Receptor binding of allylestrenol, a progestagen of the 19-nortestosterone series without androgenic properties

Allylestrenol (17 alpha-allyl-17 beta-hydroxy-4-estren) is an orally active progestagen of the 19-nortestosterone series resembling progesterone since it has no detectable androgenic activity in animal studies and in the human. In the present study, the affinity of its 3-keto metabolite for the transformed progesterone receptor in intact MCF-7 cells was about twice that of progesterone and cyproterone acetate and about 2-3 times less than that of medroxyprogesterone acetate and norethisterone, reflecting the known progestational activity of allylestrenol. The affinity of 3-ketoallylestrenol for the transformed androgen receptor in intact MCF-7 cells was weak (like other progestagens lacking androgenic activity or possessing anti-androgenic activity) and lower than that of weakly androgenic progestagens. On the other hand, the relatively high affinity of 3-keto-allylestrenol for the non-transformed androgen receptor at 4 degrees C in the cytosol fraction did not reflect the known lack of androgenic activity of allylestrenol. Thus competitive studies carried out with transformed receptor complexes in intact cells at 37 degrees C and non-transformed complexes in cytosol distinguish progestagen with weak androgenic activity (e.g. norethisterone) from those displaying no androgenic activity or possessing anti-androgenic activity (e.g. 3-keto-allylestrenol, progesterone, cyproterone acetate and spironolactone).     

Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [³H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 10⁸ M⁻¹ at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [³H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [³H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [³H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [³H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C . Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [³H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K⁻¹ · mol⁻¹.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight.     

Binding of a contraceptive progestogen ORG 2969 and its metabolites to receptor proteins and human sex hormone binding globulin

Org 2969 is an orally active progestogen which can be used in oral contraceptives because of its strong ovulation-inhibiting activity and acceptability. The present study examines tha binding of Org 2969, its metabolites and reference coupounds to the progesterone and estrogen receptors in human and rabbit myometrium, the rat prostate androgen receptor and human SHBG (sex hormone binding globulin). At 4 degrees Centigrade, the affinite of the major metabolite 3-keto-Org 2969 was similar to that of levonorgestrel and 6 and 2 times higher than that of progesterone and norethisterone, respectively. At 30 degrees, the binding affinity of 3-keto-Org 2969 was twice that of levonorgestrel and 25 times that of progesterone. Other metabolites and derivatives tested displayed low but measurable affinities under equilibrium and non-equilibrium conditions. The binding characteristics of Org 2969 and its metabolites differ from those of other progestogens. The major metabolite 3-keto-Org 2969 binds strongly to the progesterone receptor and relatively weakly to the androgen receptor and human SHBG and shows little or no affinity for the estrogen receptor. Org 2969 is a strong and specific progestogen and its use in oral contraceptives will induce a low incidence of androgenic side effects.     

Effects of progestagens and Org OD14 in in vitro and in vivo tumor models

Sex steroids, in particular estradiol (E2) and progesterone (P4), play, together with other hormones and growth factors, a role in the development of normal breast tissue. The effect of four progestagens (norethisterone, 3-ketodesogestrel, gestodene and P4) and Org OD14, a steroid with weak estrogenic, progestagenic and androgenic properties were studied on growth of breast tumor cells in vitro using two subclones of MCF-7 (H and A) and T47D (S and A) cells. In addition, we investigated the effects of 3-ketodesogestrel, gestodene and Org OD14 on the growth of 7,12-dimethyl-benz(a)anthracene(DMBA)-induced mammary tumors in rats. In the in vitro assays with MCF-7 cells norethisterone, 3-ketodesogestrel and gestodene stimulated growth only at high doses (10⁻⁷ M), whereas P4 had no effect. Gestodene was more potent than 3-ketodesogestrel and norethisterone. Org OD14, stimulated cell growth at a dose of 10⁻⁸ M, while E2 is active at 10⁻¹⁰ M. In T47D-A cells similar effects were found, but the subclone S did not respond to the progestagens and Org OD14. The two T47D subclones also reacted differently to progestagens during growth stimulation with E2. In T47D-S the progestagens and Org OD14 inhibited, while in T47D-A these compounds did not modulate the effect of E2. In the DMBA model we found that gestodene and 3-ketodesogestrel were able to inhibit tumor growth to the same extent. Surprisingly, Org OD14 was even more effective in the DMBA model using the therapeutic approach. Using the prophylaxic approach tumor development was delayed and tumor growth was strongly suppressed. The inhibitory effects of Org OD14 on tumor growth in the DMBA model may be attributed to its mixed hormonal profile. From these studies we conclude that different cell lines and even subclones thereof respond quite differently to steroids. Both in vitro and in vivo studies are required to judge whether synthetic steroids might be involved in an increased risk for the development of breast tumors.     

A monoclonal antibody to a 48 K antigen in oestrogen-dependent human breast cancer cells

A mouse was immunised with an antigen(s) purified by oestradiol-Sepharose affinity chromatography of pooled oestrogen-receptor positive cytosols from human breast cancer tissue. One antibody secreting clone was identified which precipitated labelled antigen and which also stained MCF-7 cells. Culture supernatant and ascites fluid were used for immunofluorescence, SDS-PAGE-Western blotting, photoaffinity labelling and binding studies. The antibody staining of MCF-7 cells was inhibited by preincubation in oestrogen-receptor positive cytosol but was unaffected by oestrogen-receptor negative cytosol. MCF-7 cells stained whether cultured in the presence or absence of oestradiol. The oestrogen-receptor negative cell lines MDA-MB-231 and MDA-MB-330 did not stain. Binding studies with 16-alpha-iodooestradiol using breast cancer tissue cytosols followed by immunoprecipitation showed activity only with oestrogen-receptor positive cytosols with optimal binding activity at 4 degrees C, unaffected by molybdate, but reduced at 25 degrees C or in the presence of 0.4 M KCl. Binding studies with MCF-7, MDA-MB-231 and MDA-MB-330 cytosols and nuclear fractions only showed activity with the MCF-7 cytosol and MCF-7 particulate fractions. The antibody recognised a 48 K species in both MCF-7 cytosol and nuclear fractions but not in the cytosol and nuclear extracts of oestrogen-receptor negative cell lines. Photoaffinity labelling using 16 alpha-iodooestradiol suggests the 48 K antigen does not bind oestradiol directly. The relationship of this antigen to the classical oestrogen-receptor and receptor complex awaits further clarification.     

Progestins induce catalase activities in breast cancer cells through PRB isoform: Correlation with cell growth inhibition

Reactive oxygen species (ROS) have been suggested to participate in tumor emergence due to their mitogenic and apoptotic signaling, and as contributors to DNA structural damage. Here we report that progesterone and various synthetic steroids with progestin potencies (norethisterone acetate, MPA, and Tibolone) counteract cell growth induced by hydrogen peroxide (H₂O₂), through a potent induction of catalase activities, in breast cancer cells and normal human epithelial breast cells. At physiological concentrations, progesterone and the pure progestin, Org2058, displayed the most potent H₂O₂ detoxification ability suggesting its effect was characteristic of its progestin potency. We also report on the enhancement of catalase activities by progesterone receptor isoform B (PRB), as determined from experiments using antiprogestins and MDA-MB-231, cells engineered for the selective expression of progesterone receptor isoform A or B. The potent action of progesterone on catalase activities indicates its contribution to a beneficial role in breast cell homeostasis.     

An overview of nomegestrol acetate selective receptor binding and lack of estrogenic action on hormone-dependent cancer cells

The specific pharmacological profile of the 19-norprogestin nomegestrol acetate (NOMAC) is, at least in part, defined by its pattern of binding affinities to the different steroid hormone receptors. In the present study, its affinity to the progesterone receptor (PgR), the androgen receptor (AR) and the estrogen receptor (ER) was re-evaluated and compared to those obtained for progesterone (P) and several progestins. The characteristics of binding to the PgR in rat uterus were determined and Ki were found to be roughly similar with 22.8 and 34.3 nM for NOMAC and P, respectively. The binding characteristics of 3H-NOMAC were also determined and compared to that of 3H-ORG2058 with Kd of 5 and 0.6 nM, respectively for rat uterus and 4 and 3 nM, respectively for human T47-D cells. Structure-affinity and -activity relationships were studied on a variety of compounds related to NOMAC in order to assess its specificity as a progestin. The effects of NOMAC on the binding of androgen to the AR were investigated, using rat ventral prostate as target model. Contrary to what was observed for MPA, the RBA of NOMAC was found to decline with time, showing anti-androgenic rather than androgenic potential, a result that was confirmed in vivo. Regarding the ER, since none of the progestins were able to compete with estrogen for binding in rat uterus as well as in Ishikawa cells, the induction of alkaline phosphatase activity (APase) was used as an estrogen-specific response. It confirmed the intrinsic estrogenicity of progestins derived from 19-nor-testosterone (19NT), norethisterone acetate (NETA), levonorgestrel (LNG) or norgestimate (NGM) and others. In contrast, all P and 19-norP derivatives remained inactive. Finally, to complete this overview of NOMAC at the sex steroid receptor levels, the lack of estrogenic or estrogenic-like activity was checked out in different in vitro models. Data from this study have demonstrated that NOMAC is a progestin that has greater steroid receptor selectivity compared to MPA or some other synthetic progestins. It may provide a better pharmacological profile than those progestins currently in use in HRT and OC.     

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

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: affinity chromatography, desalting of eluate on Sephadex G-25, 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 with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Effect of tibolone (Org OD14) and its metabolites on aromatase and estrone sulfatase activity in human breast adipose stromal cells and in MCF-7 and T47D breast cancer cells

Tibolone (Org OD14) is a synthetic steroid used for post-menopausal hormone replacement therapy (HRT). Since HRT might increase breast cancer risk, it is important to determine the possible effects of tibolone on breast tissues. Tibolone and its metabolites Org 4094, Org 30126 and Org OM38 have been reported to inhibit estrone sulfatase activity in MCF-7 and T47D breast cancer cell lines, which suggest beneficial effects on hormone dependent breast cancer by reducing local production of free estrogens. Breast adipose stromal cells (ASCs) contain aromatase activity-an obligatory step in the biosynthesis of estrogens-and possibly contain sulfatase activity. We investigated the effects of tibolone, its metabolites and the pure progestin Org 2058 on PGE(2)-stimulated aromatase activity and on sulfatase activity in human ASC primary cultures and on sulfatase activity in MCF-7 and T47D cell lines. In MCF-7, tibolone and metabolites, but not Org 2058, were found to inhibit sulfatase activity. In T47D, tibolone inhibited sulfatase only at 10(-6)M, although weakly. ASC had high sulfatase activity, which was inhibited by 10(-6)M of tibolone, Org 4094 and Org 30126, but not by Org OM38 or Org 2058. Surprisingly, aromatase activity in ASC was increased by both tibolone and Org 2058 at 10(-6)M. As ligand binding assay results and immunohistochemistry indicated the absence of progesterone and estrogen receptors in ASC, these effects on aromatase and sulfatase activity in ASC likely take place by other routes. Because tibolone and its metabolites inhibit sulfatase activity, and because tibolone only increases aromatase activity at a high concentration, we conclude that effects of tibolone on the breast are probably safe.     

Novel antiprogestins Org 31806 and 31710: interaction with mammalian progesterone receptor and DNA binding of antisteroid receptor complexes.

We have examined steroid binding parameters and transformation of calf uterine progesterone receptor (PR) liganded with progestins (progesterone and R5020) and the newly synthesized antiprogestins (Org 31806 and 31710). Species specificity analysis indicated that [3H]R5020 binding in the chicken oviduct cytosol could be eliminated in the presence of 100-fold excess radioinert progesterone and R5020 but not Org 31806 and 31710. In the calf uterine cytosol, the progestins and the antiprogestins appeared to interact with the same PR as revealed by the displacement of [3H]R5020 by all of the above steroids. When the extent of [3H]R5020 binding was examined in the presence of different concentrations of radioinert steroids, the relative affinity with which these compounds interacted with the uterine PR was found to be comparable. A 23 degrees C incubation of cytosol transformed the progestin-bound PR complexes increasing their binding to DNA-cellulose from 5 (0 degrees C, nontransformed) to 35%. Under these conditions, 20% Org 31710- and RU486-occupied PR complexes bound to DNA-cellulose whereas only 10% Org 31806-receptor complexes were retained by the resin. Transformation (23 degrees C) of cytosol receptor caused a loss of the larger 8 S form and an increase in the smaller 4 S form. In its unliganded state or when it was complexed with R5020 or the antiprogestins, incubation of PR at 23 degrees C led to dissociation of the receptor-associated 90 kDa heat-shock protein (hsp90). The PR-hsp90 association was stabilized in the presence of 10 mM iodoacetamide when the ligand binding site was occupied by Org 31806 and 31710. The R5020-receptor complexes, however, allowed release of hsp90 under the above transforming conditions. Our results indicate that although Org 31806 and 31710 show no affinity for the avian PR, these steroids interact with the mammalian PR. We propose that the reported antiprogestational effects of Org 31806 and 31710 are mediated via their interaction with PR which appears similar to one that exists between PR and RU486.     

Evidence for a Leydig cell progesterone receptor in the rat

Tritiated promegestone [3H] R 5020 is bound with high affinity by charcoal-treated cytosol prepared from purified Leydig cells. The binding is characterized by high affinity (Kd = 2 x 10(-9) M) and specificity (R 5020 = progesterone greater than testosterone = dehydrotestosterone greater than hydroxyprogesterone greater than cortisol = dexamethasone greater than estradiol) appropriate for progesterone receptors. In vitro, progestin-bound cytosol was quantitatively translocated to nuclei fractions, only if cytosol samples were previously labeled at 25 degrees C. However no translocation of binding activity was observed when previous cytosol labeling was done in the presence of sodium molybdate. Effects of glucocorticoids, androgens and estrogens on the Leydig cell are well documented, the demonstration of a putative progesterone receptor raises the possibility of direct effect of progesterone on the Leydig cell.     

Effects of Temperature,Nucleotides and Sodium Molybdate on Activation and DNA Binding of Estrogen,Glucocorticoid, Progesterone and Androgen Receptors In MCF-7 Human Cancer Cells

Abstract

We studied the effects of temperature, ribonucleotides and sodium molybdate on the activation and DNA cellulose binding of estrogen, glucocorticoid, progesterone and androgen receptor complexes in MCF-7 cells. Using DNA cellulose binding as a measure of receptor activation, we found that ribonucleotides activated all four of these receptor complexes. Temperature also activated glucocorticoid receptor complexes efficiently but activated progesterone and androgen receptor complexes less well. Temperature did not activate estrogen receptor complexes. Sodium molybdate blocked either ATP or temperature induced activation of glucocorticoid, progesterone and androgen receptor complexes but only partially blocked estrogen activation. Sodium molybdate also prevented the formation of multiple forms of estrogen and glucocorticoid receptor complexes seen on DEAE cellulose and hydroxylapatite chromatography of crude cytosol. The mechanism by which ribonucleotide enhances and molybdate inhibits activation are discussed.     

The effects of oestrogens and progesterone on oestrogen receptors in female rat liver.

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.     

Degradation without apparent change in size of molybdate-stabilized nonactivated glucocorticoid-receptor complexes in rat thymus cytosol

We have investigated the stability of the [3H]dexamethasone 21-mesylate-labeled nonactivated glucocorticoid-receptor complex in rat thymus cytosol containing 20 mM sodium molybdate. Cytosol complexes were analyzed under nondenaturing conditions by gel filtration chromatography in the presence of molybdate and under denaturing conditions by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. When analyzed under nondenaturing conditions, complexes from fresh cytosol and from cytosol left for 2 h at 3 degrees C eluted from gel filtration as a single peak of radioactivity with a Stokes radius of approximately 7.7 nm, suggesting that no proteolysis of the complexes had occurred in either cytosol. When analyzed under denaturing conditions, however, whereas the fresh cytosol gave a receptor band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr approximately 90,000 (corresponding to the intact complex), the cytosol that had been left for 2 h at 3 degrees C gave only a fragment (Mr approximately 50,000). This fragment, just as the intact complex, could be thermally activated to a DNA-binding form. Proteolysis of the receptor could be blocked by preparing the cytosol in the presence of EGTA, leupeptin, or a heat-stable factor present in the cytosol of rat liver and WEHI-7 mouse thymoma cells. From these results we conclude: (i) 20 mM molybdate does not protect the nonactivated glucocorticoid-receptor complex present in rat thymus cytosol against proteolysis under conditions which are commonly used for cell-free labeling of the receptor, and (ii) the demonstration of a Stokes radius of approximately 8 nm for the nonactivated glucocorticoid-receptor complex is not sufficient to indicate that the receptor complex is present in its intact form.     

New method for the determination of estrogen and progesterone receptors in human breast cell lines

A method for the determination of estrogen and progesterone receptor levels in human mammary cell lines (MCF-7, Cama-1, ZR-75-1, Evsa-T and HBL-100) is described. Cells cultured as monolayers were incubated with the tritiated steroids, [3H]-17 beta-Estradiol or [3H] ORG-2058. Binding of steroids to receptors was a function of cellular uptake. Incubation periods of 50 min were sufficient to attain maximum intracellular incorporation. The binding of 17 beta-E2 and ORG-2058 to MCF-7 cells, a phenomenon which is saturable at low concentrations for the radioactive ligand, is a linear function of the number of cells assayed (Interval: 2.5 X 10(4) to 1.5 X 10(6) cells per well). Binding data and their Scatchard plot allowed for the calculation of affinity and capacity values. Thus, for ER, Kd = 2.0 +/- 0.5 X 10(-10) M and n = 3.76 +/- 0.91 Fmol/microgram DNA, and for PgR Kd = 2.0 +/- 0.2 X 10(-10) M and n = 14.02 +/- 2.30 Fmol/microgram DNA (Mean +/- SD). Binding specificity of 17 beta-Estradiol and ORG-2058 to MCF-7 cells was analysed by means of study on the inhibitory effect of increasing concentrations of unlabelled competitors: 17 beta-Estradiol, ORG-2058, Estrone, DES, R-5020, Cortisol, Androsterone and Testosterone. Only pharmacological doses of some of the mentioned molecules produce displacement of the hormonereceptor binding. This phenomenon appears to be related to the affinity of these chemical compounds for the receptor macromolecules to which estrogens and progesterone bind.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation

Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits.     

Nestorone: a progestin with a unique pharmacological profile

Nestorone(R) (Nestorone 16-methylene-17alpha-acetoxy-19-norpregn-4-ene-3,20-dione), formerly referred to as ST 1435, is a potent progestin when given parenterally via sustained release formulations. The pharmacological profile of Nestorone was compared with that of levonorgestrel and 3-keto-desogestrel by steroid receptor binding studies and by in vivo bioassays in rats and rabbits. 3-Keto-desogestrel showed the highest binding affinity to progesterone receptors (PR) followed by Nestorone, levonorgestrel, and progesterone. The binding affinity of Nestorone to androgen receptors (AR) was 500- to 600-fold less than that of testosterone. However, both levonorgestrel and 3-keto-desogestrel showed significant binding (40 to 70% of testosterone) to AR. None of the progestins bound to estrogen receptors (ER). The progestational activity of Nestorone, levonorgestrel, and progesterone was compared using McPhail index in immature rabbits and pregnancy maintenance and ovulation inhibition tests in rats after subcutaneous (s.c.) administration. In all three tests, Nestorone was the most potent progestin. The progestational activity of Nestorone was also compared after oral and s.c. administration in rabbits. The potency of Nestorone was over 100-fold higher upon s.c. administration than via the oral route. The androgenic activity of progestins, based on the stimulation of ventral prostate (androgenic target) and levator ani (anabolic target) growth in castrated immature rats, showed good correlation with their binding affinity to AR. Nestorone showed no androgenic or anabolic activity. Nestorone did not bind to sex hormone binding globulin (SHBG), whereas both levonorgestrel and 3-keto-desogestrel showed significant binding to SHBG. The estrogenic/antiestrogenic activity of Nestorone was investigated in immature ovariectomized rats. In contrast to estradiol and levonorgestrel, Nestorone showed no uterotropic activity in ovariectomized rats. Despite significant binding to glucocorticoid receptors (GR), Nestorone showed no glucocorticoid activity in vivo. It is concluded that a strong progestational activity, combined with lack of androgenic, estrogenic, and glucocorticoid-like activities, confer special advantages to Nestorone for use in contraception and hormone replacement therapy.     

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.