional epithelium (mucosa) and subjacent stroma (submucosa) of the prostatic urethra in the male dog and of the proximal urethra in the female dog. In both sexes there was a gradient of ER staining intensity along these urethral segments. In the male, ER staining intensity was highest in the region of the verumontanum. The pattern and intensity of staining were similar in the male prostatic urethra and female proximal urethra, indicating a similar concentration of ER in these tissues, which have the same embryological origin. No specific staining was found in the kidney, ureter, bladder, or distal urethra of either sex. In the normal prostate, specific immunocytochemical ER staining was confined to nuclei of the prostatic stroma and prostatic ductal epithelium. Specific staining intensity appeared to be higher in the periurethral region of the prostate than in the periphery. No specific staining was found in the acinar epithelium of the prostate. Based on overall staining intensity there appeared to be a higher concentration of ER in the urethra than in the prostate. Scatchard analysis of [3H]estradiol binding confirmed a similar ER content in the urethra of male and female dogs and a higher ER content in the prostatic urethra than in the prostate itself (P less than 0.001). The location of ER in the normal canine prostate and prostatic urethra is consistent with the location of histological changes induced by estrogen administration, indicating that these immunoreactive ER probably represent biologically functional receptors.

Animals↗
PubMed1985

Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy.

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

Adenocarcinoma↗
PubMed1984

The relationship of androgen receptor levels to androgen responsiveness in the Dunning R3327 rat prostate tumor sublines.

The objective of this study was to determine whether androgen receptor levels in a transplantable animal model of prostatic adenocarcinoma correlated with androgen responsiveness of the tumor. This is the first comparative study of androgen receptor levels in 3 subcellular compartments (cytosol, nuclear salt-extractable and nuclear salt-resistant fractions) of 4 Dunning R3327 rat prostatic adenocarcinoma sublines that vary in their response to androgen ablation. Tumors were harvested from intact adult male rats in order to best approximate the human clinical setting in which receptor levels are quantitated prior to androgen ablative therapy. Only the nuclear salt-resistant (nuclear matrix) and total nuclear androgen receptor contents were significantly different among all tumor sublines. The properties of the tumors studied and their nuclear salt-resistant androgen receptor levels were as follows: H tumor--well-differentiated, slow growing, androgen-dependent, 63 +/- 11 fmol./mg. DNA; HI tumor--well-differentiated, slow growing, androgen-insensitive, 19 +/- 8 fmol./mg. DNA; G tumor--poorly-differentiated, fast growing, androgen-sensitive, 195 +/- 42 fmol./mg. DNA; and AT-2 tumor--anaplastic, fast growing, androgen-insensitive, no detectable receptors. There was no apparent quantitative relationship between androgen receptor content and tumor growth rates, which varied considerably irrespective of the androgen responsiveness of the tumor. However, there was a qualitative relationship between nuclear salt-resistant or total nuclear receptor content and androgen responsiveness. Higher levels of receptor (H and G tumor sublines) were associated with responsiveness to androgen ablation (cessation or slowing of growth, respectively), whereas lower levels of receptor (HI and AT-2 sublines) were associated with androgen insensitivity. These observations, based on relatively homogeneous tumors, may have important implications for human prostatic cancers which appear to be composed of heterogeneous cell populations.

Adenocarcinoma↗
PubMed1983

The nuclear matrix of the prostate contains acceptor sites for androgen receptors.

In cell-free binding experiments, androgen receptors of rat ventral prostate cytosol bound with high affinity (Kd = 10(-10) M) to a saturable number of sites (acceptors) associated with the nuclear matrix of the prostate. This binding was dependent on the presence of an activated androgen-receptor complex. In addition, the binding was tissue-specific, since liver nuclear matrix, in contrast, contained only 15-20% as many acceptors for androgen receptors as the prostate nuclear matrix. The prostate nuclear matrix contained approximately 75% of the number of total nuclear acceptor sites. Matrix-associated acceptor sites appear to be associated with the internal RNA-protein network of the nuclear matrix since the peripheral lamina component of the matrix bound only 30% as many receptors as the intact nuclear matrix.

Animals↗
PubMed1983

Estrogen receptors in human prostate: evidence for multiple binding sites.

The existence of estrogen receptors in the human prostate has long been a controversial issue. This may be explained partly by the apparent heterogeneity of estrogen-binding sites in prostatic tissue. We herein report on multiple binding sites for estrogens in cytosol as well as nuclear preparations of human prostatic tissues. One class of binding sites corresponds to the classical, high affinity estrogen receptor; the Kd for [3H]estradiol binding to the receptor was approximately 0.10 nM and the binding was specific for estrogens. The second class of binding sites appeared to have a Kd for [3H]estradiol in the range of 5-10 nM. This second, lower affinity class of binding sites markedly influenced studies of the classical receptor even at low ligand concentrations. Saturation analysis should be performed over a wide range of ligand concentrations (0.05-10 nM) to allow separation of the two binding components. Quantitation of estrogen receptor by a single point assay cannot be carried out accurately unless the low affinity binding component can be blocked. Multiple binding sites for estradiol were observed in the cytosol as well as in the nuclear salt extractable and salt-resistant compartments of normal, benign hyperplastic, and cancerous human prostates. Normal peripheral and cancerous prostates contained significantly (P less than 0.01) higher amounts of cytosol estrogen receptor compared to benign hyperplastic tissue.

Binding, Competitive↗
PubMed1983

Subcellular distribution of androgen receptors in human normal, benign hyperplastic, and malignant prostatic tissues: characterization of nuclear salt-resistant receptors.

Two populations of nuclear androgen receptors have been characterized in human prostatic tissue, and the levels and proportions of each were found to differ in normal prostates, benign hyperplastic prostates (BPH), and malignant prostates. A significant percentage (35 to 50%) of total nuclear androgen receptors was associated with the salt-resistant nuclear matrix fraction. The remainder were easily extracted from nuclei by 0.6 M KCl. Optimal conditions for measuring receptors in both compartments involved the use of an inhibitor of proteolysis (phenylmethylsulfonyl fluoride) and the omission of dithiothreitol from buffers. In the presence of dithiothreitol, most of the nuclear salt-resistant receptors were rendered salt extractable. Cytosol androgen receptor levels were not significantly different in normal, BPH, or malignant prostatic tissues. In contrast, the levels and distribution of nuclear salt-extractable and salt-resistant androgen receptors exhibited characteristic patterns. Compared to normal prostatic tissue, nuclear salt-extractable receptors were significantly elevated in both BPH and cancer, whereas nuclear salt-resistant receptors were elevated in BPH but not in cancer. The ratio of salt-extractable to salt-resistant receptors was approximately 1:1 in both normal and BPH tissues and 2:1 in cancer. In addition, a microassay has been developed for the measurement of androgen receptors in the three subcellular compartments of needle biopsy specimens of prostatic cancer. Studies are in progress to determine whether the measurement of both nuclear salt-extractable and salt-resistant receptors may improve the usefulness of receptor levels to predict the hormonal responsiveness of prostatic cancer.

Cell Nucleus↗
PubMed1982

Simultaneous measurement of progesterone and androgen receptors in human prostate: a microassay.

The characteristics of binding of radiolabeled progesterone, promegestone [17 alpha,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020)], medroxyprogesterone acetate (4-pregnen-6 alpha-methyl-17 alpha-ol-3,20-dione acetate), and methyltrienolone [17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one (MT)] to the progesterone receptor in human prostatic cytosol have been compared. MT binds to both androgen and progesterone receptors with high affinity (Kd = 0.9 and 0.6 nM, respectively). The binding of MT to the progesterone receptor can be blocked by adding an excess of unlabeled triamcinolone acetonide [9 alpha-fluoro-11 beta, 16 alpha, 17 alpha,21-tetrahydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide (TAC)]. The difference between the binding of [3H]MT in the absence and presence of TAC (i.e. [MT - (MT + TAC)] represents specific binding of MT to the progesterone receptor. Ligand specificity studies demonstrated that this binding was typical of a progesterone receptor. Furthermore, progesterone receptor levels measured in this way were comparable to those obtained using progesterone, R5020, or medroxyprogesterone acetate as labeled ligands. Progesterone receptor quantitation from the difference MT - (MT + TAC) is of particular advantage when simultaneous quantitation of progesterone and androgen receptors is desired in small tissue specimens since only three sets of incubations are required: [3H]MT, [3H]MT plus unlabeled TAC, and [3H]MT plus unlabeled MT (to measure nonspecific binding). Conditions are described for the application of this methodology to a microassay. A marked underestimate of progesterone receptor content was observed when incubation was terminated with hydroxylapatite compared to that measured when dextran-coated charcoal was used. The presence of comparable amounts of progesterone and androgen receptors in human prostatic cytosol deserves further investigation.

Binding, Competitive↗
PubMed1981

Mitogenesis in normal human fibroblasts by polyinosinic . polycytidylic acid and other synthetic acidic polymers: enhancement of action by glucocorticoids.

The ability of specific synthetic polyelectrolytes to act as mitogens for quiescent normal human fibroblasts in cultures is described. Of several acidic polymers tested, polyinosinic acid .polycytidylic acid (poly I.poly C) and dextran sulfate were the most effective in stimulating 3H]thymidine incorporation (2-to 10-fold). The concentration for a half-maximal effect (ED50) was 0.4 microgram/ml (0.8 nM) for poly I.poly C, and 1.7 microgram/ml (3.4 nM) for dextran sulfate. Single-stranded polyinosinic acid or polycytidylic acid had no effect. The time course of stimulation of DNA synthesis by these acidic polymers was similar to that for naturally occurring mitogens such as epidermal growth factor, beginning at about 18 hours and reaching a maximum rate 26 to 30 hours after the addition of polymer. Glucocorticoids that have an 11-beta hydroxyl group (e.g., dexamethasone) had no effect on DNA synthesis alone, but enhanced several-fold the mitogenic activity of poly I.poly C or dextran sulfate; the ED50 for dexamethasone was 0.75 ng/ml (1.9 nM). Glucocorticoids with an 11-keto group were inactive in this respect. The labeling index following treatment of cultures with poly I.poly C and dexamethasone was 14%, compared with a labeling index of 25% following stimulation by fetal calf serum. The extent of stimulation of DNA synthesis by poly I.poly C and dexamethasone was comparable to that induced by epidermal growth factor. It appears that both the poly I.poly C and dexamethasone are required for only a short period of time (approximately 3 hours) in order to produce maximal stimulation of DNA synthesis 30 hours later.

Anions↗
PubMed1980

The specific binding of estrogens and androgens to the nuclear matrix of sex hormone responsive tissues.

Specific sex steroid hormone binding sites have been identified in a discrete nuclear subfraction that is chromatin-depleted and salt-insoluble. This structure, referred to as the nuclear matrix, contains a residual pore complex-lamina, remnants of an internal network, and a residual nucleolus. The nuclear matrix of an estrogen-responsive tissue (chicken liver) and of an androgen target tissue (rat ventral prostate) contains binding sites for estradiol and dihydrotestosterone, respectively. The binding of steroids to these sites is saturable, high affinity (Kd approximately 10(-9) M), steroid-specific (liver binds estrogens, prostate binds androgens), and heat- and pronase-sensitive. The levels of these matrix-associated steroid binding sites change in response to manipulation of the hormonal status of the animal. Thus, the liver nuclear matrix of laying hens contains a significant number of estradiol binding sites, whereas that of untreated chicks or roosters contains very few sites. Treatment of chicks or roosters with a dose of estrogen that stimulates the liver to synthesize vitellogenin leads to a marked increase in the number of estradiol binding sites associated with the liver nuclear matrix. In the rat ventral prostate, the nuclear matrix-associated binding sites for dihydrotestosterone that are present in intact adults essentially disappear within 24 h after castration. Androgen replacement therapy restores, within 1 h, the number of matrix binding sites for dihydrotestosterone to normal levels. Inhibitors of proteolysis (phenylmethylsulfonyl fluoride) are essential for the protection of these androgen binding sites. Conditions that lead to the solubilization of the internal network material of the nuclear matrix also result in the extraction of 70 to 85% of the specific dihydrotestosterone binding sites from the matrix; a limited number of binding sites still remain associated with the peripheral lamina.

Animals↗
PubMed1979

The specific binding of estradiol to the nuclear matrix.

Recent studies on the eukaryotic nucleus have described the presence of a structural component termed the nuclear matrix, that appears to be involved in nucleic acid synthesis. This study will present evidence that the nuclear matrix of both the rat uterus and chick liver contains specific, high affinity, saturable estradiol binding sites.

Animals↗