Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881)

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.     

Binding of methyltrienolone to various androgen-dependent and androgen-responsive tissues in four animal species.

Comparative sucrose gradient studies of the in vitro binding of dihydrotestosterone (DHT) and of a synthetic androgen, methyltrienolone (R 1881), have been done with the cytosols of various tissues of the rat, mouse, cock and man. With rat prostate cytosol, the amount of R 1881 and DHT binding in the 8-9S region of the gradient was found to be comparable. Specific 8-9S peaks of R 1881 were also found in rat levator ani/bulbocavernosus and skeletal muscles and in the mouse kidney. Only 4-5S peaks could be demonstrated in the cock's comb while DHT under the same conditions showed both 8-9S and 4-5S binding. Binding of R 1881 to the cytosol of the hyperplastic prostate was polydispersed, and showed evidence of the presence of aggregates. Evidence was also found that R 1881 could bind to the progesterone receptor in rat uterus. Our study supports the theory that in a given species the androgen receptors are similar if not identical in all the tissues. The synthetic androgen R 1881 appears to be a useful tool for androgen receptor studies in various animal species provided that the tissue under study contains no progesterone receptor.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

Assay of androgen binding sites by exchange with methyltrienolone (R 1881).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) binds specifically to androgen receptor in rat prostate cytosol where, unlike androstanolone, it is not metabolized. By exchanging bound endogenous hormone in rat prostate cytosol with labelled R 1881, it is possible to measure total (free anc occupied) binding sites. This assay method has also been applied to the measurement of androgen receptor sites in human benign prostatic hypertrophy where R 1881 has the added advantage of not being bound by any contaminating plasma protein (sex hormone binding protein).     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Characteristics of androgen binding in rat adrenal tissue using [3H]methyltrienolone (R1881) as tracer

A high level of binding of [3H]methyltrienolone (R1881 = 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [3H]R1881 was of high affinity (DK = 6.2 nM) and highly specific for androgens. The [3H]R1881 complex migrates at 7-9S on sucrose gradients in low ionic strength buffer and at 4-5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.     

In situ photolabelling of the human androgen receptor

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.     

Methyltrienolone, a specific ligand for cellular androgen receptors.

Methyltrienolone (R 1881), 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one, a very active androgen, binds specifically to rat prostate cytosol with a higher affinity than androstanolone. Unlike the physiological hormone, however, it is not bound by human sex steroid plasma binding protein, SBP. This specific ligand is thus a useful tool for the detection of elusive androgen receptors and for their study, for instance, in human tumors where interference from plasma contamination has to be circumvented.     

Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity

In the present study, calf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl2. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [3H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5 while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [3H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.     

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity (Kd 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [3H]R1881 as tracer showed a 200-fold excess of testosterone to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.     

The synthesis and testing of E-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone and Z-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone as gamma-emitting ligands for the androgen receptor

Two iodinated steroids, E-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone and Z-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone were synthesized in a search for a gamma-emitting androgen that binds with high affinity to the androgen receptor. Such compounds would be extremely useful research tools for studies of androgen responsive tissues and as in vivo probes of androgen responsive tumors such as prostate cancer. These 17 alpha-iodovinyl steroids were synthesized because many 17 alpha-substituents do not interfere markedly with binding to the androgen receptor and because similar analogs of other steroids, estrogens and progestins, have been shown to have the requisite properties for ligands to those receptors. Both of these potential ligands were tested for their ability to compete with [3H]R1881 for binding to the androgen receptor in cytosols from prostate, hypothalamus and pituitary. The relative binding affinities ranged between 5 and 20%, depending upon the tissue and steroid. In order to test the two ligands directly, they were both synthesized labelled with 125I and tested for binding to the androgen receptor in prostatic cytosol and in vivo for specific concentration in androgen responsive tissues. While there was considerable binding in the prostatic cytosol, it was not specific because 5 alpha-dihydrotestosterone did not compete. Likewise in the in vivo experiment there was no evidence for androgen receptor mediated concentration of the tracers. While on the basis of relative binding affinity, these 2 steroids appeared to be good candidates for androgen receptor ligands, neither were useful for this purpose. These results contribute new information which will be valuable in the design of other gamma-emitting androgens and emphasises that, in this process, other factors such as metabolism and nonspecific binding must be considered.     

Purification and characterization of the untransformed androgen receptor in rat prostate

The isolation and characterization of the untransformed form of androgen receptors has not yet been successful, owing to their inherent lability as well as to their ready proteolysis. In this study, we have stabilized rat prostate androgen receptors by sodium molybdate and by rapid filtration on phosphocellulose. Proteases were inhibited by bacitracin, aprotinin, leupeptin and PMSF. Under these conditions the untransformed complex was purified approx 3000-fold, corresponding to 18% yield, by differential chromatography on DEAE cellulose and phosphocellulose gels. The partially purified receptor has the same ionic characteristics as the original untransformed receptor of crude cytosol; in addition, it possesses a Stokes' radius of 75 A, as determined by Sephacryl S-300 gel filtration, a sedimentation coefficient of 8.8S, a calculated molecular weight of 275 kDa and a friction coefficient of 1.6. The [3H]R1881 receptor complex was specific to androgens since unlabelled R1881 and dihydrotestosterone were able to completely displace bound [3H]R1881, whereas estradiol, cortisol, and triamcinolone acetonide did not compete. The purified complex was a multimer dissociable by 0.6 M KCl, resulting in a form migrating in the 4S area on sucrose density gradient. After treatment with 0.5% formaldehyde, three forms were obtained, migrating in the areas of 8-9, 5-6 and 3-4S respectively, of a sucrose density gradient containing 0.6 M KCl. This is the first step towards the purification to homogeneity of the untransformed androgen receptor.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

Mechanism of androgen-receptor augmentation. Analysis of receptor synthesis and degradation by the density-shift technique

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) contains receptors for, and is stimulated by, androgens. Cells cultured in the absence of androgens maintain a basal level of androgen receptors. Following incubation with various concentrations of the synthetic androgen methyltrienolone (R1881) for 1-6 h, the concentration of these receptors increased from 6.0 to 12.2 fmol/micrograms of DNA, while the equilibrium dissociation constant (Kd) of 0.5 nM for this steroid remained unchanged. The steroid-induced increase in androgen receptor levels was specific for androgens and dependent upon protein synthesis. The mechanism of receptor augmentation was examined by utilization of isotopically dense amino acids to determine rates of receptor appearance and degradation in the presence or absence of [3H]R1881. In the absence of androgens, the half-life of the androgen receptor was 3.1 h, with a rate constant (kD) of 0.22/h. In the presence of 1 nM [3H]R1881, however, the half-life was 6.6 h, with kD = 0.11/h. The rate constant for receptor synthesis (ks) in the absence or presence of [3H]R1881 was calculated to be 1.35 and 2.23 fmol/micrograms of DNA/h, respectively. Thus, androgen-induced androgen-receptor augmentation is explained by an increase both in receptor half-life and in rates of receptor synthesis.     

Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity

In the present study, culf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl₂. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [³H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5, while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [³H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.     

Androgen receptors in ventral prostate glands of zinc deficient rats

Androgen binding has been studied in the prostate cytosol of zinc deficient rats by charcoal assays. Rats were housed individually in plastic cages and maintained on a zinc deficient diet for 3 months. The cytosol fraction of prostate gland was incubated with various concentrations of tritiated methyltrienolone (3H-R1881, a synthetic androgen) alone or in the presence of 500-fold excess of radioinert R1881. Scatchard analysis of the data revealed that the number of androgen binding sites in the cytosol fraction of the zinc deficient rat prostate was 31 +/- 5.2 fmol/mg cytosol protein. This was significantly lower than that (84 +/- 11.5 fmol/mg protein) of the controls. Their dissociation constant (Kd = 1.6 +/- 0.6 nM) on the other hand was not different from that (1.7 +/- 0.7 nM) of control animals. This decrease in the concentration of cytosol receptor sites in the zinc deficient state suggests that this metal is involved in the androgen-binding process in the target cells.     

Effect of 16 beta-ethyl-17 beta-hydroxy-4-estren-3-one (TSAA-291) on the binding of promegestone (R5020) and methyltrienolone (R1881) to hyperplastic and neoplastic human prostate

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.     

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.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.