Nuclear androgen receptors in the prostate of male Praomys (Mastomys) Natalensis

Summary The binding of dihydrotestosterone (DHT) within the nuclear fraction of the prostate of male Praomys (Mastomys) Natalensis has been investigated. Using in vivo or in vitro labelling with ³H-DHT, the presence of a receptor protein having a sedimentation coefficient of 3.0 S was demonstrated. The binding was shown to be specific towards DHT and could not be found in control tissue. Analysis of radiometabolites associated with the steroid receptor complex demonstrated that the majority of the bound steroid was DHT. The similarity between this steroid receptor complex and that of rat ventral prostate, together with the potential use of this experimental model are discussed.     

Chemical demonstration of nuclear androgen receptor following affinity chromatography with immobilized ligands

With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate. After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl. Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining. To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate. Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein. About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml). The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated. Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis. In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10%. The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection. We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale.     

Androgen steroid-receptor binding in the canine epididymis

The epididymis of the dog has been studied with regard to its ability to bind androgens with a high affinity and low capacity. A proteinaceous molecule was demonstrated in the high-speed supernatant fraction of disrupted cells that bound DHT and sedimented in a sucrose density gradient with a sedimentation coefficient of 8S with respect to bovine serum albumin. This binding protein was able to transport DHT to the nucleus at elevated temperatures where it sedimented as a 4S in a 5-20% sucrose density gradient. Even though this protein appears to be similar to other androgen receptors in other accessory sex organs in a variety of mammalian tissue, it is different from androgen-binding protein (ABP). The apparent specificity of the receptor protein for androgens was demonstrated using competition studies. The molecular configuration of the steroids was found to be instrumented in this selectivity. A reduced affinity of the androgen receptor was found when the steroid did not possess a hydroxyl group at the 17 beta-position and a 3-oxo-group irrespective of the degree of saturation of the A-ring. Studies concerning the physical properties of the androgen binder revealed a protein with a molecular weight of 220,000, a Stokes' radius of 54A, and a frictional ratio of 1.35.     

Assay of nuclear androgen receptor in human prostate.

Nuclei were recovered from normal and hyperplastic human prostates and from well differentiated carcinoma using triton N-101 and discontinuous sucrose density gradient centrifugation. The nuclei were sonicated and dissolved in 2-([2-hydroxy-1,1-bis(hydroxymethyl)ehtyl]amino)ethane sulfonic acid buffer, pH 7.0, containing 0.6 M. sodium chloride. After incubation of the nuclear extract in the presence of 2 to 20 nM. 3H-dihydrotestosterone at 4C for 18 hours, an androgen receptor was isolated by Sephadex G-25/G-200 dual-column chromatography. The receptor demonstrated greater specificity for testosterone and dihydrotestosterone than for cortisol, progesterone and 17beta-estradiol. It was characterized by a sedimentation coefficient of 3 S and a Kd of 4.5 x 10(-9) M. The mean concentration of the nuclear androgen receptor, in terms of molecules per nucleus, was normal prostate-1,000, hyperplastic prostate-1,400 and well differentiated carcinoma-1,900. With this assay the problems associated with the measurement of a cytoplasmic androgen receptor can be avoided.     

Finasteride does not prevent bladder cancer: A secondary analysis of the Medical Therapy for Prostatic Symptoms Study

Background

Preclinical models have demonstrated that androgen receptor modulation can influence bladder carcinogenesis with an inverse association observed between serum androgen levels and bladder cancer (BC) incidence. It is still unclear whether 5α-reductase inhibitors, by preventing the conversion of testosterone to dihydrotestosterone, have a similar effect. This study aims to evaluate whether dihydrotestosterone-mediated androgen activity has an impact on BC incidence in a cohort of men included in a clinical trial of finasteride vs. placebo with rigorous compliance monitoring.

Androgen and glucocorticoid receptors in the pollard prostate adenocarcinoma cell lines

Both the androgen and glucocorticoid receptors of the Pollard prostate adenocarcinoma cell lines (PA-II and PA-III) have been examined and characterized. Both cell lines contain distinct binding sites for 5α-dihydrotestosterone (DHT) and dexamethasone (DM). The specific binding of the ligands to the whole cell was saturable, and Scatchard analysis showed a single class of high-affinity and low-capacity sites. (³H)-DHT was bound to a cytosolic macromolecule which sedimented at 7S after sucrose density gradient centrifugation in low salt buffer, and was completely displaced by radioinert DHT. By means of competition studies, the androgen receptor could be distinguished from that for the glucocorticoids. The DNA and protein synthesis of both tumor lines were significantly enhanced after exposure to DHT.     

Estrogen Steroid-Receptor Binding in the Canine Epididymis

The epididymis of the dog has been studied with regard to its ability to bind estrogens. A proteinaceous molecule was demonstrated in the high-speed supernatant friction of disrupted cells that bound E₂β and sedimented in a low salt sucrose density gradient with a coefficient of variation of 7-8S with respect to bovine serum albumin. This binding protein was able to transport E₂β to the nucleus at elevated temperatures, where it is sedimented as a 4S in a 5–20% sucrose density gradient. The specificity of the receptor protein for estrogens was demonstrated using competition analysis. Studies concerning the physical properties of the estrogen binder revealed a protein of molecular weight of 200,000, Stokes' radius of 51 Å and a fractional ratio of 1.32.
Östrogen-Steroid-Rezeptorbindung im Nebenhoden des Hundes

Zusammenfassung

Der Nebenhoden des Hundes wurde im Hinblick auf seine Fähigkeit, Östrogene zu binden, untersucht. Im Überstand hochtourig zentrifugierter zertrümmerter Zellen, die E₂β gebunden hatten, wurde ein proteinartiges Molekül nachgewiesen, das in einem niedrigen Salz-Saccharose-Dichtegradienten mit einem Sedimentationskoeffizienten von 7-8S bezogen auf Rinderserumalbumin sedimentiert wurde. Dieses Bindungsprotein war in der Lage, E₂β bei erhöhten Temperaturen zum Kern zu transportieren, wo es in einem 5–20% Saccharose-Dichtegradienten bei 4S sedimentierte.
Die Spezifität des Rezeptorproteins für Östrogene wurde mittels Kompetitionsanalyse nachgewiesen. Die Untersuchungen bezüglich der physikalischen Eigenschaften ergaben ein Protein mit dem MG 200.000, einem Radius von 51 Å und einem Friktionsverhältnis von 1.32.     

Growth inhibition by diethylstilbestrol and relapse of the Noble rat prostatic tumor

The response of androgen-sensitive Noble (Nb) rat prostatic adenocarcinoma [2Pr-121D (1)] to varying doses (50 approximately 1,000 micrograms/kg body weight) of diethylstilbestrol (DES) was investigated and characterized with respect to cytosol and nuclear androgen binding profiles, histology and pattern of relapse. Inhibition of tumor growth was closely related to the dose of DES. Treatment at all but the lowest dose (50 micrograms/kg) initially caused tumor regression, whereas serum testosterone concentrations in all groups, including that receiving the lowest dose, were decreased to castrate levels. Histologically, while extensive cellular destruction was clearly evident at higher doses of DES, some active tumor cells appeared to survive. Tumors eventually relapsed when higher doses of DES were stopped or with the continued administration of low doses. The cytosol dihydrotestosterone (DHT) receptor in this tumor line, as determined by sucrose density gradient, dextran charcoal and Sephadex column methods, was negative. Nuclear binding, however, was positive. Salt-extractable and salt-resistant fractions of nuclei derived from the untreated primary tumor and relapsed tumor following DES treatment contained high affinity androgen receptor. Comparison of binding constants revealed no significant differences. Our findings, based on the Nb rat prostatic tumor model, indicate that DES acts not only by eliminating circulating testosterone, but also by a direct cytotoxic effect on malignant cells. The results also suggest the lack of an apparent relationship between the loss of hormone responsiveness associated with recurrence of prostatic tumor growth and nuclear androgen binding parameters.     

Purification of the human prostate androgen receptor. II. Characterization of the human prostate androgen receptor

For improved purification of human prostate androgen receptors (AR), the effect of molybdate, NaSCN and heparin on the binding capacity of human prostate cytosol AR was investigated. When 10 mM of molybdate in final concentration was added to cytosol, the increase in the maximum binding sites of AR to 3H-R1881 was approximately 60%, while the dissociation constant was rather increased. With sucrose density gradient centrifugation, the cytosol with molybdate added showed a constant peak of 8 approximately 9 S. However, cytosol not enriched with molybdate did not show any significant peak. These results constitute evidence that molybdate has a stabilizing effect of the AR. When 0.1 approximately 0.5 M of NaSCN was added to the cytosol, the dissociation as well as the association of the 3H-R1881-AR complex showed a tendency to accelerate. However, at a concentration of 0.3M and over NaSCN, the binding capacity of the AR rapidly disappeared. When the NaSCN was removed with gel filtration, the binding capacity of the AR recovered partially. The effect of NaSCN on the AR was therefore different from that on the estrogen receptors. Four mg/ml of heparin accelerated the dissociation of the 3H-R1881-AR complex. Furthermore, with the addition of 4 mg/ml of heparin, the maximum binding sites of AR decreased to 56%. The above results make it clear that molybdate, NaSCN and heparin have various effects on human prostate cytosol AR, which might be applied to the purification of AR.     

Microassays for androgen and progesterone receptor quantitation as compared with standard saturation analyses in human prostatic tissues

Summary Simultaneous measurement of androgen and progesterone receptor content in cytosol and salt extractable nuclear subcompartments of benign hyperplastic prostatic tissue was carried out with various microassay techniques and compared to the results from analyses on bulky tissue from the same tissue specimens. The microassays were carried out as modified saturation analyses or as single concentration assays at various degrees of dilution with tris-EDTA-glycerol (TEG) buffer. Tissue samples for the standard assay weighed between 1.76 and 3.22 g, whereas the microassay samples weighed between 0.14 and 0.47 g. When considering the results of the standard assay as the true value, the microassays on the same tissue samples tended to underestimate both the androgen and progesterone receptor contents. Data from the microassays showed a wide variation of the androgen and progesterone receptor content in cytosol and nuclei. With the standard assay technique no detectable amount of progesterone receptor was found in the nuclei, whereas the microassays often indicated false-positive progesterone receptor content in this subcompartment. Therefore, the measurements of steroid receptors using biochemical microassays in prostatic tissue are unreliable and not suitable for clinical use, at least with the techniques available today. Reports in the literature based on such assays should therefore be interpreted with great caution.     

Specific binding protein for 17 beta-estradiol in prostate with adenocarcinoma.

A specific binding protein for 17beta-estradiol has been detected in prostates of patients with prostatic adenocarcinoma, who had no endocrine manipulation prior to the removal of their prostates. Both the sucrose density gradient centrifugation and dextran-coated charcoal techniques were employed. The 17beta-estradiol binding protein has an approximate sedimentation coefficient of 3.6S and is distinct from cystolic dihydrotestosterone binding protein.     

Approaches to prostatic cancer chemotherapy using the Dunning R3327H prostatic adenocarcinoma

Androgen-responsive cells: To determine if testosterone or dihydrotestosterone is the main trophic hormone of prostatic adenocarcinoma, we have treated Dunning R3327H prostatic adenocarcinoma-bearing rats with 6-methylene progesterone, which blocks conversion of testosterone to dihydrotestosterone. Copenhagen-Fisher rats were treated with steroid (20 mg/Kg daily) immediately following implantation of tumor and thereafter for 117 days. There was a 92% inhibition of growth of tumors and a lesser effect upon prostate and seminal vesicles. Tumor-free body weights remained unchanged. Both treated and untreated tumors had equivalent DNA content on a per weight basis. This result supports the thesis that prostatic adenocarcinoma requires dihydrotestosterone for growth. Androgen-insensitive cells: Advanced prostate cancer does not respond to endocrine therapy but is temporarily controlled by the cytotoxic steroid estramustine. The latter shows significant selective binding to prostatic protein. To develop chemotherapeutic agents that will control androgen-insensitive cells and possess improved selectivity for prostatic protein, we have studied a number of steroids for their ability to displace 3H-labeled estramustine from prostatic cytosolic proteins. Surprisingly, a carbamido substituent at the C17 position was found to confer significant binding affinity for prostatic estramustine-binding protein. Extension of this structural characteristic to the estramustine type of molecule is being studied.     

Characterization of steroid receptors in human prostate using mibolerone

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.     

Concentration of the endogenous antiandrogen epitestosterone and androgenic C19-steroids in hyperplastic prostatic tissue

Epitestosterone (epiT, 17α-hydroxy-4-androsten-3-one), an endogenous C19-steroid in humans, was considered for a long as a physiologically inactive steroid. Recently, its antiandrogenic properties have been discovered. For the evaluation of its biological availability in the target organs the tissue concentration is of importance. EpiT, testosterone, dihydrotestosterone, and androstenedione concentrations in prostatic tissue were determined in 15 prostate samples obtained by suprapubic prostatectomy for benign prostatic hyperplasia. The steroids were extracted and separated by high-pressure liquid chromatography and determined by specific radioimmunoassay (RIA) methods. The concentration of epiT (mean 58.4 ± 40.4 SD, range 14.0–144.0 fmol/mg protein) exceeded that of testosterone and was approximately as high as that of dihydrotestosterone. EpiT level increased with age and the correlation was significant (P < 0.05). It did not correlate significantly with testosterone but did with androstenedione and dihydrotestosterone (P < 0.05, each). As expected, a positive correlation was found between testosterone and dihydrotestosterone levels. © 1996 Wiley-Liss, Inc.     

Hormonal consequences of orchidectomy for carcinoma of the prostate. With special reference to the measurement of free testosterone in saliva and prostatic dihydrotestosterone levels

22 patients with metastatic carcinoma of the prostate were treated by subcapsular orchidectomy and followed by regular determinations of plasma testosterone, dihydrotestosterone, delta 4-androstenedione, dehydroepiandrosterone sulfate and testosterone estradiol-binding globulin as well as salivary testosterone, which reflects free plasma testosterone. Subcapsular orchidectomy constantly induced a dramatic and stable decrease in testicular androgens without modification of adrenal androgens or testosterone estradiol-binding globulin. Free testosterone levels vary widely for a given value of plasma testosterone, probably due to individual variations of testosterone estradiol-binding globulin. Salivary testosterone is preferable to plasma testosterone for monitoring hormonal therapy for metastatic carcinoma of the prostate. In another group of orchidectomized stage-D2 patients, obstructive symptoms motivated transurethral resection of the prostate allowing the assay of intraprostatic dihydrotestosterone which was constantly below the dihydrotestosterone level of androgen-independent tissues (below 2.5 ng/g).     

3－酮－地索高诺酮对雌、孕及雄激素受体亲和力选择性的实验研究

采用体外放射配体受体结合法测定３－酮－地索高诺酮（ＫＤＧ）与兔子宫胞浆雌激素受体（ＥｃＲ）、孕激素受体（ＰｃＲ）和大鼠腹侧前列腺胞浆雄激素受体（ＡｃＲ）结合的亲和力，并以左旋１８－甲基炔诺酮（ＬＮＧ）作对照，从受体水平探讨ＫＤＧ对当体激素受体的选择性。结果表明：ＫＤＧ对ＰｃＲ有很高的亲和力，是ＬＮＧ的１．５倍；对ＡｃＲ的亲和力很低，仅为ＬＮＧ的０．７４倍；对ＥｃＲ几乎没有亲和力，受体亲和力选择系数也明显高于ＬＮＧ。由此可见，在ＬＮＧ甾核结构中Ｃ１１位引入次甲基合成的ＫＤＧ，其受体亲和力选择性更专一。     

Serum testosterone levels after medical or surgical androgen deprivation: A comprehensive review of the literature

Androgens and the androgen receptor play a role in the progression of prostate cancer. Androgen deprivation therapy (ADT) is a mainstay in the treatment of metastatic prostate cancer. ADT is expected to reduce serum testosterone levels from a normal level of about 500 to 600 ng/dl (17.3–20.8 nmol) down to castration levels. Traditionally, castration was considered to be achieved if testosterone levels were lowered to a threshold of 50 ng/dl (1.73 nmol/l), a definition determined more by measurement methods derived from the use of old assay methods than by evidence. Serum testosterone levels in three-quarter patients after surgical castration drop to less than 20 ng/dl (0.69 nmol/l). Ineffective suppression of testosterone is currently poorly recognized and may possibly have an effect of prostate cancer mortality. Persistent levels of serum testosterone after castration are mainly derived from adrenal androgens. Furthermore, the arrival of new therapies targeting androgen synthesis and androgen receptor activity has renewed interest on serum testosterone. This review discusses the biosynthetic pathway for androgen synthesis in humans and provides a comprehensive review of serum testosterone levels after surgical or medical castration. This review assesses serum testosterone levels after surgical castration and different pharmacologic castration in patients with prostate cancer under ADT, and ineffective testosterone suppression. The author proposes methods to better lower serum testosterone levels during ADT.     

The effect of antiandrogens and stilboestrol on the cytosol receptor in rat prostate.

The effects of 4 antiandrogens and stilboestrol on the cytosol receptor of the rat ventral prostate have been compared. Equimolar doses of cyproterone acetate, chlormadinone acetate, SC 9022 and flutamide inhibited the binding of dihydrotestosterone to the receptor complex in vivo by 44 to 50% whereas stilboestrol had no effect. In the in vitro studies an equimolar concentration produced similar effects to those observed in vivo with the exception of flutamide which had no effect. This technique provides a method for assessing and comparing the effectiveness of antiandrogens on the androgen receptor complex of the prostate.     

Specific binding protein for 17β-estradiol in prostate with adenocarcinoma

A specific binding protein for 17β-estradiol has been detected in prostates of patients with prostatic adenocarcinoma, who had no endocrine manipulation prior to the removal of their prostates. Both the sucrose density gradient centrifugation and dextran-coated charcoal techniques were employed. The 17β-estradiol binding protein has an approximate sedimentation coefficient of 3.6S and is distinct from cytosolic dihydrotestosterone binding protein.     

Effect of prolactin and the anti-prolactin bromocriptin on the testosterone uptake and metabolism in androgen-sensitive and insensitive canine organs

Summary Prolactin promotes the growth and function of the prostate in low doses, whereas high doses or previous castration reduce this effect. The antiprolactin bromocriptin should reverse the prolactin action. In the castrated dog the highest accumulation of H³-testosterone given i. v. occured in the prostate as compared with muscle, urethra, penis, liver and kidney. Prolactin pretreatment increased the radiosteroid uptake only in the liver. Conversely, bromocriptin suppressed the tracer incorporation into the liver, but increased prostatic accumulation. The highest testosterone reduction occurred in the prostate of the untreated castrated dogs as compared with other organs. Prolactin suppressed 5-dihydrotestosterone formation but otherwise did not significantly influence testosterone turnover. Bromocriptin, however, stimulated dihydrotestosterone formation in the prostate and caused complete inhibition of hepatic testosterone reduction.Parlodel^R from Sandoz AG, CH-4000 BaselSupported by the Helmut Horten-Foundation