Steroid hormone receptors in human melanoma.

Human melanomas were investigated for the presence of high-affinity 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 …

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 …

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 3 H-estradiol and 3 H-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 0.4M 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 4-5S. The number of cytoplasmic and nuclear binding sites for both estradiol and R5020 varied dramatically throughout the menstrual cycle: the estradiol and progesterone receptor concentrati…

Basic and Clinical Relevance of Hormonal Influence in Breast Cancer

In the Federal Republic of Germany breast cancer has a leading place among malignant tumors in women. Genital and breast cancers account for 32% of all female deaths from malignant neoplasias. The distribution of types of cancer is as follows: breast 52%, uterine cervix 9%, corpus uteri 4.5%, adnexae 19%, and other sex organs 15.5% (Maas and Sachs 1972; Schmidt-Matthiesen 1975, Vorherr 1980). Besides general epidemiological factors such as geographical distribution, dietary factors, age distribution, familial disposition, and socioeconomic influences, possible hormonal components are discussed in connection with the etiology of breast cancer (Fischedick and Lux 1977; Henderson et al. 1974; …

Application of liquid-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 Mon…

Synthesis of a New Disulfide Affinity Adsorbent for Purification of Human Uterine Progesterone Receptor

For purification of the human uterine progesterone receptor, an affinity adsorbent was synthesized in which the specific ligand (16 alpha-ethyl-3-oxo-19nor-androst-4-ene 17 beta-carboxylic acid) was bound to derivatized celulose using a disulfide-group-containing spacer. The purified receptor protein, isolated by reductive cleavage of the disulfide bond, bound the synthetic gestagen R5020 with high affinity (Kd 12.2 nmol/l). The affinity gel was highly efficient. A 24000-fold purification of progesterone receptor with a recovery of 40% could be achieved in a single step within 6 h. By means of dodecyl sulphate/polyacrylamide gel electrophoresis two main polypeptides with molecular weights o…

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.

Synthesis of biotin-labelled dexamethasone derivatives. Novel hormone-affinity probes.

A new, general methodology for 'sandwich' affinity chromatography of steroid hormone receptors is proposed, the part purification of the human spleen tumor glucocorticoid receptor is quoted as an illustration. 9-Fluoro-16 alpha-methyl-11 beta, 17-dihydroxy-1,4-androstadiene-3-one-17 beta-carboxylic acid was coupled to biotin using pentamethylenediamine (BioDex 1) as a spacer. The bifunctional derivative binds to glucocorticoid receptors and avidin-Sepharose and efficiently protects the glucocorticoid receptor against inactivation when previously added during homogenisation. We have standardized the capacity and optimum conditions for elution of receptor-BioDex-1 complexes which are bound to…

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies.

Abstract This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: (1) affinity chromatography, (2) desalting of eluate on Sephadex G-25, (3) anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85–95%. Investigations wi…

16α-Iodo-3,17β-estradiol: A stable ligand for estrogen receptor determinations in tissues with high 17β-hydroxysteroid dehydrogenase activity

Recently, the successful synthesis of radioiodinated 16 alpha-iodo-3,17 beta-estradiol-[125I] [125I]E2 was reported [1]. This new ligand has similar binding characteristics to the estrogen receptor (ER) [2-5] as the currently used tritium labeled estradiol [3H]E2. However, it offers several advantageous features: (a) high specific activity (theoretically 2,000 Ci/mmol) [1]; (b) minor problems with radioactive waste due to its short half life and (c) the possibility of simultaneous determination of ER and progesterone receptors (PgR) by double labeling with [125I]E2 and [3H]R5020 [6, 7]. As we are presently trying to determine ER and PgR in human placental cytosols we were interested in the …

3H-estradiol and3H-R5020 binding in cytosols of normal and neoplastic human ovarian tissue

High-affinity cytoplasmic estrogen and progesterone receptors in normal and abnormal ovarian tissues were studied. Estradiol receptor was detectable in 65% and progesterone receptor in 36% of the malignant tumors; 39% of all malignant ovarian tissues were estradiol- as well as progesterone-receptor-positive. Tumors were said to be receptor-positive when the receptors bound greater than 5 fM steroid/mg cytosol protein. No correlations were found between receptor status and histopathological diagnosis. In normal ovarian tissues collected at various phases of the menstrual cycle no changes in [3H]-estradiol and [3H]-R5020 binding could be detected. Analysis of the receptor concentration for bo…

Synthesis of tritium labeled 7α-methoxycarbonyl-15β,16β-methylene-3-oxo-17α-pregn-4-ene-21, 17-carbolactone, [3H]SH-D515, a highly selective tracer for the mineralocorticoid receptor

The synthesis of 7α-methoxycarbonyl-15β,16β-methylene-3-oxo-17α-pregn-4-ene-21,17-carbolactone (5, SH-D515, ZK 91587), a potent aldosterone antagonist exhibiting a strong and very selective binding to the mineralocorticoid receptor and the tritiated analog 6 is reported.

17 beta-carboxamide steroids: highly effective inhibitors of the phytohaemagglutinin mediated blastogenesis of normal human peripheral lymphocytes.

Several novel 17 beta-carboxamide analogues of dexamethasone were synthesized. The common precursor, 9-fluoro-16 alpha-methyl-11 beta,17-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxylic acid, did not bind to the glucocorticoid receptors of rat liver and human spleen tumours. In addition, no inhibition of the mitogen-induced blastogenesis of cultured human peripheral lymphocytes was observed. The 17 beta-carboxamide analogues, however, bound with similar affinities to the glucocorticoid receptors of both tissues. They inhibited the mitogen-induced blastogenesis of peripheral lymphocytes, showing the same potency and same order of binding affinity as the natural glucocorticoids.

Randomisierte Tamoxifen-Loading dose-Studie: Bestimmung der Serumspiegel von Tamoxifen und 5 Hauptmetaboliten mit einer neuen HOPLC-Methode

Je 20 Patientinnen mit Ostrogen- und/oder Progesteron-Rezeptor-positiven Mammakarzinomen oder Patientinnen mit unbekanntem Rezeptorstatus aber gunstigen Prognosekriterien wurden mit Tamofen® (Fa. Rhone-Poulence) einem der folgenden Therapieschemata unterworfen: 1.) 160 mg/Tag fur eine Woche, dann Umsetzen auf eine Dauerdosis von 20 mg/Tag; 2.) 80 mg/Tag fur eine Woche, Erhaltungsdosis 20 mg/Tag; 3.) 80 mg/Tag fur eine Woche, Erhaltungsdosis 40 mg/Tag; 4.) Dauerdosis 30 mg/Tag.

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 10(3) for 3H-R5020, 4.5 X 10(4) for 3H-progesterone and 4.0 X 10(4) 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,…

Steroid hormone receptor analysis in human melanoma and non-malignant human skin

Lack of binding of gestodene to estrogen receptor in human breast cancer tissue

Competition studies with progesterone and estradiol receptors of human myometrial tissue as well as of mammary cancer tissue showed that gestodene bound with high affinity to the progesterone receptor, as did other synthetic and natural progestogens. However, gestodene did not bind to the estradiol receptor. The relative binding affinities of all tested synthetic and natural ligands showed no organ-specific differences and no differences between neoplastically transformed and normal tissues.