H.J. Grill

A comparison of cytoplasmic and nuclear estradiol and progesterone receptors in human fallopian tube and endometrial tissue

Quantitative and qualitative aspects of the in vitro binding of 3H-estradiol and 3H-progesterone to receptor components from human endometrium and fallopian tube cytoplasmic and nuclear fractions were studied. The steroid binding macromolecules formed in vitro could be extracted from nuclei by 0.4 M KCl and detected by glycerol gradient centrifugation. Both estradiol- and progesterone-binding compounds formed only one peak (under high ionic strength conditions) with a sedimentation coefficient of about 4-5 S. The number of cytoplasmic and nuclear binding sites for both estradiol and R5020 varied dramatically throughout the menstrual cycle: the estradiol and progesterone receptor concentrations were highest during the proliferative phase and were very significantly lower in the second half of the menstrual cycle. Furthermore, measurement of both receptors in the cytosol revealed differences among the anatomic segments of the fallopian tube. The highest estradiol and progesterone binding could be detected in the ampullary region; significantly lower levels of estradiol and progesterone receptors were seen in the infundibulum and the isthmus.

Gestodene: A novel synthetic progestin — characterization of binding to receptor and serum proteins

Gestodene, 17 alpha-ethinyl-13-ethyl-17 beta-hydroxy-4,15-gonadien-3-one, is a new orally active progestational agent, which is available for clinical use in oral contraceptives. The aim of the present study is to make a broad characterization of gestodene at the receptor level and to discuss the results in comparison to those of established progestogens. Kinetic studies of 3H-gestodene uptake show a rapid increase in the amount of specific binding during the first three hours. After saturation, the amount of specifically bound 3H-gestodene remained almost constant up to 24 hours at 4 degrees C. The dissociation of 3H-gestodene from the cytoplasmic myometrial progestone receptor, measured by displacement of labeled steroid with dextran-coated charcoal treatment at 4 degrees C at various times, showed a biphasic or two-component first order dissociation curve. As anticipated, sucrose gradient centrifugation analysis of the 3H-gestodene-labeled cytosol of human myometrial tissue showed that the gestodene binding components sedimented in the 4S and 8S region. A 200-fold molar excess of nonradioactive gestodene reduced only the 8S binding of 3H-gestodene. 4S binding of 3H-gestodene was not reduced, which indicate the existence of a second high capacity binding component. In biological test systems, such as the Clauberg test or Kaufmann test, gestodene has proved to be a very effective progestogen. Among nortestosterone derivatives it is one of the most potent and resembles progesterone biologically in its progestogenic effects. This biologically identical gestagenic activity of gestodene and progesterone is reflected by a very similar behavior in vitro in terms of binding to progesterone receptors of human uterus cytosol. Furthermore, competitive studies indicated that gestodene like other synthetic progestagens also displays some affinity for androgen and glucocorticoid receptors but no measurable affinity for the estrogen receptor. Remarkable is the high binding affinity of gestodene to the binding sites of the mineralocorticoid receptor of rat kidney with a RBA value of 350% compared to aldosterone.

Criteria for the establishment of a double-labeling assay for simultaneous determination of estrogen and progesterone receptors.

The availability of [125I]-16 alpha-iodo-3,17 beta-estradiol ( [125I]-E2) with binding characteristics similar to estrogen receptor (ER) enabled us to establish a double-labeling assay for the simultaneous determination of ER and progesterone receptors (PgR) using 125I-E2 and [3H]-R5020. The criteria for the establishment of such a double-labeling assay are described. 150 human mammary tumor cytosols have been investigated with the standard routine receptor assay for ER as well as PgR and the results were compared to those obtained by the double-labeling assay. ER: a coefficient of correlation of 0.691 was obtained, the parameters of the regression line were Y = 1.025 X X-0.036. When referring to the standard assay, 3 determinations were false-positive and 4 false-negative. PgR: a correlation coefficient of 0.984 was found, the parameters of the regression line were Y = 0.960 X X + 12.16. One value was false-negative and 1 false-positive. An equivalence of the two methods could be demonstrated. This new assay reduces by half the amount of tissue necessary for a valid 4- to 6-point saturation analysis, the time required for performing the assay and its cost.

Steroid side-chain modification and receptor affinity: Binding of synthetic derivatives of corticoids to human spleen tumor and rat liver glucocorticoid receptors

Abstract Human spleen tumors (Hodgkins disease I) contain varying amounts (40–150 fmol/mg protein) of glucocorticoid receptors. The apparent K diss at 0°C is 6.8 ± 2.1 nmol/1. A second glucocorticoid binder, presumably the plasma contaminant human corticosteroid binding globulin (CBG), is shown by a newly developed [ 3 H]-hydrocortisone assay. A series of side-chain modified natural and synthetic corticoids bind with similar affinities to human spleen tumor and rat liver glucocorticoid receptors. 17β-Carboxylic acid derivatives do not bind to both receptors whereas methylation or amidation of the acids partially restore their binding affinities. The one exception, the inactive methyl 17β-carboxester of betamethasone, which bears a 16β-methyl substitution, confirms the hypothesis that receptor binding involves a specific conformation of the side-chain.

Application ofliquid—liquid partition chromatography in the simultaneous purification of sex-hormone-binding globulin and corticosteroid-binding globulin

Two human serum proteins, corticosteroid-binding globulin (CBG) and sex-hormone-binding globulin (SHBG), were purified to homogeneity by the application of a combination of three different modes of chromatography. Human pregnancy serum was fractionated with ammonium sulphate. SHBG (50% pellet) and CBG (80% pellet) were then purified by affinity chromatography on tresyl-activated Sepharose with 15-aminopentadecanoic acid (for SHBG) and 1,12-diaminododecane (for CBG) as spacers and 17 zeta-aminoethyl-5 alpha-androstan-3 beta,17-diol (for SHBG) and 17 alpha-hydroxy-4-androsten-3-one-17 beta-carboxylic acid (for CBG) as specific ligands for these two proteins. The eluate was injected into a Mono Q anion-exchange column. Fractions containing SHBG or CBG were finally purified by liquid-liquid chromatography on Lipar-Gel 750. This chromatographic sequence clearly separated SHBG and CBG from other proteins, mainly serum albumin, without a loss of protein or binding activity.

Steroid hormone receptors in human melanoma

SummaryHuman melanomas were investigated for the presence of highaffinity estrogen-, gestagen-, and glucocorticoid-binding proteins. A statistically significant difference was found for mean estrogen receptor (ER) concentrations in melanomas of male versus female origin: female origin 37.6 (0–107) fmol/mg protein, male origin 3.9 (0–8.3) fmol/mg protein. No significant difference between sexes was found for gestragen receptors: 41.5 (0–194) fmol/mg protein for melanomas of female origin versus 99 (0–362) fmol/mg protein for male.Sucrose density gradient analyses revealed specific binding for both receptor types in the 4–5 S region as well as in the 8 S region. The binding affinities were in the same order of magnitude as reported for receptors found in typical steroid target organs.No significant difference in receptor values depending on sex was found for the glucocorticoid receptor: 19.2 (0–43) fmol/mg protein.

High-performance and ion-exchange chromatography and chromatofocusing of the human uterine progesterone receptor: its application to the identification of 21-[3H]dehydro Org 2058-labelled receptor.

Two independent lines of evidence were used to identify the human uterine progesterone receptor. First, three differently tritiated progestogens (Org 2058, R 5020, progesterone) were used for reversible labelling of the receptor. Secondly, the highly potent affinity label 21-[3H]dehydro Org 2058 was used to label covalently the steroid-specific binding site of the receptor. The labelled cytosols were chromatographed on a Mono Q high-performance anion-exchange column in the absence or presence of a high molar excess of the respective unlabelled competitor steroids. In the case of 21-[3H]dehydro Org 2058, Org 2058 was used as the unlabelled competitor. After elution with a NaCl gradient, the radioactivity was determined in each fraction and the elution profiles (absorption, A at 280 nm; radioactivity, dpm) were superimposed. Free steroid was eluted with the washing buffer. When the NaCl gradient was performed, two peaks of radioactivity were located. The specifically protein-bound radioactivity was eluted at 0.08 M NaCl. Two non-specific steroid-binding entities were eluted at 0.1 and 0.22 M NaCl, the second of which was identified as albumin. The elution profiles of tritiated progesterone, R 5020, Org 2058 and the affinity label 21-dehydro Org 2058 were identical. In a second set of experiments, Org 2058- and 21-dehydro Org 2058-labelled cytosols were subjected to high-performance liquid chromatography on a Mono P high-performance chromatofocusing column in the absence or presence of a high molar excess of unlabelled Org 2058. After elution with Polybuffer 74, only one specifically labelled protein (pH 6.4) was detected. When the Mono P-purified receptor was submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis, two labelled polypeptides with Mr = 45,000 and 27,000 were detectable.

3H-ZK 98,734, a new 11β-aryl substituted antigestagen: Binding characteristics to receptor and serum proteins

Recently, in the laboratories of Schering in Germany a competitive progesterone antagonist, ZK 98,734, was synthetized, which is characterized by a similar antigestagenic activity as RU 38,486, synthezised by Roussel-Uclaf in France, as assessed by inhibition of nidation tests in rats and guinea pigs. However, this compound has a substantially lower antiglucocorticoid activity measured in cell culture systems than RU 38,486. The purpose of this study was to present a comparison of biochemical and physical properties of the complexes formed by the human uterine progesterone receptor with 3H-ZK 98,734 on one hand and with other well-established progestins on the other hand. ZK 98,734 competed in the same order of magnitude as progesterone or RU 38,486 for the 3H-R5020 binding site of progestin receptor, whereas R5020, Org 2058 or progesterone were unable to compete against 3H-ZK 98,734. This apparent contradiction could be explained by means of FPLC-chromatography and sucrose density centrifugation technique. FPLC-chromatography with an anion exchange column (Mono Q, Pharmacia, Uppsala, Sweden) showed that 3H-ZK 98,734 forms at least two stable complexes with uterine cytosol, on one hand with serum albumin, which presents almost 90% of bound radioactivity, and on the other hand with the two native progestin receptor forms, corresponding to 4S and 8S receptor forms in sucrose density gradient analysis. Competition experiments in liver cytosol of adrenalectomized rats with increasing concentrations of unlabeled ligands other than dexamethasone showed that ZK 98,734, RU 38,486 and cortisol displaced 3H-dexamethasone efficiently from the binding sites in the cytosol. Furthermore, results concerning the specificity of 3H-cortisol binding to serum proteins in diluted pregnant serum demonstrated that ZK 98,734 did not compete with 3H-cortisol for serum binding.

3H-cyproterone acetate: binding characteristics to human uterine progestagen receptors

The availability of tritium labeled cyproterone acetate (CPA) facilitated the systematic investigation of the binding characteristics of this compound for human uterine progesterone receptors (PgR). The binding parameters of 3H-CPA are compared to those of 3H-R5020 and 3H-progesterone. The rate constants of association (k1M-1sec-1) to PgR were 7.8 x 103 for 3H-R5020, 4.5 x 104 for 3H-progesterone and 4.0 x 104 for 3H-CPA. The rate constants of dissociation (k-1, sec-1) were 3.6 x 10-5 for 3H-R5020, 21.3 x 10-5 for 3H-progesterone and 17.8 x 10-5 for 3H-CPA. The Kd-values (M), as obtained by titration analysis and subsequent Scatchard plot analysis were 1.2 x 10-9 for 3H-R5020, 6.0 x 10-9 for 3H-progesterone and 5.2 x 10-9 for 3H-CPA. On sucrose density gradient analysis binding in the 3.5, 5 and 8 S area could be observed using 3H-R5020. For 3H-progesterone and 3H-CPA binding was exclusively found in the 5 S area. The specificity of the steroid binding site of PgR is identical for 3H-R5020 and 3H-CPA. The order of potency of binding for various competitors decreases identically for both radioactive ligands: R5020 < progesterone < R1881< CPA < dihydrotestosterone < dexamethasone < cyproterone < 15β-OH-cyproterone. CPA resembles progesterone very closely in its binding characteristics to human uterine PgR.

Vergleichende Untersuchungen der synthetischen Antigestagene RU 38486, ZK 98734 und ZK 98299 auf der Rezeptorebene

Csapo formulierte das Konzept, das der Entzug von Progesteron unter der Schwangerschaft zwangslaufig zu einer Beendigung dieser fuhren mus. Seitdem Antigestagene vom RU-486-Typ existieren, die als kompetitive Progesteron-Antagonisten die Wirkung von Progesteron dosisabhangig am Uterus blockieren, ist es moglich, Csapo’s Hypothese sowohl klinisch als auch auf der tierexperimentellen wie molekularbiologischen Ebene kritisch zu uberprufen.