metribolone and 16-alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3-20-dione

Drug-receptor binding thermodynamics has proved to be a valid tool for pharmacological and pharmaceutical characterization of molecular mechanisms of receptor-recognition phenomena. The large number of membrane receptors so far studied has led to the discovery of enthalpy-entropy compensation effects in drug-receptor binding and discrimination between agonists and antagonists by thermodynamic methods. Since a single thermodynamic study on cytoplasmic receptors was known, this paper reports on binding thermodynamics of estradiol, ORG2058, and R1881 bound to estrogen, progesterone, and androgen steroid/nuclear receptors, respectively, as determined by variable-temperature binding constant measurements. The binding at 25 degrees C appears enthalpy/entropy-driven (-53.0

Topics: Estradiol; Female; Humans; In Vitro Techniques; Male; Metribolone; Pregnenediones; Prostate; Receptors, Androgen; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Progesterone; Receptors, Steroid; Thermodynamics; Uterus

Enhanced expression of fatty acid synthase and other lipogenic enzymes has been observed in a subset of breast cancers with poor prognosis. This phenomenon has been related to amplification of a gene on chromosome region 11q13 encoding Spot 14, a putative regulator of lipogenic enzyme expression. In this paper we demonstrate that the induction of lipogenesis by progestins and androgens in the breast cancer cell line T47-D is accompanied by a marked increase in the expression of Spot 14. These data corroborate the correlation between Spot 14 expression and increased lipogenesis. Moreover they show that apart from gene amplification there is another steroid-regulated pathway that may enhance Spot 14 expression and lipogenesis in tumor cells.

Topics: Breast Neoplasms; Chromosomes, Human, Pair 11; Dihydrotestosterone; Female; Gene Expression; Humans; Lipids; Metribolone; Neoplasms, Hormone-Dependent; Nuclear Proteins; Pregnenediones; Progesterone; Progesterone Congeners; Proteins; Testosterone Congeners; Transcription Factors; Tumor Cells, Cultured

A vast number of substances have been suggested as possibly contributing to perturbation of the endocrine system. Several have been tested with different approaches ranging from yeast expression system of human oestrogenic receptors to human breast cancer cells assays. Surprisingly, no inhibition-binding experiments to steroid receptors on healthy human tissue have been performed so far. Our study provides inhibition binding experiments to oestrogens, progesterone, testosterone and retinoic acid receptors in prostate and uterine human tissue of organochlorine pesticides, phthalate esters, oestrogenic constituents derived from plants and phenol derivates. Affinities of significant extent of phthalates on oestrogenic, progestinic and androgenic receptors have not been detected. As for retinoic acid receptors, mono(2-ethylexyl)phthalate provokes a notable reduction of the binding of the tritiated retinoic acid, phtalic acid ethyl-n-butyl ester and 4-octylphenol show an affinity comparable to that of isoflavonoid genistein, whereas 4-nonylphenol reduces the binding of retinoic acid in prostate.

Topics: Androgen Receptor Antagonists; Binding, Competitive; Culture Techniques; Environmental Pollutants; Estradiol; Female; Humans; Male; Metribolone; Phthalic Acids; Pregnenediones; Prostate; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Receptors, Retinoic Acid; Testosterone Congeners; Tretinoin; Tritium; Uterus

In order to elucidate the mechanism of androgen-regulation of genes expressed only in the prostate gland, the effects of steroid hormones on the biosynthesis and secretion of human prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were studied in the human prostatic carcinoma cell line, LNCaP. This cell line produces PAP and PSA, both of which were found to be similar to the proteins purified from and located in human prostatic tissue, as shown by Western blot analysis. The synthetic androgen, R1881, regulated the biosynthesis of these two important tumour marker proteins inversely: the amount of PSA released into the medium was increased to 506% +/- 100 of the control levels, while that of PAP was decreased to 26% +/- 3, in 7 days. These effects were dependent on the concentration of the steroid in the growth medium. The androgen-dependent changes observed in the amounts of the secreted proteins were correlated with alterations in their intra-cellular levels. LNCaP cells were found to have very different capacities for secreting PAP and PSA. Whereas the measurable, cellular amounts of PSA and PAP were of similar magnitudes, much larger amounts of PSA than PAP were secreted into the medium. PSA was also found to be more stable than PAP in the culture medium of the LNCaP cells. Other steroids could elicit effects on PAP and PSA biosynthesis similar to those induced by R1881, and the combined effects of effective concentrations of these steroids were undistinguishable from those caused by each one of them separately, suggesting that all these compounds compete for binding to the same modified androgen receptors of the LNCaP cells. Thus, our results confirm the observations of the altered nature of the LNCaP androgen receptors, and demonstrate the ability of these ligands to produce changes in the expression of androgen-dependent prostatic genes. The fact that the changes observed at the protein level were accompanied by increased levels of PSA mRNAs and by decreased levels of PAP mRNA in steroid-treated cells, suggests that one of the targets of androgen and steroid action in the regulation of these genes is at the mRNA level.

Topics: Acid Phosphatase; Antigens, Neoplasm; Biomarkers, Tumor; Cell Line; Cycloheximide; Estradiol; Humans; Kinetics; Male; Metribolone; Pregnenediones; Progesterone; Progesterone Congeners; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; Tumor Cells, Cultured

It is firmly believed that sexual differentiation of the brain is linked with external genital differentiation in timing as an in utero event in the human. An extensive search for oestrogen, androgen and progestin receptors failed to show their presence despite adequate controls in cytosols from human fetal brain of gestational ages 14-20 weeks. It is possible that the receptors are present in levels so low that they are undetectable by present-day methods. Our results would indicate that hormonally influenced in utero brain sexual differentiation is most unlikely to occur as a mid-trimester event.

Topics: Animals; Brain; Brain Chemistry; Cytosol; Diethylstilbestrol; Estradiol; Estradiol Congeners; Estrenes; Ethinyl Estradiol; Female; Gestational Age; Humans; Ligands; Male; Metribolone; Pregnenediones; Progesterone Congeners; Rats; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Sex Differentiation; Testosterone; Testosterone Congeners

Estradiol (ER), progesterone (PR), androgen (AR) and glucocorticoid receptor (GR) levels were assayed in 25 breast cancer tumors. The tissue was pulverized and homogenized in buffer, then divided into two parts: one was assayed by the standard dextran-coated charcoal method (DCC), with Scatchard plot analysis, the other was assayed by a micromethod developed in our laboratory, as described below: --incubation of the cytosol with several ligands (labelled and unlabelled) selected to avoid unwanted cross-reactions --DCC separation, followed by extraction of all receptor-bound steroids by precipitation of proteins with methanol/TCA --separation of these steroids on a high pressure liquid chromatography (HPLC) column using a methanol/water solvent --collection of the fractions of the column outlet and counting. Use of three labelled ligands and appropriate unlabelled ligands allowed assays of the four receptors. This micromethod was highly correlated with the standard method: ER = 0.985 (P less than 0.001); PR = 0.999 (P greater than 0.001); AR = 0.989 (P less than 0.001); GR = 0.867 (P less than 0.001). Thresholds of positivity were not modified. This micromethod allowed simultaneous measurement of several receptors in 40 mg biopsy specimens and can be applied to other hormone-dependent tissues.

Topics: Breast Neoplasms; Chromatography, High Pressure Liquid; Estrenes; Female; Humans; Ligands; Metribolone; Pregnenediones; Receptors, Androgen; Receptors, Estradiol; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone

In rat uterus and prostate, 7 alpha, 17 alpha-dimethyl-19-nortestosterone (DMNT) binds to the androgen receptor specifically and with high affinity. However, this steroid does not bind to glucocorticoid receptors, since it does not displace binding of [3H]triamcinolone acetonide in calf thymus cytosol. In calf uterine and human breast tumor cytosols DMNT binds to the androgen and progesterone receptors, since binding of [3H] DMNT is displaced by unlabeled 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3, 20-dione triamcinolone acetonide, and 5 alpha-dihydrotestosterone (DHT). Conversely, binding of [3H]16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione is effectively competed for by unlabeled DMNT but not by DHT. The observed differences in binding of [3H]DMNT to rat and calf uterine cytosols suggest the species specificity of progesterone receptors. Unlike DHT, DMNT has no appreciable binding to human sex-steroid binding globulin. These findings suggest DMNT as a suitable ligand for measurement and characterization of androgen receptors in rat and human prostate.

Topics: Animals; Blood Proteins; Breast; Cattle; Cell Nucleus; Cytosol; Dihydrotestosterone; Estrenes; Female; Humans; Male; Metribolone; Nandrolone; Pregnenediones; Prostate; Prostatic Hyperplasia; Rats; Rats, Inbred Strains; Receptors, Androgen; Receptors, Progesterone; Substrate Specificity; Uterus

The interaction of norethisterone (NET) and four A-ring reduced metabolites of NET with cytosol receptors for progesterone (PR), androgen (AR), and estrogen (ER) was investigated. Cytosol preparations from: uteri of adult estrogen-primed castrated rats, ventral prostates of adult castrated rats and uteri of immature rats were used as the source of PR, AR, and ER respectively. 3H-Labeled ORG-2058, R-1881, and 17 beta-estradiol were used as the radioligands. The results of competitive studies disclosed that: the most efficient competitor for PR binding sites was NET (Ki = 1.1 X 10(-7) M) followed by 5 alpha-dihydro NET (5 alpha-NET), whereas the 3 alpha,5 alpha; 3 beta,5 alpha and 3 alpha,5 beta-tetrahydro NET derivatives were ineffective the most efficient competitor for AR binding sites was 5 alpha-NET (Ki = 1 X 10(-8), immediately followed by NET, while the three tetrahydro NET derivatives were not competitors and remarkable competition for ER binding sites was only exhibited by the 3 beta,5 alpha-tetrahydro NET derivative (Ki = 4.6 X 10(-8) M) and to a lesser extent by its 3 alpha,5 alpha-epimeric alcohol, while NET and 5 alpha-NET were completely ineffective. These findings demonstrate the stereospecificity of the intracellular binding of NET and its reduced metabolites with cytosol steroid putative receptors, and provide biochemical support to the understanding of the variety of hormone-like effects observed after the in vivo administration of NET.

Topics: Animals; Binding, Competitive; Cytosol; Estradiol; Estrenes; Female; Male; Metribolone; Norethindrone; Pregnenediones; Prostate; Rats; Rats, Inbred Strains; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Receptors, Steroid; Structure-Activity Relationship; Uterus

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.

Topics: Animals; Binding Sites; Cell Line; Dexamethasone; Diethylstilbestrol; Dihydrotestosterone; Electrophoresis, Polyacrylamide Gel; Estradiol; Estrenes; Kidney Neoplasms; Metribolone; Mice; Pregnenediones; Steroids

Topics: Binding, Competitive; Cytosol; Endometrium; Estrenes; Female; Humans; Metribolone; Pregnenediones; Progesterone; Progesterone Congeners; Receptors, Progesterone; Steroids; Tritium