Antagonism of glucocorticoid action in cultured hepatoma cells

We report here our recent data on the effects of antiglucocorticoids in two established cell lines (HTC, FAZA). These steroid hormone target tissues were designed to consider the problem of differential antagonism and lack of correlation between antiglucocorticoid activity and competition for the glucocorticoid receptor. In the systems chosen, several responses were considered which may be differentially antagonized: induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and tryptophan oxygenase (TPO). By testing the anti-inducing capacities of a number of steroids, we found a few synthetic compounds like promegestone and R 25055 which exert a stronger antagonism against TAT and AAT induction than natural steroids like progesterone. The availability of highly radioactive antagonists (promegestone, progesterone) has greatly facilitated our "whole cell" study and allowed us to detect the antagonists in isolated nuclei whose purity and morphological integrity were controlled by specific criteria; our results suggest that the binding of the antagonists to the nucleus proceeds via the glucocorticoid receptor. The appearance of promegestone and progesterone in the nucleus suggests that the nucleus may be involved in the mechanism of action of anti-glucocorticoids.     

Effects of progestins and antiprogestins on mitochondria in uterine glandular cells in the rat

Summary The administration of progesterone to ovariectomized rats induces an increase in the volume density (Vv) of the mitochondria and the appearance of giant mitochondria in the uterine glandular cells. This experimental model, including a stereological analysis, allowed us to investigate and quantify a direct effect of progesterone on a well-defined cellular structure without the intervention of estrogen in a priming phase. Synthetic compounds, promegestone, gestrinone and RU 38486, were tested in this model either in place of progesterone or simultaneously with progesterone. The potent progestomimetic activity of promegestone was confirmed by the proliferation of giant mitochondria and a high Vv value for the mitochondria, the two other compounds being inactive even at higher doses. At lower doses, gestrinone and RU 38486 partially inhibit the action of progesterone and at higher doses they both show a complete antagonist effect by preventing the development of the mitochondria.     

Metabolism and antiproliferative effect of the glucocorticoid antagonist RU38486 in cultured liver and hepatoma cells

In an attempt to elucidate the relationship between the antiglucocorticoid effect and the state of differentiation of the target cells, we studied the metabolism of the potent antagonist in cultured liver and hepatoma cells (HTC, FAZA). After incubation of [3H]RU38486 with the cells for different periods of time, the native steroid and its metabolites were extracted and analyzed by thin layer chromatography. We observed that RU38486 was not metabolized in the transformed cell lines after a 3 h incubation. In contrast RU38486 was extensively metabolized in cultured liver cells. The observed degration could help explain why RU38486 inhibited tyrosine aminotransferase induction in hepatoma cells at a concentration 100 times lower than that needed in liver cells. Moreover this catabolism concerned specifically the antagonist RU38486 since other steroids tested (dexamethasone, promegestone) underwent a much slower degradation. Indirect experiments suggest that the alterations of the RU38486 molecule might be at least partially related to the cytochrome P-450 which is very active in the hepatocytes. This study was paralleled by testing the effect of the antagonist on the growth of hepatoma cells. RU38486 exerted an antiproliferative effect in absence of serum. On the basis of the low metabolism of RU38486 and of its antiproliferative effect in hepatoma cells. one can emphasize that RU38486 might represent a potential drug for use in cancer therapy.     

Active immunization of female rats against 17 beta-estradiol. Preliminary studies on 17 beta-estradiol binding in uterine and pituitary cytosols

Female rats were immunized with 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin. They developed antibodies to estradiol and, to a very low extent, antibodies to BSA. Anti-estradiol antibodies possessed tight specificity to estradiol-17 beta, without cross-reactivities with other estrogens. It was demonstrated that the specific estradiol binding in uterine and pituitary cytosols gradually decreased when antiserum titres increased. In uterine cytosols, the presence of progesterone receptor was studied using promegestone (R50 20) as ligand. No significant variations in promegestone binding were observed. Competition experiments however, questioned the permanence in immunized rats of the actual progesterone receptor or of a promegestone binding protein.     

Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.     

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.     

Progestagen-concentrating cells in the brain, uterus, vagina and mammary glands of the galago (Galago senegalensis)

Progestagen-concentrating cells were localized in the oestrogen-primed ovariectomized galago by radioautography after injection of [3H]promegestone (R5020). In the brain, radioactivity was concentrated in the nuclei of neurones in the preoptic region and in the mediobasal hypothalamus. Labelled cells were also observed in the anterior pituitary. In the uterus (uterine horns and cervix), the muscle and stromal cells showed greater labelling than did the glandular and luminal epithelia. Labelled cells were present in the different cell layers of the vagina. The majority of glandular epithelial cells of the mammary glands exhibited a high degree of labelling. Pretreatment with an excess of unlabelled promegestone but not with an excess of nonradioactive testosterone reduced the nuclear concentration of radioactivity in these target tissues. These results show that there are no major differences in the distribution of progestagen-concentrating cells in rodents and galago.     

Progestin effects on growth in the human breast cancer cell line T-47D--possible therapeutic implications

In order to determine growth effects of the progestin R5020, (promegestone), we have utilized the progesterone-receptor rich human breast cancer cell line T-47D, growing the cells in the absence of the pH indicator phenol red, which has recently been found to be estrogenic. In contrast to reports on cells grown in the presence of phenol red, we find that promegestone alone, at physiological progestin concentration, significantly stimulates growth. Estradiol alone, at physiological concentration, stimulates growth much more. Promegestone in combination with estradiol is antiestrogenic for growth; that is, it significantly decreases the growth stimulatory effect of estradiol. These results raise the possibility that estrogen receptor and progesterone receptor-rich breast cancer patients might benefit more from a combination of anti-progestin and anti-estrogen therapy than from anti-estrogens alone.     

Conditionally adherent growth of serum-independent CHO cells for automated drug screening and biopharmaceutical production.

SSF3 is a CHO cell line adapted for growth in protein-free medium. It grows in suspension unless serum-derived attachment factors such as vitronectin are added to the medium. Serum-independent cell lines, which adhere to the substrate after induction with dexamethasone or constitutively, were created by transfection with a human vitronectin gene under control of the mouse mammary tumor-virus promoter. Substrate attachment and SSF3VN-cell spreading could be prevented with an RGD peptide (arginine-glycine-aspartic acid) confirming that attachment is mediated by an intregrin receptor. Hormone-inducible attachment could be blocked by glucocorticoid antagonist promegestone. All steps in the isolation of stable transfected SSF3VN cell lines could be done in a chemically defined medium avoiding the risk of introduction of serum-derived infectious agents. SSF3VN cells could be grown in protein-free medium in solid-phase large-scale bioreactors. Application in microplates as used in high-throughput screening was demonstrated in an assay of Ca(2+) release from internal stores induced by agonist-binding to recombinant human metabotropic glutamate receptor hmGluR1b.     

Failure of the platelet-activating-factor antagonist WEB 2086 BS for treatment of chronic autoimmune thrombocytopenia

Thirteen patients with chronic autoimmune thrombocytopenia (AITP) were treated for 14 days with daily oral doses of 120 mg of the novel platelet-activating-factor (PAF) antagonist WEB 2086 BS. Clinical bleeding symptoms remained essentially unchanged in 9 patients and became more pronounced in the post-treatment period in 4 patients. In no case was an increase in platelet counts observed. While the PAF antagonist was well tolerated subjectively during treatment, most patients exhibited a prolongation of the sensitive "hemostasis time" (a modified bleeding time test) after treatment, but the Duke bleeding time was not changed. We conclude that the PAF antagonist WEB 2086 BS is ineffective for treatment of chronic AITP and should be used with caution in thrombocytopenic patients.     

Effects of progesterone, promegestone and RU 486 on glucocorticoid receptor levels in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain glucocorticoid receptors whose levels are modulated by a variety of steroids. In the absence of any added steroid to the cell culture medium, the levels of glucocorticoid receptors in the cells decline during culture, which is counteracted by the addition of a variety of glucocorticoid agonists. The effectiveness of the glucocorticoid in preventing the loss of glucocorticoid receptors is in turn counteracted by the addition of the synthetic progestin promegestone and the synthetic antiglucocorticoid RU 486. Of the two, RU 486 is the most potent in antagonizing the effect of cortisol on the GR levels. Promegestone antagonizes the effect of cortisol, too, although higher concentrations are necessary. Progesterone was without a clear effect either as a glucocorticoid agonist or an antagonist. Progesterone, however, was extensively metabolized by mammary epithelial cells in culture. Based on these observations we conclude that in mammary epithelial cells glucocorticoids positively regulate the metabolism of their own receptors and that antiglucocorticoids, such as RU 486 and progestins, can antagonize that effect.     

The selective estrogen enzyme modulator (SEEM) in breast cancer

Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17β-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol biosynthesis. This tissue also contains sulfotransferase for the formation of the biologically inactive estrogen sulfates. In the last years, it was demonstrated that various progestins (promegestone, nomegestrol acetate, medrogestone), as well as tibolone and its metabolites are potent inhibitors of sulfatase and 17β-hydroxysteroid dehydrogenase activities. It was also shown that medrogestone, nomegestrol acetate, promegestone or tibolone can stimulate the sulfotransferase activity for the local production of estrogen sulfates. All these data, in addition to numerous agents, which can block the aromatase action, lead to the new concept of selective estrogen enzyme modulators (SEEM), which can largely apply to breast cancer tissue. The exploration of various progestins and other active agents in trials with breast cancer patients, showing an inhibitory effect on sulfatase and 17β-hydroxysteroid dehydrogenase, or a stimulatory effect on sulfotransferase, will provide a new possibility in the treatment of this disease.     

Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery

We examined the contributions of the cotransmitters norepinephrine (NE), ATP, and neuropeptide Y (NPY) to sympathetically evoked vasoconstriction in the rat tail artery in isolated vascular rings by using 1-100 stimulation impulses at 20 Hz. Phentolamine (2 microM), the alpha-adrenoceptor antagonist, markedly reduced responses to all stimuli, although responses to lower impulse numbers were reduced less than responses to longer trains. The purinergic receptor antagonist suramin (100 microM) reduced all responses, but to a much greater extent with few impulse trains. Responses were further reduced or abolished by addition of the second antagonist. Any remaining responses were abolished by the NPY-Y(1) receptor antagonist BIBP-3226 (75 nM). NPY had a direct agonist action and potentiated sympathetically mediated responses. NPY (75 nM) potentiated responses and BIBP-3226 decreased responses to 2- and 20-impulse trains. Both affected responses from 2 impulses to >20 impulses, but there was no preferential effect on purinergic contributions to responses because neurally released NPY potentiated both "pure" NE and ATP responses equally. We conclude that all three cotransmitters contribute significantly to vascular responses and their contribution varies markedly with impulse numbers. There is considerable synergy between cotransmitters, especially with lower impulse numbers where NPY contributions are greater than expected.     

Naloxone-6α-spirohydantoin: A long-acting opiate antagonist

We have synthesized a novel derivative of naloxone, naloxyl-6-spirohydantoin. The compound was rigorously purified and its stereochemical structure characterized. In tests in vivo in mice, it was less toxic than the parent compound and it showed weak anticonvulsive activity. It exerted antagonist effects comparable to those of naloxone but its effects were longer lasting, persisting for a week.     

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.     

Reduction in Thermal Hyperalgesia by Intrathecal Administration of Glycine and Related Compounds

We have previously shown in animal models that enhanced segmental glycine release is produced by neuroaugmentation techniques commonly used to control pain in humans. Our current hypothesis is that glycine administered intrathecally reduces the pain response evoked by the hotplate analgesia meter method. Neuropathic rats created by unilateral partial ligation of the sciatic nerve were treated with intrathecal infusion of glycine, strychnine, MK-801, or 5–7 DKA at 0.1 mol for 2 hours at a rate of 10 l/min. Time required for limb withdrawal at 42°C was significantly increased after glycine administration but not altered by strychnine, a specific glycine receptor antagonist. Administration of the NMDA receptor antagonist, MK-801, blocked the influence of glycine, with a less obvious antagonistic response from 5,7 DKA. Our results provide evidence that glycine and related compounds significantly modify thermal hyperalgesia, and may operate primarily through the NMDA receptor complex.