metribolone and Prostatic-Hyperplasia

A transdermal SARM has a potential to have therapeutic benefit through anabolic activity in muscle while sparing undesired effects of benign prostate hyperplasia (BPH) and liver-mediated decrease in HDL-C. 2-Chloro-4-[(2-hydroxy-2-methyl-cyclopentyl)amino]-3-methyl-benzonitrile 6 showed the desired muscle and prostate effects in a preclinical ORX rat model. Compound 6 had minimal effect on HDL-C levels in cynomolgus monkeys and showed human cadaver skin permeability, thus making it an effective tool for proof-of-concept studies in a clinical setting.

Topics: Administration, Cutaneous; Anabolic Agents; Androgen Antagonists; Aniline Compounds; Animals; Cholesterol, HDL; Humans; Hypercholesterolemia; In Vitro Techniques; Liver; Macaca fascicularis; Male; Models, Molecular; Muscular Atrophy; Nitriles; Orchiectomy; Prostatic Hyperplasia; Rats; Skin Absorption; Structure-Activity Relationship

Androgen and progesterone receptors (AR and PR) are two determining factors in gonadal differentiation that are highly expressed in developing and mature gonads. Loss of AR results in XY sex reversal and mutations causing reduced AR activity lead to varying degrees of defects in masculinization. Female PR knockout mice are infertile due to ovarian defects. While much has been discovered about positive regulation of these receptors by coactivators little is known about repression of the transcriptional activity of AR and PR in the presence of agonists. In this study we assessed the effect of SMRT and DAX-1 on AR and PR activity in the presence of both agonists and partial antagonists. We show that SMRT and DAX-1 repress agonist-dependent activity of both receptors, and the mechanism of repression includes disruption of the receptor dimer interactions rather than recruitment of histone deacetylases. We demonstrate that endogenous agonist-bound PR and DAX-1 in T47D breast cancer cells and endogenous AR and DAX-1 in LNCaP prostate cancer cells can be coimmunoprecipitated suggesting that the interaction is physiological. Surprisingly, although DAX-1 represses partial antagonist activity of AR, it was ineffective in repressing partial antagonist induced activity of PR. In contrast to most reported repressors, the expression of DAX-1 is restricted. We found that although DAX-1 is expressed in normal human prostate, its expression is strongly reduced in benign prostatic hyperplasia suggesting that DAX-1 plays a role in limiting AR activity in prostate.

Topics: Animals; Binding Sites; Breast Neoplasms; COS Cells; DAX-1 Orphan Nuclear Receptor; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Hormone Antagonists; Humans; Hydroxamic Acids; Male; Metribolone; Mifepristone; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2; Promoter Regions, Genetic; Prostate; Prostatic Hyperplasia; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Receptors, Androgen; Receptors, Calcitriol; Receptors, Interferon; Receptors, Progesterone; Receptors, Retinoic Acid; Repressor Proteins; Testosterone Congeners; Tumor Suppressor Protein p53

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Topics: Adenocarcinoma; Calcitriol; Cell Division; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 3; Male; Metribolone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Retinoids; Testosterone Congeners; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Steroid autoradiographic techniques were used to visualize the distribution of androgen receptor-positive cells in human benign prostatic hyperplasia grown in organ culture. The tissue consisted of alveoli embedded in dense fibromuscular stroma and lined either with one row of cuboidal secretory cells or with several layers of epithelial cells. In some explants alveoli were seen lined with spindle-shaped cells. The autoradiographs showed that in most explants both epithelium and stroma contained androgen receptor-positive cells but that the uptake was less pronounced in the stromal cells. In alveoli lined with several cell layers the basal cells seemed to incorporate less androgen than secretory cells on the luminal side. In alveoli lined with spindle-shaped cells, these were always androgen receptor-negative.

Topics: Aged; Aged, 80 and over; Autoradiography; Dihydrotestosterone; Estrenes; Humans; Male; Metribolone; Middle Aged; Prostatic Hyperplasia; Receptors, Androgen; Testosterone Congeners

In order to delineate optimal conditions for the determination of androgen specific binding in human benign prostatic hypertrophy tissues (BPH), extracts of these tissues were prepared using buffers of different compositions. The binding of 5 alpha-dihydrotestosterone (DHT) and of methyltrienolone (R 1881) was analyzed using agar gel electrophoresis. This method allowed the detection of 3 different high affinity tissular binding peaks with similar specificity. Moreover, the inhibition by each of the competitors was also the same for both ligands. It could be demonstrated that none of the observed peaks resulted from the binding of 1 of the ligands to sex hormone binding globulin (SHBG) or to a progesterone receptor. Hypotheses about the possible origin of these peaks are discussed.

Topics: Binding, Competitive; Buffers; Charcoal; Cytosol; Dextrans; Dihydrotestosterone; Electrophoresis, Agar Gel; Enzyme Activation; Estrenes; Humans; Male; Metribolone; Molybdenum; Progesterone; Prostatic Hyperplasia; Protease Inhibitors; Receptors, Androgen; Tartrates; Temperature; Testosterone Congeners

Existing techniques for androgen receptor (AR) assay are complicated by cross-reactivity of ligand binding affinities that can lead to incorrect estimation of receptor concentration. Two most frequently used ligands are [3H]dihydrotestosterone [( 3H]DHT) and [3H]methyltrienolone [( 3H]R1881), which in addition to binding to AR also bind to sex hormone binding globulin (SHBG; Kd = 1.5 nM) and progesterone receptors (PgR; Human Kd = 1 nM, rat Kd = 6 nM) respectively. Triamcinolone acetonide (TMA) is commonly used to block binding of [3H]R1881 to PgR, however at high concentrations TMA itself will bind AR (Kd = 7 microM). We have developed a hybrid ligand method for the measurement of AR in the presence of SHBG and PgR. This method used [3H]R1881 as the high specific activity labelled tracer and DHT as the unlabelled competitor of specific AR binding. Using this assay, 20% of human colorectal carcinomas were found to contain AR.

Topics: Animals; Binding, Competitive; Colonic Neoplasms; Cytosol; Dihydrotestosterone; Estrenes; False Negative Reactions; Female; Humans; Male; Metribolone; Orchiectomy; Prostate; Prostatic Hyperplasia; Rats; Rats, Inbred Strains; Receptors, Androgen; Rectal Neoplasms; Sex Hormone-Binding Globulin; Triamcinolone Acetonide; Tritium; Uterus

We have used a novel receptor labeling and autoradiographic technique to identify the cell types in human benign prostatic hyperplasia (BPH) that contain androgen receptors, and we have found that androgen receptor localization is heterogeneous. Prostatic androgen receptors were labeled by incubating slide-mounted frozen tissue sections (10 micron thickness) with [3H]R1881 in vitro. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. Some of the sections were wiped from the slides for scintillation counting to validate that the procedure indeed measures total cellular androgen receptors of appropriate high affinity and androgen steroid specificity. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Autoradiograms were developed, fixed, and stained; silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis of human glandular BPH demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We anticipate that data obtained using this new method of steroid receptor autoradiography may provide fresh insight into the mechanism of hormonal regulation of the prostate.

Topics: Autoradiography; Cell Nucleus; Epithelium; Estrenes; Humans; Male; Metribolone; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Tritium

In rat uterus and prostate, 7 alpha, 17 alpha-dimethyl-19-nortestosterone (DMNT) binds to the androgen receptor specifically and with high affinity. However, this steroid does not bind to glucocorticoid receptors, since it does not displace binding of [3H]triamcinolone acetonide in calf thymus cytosol. In calf uterine and human breast tumor cytosols DMNT binds to the androgen and progesterone receptors, since binding of [3H] DMNT is displaced by unlabeled 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3, 20-dione triamcinolone acetonide, and 5 alpha-dihydrotestosterone (DHT). Conversely, binding of [3H]16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione is effectively competed for by unlabeled DMNT but not by DHT. The observed differences in binding of [3H]DMNT to rat and calf uterine cytosols suggest the species specificity of progesterone receptors. Unlike DHT, DMNT has no appreciable binding to human sex-steroid binding globulin. These findings suggest DMNT as a suitable ligand for measurement and characterization of androgen receptors in rat and human prostate.

Topics: Animals; Blood Proteins; Breast; Cattle; Cell Nucleus; Cytosol; Dihydrotestosterone; Estrenes; Female; Humans; Male; Metribolone; Nandrolone; Pregnenediones; Prostate; Prostatic Hyperplasia; Rats; Rats, Inbred Strains; Receptors, Androgen; Receptors, Progesterone; Substrate Specificity; Uterus

The heterogeneous histology of the normal and diseased human prostate is well established. This study has investigated variations in cytosol androgen receptor (AR) content throughout the diseased gland to establish the most suitable sites to obtain tissue for AR assay. Only then can AR be investigated as a potential predictor of response to endocrine treatment. In a transverse slice of an enucleated prostate showing benign prostatic hyperplasia (BPH) AR levels in 1-g segments varied from 109 to 1,212 fmol/g tissue (mean 483 +/- 273; median 406), with no negative areas. Areas of higher receptor concentrations corresponded to the glandular regions of sections obtained from a slice taken in juxtaposition; areas of low receptor concentrations corresponded to the stromal regions. A significant correlation (P less than .02) was observed between AR concentration and the proportion of glandular components of each segment. Specimens were also obtained from each of three sites from 38 prostates; 45% of all specimens contained AR; however, distribution of receptor throughout the prostate was uneven. AR were significantly more likely to be measured in the peripheral zone (71% positive) than in periurethral tissue (39% positive) whilst only 24% of specimens taken from the limit of the resection possessed AR binding capacity. Similar distribution patterns were observed in both benign and malignant prostates, although 16% more specimens from carcinomatous prostates contained receptor than did those from benign glands; this difference was maintained at each site. In addition receptor levels were consistently lower in benign than in malignant specimens. It is therefore desirable to know the histological composition of specimens used for AR measurement.

Topics: Cytosol; Estrenes; Humans; Male; Metribolone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Estrogen; Testosterone Congeners; Tritium

Accurate quantitation of androgen receptors requires a radioactive ligand which has affinity and specificity for the receptor and which is stable to metabolic enzymes. In this report, we have characterized the properties of 7 alpha,17 alpha-dimethyl-17 beta-hydroxy-4-estren-3-one (mibolerone) in human benign hyperplastic prostate cytosol and compared them to those of 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881). Mibolerone was found to have an affinity (Kd = 1.5 nM) greater than R1881. (Kd = 2.3 nM) for the androgen receptor in human prostate tissue. Surprisingly, mibolerone was found to bind with high affinity to the progesterone receptor in both human prostate (Kd = 5.9 nM) and rabbit uterus (Kd = 1.1 nM). However, binding to this receptor in both species could be blocked with a 500-fold excess of triamcinolone acetonide. [3H]Mibolerone binding to the androgen receptor was competed effectively with unlabeled dihydrotestosterone, R1881, and mibolerone but not by progesterone, diethylstilbestrol or R5020, in the presence of triamcinolone acetonide. Interestingly, mibolerone was more resistant to metabolism than R1881 in prostate cytosol when exposed to elevated temperatures (30 degrees C) for extended periods of time. However, when exposed to high-intensity ultraviolet irradiation, both compounds lost 50% of their binding ability in about 30 minutes. Mibolerone was found to have a very low affinity (Ki = 540 nM) for human sex steroid binding protein. These studies demonstrate that mibolerone is a useful ligand for androgen receptor assays. They also emphasize the need for including competitors of progesterone receptor binding in assays utilizing this steroid for androgen receptor measurements.

Topics: Animals; Chemical Phenomena; Chemistry; Dihydrotestosterone; Estrenes; Female; Humans; Male; Metribolone; Nandrolone; Promegestone; Prostate; Prostatic Hyperplasia; Rabbits; Receptors, Androgen; Receptors, Progesterone; Testosterone

Nuclear and cytosolic androgen receptor concentrations in tissues of human benign prostatic hypertrophy (BPH) were determined by use of methyltrienolone (R-1881) and 7 alpha,17 alpha-dimethyl-19-nortestosterone (DMNT) as radiolabeled ligands. Cytosolic R-1881-binding sites were 46.1 +/- 43 fmol/mg protein and nuclear R-1881-binding sites were 51.8 +/- 42 fmol/mg protein. DMNT-binding sites in cytosol were 44.3 +/- 38 fmol/mg protein and in nuclear extract 73.4 +/- 64 fmol/mg protein. No significant correlation was found between the number of R-1881- and DMNT-binding sites in either cytosol or nuclear extracts. Cytosolic or nuclear androgen receptor content was not significantly correlated with the percentage of epithelial or stromal cells as determined from the corresponding histological sections. In BPH tissue with marked cystic degeneration, very low androgen receptor levels were found.

Topics: Cell Nucleus; Cytoplasm; Cytosol; Epithelium; Estrenes; Humans; Male; Metribolone; Nandrolone; Promegestone; Prostate; Prostatic Hyperplasia; Radioligand Assay; Receptors, Androgen; Receptors, Progesterone; Testosterone Congeners

RU 27987 is a new ligand for progesterone receptor and binds in high affinity to nuclei of target tissues of progesterone. Using this compound, progestin-binding components in the benign hypertrophic human prostate were studied, and compared with those examined with R 5020, a conventional ligand, in the study of progesterone receptor. In cytosols, the binding affinity of RU 27987 was higher than that of R 5020, and the number of maximum binding sites for RU 27987 seemed to be large but correlated well with those of R 5020. The binder for RU 27987 sedimented at 8.6 S, and the binding was specific to progestational steroids, indicating that binding properties of this binder in the cytosols are identical to those for R 5020. Although there was no binding with R 5020 in the nuclear extract, a small amount of specific binding with RU 27987 was detected. However, the cytosol bound with RU 27987 was not retained in DNA Sepharose and no specific binder for RU 27987 in the nuclear extract was observed in a sucrose density gradient centrifugation. From these observations, it was assumed that the nuclear binding observed was attributable to contamination of the cytosolic binder. The results obtained in the present study suggest that the progestin-binding component in the benign prostatic hypertrophy is not the progesterone receptor but a high affinity binder for progestins whose physiological role is not clear at present.

Topics: Alpha-Globulins; Binding, Competitive; Cytosol; Estrenes; Humans; Kinetics; Male; Metribolone; Norpregnadienes; Progesterone Congeners; Progesterone-Binding Globulin; Promegestone; Prostate; Prostatic Hyperplasia; Steroids

The effect of a synthetic steroidal compound TSAA-291 (16 beta-ethyl-17 beta-hydroxy-4-estren-3-one) on the binding of methyltrienolone (R1881) and promegestone (R5020) to hyperplastic and neoplastic human prostate was investigated. TSAA-291 inhibits both androgen and progestogen binding to hyperplastic and neoplastic human prostate. Glycerol density gradient analysis revealed that the inhibition of promegestone (R5020) binding by TSAA-291 was significantly greater than that of methyltrienolone (R1881) in both hyperplastic and neoplastic human prostate. The nature of the inhibition was competitive as determined by Scatchard analysis and double reciprocal plots. Comparison of the Ki values for the inhibition by TSAA-291 of R1881 binding (3.2 X 10(-7) M) and of R5020 binding (2.0 X 10(-8) M) suggests that TSAA-291 binds to progesterone receptor with a greater affinity than to androgen receptor. Our results suggest that the effectiveness of the drug in the treatment of benign hyperplasia might be due not only to its anti-androgenic properties but also due to its ability to inhibit progesterone receptor.

Topics: Binding, Competitive; Centrifugation, Density Gradient; Cyproterone; Cyproterone Acetate; Dihydrotestosterone; Estrenes; Humans; Male; Mathematics; Metribolone; Nandrolone; Norpregnadienes; Promegestone; Prostatic Hyperplasia

The binding nature of mibolerone in cytosols and nuclear extracts from hypertrophic human prostate was examined in comparison with that of R 1881. The binding of mibolerone in the cytosol and nuclear extract was single and of high affinity when evaluated by the method of Scatchard (1949). Binding of mibolerone with testosterone-binding globulin was not detected. The sedimentation coefficients of the binder for mibolerone in the cytosol and nuclear extract were 10.6 S and 3.6 S, respectively. When triamcinolone acetonide was induced in the binding medium, inhibition of mibolerone binding in the cytosol by testosterone and dihydrotestosterone was potentiated and this may imply that the binding observed in the presence of triamcinolone acetonide was responsible for the binding of the androgen receptor. In the nuclear extract, the binding was attributable mainly to the androgen receptor irrespective of the presence or absence of triamcinolone acetonide. These properties of the binding observed in the hypertrophic human prostate were almost same as those of the binding with R 1881. Although maximum binding sites measured using mibolerone were correlated with those using R 1881 in the cytosols as well as in the nuclear extracts, the values obtained with mibolerone were slightly greater than those with R 1881. Thus, mibolerone seems to be a suitable ligand for measuring the androgen receptor, but when compared with R 1881 no special merits in using mibolerone were detected.

Topics: Cell Nucleus; Cytosol; Estrenes; Humans; Male; Metribolone; Nandrolone; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid

Androgen binding (cytosol and nucleus) was measured in tissue obtained from 223 untreated patients with proven prostate cancer (199 primary tumor, 24 malignant lymph nodes), 19 patients with hormone refractory cancer, and 46 patients with benign prostatic hyperplasia (BPH). The mean binding in both the cytosol and nucleus was significantly higher for patients with cancer than for those with BPH. Binding appeared to correlate with tumor stage. Androgen binding in malignant nodes can differ from that in the primary tissue and can vary from node to node in the same patient. Results obtained from an assay using a single saturating concentration of R1881 correlated well with those calculated from a full six-point Scatchard analysis when an adequate amount (500 mg) of tissue was available. However, binding results obtained from a single-point analysis performed on needle biopsy specimens (about 50 mg) obtained before complete surgical removal of the prostate correlated poorly with those derived from a full six-point analysis performed on tissue (500-1000 mg) removed from the center of the malignancy. Androgen binding in nuclear extracts of histologically benign tissue adjacent to the malignancy was significantly higher than in nuclear extracts of BPH tissue. Cytosolic androgen binding in tissue removed from patients who were refractory to hormonal therapy was higher than in tissue from untreated cancer patients. The binding of estradiol by extracts of benign and malignant prostate tissue was low or absent and, thus, did not appear to be a significant phenomenon.

Topics: Androgens; Biopsy; Cell Nucleus; Cytosol; Estradiol; Estrenes; Humans; Lymphatic Metastasis; Male; Metribolone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Steroid

The assay of hormone receptors in neoplastic tissues recognizes their sensitivity or autonomy with regard to the hormonal action. The hormone dependency of the activity of neoplastic tissues can be employed to select the cases having the highest probability to be benefited by the treatment with anti-hormones, castration or hypophysectomy, as in breast and prostatic cancers. Examining the case for prostatic cancer, the authors indicate that there is a good correlation between the predictive assay and the clinical evolution of the treated prostatic carcinoma employing the determination of cell receptors for 5-alpha-dihydrotestosterone (DHT) by exchange at 15 degrees C with the synthetic steroid methyltrienolone. The electrophoretic determination of binding of DHT to cell receptor a 4 degree C is a much less efficacious procedure.

Topics: Diethylstilbestrol; Electrophoresis, Agar Gel; Estramustine; Estrenes; Humans; Male; Metribolone; Orchiectomy; Prognosis; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Estradiol

To further characterize human prostatic androgen receptor, nuclei were isolated from normal prostate (no. = 3) and benign prostatic hyperplasia specimens (no. = 10). High ionic strength (0.6 M KCl) treatment of nuclei released nuclear extractable androgen receptor and DNase I digestion then yielded nuclear matrices. Androgen receptor was quantified in the nuclear extract and nuclear matrix preparations by Scatchard analysis of specific R1881 binding. Only 1 of the 3 normal tissues had extractable androgen receptor (113 fmol. per gm. of tissue) while the mean concentration of extractable androgen receptor for BPH was 189 fmol. per gm. of tissue. The mean concentrations of matrix-bound androgen receptor were 325 fmol. per gm. of tissue and 548 fmol. per gm. of tissue for normal and hyperplastic prostate, respectively. The androgen binding sites on nuclear matrix may represent the functional intranuclear androgen receptor and a characterization of these sites may provide an understanding of the etiology of BPH.

Topics: Adult; Binding Sites; Cell Nucleus; Estrenes; Humans; Male; Metribolone; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Testosterone Congeners

The benign hyperplasia of the prostate is a manifestation of aging, involving the accumulation, within the gland, of dihydrotestosterone, the probable mediator of the hyperplasia. Binding studies were performed on the cytosolic androgenic receptor of the rat prostate using [3H]methyltrienolone as a ligand. The binding of [3H]methyltrienolone at 5 nM, was inhibited by various drugs, such as methyltrienolone and cyproterone acetate. Permixon, a liposterolic extract of the plant, Serenoa Repens B, inhibits competitively the binding to the cytosolic receptor of the rat prostate. Various vegetable and mineral oils, the plant steroid: beta sitosterol and the antiprostatic drug: Tadenan, were all found to be inactive. The antiprostatic activity of Permixon shown in animal studies and controlled clinical trials, may thus result from a direct action at the cytosolic receptor.

Topics: Animals; Cytosol; Estrenes; Kinetics; Male; Metribolone; Plant Extracts; Prostate; Prostatic Hyperplasia; Rats; Receptors, Androgen; Receptors, Steroid; Serenoa

Topics: Adult; Aged; Cell Nucleus; Cytosol; Dihydrotestosterone; Estrenes; Humans; Male; Metribolone; Middle Aged; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Subcellular Fractions

Several publications indicate the dihydrotestosterone content of hyperplastic prostatic tissue from man and dog is increased. There is limited information concerning an abnormality in the androgen receptor content in this disorder. In men, we determined the androgen receptor concentration in cytosol, nuclei, the nuclear matrix, and nuclease solubilized salt extract fractions from benign prostatic hyperplasia (BPH) and normal prostate. Nine BPH cases (age 61 +/- 7, mean +/- SD) and seven controls (age 29 +/- 10.6) were compared. Prostates were obtained during autopsy performed within 12 h after death and frozen at -70 degrees C until analysis. Four grams of tissue were homogenized and centrifuged for separation of nuclei and cytosol. Androgen receptors were estimated with an improved assay procedure. The androgen receptor content per mg of DNA in whole nuclei and in the salt extract fraction (P less than 0.01 and less than 0.05 respectively) was higher in BPH cases than in controls. Cytosol did not show a significant difference. For both groups, receptors were undetectable in the nuclear matrix. We speculate that increased androgen receptor bound to chromatin of hyperplastic tissue may be causally related to the development of BPH in aging man.

Topics: Adult; Aged; Aging; Cell Nucleus; Cytosol; Estrenes; Humans; Kinetics; Male; Metribolone; Middle Aged; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Testosterone Congeners

During the course of another investigation, three dogs had been castrated 3 months previously. Upon completion of the experiment, it was discovered that one dog presented a spontaneous prostatic adenocarcinoma of intraalveolar proliferative type at histology. Prostate weight of this dog before castration was estimated to be 22 g by tridimensional measurement at laparotomy and remained relatively constant (19 g) 3 months after castration. These results indicate that if regression had occurred in some cell populations (androgen-dependent) it was only partial and masked by growth of androgen-independent cells. Analysis of 12 individual steroids in peripheral blood and in prostatic tissue attested of a normal adrenal secretory activity. A series of 15 hydrolytic enzymes along with receptors for androgen, estrogen, and progesterone, were determined in prostatic tissue obtained at sacrifice. Enzymatic activities were those of typical epithelial cells, and most of them remained relatively high despite low levels of circulating testosterone. However, two markers of androgen action in dog prostate, acid phosphatase and arginine esterase, were significantly reduced. Receptor levels were similar to those of castrated animals. Thus, cancer cells had probably retained some androgen sensitivity.

Topics: Adenocarcinoma; Animals; Castration; Cell Nucleus; Cytosol; Disease Models, Animal; Dogs; Estradiol; Estrenes; Male; Metribolone; Promegestone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Steroid; Testosterone Congeners; Time Factors

In this manuscript we have characterized the androgen receptor from human benign prostatic hypertrophied (BPH) tissue. BPH tissue was obtained fresh from surgery, ground, and frozen in liquid nitrogen. Tissue was homogenized in 10 mM Tes buffer (pH 7.4 at 25 degrees C) containing 0.25 M sucrose, 20 mM Na2MoO4, 12 mM monothioglycerol, and 1.5 mM EDTA. Cytosol was labeled with (3H)-R1881 (0.5-10 nM) +/- a 100-fold excess of R1881 and a 500-fold excess of triamcinolone acetonide to identify specific androgen receptor sites. An exchange assay was developed, whereby maximum binding was achieved by raising the temperature to 30 degrees C for 15 min. A high-affinity binding protein was detected (Kd = 1.3 +/- 0.8 nM) which had a binding capacity of 15 +/- 7 fmol/mg protein. Binding was specific for androgens with the following Ki (nM) values: R1881 (0.09), dihydrotestosterone (6), testosterone (50), progesterone (92), estradiol (100), epitestosterone (860), and cortisol (greater than 1,000). Under high ionic conditions the sedimentation coefficient of the receptor was 4.1 S. Higher S values were not observed in the presence of the following protein inhibitors: leupeptin (20 or 150 microM), iodoacetamide (10 mM), DFP (5 mM), PMSF (1 mM), bacitracin (0.1 mM), antipain (0.1 mM), aprotinin (1 TIU/ml). Gel filtration analysis revealed a Stokes radius of 6.5 nm. These data indicate a Mr of 120,000 and a f/fo of 2.01. The receptor eluted from a chromatofocusing column at a pH of 6.8. The receptor bound to phosphocellulose and DNA cellulose after being heat-treated for 15 min at 30 degrees C. These results suggest that BPH tissue contains an androgen receptor which is similar to receptors in other androgen-responsive tissues.

Topics: Binding, Competitive; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry, Physical; Dihydrotestosterone; Estrenes; Humans; Male; Metribolone; Molecular Weight; Prostatic Hyperplasia; Protease Inhibitors; Receptors, Androgen; Receptors, Steroid; Testosterone; Triamcinolone Acetonide

Direct measurement of the binding of endogenous androgens to the androgen receptor of human tissues has not been possible because of contamination of tissue with traces of plasma proteins, such as testosterone-binding globulin (TeBG), that contain more androgen-binding capacity than does the receptor itself. Molybdate is known to stabilize the 8-9S forms of other steroid hormone receptors. We took advantage of this phenomenon to characterize the androgen receptor of hyperplastic prostates removed at surgery, using sucrose density gradient centrifugation in a vertical rotor. In 10 mM sodium molybdate, the androgen receptor sediments as a distinct 9.2 +/- 0.5S moiety, easily separable from TeBG. Unlike TeBG, the 9S receptor is not removed by absorption with Concanavalin A. [3H]Dihydrotestosterone (3H-labeled 17 beta-hydroxy-5-alpha-androstan-3-one) binding to the 9S receptor is not removed by absorption with Concanavalin A. [3H]Dihydrotestosterone (3H-labeled 17 beta-hydroxy-5 alpha-androstan-3-one) binding to the 9S receptor is not competed for by excess triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetonide) or promegestone (17,21-dimethyl-19-non-pregna-4,9-diene-3,-20-dione), which are known to bind to the progestin receptor. In contrast, [3H]methyltrienolone (3H-labeled 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binds to both androgen and progestin receptors, and consequently, the binding of this ligand to the androgen receptor was assessed in the presence of a 500-fold excess of triamcinolone acetonide. The amounts of 9S binding (7.8 and 5.8 fmol/mg protein) are similar for dihydrotestosterone and methyltrienolone. The amount of 9S binding of testosterone to the receptor was also similar to that of dihydrotestosterone, but the affinity of testosterone for the 9S receptor was only a fifth or less of that for dihydrotestosterone. The observation that testosterone binds less avidly than dihydrotestosterone to the receptor may explain the role of dihydrotestosterone formation in androgen physiology.

Topics: Centrifugation, Density Gradient; Cytosol; Dihydrotestosterone; Estrenes; Humans; Kinetics; Male; Metribolone; Molybdenum; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Testosterone; Triamcinolone Acetonide

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.

Topics: Cell Nucleus; Deoxyribonuclease I; Endodeoxyribonucleases; Estrenes; Humans; Male; Metribolone; Microchemistry; Molybdenum; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Steroid; Tissue Distribution

Topics: Aged; Binding, Competitive; Cytosol; Estrenes; Humans; Male; Metribolone; Middle Aged; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid

No difference was found in the values of the equal constants of dissociation and the number of sites specifically binding [3H] DHT and [3H] R1881 in the cytosols of tumor cells and human prostatic nodular hyperplasia. The above data suggest that in the determination of cytoplasmic androgen receptors in healthy and tumorous tissues of the target organs one can use both natural (DHT) and synthetic (R1881) steroids precipitating the hormone-receptor complex with protamin sulphate.

Topics: Dihydrotestosterone; Estrenes; Humans; Male; Metribolone; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Steroid; Testosterone Congeners

Androgen-binding proteins in the cytosol of human benign prostatic hypertrophic tissue were studied with 3H-dihydrotestosterone (DHT) and 3H-methyltrienolone (R 1881) as ligands. In the prostatic cytosol, saturable binding proteins with high affinity to 3H-DHT or 3H-R1881 were found by Scatchard analysis. The dissociation constant (Kd) for 3H-DHT was 9.4 X 10(-10)M, and the maximum number of binding sites (B max) was 55 fmoles/mg protein; the Kd for 3H-R1881 was 5.1 X 10(-9)M, and B max was 25.5 fmoles/mg protein. Competitive binding assay for each binding protein with various non-labeled steroids, demonstrated a specificity for DHT or R1881. The binding of prostatic cytosol and 3H-DHT was eluted by Sephadex G-200 column chromatography in 2 places, one in the void volume and one near the IgG fraction. The binding with 3H-R1881 was eluted in the void volume, the elution near the IgG fraction being very small, which suggests a non-specific binding. The binding of prostatic cytosol and 3H-DHT increased with increase in the concentration of Ca2+ or Mg2+ (0-12 mM), but the binding with 3H-R1881 decreased conversely. This change was studied with Sephadex G-200 column chromatography; the binding with 3H-DHT showed a decrease of the peak in the void volume and marked increase of the peak near the IgG fraction with increase in the concentration of both ions, whereas the binding with 3H-R1881 only showed a decrease of the peak in the void volume. Ca2+ had a greater influence than Mg2+ on the change of these bindings. These results indicate a change of binding protein due to ionic action, suggesting also the possibility of the presence of a substance in the cytosol which changes the properties of the binding protein dependent on Ca2+.

Topics: Androgen-Binding Protein; Calcium; Carrier Proteins; Cytosol; Dihydrotestosterone; Estrenes; Humans; Ions; Magnesium; Male; Metribolone; Prostate; Prostatic Hyperplasia

In an effort to determine whether human benign prostatic hyperplasia (BPH) is characterized by an increase in androgen receptor content, the levels of nuclear and cytosolic androgen receptors were quantitated in normal prostatic tissue obtained from five young men (mean age /+- SEM, 26 +/- 3 yr) and in hyperplastic (periurethral) and peripheral prostatic tissues obtained from nine older men (mean age, 62 +/- 2 yr). There was no significant difference between the cytosolic or nuclear androgen receptor content of the hyperplastic, peripheral, or normal prostatic tissue. Thus, in this study we were unable to identify an increase in androgen receptor content in BPH. These findings fail to support the hypothesis that increases in androgen receptor content are involved in the pathogenesis of human BPH.

Topics: Adult; Aged; Cell Nucleus; Cytosol; Dihydrotestosterone; Estrenes; Humans; Kinetics; Male; Metribolone; Middle Aged; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Testosterone Congeners

Cytosol of the benign hypertrophic human prostate was prepared in a low salt medium and then the concentration of salt was increased to 0.4 M with KCl (0.4 M KCl-cytosol). This preparation showed a high affinity binding to R 1881 and the binding was specific for androgens. These results suggest that the binding of the preparation to R 1881 was due mainly to the cytosolic androgen receptor. The R 1881 binding component in the 0.4 M KCl-cytosol was sedimented at 3S by sucrose density gradient centrifugation. The small sedimentation coefficient of the binder seems to be due to the high concentration of salt and not to degradation by proteolytic enzymes in the preparation. The molecular weight, Stokes radius and frictional ratio of this binding component were 32,000, 25.9 A and 1.24, respectively.

Topics: Cytosol; Estrenes; Humans; Male; Metribolone; Molecular Weight; Potassium Chloride; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Time Factors

A 3-carboxymethyloxime conjugate of R 1881 was conjugated with bovine serum albumin and reacted with fluorescein isothiocyanate. Tissue sections from patients with benign prostatic hypertrophy were incubated with this compound. Fluorescence was observed in the cytoplasm of gland cells, although both positive and negative cells were distributed evenly in some areas. Neither the stroma nor the nuclei of epithelial cells were fluorescent. Preincubation with a 20 fold molar excess of R 1881-carboxymethyloxime-bovine serum albumin, 1 microM of R 1881, or 1 microM of triamcinolone acetonide diminished fluorescence of the section. Bovine serum albumin conjugated with fluorescein isothiocyanate did not stain the section. Therefore, the fluorescence observed in the sections seems to be attributable to the high affinity binding of R 1881. The intensity and number of fluorescent cells and the R 1881 binding estimated by a biochemical method were well correlated.

Topics: Androgen-Binding Protein; Androgens; Binding Sites; Carrier Proteins; Cell Division; Cytoplasm; Cytosol; Estrenes; Fluorescence; Histocytochemistry; Humans; Male; Metribolone; Prostate; Prostatic Hyperplasia; Testosterone Congeners

The binding of dihydrotestosterone, R 1881 and R 5020 was examined in cytosols of normal, benign hypertrophic and cancerous tissues of the human prostates. Almost all samples obtained by open operation showed high affinity binding to these ligands. Dissociation constants of the binding to these ligands were approximately 10(-9) M irrespective of the pathological state of the prostates. Maximum binding sites for dihydrotestosterone seemed to be greater in normal tissues than in the pathological ones. However, maximum binding sites for R 1881 and R 5020 were not significantly different among the normal and pathological prostates examined in the present study. Moreover, some correlation was observed between the maximum binding sites for R 1881 and those for R 5020. The samples resected by TUR seemed to be inadequate for analyses of androgen binding.

Topics: Cytosol; Dihydrotestosterone; Estrenes; Humans; In Vitro Techniques; Male; Metribolone; Norpregnadienes; Promegestone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Testosterone Congeners

Topics: Cell Nucleus; Cytoplasm; Dihydrotestosterone; Estrenes; Glycine; Heparin; Humans; Isoelectric Focusing; Male; Metribolone; Prostate; Prostatic Hyperplasia; Receptors, Androgen; Receptors, Steroid; Trypsin

Dihydrotestosterone-binding protein in cytosols from human prostates was different from the R1881- and R5020-binding proteins. The R1881- and R5020-binding proteins in the cytosol were very similar and most of the binding sites for R1881 were capable of binding R5020. However, nuclear extracts showed equal binding to dihydrotestosterone and R1881, but not to R5020, suggesting that there might not be a progestin receptor in the human prostate. Maximum binding sites to dihydrotestosterone, R1881, and R5020 in cytosols were very similar among "normal" and pathological prostates except that dihydrotestosterone binding was higher in "normal" prostates than in the pathological ones. Histochemically, R1881-binding was observed in the epithelial cells and in malignant cells, but not in the stroma.

Topics: Androgen-Binding Protein; Carrier Proteins; Cell Nucleus; Cytosol; Dihydrotestosterone; Estrenes; Humans; Male; Metribolone; Promegestone; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms

The binding of R 1881 in cytosols from the normal, benign hypertrophic and cancerous human prostates was examined in the presence of molybdate and triamcinolone acetonide. The addition of 10 mM Na2MoO4 resulted in an increase in the stability of the binder without any change in Kd. Triamcinolone acetonide added to the incubation of cytosol with R 1881 modified the inhibition pattern by other steroids, and the binding in the presence of triamcinolone acetonide exhibited the characteristics of androgen receptor. The complex of cytosol and R 1881 formed in the incubation in the presence of triamcinolone acetonide was sedimented at 8.5S. Kd of the binding to R 1881 of the normal and pathological prostates was almost identical, but maximum binding sites of the androgen receptor were larger in benign hypertrophic and cancerous prostates than in normal tissues. For the assay of binding capacity in needle biopsy specimens, one point determinations were performed using 2.5 nM R 1881 as the ligand. The number of binding sites obtained by this method were well correlated with those obtained by the Scatchard plot in various prostatic tissues.

Topics: Cytosol; Dihydrotestosterone; Estrenes; Humans; Kinetics; Male; Metribolone; Molybdenum; Progesterone Congeners; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Steroid; Triamcinolone Acetonide

Topics: Adenocarcinoma; Binding, Competitive; Cell Nucleus; Cytosol; Estrenes; Humans; Kinetics; Lymph Nodes; Male; Metribolone; Neoplasm Metastasis; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Receptors, Androgen; Receptors, Steroid

Cytosol of human benign prostatic hypertrophy bound to R 1881 in a high affinity manner. Most of the protein which bound to R 1881 was recovered in the precipitate of a 0-30% saturation of ammonium sulfate, and was eluted in the void volume on a Sephadex G-200 column. The binding of cytosol to R 1881 was more inhibited by progestins than by dihydrotestosterone and estradiol-17 beta. The binder therefore seemed to be different from dihydrotestosterone-binding protein. The R 1881-binding component extracted from the nuclei by 0.4M KCl bound also to dihydrotestosterone in a high affinity manner. Cytosol prelabeled with R 1881 was bound to the nuclei in a nonsaturable process, and the extraction pattern of R 1881 by 0.4M KCl from the nuclei was almost identical to that in the case of dihydrotestosterone as the ligand. These results suggested that a part of the cytosolic protein which bound to R 1881 entered the nuclei where it bound to nuclear such components as dihydrotestosterone-binding protein.

Topics: Cell Extracts; Chromatography, Gel; Cyproterone; Cytosol; Dihydrotestosterone; Estrenes; Humans; Male; Metribolone; Prostatic Hyperplasia; Protein Binding; Tritium