A hybrid ligand method for androgen receptor measurement

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.     

NAD-sensitive hydrogel for the release of macromolecules

A hydrogel membrane containing immobilized ligands and receptors was synthesized and investigated for the controlled diffusion of test proteins (cytochrome C and hemoglobin). Both Cibacron blue (ligand) and lysozyme (receptor) were covalently linked to dextran molecules that were subsequently crosslinked to form a gel. The resulting stable hydrogels contained both covalent and affinity crosslinks such that their intrinsic porosities were sensitive to competitive displacers of the affinity interaction between lysozyme and Cibacron blue. Transport experiments in a twin chamber diffusion cell showed that as NAD was added to the donor side, the dissociation of the binding sites between the Cibacron blue and the lysozyme led to an increase in protein diffusion through the hydrogel. The results showed that addition of NAD caused a saturable concentration-dependent increase in the transport of both cytochrome C and hemoglobin. This effect was shown to be both specific and reversible.     

Interaction of alpha 2-HS-glycoprotein with immobilized triazine dyes

We studied the interaction of alpha 2-HS-glycoprotein with immobilized Cibacron blue F3-GA (Blue A) and Procion red HE-3B (Red A). When whole plasma was applied on the Blue A, alpha 2-HS-glycoprotein remained unbound, together with other plasma proteins. In contrast, when this fraction was applied on the Red A, alpha 2-HS-glycoprotein was shown to bind tightly and was eluted with a linear sodium chloride gradient between 0.5 and 0.8 M. This proved to be a useful two-step technique for the purification of alpha 2-HS-glycoprotein. Further characterization revealed that the protein appeared homogeneous by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis with yields greater than 30%. A small (less than 5%) but significant fraction of alpha 2-HS-glycoprotein with a same molecular weight as the native protein was consistently found in the wash of the Red A column, and may correspond to alpha 2-HS-glycoprotein bound to a yet unidentified ligand.     

Endogenous C19-steroids and oestradiol levels in human primary breast tumour tissues and their correlation with androgen and oestrogen receptors

Endogenous levels of testosterone, 5 alpha-dihydrotestosterone (5 alpha-DHT), androstenedione and oestradiol as well as levels of androgen (AR) and oestrogen (ER) receptors were measured in human primary breast tumour samples. The purification procedure developed allowed simultaneous quantitation of the four steroids, by radioimmunoassay, in small samples with adequate precision, sensitivity and accuracy. The majority of the tumours analysed contained detectable levels of the four steroids in the homogenate or cytosol fractions. There was no significant correlation between steroid content of the tissue and the age of the patient for any of the four steroids. A positive correlation (r = 0.71) was found between the levels of 5 alpha-DHT and testosterone in tumours. In general, tissue steroid concentrations decreased with an increase in dedifferentiation. Fifty-two per cent of the tumours analysed for receptor content were found to be ER positive, and a similar proportion were AR positive. No relationship was observed between AR status and age although receptor concentration was significantly (P = 0.004) higher in post-menopausal women when only receptor positive tumours were evaluated. The mean values for AR and ER were higher in tumours containing both receptors than in tumours showing either receptor alone; there was, however, no significant relationship between concentrations of the two receptors. No correlation was observed between tumour AR or ER status and any of the four steroids measured in either fraction. In addition, the ratio between the combined levels of 5 alpha-DHT and testosterone compared to oestradiol in the same tumour, only showed a maximum value of 40. Thus, in vivo these two androgens are unlikely to influence oestrogen action in human primary breast tumours by interfering with the association of oestradiol with its receptor.     

Expression of sex steroid receptors and IGF-1 mRNA in breast tissue--effects of hormonal treatment

The mechanisms behind increased breast tissue proliferation and a possibly increased breast cancer risk in women using hormonal contraception (HC) and hormonal replacement therapy (HRT) are incompletely understood. We analyzed breast tissue from 20 premenopausal and seven postmenopausal women undergoing reduction mammoplasties for estrogen receptor (ER) and progesterone receptor (PR) content as well as mRNA levels for ER, PR and insulin-like growth factor-1 (IGF-1). The receptor values were correlated to IGF-1 mRNA concentrations and levels of steroid and peptide hormones and SHBG. In women using HC, we found significantly lower ER values (p=0.02) but non-significantly lower ER mRNA levels compared to those in naturally cycling women. PR and PR mRNA were no different. Women on HC displayed a higher breast tissue proliferation (p=0.05) expressed as Ki-67, MIB-1 positivity, which was correlated with IGF-1 mRNA (r_s=0.82, p=0.04). Since the concentration of sex steroid receptors in breast tissue is comparatively low and steroid receptors are down-regulated during hormonal treatment, mechanisms other than direct sex steroid receptor action are likely to be present. Our results suggest a role for IGF-1 in the proliferative response of breast tissue during exogenous hormonal treatment.     

An improved micromethod for the determination of biochemically active estrogen and progesterone receptors in parallel to comparative histological examination of a single piece of tissue

An improved radioreceptor assay of unfixed cryostat sections of human target tissues has been developed. Sections collected on glass coverslips were immediately incubated with 5 nM concentrations of either tritiated estradiol-17 beta for estrogen receptor (ER) or ORG 2058 for progesterone receptor (PR) determination. For quantitation, receptor-bound and free hormone were separated by isoelectric focusing (IEF). The assay allows the determination of steroid hormone receptors and comparative histological examinations in immediately neighbouring serial sections of a single piece of tissue. Biochemically, the validity of the assay procedure was evidenced by Scatchard analysis, by ligand and tissue specificities, by the linear relations of receptor and protein concentrations and the number of sections per test tube. Diagnostically, we compared the routine (6 point DCC-Scatchard) procedure for breast cancer analysis with the section method. A good correlation for ER and a less pronounced correlation for PR was found. Statistically, the precision of the method was verified by low deviations of duplicate determinations, low day-to-day variations and low inter-assay variations.     

Determination of cytoplasmic receptors for specific steroid hormones. II) Androgen receptor

Cytoplasmic receptors for 5 alpha-dihydrotestosterone (3H-DHT) were determined in normal and hypertrophic human prostate using the slightly modified DCC method we previously standardized for 17beta-estradiol-receptor. Incubations were always performed at 0 degree C for 1 hr. Discrimination between 3H-DHT binding to cytoplasmic receptor and to Sex Hormone Binding Globulin (SHBG) was achieved on the basis of binding affinity, thermolability and pattern of specificity by various steroid hormones. In particular, 5 beta-DHT did not bind to cytoplasmic receptor, while it did to SHBG.     

Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor

Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed, FAD-containing enzyme that catalyzes the obligatory two-electron reduction of quinones. Cibacron Blue is an inhibitor of this enzyme comparable in potency to dicoumarol. Pure quinone reductase was obtained from the livers of Sudan II (1-[2,4-dimethylphenylazo]-2-naphthol)-treated rats in a single step by Cibacron Blue-agarose chromatography. Cibacron Blue is a competitive inhibitor with respect to NADH (Ki = 170 nM) and is a noncompetitive inhibitor with respect to menadione (Ki = 540 nM). Addition of Cibacron Blue to quinone reductase resulted in a decrease and red shift of the enzyme-bound FAD peak at 450 nm. The titration of the absorbance changes for both FAD and Cibacron Blue could be fitted to curves describing an equilibrium binding equation with a KD of 300 nM and one binding site per enzyme subunit. Furthermore, the Cibacron Blue difference spectrum that resulted from binding to quinone reductase was abolished by dicoumarol. Significant amino acid homology between quinone reductase and the nucleotide binding regions of enzymes that bind to Cibacron Blue was found. These data indicate that Cibacron Blue is a useful ligand for the purification of quinone reductase and a new probe for its NAD(P)H binding site. Conditions for crystallizing rat liver quinone reductase are also described.     

Purification of immunotoxins containing ricin A-chain and abrin A-chain using Blue Sepharose CL-6B

We describe a method for separating antibody from immunotoxins by affinity chromatography on Cibacron blue F3GA coupled to Sepharose (Blue Sepharose). The antibody did not bind to the gel. The immunotoxins were bound by their ricin A-chain or abrin A-chain moiety and could be recovered in high yield and purity using mild elution conditions. The method is suitable for the large-scale purification of immunotoxins.     

The Interaction of Mammalian Interferons with Immobilized Cibacron Blue F3Ga: Modulation of Binding Strength

Mouse, hamster, rabbit, horse, and human interferons bind to immobilized Cibacron Blue F3GA under appropriate solvent conditions. Three forms of the immobilized ligand have been investigated: Cibacron Blue F3GA-Sepharose 4B, Blue Dextran-Sepharose 4B and Blue Sepharose CL-6B. The strength of binding of an interferon depends critically on the sorbent: Cibacron Blue F3GA-Sepharose 4B is the weakest in the series and Blue Sepharose CL-6B the strongest. The use of Blue Dextran-Sepharose 4B - a sorbent of intermediate binding properties - allows the complete separation of hamster, mouse and human fibroblast interferons in a single chromatographic step. Indeed, both the resolution, as well as the recovery, of those interferons is complete - regardless of the relative complexity of the chromatographed preparation (containing either crude or purified interferons). Thus, these ligands should prove of considerable use     

Identification and characterization of a ligand-regulated nuclear export signal in androgen receptor

Androgen receptor (AR) belongs to the steroid receptor superfamily that regulates gene expression in a ligand-dependent fashion. AR is localized to the cytoplasm in the absence of androgen and translocates into the nuclei to activate gene expression in the presence of ligand. Regulation of AR nuclear import and export represents an essential step in androgen action. A nuclear localization signal (NLS) has been identified in the DNA-binding domain and hinge region of AR and other steroid receptors. Studies on nuclear export of AR, however, are limited, and what might be the underlying mechanism regulating the intracellular localization of steroid receptors is unclear. Our studies have identified a leptomycin B-insensitive nuclear export signal (NESAR) in the ligand-binding domain of AR, which is active in the absence of androgen and repressed upon ligand binding. Consistent with its androgen-sensitivity, NESAR contains amino acid residues in the immediate vicinity of the bound ligand. NESAR is necessary for AR nuclear export and is dominant over the NLS in the DNA-binding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NESAR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/cytoplasmic shuttling. Estrogen receptor alpha and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors.     

An allosteric synthetic DNA

Allosteric DNA oligonucleotides are potentially useful diagnostic reagents. Here we develop a model system for the study of allosteric interactions in DNAs. A DNA that binds either Cibacron blue or cholic acid was isolated and partially characterized. Isolation was performed using a multi-stage SELEX. First, short oligos that bind either Cibacron blue or cholic acid were enriched from random oligonucleotide pools. Then, members of the two pools were fused to form longer oligos, which were then selected for theability to bind Cibacron blue columns and elute with cholic acid. One resulting isolate (A22) was studied. Dye- and cholate-binding functions can be separated on sequences from the 5'- and 3'-regions, respectively. Ligand-column affinity assays indicate that each domain binds only its respective ligand. However, the full-length A22 will bind either dye or cholate columns and elute with the other ligand, as if binding by the ligands is mutually exclusive. Furthermore, S1 nuclease protection assays show that Cibacron blue causes a structural change in A22 and that cholic acid inhibits this change. This system will be useful for elucidating mechanisms of allosteric interactions in synthetic DNAs.     

Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.