Transient transcriptional inhibition of the transferrin gene by cyclic AMP.

Transferrin mRNA content and gene transcription rate were measured in the liver of rats submitted to iron overload or depletion, castration, treatment with sexual steroid hormones, glucagon and cyclic AMP. The influence of puberty in males and females and of pregnancy was also analysed. Glucagon and cyclic AMP reduced mRNA level by about 50% at the 12th hour of treatment and transferrin gene transcription by as much as 95% at the 30th minute of drug infusion, with a secondary increase of the transcription rate for a protracted treatment. None of the other hormones tested had any detectable effect on transferrin gene expression, the same being true for iron overload or depletion.

Binding and action of glucocorticoids and mineralocorticoids in rabbit mammary gland. Exclusive participation of glucocorticoid type II receptors for stimulation of casein synthesis.

In order to ascertain whether the effect of corticoids upon casein synthesis in pregnant rabbit mammary gland culture is due to interactions with classical glucocorticoid or type I (mineralocorticoid) receptors we have demonstrated the existence of both types of receptors in the tissue and have studied the effects of aldosterone and the specific glucocorticoid agonist RU 28362 upon casein synthesis in tissue culture. Both compounds significantly stimulated prolactin-induced casein synthesis. On dose-response studies RU 28362 proved to be as active as dexamethasone, cortisol was active at intermediate concentrations and aldosterone was the least active. The three glucocorticoids were able to stimulate DNA synthesis in the tissue, but aldosterone had no effect. Finally, RU 486, a potent glucocorticoid antagonist, blocked the action of aldosterone and the other corticoids upon casein synthesis, whereas spironolactone, a mineralocorticoid antagonist, was unable to do so. These results demonstrate that the stimulatory effect of corticoids upon casein synthesis in pregnant rabbit mammary tissue culture is mediated through classical (type II) glucocorticoid receptors. Transferrin accumulation in the tissue was not modified by any treatment, indicating that the action of the steroids was specific for casein, and not a general stimulation of protein synthesis.

Pharmacology of antiandrogens and value of combining androgen suppression with antiandrogen therapy.

Antiandrogens are compounds able to block the effect of androgens directly on their target cells by inhibiting their binding to the androgen receptor (AR). Two chemical classes of antiandrogens are presently on the market or in clinical trials: steroids (cyproterone, megestrol acetates), and nonsteroids (flutamide, nilutamide). Steroid antiandrogens interact not only with AR but also with progestin and glucocorticoid receptors and thus give rise to progestin and glucocorticoid effects. By contrast, nonsteroid antiandrogens interact only with AR and are thus devoid of other hormonal or antihormonal activities. Nilutamide does not need to be transformed into an active metabolite, unlike flutamide, and interacts with dog, rat, and human prostate AR in vitro. Its kinetics lead to a prolonged interaction with AR in vivo after administration to rats. In prostate cancer treatment, it is necessary to combine an antiandrogen to surgical or chemical (estrogens, LH-RH agonists) castration to obtain a complete suppression of androgens. The antiandrogen will block specifically, at the target site, the trophic effect of adrenal androgens left intact by castration, and the secretion of which can only be suppressed by treatments (adrenalectomy, aminoglutethimide, ketoconazole) that also suppress corticoid synthesis. We have shown that nilutamide counteracts the trophic effect, on the prostate of castrated rats, of adrenal androgens administered continuously (minipumps) at circulating levels similar to those recorded in castrated men. Nilutamide will also impede the flare-up effect of the testosterone increase induced by LH-RH agonists at the beginning of treatment. We have shown in the rat treated with buserelin that the increase in prostate weight observed during the initial days of treatment by the LH-RH agonist can be inhibited by a combined treatment with nilutamide. This combined treatment "nilutamide plus castration" has been tested in an experimental androgen-dependent cancer model, the Shionogi tumor. The administration of nilutamide to mice, castrated twenty-four hours before the inoculation of tumor cells, delayed the appearance of tumors and reduced their number. Finally, the absence of androgen effect and the antiandrogen activity of the product were also demonstrated in human tumor cells in culture (T-47 D cells) transfected with the MMTV androgen-dependent promoter coupled with the CAT reporter gene.

Estrogen-sensitive progestin-binding sites in the female rat brain and pituitary.

The following properties of the cytoplasmic progestin receptor were studied in the hypothalamus, cortex, pituitary and uterus of the estrogen-primed castrated adult female rat using the highly potent progestin R 5020 (promegestone). (a) Sedimentation pattern. In sucrose density gradients, the R 5020-progestin receptor complex sedimented with a coefficient of about 6 to 7S. (b) Binding parameters. R 5020 bound to the progestin receptor with an intrinsic dissociation constant of about 10(-9) M as measured by a Dextran-coated charcoal (DCC) technique. The number of binding sites, however, differed widely. (c) Specificity. Only progestins competed for [3H]R 5020 binding. (d) Estrogen-dependency. In both immature and castrated adult rats, estrogen administration increased the number of R 5020-specific binding sites, assayed in vitro by a DCC technique, in the uterus, pituitary and hypothalamus, but not in the amygdala, hippocampus nor in the cortex. The increase was maximum between 40 and 48 h after priming with the potent estrogen, moxestrol, and could not be induced by androgens nor by progestins.

A progesterone metabolite enhances the activity of the GABAA receptor complex at the pituitary level.

The interaction of 5 alpha-pregnane-3 alpha-ol-20-one (5 alpha 3 alpha P), a progesterone metabolite, with the GABAA receptor chloride channel complex was investigated at the pituitary level. In nanomolar concentrations this steroid potentiated the inhibitory effect of muscimol (a GABAA agonist) on prolactin release from rat pituitary cells in culture. In micromolar concentrations 5 alpha 3 alpha P had a direct inhibitory effect, similar to that of muscimol, with an IC50 value of 370 nM. This effect was antagonized by bicuculline, a GABAA antagonist, and by picrotoxin, a chloride ion channel blocker. Its reduced isomer, 5 alpha 3 beta P, and progesterone (Pg) were devoid of activity. Using [35S]t-butylbicyclophosphorothionate ([35S]TBPS) as a ligand, we demonstrated, for the first time, specific barbiturate sites on pituitary membranes, similar to those of the central nervous system, with a Kd value of 25 nM and a Bmax value of 62 fmol/mg protein. 5 alpha 3 alpha P inhibited the binding of [35S]TBPS. In contrast, its 3 beta isomer was inactive. These data show that 5 alpha 3 alpha P enhanced the activity of the GABAA receptor complex at the pituitary level and suggest that its inhibitory effect on prolactin release might be mediated by the barbiturate site or by a closely related site.

Inhibition of progesterone secretion during the luteal phase by two luteinizing hormone-releasing hormone agonists in Macaca fascicularis.

The administration of 200 microgram of the potent luteinizing hormone-releasing hormone (LHRH) agonist [D-Leu6,des-Gly-NH2(10)]LHRH ethylamide on day 6 following the plasma estradiol peak to 11 female monkeys (Macaca fascicularis) during two consecutive menstrual cycles decreased plasma progesterone levels by 40.0% +/- 3.9% as compared with previous control cycles. The plasma estradiol profile and the cycle length were not affected significantly by the treatment. Similar results were obtained with 25 microgram of [D-Ser(TBU)6,des-Gly-NH2(10)]LHRH ethylamide administered to one monkey at the same period of the cycle, such treatment leading to a 41% inhibition of circulating progesterone levels. Although plasma progesterone levels were still reduced in the two post-treatment cycles in monkeys treated with the high dose (200 microgram) of [D-Leu6,des-Gly-NH2(10)]LHRH ethylamide, the recovery cycle was normal after the administration of a lower dose (25 microgram) of [D-Ser(TBU)6, des-Gly-NH2(10)] LHRH ethylamide. The M. fascicularis monkey thus appears as a valid model with which to study the inhibitory effects of LHRH agonists on luteal function.

Autoradiographic localization of glucocorticosteroid binding sites in rat brain after in vivo injection of [3H]RU 28362.

The autoradiographic distribution of glucocorticosteroid binding sites in the brain of adrenalectomized rats was studied following in vivo injection of a potent synthetic glucocorticosteroid agonist [3H]RU 28362. Analysis of the autoradiograms revealed a specific and dense labeling in the pyramidal cell layer of the Ammon's horn and in the granular cell layer of the dentate gyrus of the hippocampus. In the hypothalamus, the labeling was particularly high in the paraventricular nucleus (site of CRF synthesis), the arcuate, periventricular and the supraoptic nuclei as well as in the median eminence. Autoradiograms also revealed the presence of [3H]RU 28362 binding sites in several brain regions including the amygdala, the pineal gland, the entorhinal cortex, the interpeduncular, interfascicular and dorsal raphe nuclei, the central grey and the substantia nigra suggesting possible effects of glucocorticosteroids in these structures.

Comparison between the interaction of steroids with [35S]TBPS binding to cerebral cortical and to pituitary membranes: correlation with inhibition of prolactin release.

It has been shown previously that 5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha P) can inhibit prolactin release from anterior pituitary gland cells in culture through an interaction with a specific modulatory site on the GABAA receptor complex in anterior pituitary gland membranes. In the present work, this receptor site has been labelled with [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to enable a study of the relative binding affinities (RBA) of different steroids for the GABAA receptor complex to be made. We have found a high correlation (r = +0.88) between the inhibition of [35S]TBPS binding to anterior pituitary membranes and the inhibition of [35S]TBPS binding to cerebral cortical membranes by nine different steroids. There was also a high correlation between the inhibition of prolactin release from anterior pituitary gland cells in culture by these steroids and the inhibition of [35S]TBPS binding to anterior pituitary membranes (r = +0.99) or to cortical membranes (r = +0.81). These observations suggest that the measurement of prolactin release from anterior pituitary gland cells in culture is a good indicator of the functional activity of drugs that bind to the allosteric modulatory TBPS-binding site on the GABAA-receptor complex.

Progestin binding sites in the rat hypothalamus pituitary and uterus.

Specific, estrogen-inducible, nuclear radioactivity uptake has been demonstrated in the uterus, pituitary and hypothalamus of immature female rats after injection of a highly potent labelled progestin, R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione). A cytoplasmic progestin "receptor" has been characterized in these target tissues by an in vitro Dextran-coated charcoal adsorption method. R 5020 binds with the same intrinsic dissociation constant to the receptor present in these tissues; it dissociates at the same rate from the uterine and pituitary receptor (k-1 = 1 multiplied by 10(-2) min (-1), but about 6 times slower than progesterone. This evidence, together with the similar hormone specificity in the uterus and pituitary, suggests that the progestin "receptor" is a similar entity in central and peripheral target tissues.

Cytosolic and nuclear sex steroid receptors in meningioma.

To determine the nature of the hormone dependency of meningioma, particularly whether progestin receptor (PR) and tumor growth are estrogen-dependent, we measured the cytosol estrogen receptor (ER) and PR separately by an exchange assay with [3H]R 2858 (for ER) and [3H]RU 27987 (for PR) in 21 meningiomas. We correlated these receptors with age, plasma estradiol and progesterone concentrations, histological subtypes, clinical data such as uterine fibromas, and finally preoperative glucocorticoid therapy. ER was detected at low levels in the cytosol and in the nucleus of a few tumors whereas PR was found in 20 of 21 cases at moderate to high levels, usually with higher concentrations in the cytosol than in the nucleus. No clear relation could be drawn between either cytosol or nuclear ER and PR levels and estradiol and progesterone plasma concentrations, or with the other parameters. On the contrary, a clear correlation could be drawn between cytosolic PR and androgen receptor (AR) concentrations (measured with [3H]R 1881 in the presence of triamcinolone acetonide), leading to several hypotheses of hormonal regulation in meningioma.

The antiandrogen anandron potentiates the castrating effect of the LH-RH agonist buserelin in the rat.

When buserelin (0.04-25 micrograms/kg/day), a potent LH-RH agonist, was administered s.c. daily for 15 days to male rats, prostate weight decreased after a transient increase in the first days of treatment but never as much as after orchidectomy, since testosterone secretion was never totally suppressed. The combination of the pure nonsteroid antiandrogen Anandron (20 mg/kg/day) with even low doses of buserelin led to an immediate decrease in prostate weight that was complete at 15 days. This could be explained (a) by an additivity of effects: inhibition by Anandron of the action of residual testosterone unsuppressed by buserelin on the prostate and prevention by buserelin of the rebound testosterone increase induced by Anandron: (b) by a potentiation by Anadron of the direct castrating effect of buserelin on the testis since testis weight and testosterone secretion were lowered to a greater extent by the combination than by buserelin alone. Anandron might increase the sensitivity of LH-RH receptors to LH-RH agonist in the testes, as has been shown in the pituitary. In contrast, the combination of the antiandrogenic steroid progestin, cyproterone acetate, with buserelin never potentiated the castrating effect of buserelin on testis weight and testosterone and only partially potentiated prostate atrophy. If such actions also exist in the human, the addition of Anandron to LH-RH agonist treatment would not only counter flare-up and adrenal androgen effects on the prostate but would also lower the doses of peptide or the time required for chemical castration.

[Cancer of the prostate: biologic bases for the use of an antiandrogen in its treatment]. / Cancer de la prostate: bases biologiques pour l'emploi d'un antiandrogène dans le traitement.

Although orchiectomy, estrogens and LHRH agonists suppress testicular androgens, they are without effect on adrenal androgens which are converted into dihydrotestosterone in the prostate. It is therefore necessary to develop substances able to block the action of all androgens, whatever their source, on target organs. The non-steroid, Anandron (RU 23908), when administered orally, gives rise to a high and sustained plasma level of intact compound that inhibits testosterone binding to its receptor. This inhibition, however, occurs not only in the prostate but also in the pituitary. The negative feedback action of androgens is thus inhibited by Anandron resulting in an increased secretion of testosterone and explaining the necessity of combining Anandron with castration (whether surgical or medical by an LHRH agonist). Anandron opposes, on the one hand, the action of adrenal androgens and, on the one other, of the testosterone surge that occurs during the early days of treatment with the LHRH analog. The efficacy of the combined treatment has been demonstrated experimentally. Clinical trials are presently underway.

[Steroid receptors in the central nervous system. Implications in neurology]. / Les récepteurs de stéroides du sytème nerveux central. Implications en neurologie.

This review deals with steroid hormones and receptors in relation to the physiology and the pathology of the central nervous system (CNS) and meninges. In recent years experiments performed in animals showed that: 1) endogenous steroid hormones cross the blood brain barrier: 2) radiolabelled steroid hormones bind in specific areas of the CNS; 3) all five classes of steroid receptors, i.e. oestrogen, progesterone, androgen, glucocorticoid and mineralocorticoid receptors (OR, PR, AR, GR, MR), are present in brain tissues, especially in the hypothalamus and the limbic system; 4) the interaction of steroid hormones and specific receptors induces the synthesis of proteins in the CNS; 5) finally, in situ metabolism of steroid hormones has been evidenced by the presence of specific enzymes. A few studies in human brain tissues have shown the presence of GR and OR as well as enzymes involved in the metabolism of sex steroid hormones. In neurology, some epidemiological and clinical data suggest the implication of steroid hormones and receptors in human CNS: 1) the influence of oestrogens in tardive dyskinesia; 2) the relevance of hormonal changes in benign intracranial hypertension; 3) the usefulness of glucocorticoid therapy in many patients with intracranial tumors and/or edema. Due to feasibility, most researches have concerned tumors: meningioma, neurinoma and glioma. Firstly, a reappraisal of biochemical and histochemical technics used to detect and characterize the receptors in tumors is presented. Then results from the recent literature are reviewed. In meningioma, PR was found in 89 p. 100 (152/177) of the cases, usually at moderate to high levels (up to 33 000 fmol/gT). In addition, PR has been fully characterized from a biochemical point of view. Furthermore, it has been hypothesized that PR may be a marker of leptomeningeal cells since it was detected at high levels in well differentiated tumors provided they had no or few psammoma. This was further supported by the discovery of PR in normal leptomeninges in human adults. OR was detected in 48 p. 100 (87/177) of the meningioma, at low levels. This is in contrast with PR but the percentage of cases with OR raises to 70 p. 100 (42/60) if one considers only tumors assayed for both cytosolic and nuclear receptors. Therefore it has been suggested that OR had translocated into the nucleus, at least in some cases, and subsequently the hypothesis of functional OR in meningioma was raised. AR was also detected in meningioma. Furthermore AR levels were found to correlate well with PR levels.(ABSTRACT TRUNCATED AT 400 WORDS)

RU 38486: potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation.

In order to explain the potent antiglucocorticoid activity of RU 38486 and the absence of agonist effect in spite of its very strong interaction with the cytoplasmic glucocorticoid receptor (GR), we investigated the compound's ability to promote GR "activation" and nuclear translocation. We have compared the dissociation-rates of the "non-activated" (molybdate stabilized) and of the "activated" (25 degrees C pre-heated) GR complexes formed either with [3H]RU 38486 or with different tritiated glucocorticoid agonists. While agonists dissociated more slowly from the "activated" than from the "non-activated" complex, RU 38486 dissociated much faster from the "activated" than from the "native" receptor. This difference of activation was confirmed in a DNA-cellulose binding assay. The affinity of the "activated" RU 38486-GR complex for DNA was much lower than that of the dexamethasone-GR complex. Finally, the in vitro nuclear uptake of [3H]RU 38486 was compared with that of [3H]dexamethasone after incubation with thymus minces at 25 or 37 degrees C. A very weak or nearly undetectable level of specific uptake of [3H]RU 38486 was observed in purified nuclei, whatever the concentration or the time of incubation used. These observations suggest that while glucocorticoid agonists form with the non-activated receptor a complex able to be activated into a more stable form (lower k-1), RU 38486 interacts strongly with the non-activated receptor (impeding the binding of DM) but the complex is "transformed" by heat to a less stable form (higher k-1), unable to translocate properly into the nucleus in order to trigger a glucocorticoid response.

Pharmacological and clinical studies of the antiandrogen Anandron.

This paper summarizes the animal and human studies with Anandron available at the time of the meeting. The following was demonstrated in the rat and confirmed in man: interaction of Anandron with the prostatic androgen receptor, antiandrogen activity against testosterone (in particular against the early transient rise induced by LHRH analogs) and adrenal androgens. Thus, as shown in 4 different double blind studies performed in stage D2 prostrate cancer patients, the combination of Anandron with surgical or chemical castration enhanced the beneficial effects of castration alone and thus seems a step forward in the hormonal treatment of prostatic carcinoma.

How the study of the biological activities of antiandrogens can be oriented towards the clinic.

Antiandrogens can be used in various androgen-dependent diseases. Depending upon the therapeutic indication, they can be administered systemically or topically. Systemic treatment with an antiandrogen will inhibit androgen action not only in the desired target site but also in all other target tissues; thus, it will block the androgen-dependent feedback regulating the secretion (hypothalamo-pituitary-testis axis) or the action (protein factors) of androgens. In contrast, topical treatment (acting through cutaneous receptors or local metabolism) should not produce systemic side effects especially in man. Pharmacological assays which can select antiandrogens irrespective of the mechanism measure changes in the final androgenic response, but they consume a great deal of time and test compound and bear little relation to therapeutic activity. Therefore, the biological strategy that we report here and which, at Roussel-Uclaf, has led to the selection of a systemic and a topical antiandrogen (RU 23908 and RU 38882) has consisted in successively performing: (1) in vitro assays which measure an effect at a specific level in the mechanism of antiandrogen action, e.g. interaction with the androgen receptor. Assessing interactions with other classes of steroid hormone receptor can be used to predict possible hormonal side-effects, (2) in vitro determinations of agonist or antagonist activity, e.g. in pituitary cells (LH response to LHRH) or mammary tumor cells (induction of androgen-dependent proteins), (3) in vivo antiandrogen assays after a single treatment (induction of mouse kidney proteins, rat prostatic binding protein) or after repeated treatment (inhibition of the growth of rat accessory glands or of hamster sebaceous glands), to determine the active dose of the compound and possibly the absence of systemic effects by the topical route, (4) assays in animal models designed to mimic a therapeutic context e.g. for prostate cancer: inhibition of the "flare-up" effect of LHRH-A or of the trophic effect of perfused adrenal androgens on rat prostate, antitumoral activity in experimental cancer models. For hyperseborrhoea and acne: histological and stereological analysis of rat skin biopsies to measure the volume density of the smooth endoplasmic reticulum vesicles of the differentiating cells of the sebaceous gland.

Pharmacology of an antiandrogen, anandron, used as an adjuvant therapy in the treatment of prostate cancer.

To improve the inhibition of prostate cancer growth obtained by surgical or chemical castration (estrogens or LHRH analogs), blockade of the action of residual androgens of adrenal origin has been proposed. Among antiandrogens acting through the androgen receptor (AR), the nonsteroid anandron (RU 23908) has several advantages over available compounds: megestrol acetate and cyproterone acetate, both steroids, bind substantially to other hormone receptors (progestin, gluco- and mineralocorticoid); and anandron binds only to AR. The nonsteroid flutamide is a prodrug converted to the active metabolite, hydroxyflutamide; anandron is well absorbed on oral administration of an active dose and intact compound disappears slowly from plasma. This may explain why, although in vitro anandron interacts very transiently with AR, in vivo a high level of untransformed anandron is present at the receptor site to induce its antiandrogenic activity. Animal experiments confirm that anandron can counteract the effect of adrenal androgens and inhibit the LHRH analog-induced initial increase in androgen ("flare-up"). Thus, in rats castrated either surgically or by buserelin or DES and supplemented with adrenal androgens (since endogenous adrenal secretion is very low in this species compared to man), anandron decreased prostate weight to control levels. The administration of buserelin to intact rats over 15 days resulted in a significant increase in prostate weight between Days 1 and 5. The addition of anandron to the buserelin inhibited this increase and, furthermore, led to a far greater decrease in prostate weight than that due to buserelin alone at 15 days, indicating a synergy of action.

[Molecular basis for the use of steroids in psychiatry (author's transl)]. / Bases moléculaires de l'emploi des stéroïdes en psychiatrie.

In animals, estrogens and progestins modulate food intake, running wheel activity and sexual behavior, androgens stimulate sexual behavior and aggression, and corticoids influence cerebral electrolyte regulation, sleep and memory. In this and other studies, we have established that soluble receptors binding steroid hormones are present, often in high concentration, in various central structures such as hypothalamus, cortex, hippocampus and amygdala and have shown that some of the effects of steroids on behavior can be related to their interaction with these receptors and the resultant biochemical events. Thus, in the rat, the induction of sexual behavior by estrogens and progesterone can be related to the increase in progestin receptor induced in the hypothalamus by estrogens. The action of glucocorticoids has been implicated in memory processes involving protein synthesis and this study shows that glucocorticoids stimulate protein synthesis in the cortex of hypophysectomized rats, a structure rich in glucocorticoid receptors. Since the hormonal specificity of receptor binding is similar in peripheral and central target tissues, it is suggested that competition experiments on a peripheral target tissue can be used as a simple test to select compounds (agonists or antagonists) able to replace endogenous hormones at the receptor site in the treatment of psychiatric disorders due to endocrine dysfunction (hormone deficit or excess).

Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute s.c. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [3H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose-dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.