ORG-2058 as a ligand in the assay of progesterone receptor in breast cancer

Tritiated [(16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione)-6,7-3H] (ORG-2058) and 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020) were compared as ligands in the assay of progesterone receptor in human and rat breast tumors. We found that ORG-2058 is a better ligand because of its low nonspecific binding. Most of the nonspecific binding of the other ligand R5020, is to proteins which bind corticosteroids. In cancerous tissue ORG-2058 binds to progesterone receptor linearly in a range of protein concentrations which are normally used in the receptor assay. On the other hand, R5020 exhibits binding linearity over a narrower protein concentration in many tumor biopsies, which may cause severe limitation in the assay procedure or frequent underestimation of receptor content.     

Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Progesterone receptor synthesis and degradation in MCF-7 human breast cancer cells as studied by dense amino acid incorporation. Evidence for a non-hormone binding receptor precursor

We have used the technique of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids to study the synthesis and degradation rates of the progesterone receptor in MCF-7 human breast cancer cells. In cells grown in the absence of progestin, sucrose gradient shift analyses reveal that it takes 17 h for the normal density progesterone receptor levels to be reduced to half the initial value, whereas in the presence of 10 nM of the synthetic progestin [3H]R5020, the receptor turns over more rapidly, such that the normal density R5020-occupied progesterone receptor complexes are reduced to half in 12 h. The accelerated progesterone receptor turnover in the presence of [3H]R5020 reflects increased turnover rates of both the A (Mr-85,000) and B (Mr-115,000) subunits, as determined by sodium dodecyl sulfate gel analyses of dense and light receptors photoaffinity labeled with [3H]R5020. In both control and progestin-exposed cells, the time course of progesterone receptor turnover shows a lag of approximately 6 h after dense (15N, 13C, 2H) amino acid exposure, before dense hormone binding receptor species are seen and before normal density progestin binding activity starts decreasing. Since our evaluations of progesterone receptor depend upon its binding of radiolabeled ligand ([3H]R5020), this lag in the density shift kinetics would be consistent with the presence of a non-hormone binding biosynthetic precursor, from which the hormone-binding form of progesterone receptor is derived. A kinetic model is used to analyze the lag-decay profiles and to determine the rate constants for progesterone receptor synthesis, activation to the hormone-binding form, and degradation.     

Specific progesterone receptors in dimethylbenzanthracene (DMBA)-induced mammary tumors.

Properties of a progesterone receptor present in the cytosol (105,000 xg supernatant) of dimethylbenzanthracene (DMBA)-induced mammary tumors were studied using the highly potent progestin [3H]R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of [3H] R 5020 is associated with components migrating at 7-8S and 4S. Low affinity binding of the synthetic progestin is eliminated by treatment with dextran-coated charcoal. [3H] R 5020 binding is highly progestin-specific since it is easily displaced by unlabelled norgestrel, R 5020 and progesterone while estradiol-17beta, dihydrotestosterone, testosterone, testosterone and diethylstilbestrol have much lower activity. Dexamethasone and cortisol have little, if any, effect on [3H] R 5020 binding.     

Characterization of a progestin-binding component in lactating rat mammary glands

Experiments were carried out to identify progestin-binding receptors in the mammary gland where casein synthesis is known to be inhibited by this hormone. A progestin-binding component with high affinity, low capacity and a sedimentation coefficient of 8.8 S was isolated from the cytosol of lactating rat mammary glands. This component strongly bound [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) with a dissociation constant of 3.9 · 10^?9 M under low-salt conditions and with that of 8.2 · 10^?10 M in the presence of 0.3 M KCl. Specificity studies showed a higher degree of progestin specificity under high salt conditions. In the absence of KCl, binding of [³H]-R5020 was inhibited by unlabeled glucocorticoid in the same degree as unlabeled progestin, but the inhibition by glucocorticoid was greatly diminished by the presence of 0.3 M KCl. These observations suggest that the [³H]R5020-binding-component is the progestin receptor and that its function may be regulated by the concentration of glucocorticoid and salt.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

Cytoplasmic progesterone receptors in the hypothalamus-preoptic area of the mouse: effect of estrogen priming

A synthetic progestin, R5020, was used to identify cytoplasmic progestin receptors in the hypothalamuspreoptic area (HPOA) of ovariectomized mice. These high-affinity receptors exhibited an apparent dissociation constant of approx. 1 nM. The receptors were specific for progestins. [3H]R5020 binding was inhibited by more than 50% with a 50-fold excess of either radioinert R5020 or progesterone. 5 alpha-Dihydroprogesterone inhibited binding to a lesser extent. 3 alpha-Hydroxy-5 alpha-pregnane-20-one and cortisol did not compete for [3H]R5020 binding. Administration of estradiol benzoate (10 micrograms), 48 h prior to death, resulted in a 54% increase in the HPOA progestin receptor concentration when compared to oil-injected controls. These data demonstrate that there are specific and saturable cytoplasmic progestin receptors in the mouse HPOA and that the concentration of these receptors is increased after estrogen treatment.     

Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.     

Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at congruent to 90 mM and congruent to 155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4 degrees C or addition of 10 mM GTP increased the proportion of the congruent to 90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 microM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.     

Uptake of three [3H]progestins by target tissues in vivo: Implications for the design of diagnostic imaging agents

We have investigated the tissue distribution of radioactivity for 0.5–4 h follwing the i.v. injection of three tritium-labeled progestins in estrogen-primed, immature rats. Whereas [³H]progesterone shows minimal uterine uptake (<0.7% injected dose per gram; %ID/g), the two higher affinity, synthetic progestins [³H]R 5020 (promegestrone) and [³H]ORG 2058 show highly selective uptake that reaches 4–5% ID/g by 1–3 h. The uterus to non-target tissue activity ratio at 2–4 h is approximately 12–20 for R 5020 and ORG 2058, but less than 2 for progesterone; the uterus to blood activity ratio for R 5020 is also high (approximately 15), but is lower for ORG 2058, possibly due to the accumulation of radiolabeled metabolites in the blood. The uterine uptake is selectively blocked by simultaneous injection of a large dose of unlabeled steroid, indicating that the uptake is mediated by a high affinity, low capacity binding system, presumably the progesterone receptor. Pronounced uptake is also observed by the liver and into fat, but is not receptor-mediated. The highly selective target tissue uptake by the two synthetic steroids, but not by progesterone, indicates that one must have ligands with sufficiently high affinity for the target tissue receptor, as well as low affinity for certain non-receptor binding proteins, in order to obtain adequate contrast between target and non-target tissues in dynamic uptake studies. These guidelines will be important in the development of suitable in vivo imaging agents based on the progesterone receptor.     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

Binding of (3H)7 alpha,17 alpha-dimethyl-19-nortestosterone (mibolerone) to progesterone receptors: comparison with binding of (3H)R5020 and (3H)ORG2058

The binding characteristics of (3H)7 alpha,17 alpha-dimethyl-19-nortestosterone [3H)DMNT) to progesterone receptors (PgR) of calf uterine tissue cytosol were determined and compared to those of the synthetic progestins (3H)ORG2058 and (3H)R5020. Scatchard plot analysis of the equilibrium binding data showed that (3H)DMNT binds to calf uterine PgR with a KD of 2.35 +/- 1.1 nM. This value is slightly higher than that of (3H)R5020 (KD = 1.16 +/- 0.4 nM) and (3H)ORG2058 (KD = 1.04 +/- 0.65 nM). Analysis of dissociation kinetics showed that (3H)DMNT dissociates much more rapidly from the receptor than the other two ligands. Competition experiments showed that ORG2058 has a lower inhibition constant (Ki) than DMNT. Sucrose density gradient (SDG) analysis of PgR showed that (3H)ORG2058-PgR complexes sediment as 8S, (3H)R5020-PgR complexes sediment as 8S and 4S, and (3H)DMNT-PgR complexes sediment as 8S entities along with dissociated (3H)steroid. These data suggest that (3H)DMNT binds to PgR with lower affinity than (3H)ORG2058 and (3H)R5020. The number of binding sites detected with (3H)DMNT are significantly lower than those measured with (3H)ORG2058.     

Progesterone receptor characterized by photoaffinity labelling in the plasma membrane of Xenopus laevis oocytes.

R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) is a synthetic analogue of progesterone, which is the physiological hormone that reinitiates germinal vesicle breakdown in Xenopus laevis oocytes. U.v.-driven photoaffinity labelling experiments were conducted with [3H]R 5020 in oocyte subcellular fractions, and covalently bound radioactivity was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In P-10000 (the pellet sedimenting between 1000 and 10000 g and which contains plasma membrane), a major radioactive band migrating as a 30kDa peptide was found. Non-radioactive progesterone competed with the [3H]R 5020 labelling of this fraction, but not with the labelling of minor [3H]R 5020-binding fractions. It displayed the required characteristics of a specific progesterone-binding membrane 'receptor', postulated from previous studies with intact oocytes and with cell-free P-10000 preparations of membrane-bound adenylate cyclase. The apparent Ki of approx. 4 microM for progesterone was compatible with the active concentration of the hormone. Binding specificity, as determined in competition studies, was highly correlated with the germinal vesicle breakdown activity of the steroids and analogues tested. The receptor was not found in the vitelline envelope, in vitelline platelets, in melanosome-enriched or microsomal fractions, in cytosol, nor in germinal vesicles of oocytes. The properties of this membrane steroid receptor are different from those of the already known soluble intracellular steroid receptors, in particular regarding ligand binding specificity and subcellular distribution.     

Modulation of brain progestin and glucocorticoid receptors by unsaturated fatty acid and phospholipid

In an attempt to learn how nonsteroidal factors modulate brain progestin and glucocorticoid receptors, the effects of saturated and unsaturated fatty acids, and phosphatidylinositol on the binding of [3H]R5020 or [3H]dexamethasone, determined by sucrose density gradient and gel filtration on LH20, were examined in the cerebral cortical cytosol from 10-day-old female rats which contain a considerable amount of progestin and glucocorticoid receptors. Unsaturated fatty acids such as oleic (C18:1), arachidonic (C20:4) and docosahexaenoic acid (C22:4) depressed the [3H]R5020 or [3H]dexamethasone binding in increasing order, but saturated fatty acids had no effect. Arachidonic and docosahexaenoic acids, which were strong inhibitors, lowered the binding dose dependently. The fatty acid inhibition on brain progestin and glucocorticoid receptors was thus a function of acid dose and degree of acid unsaturation. Interestingly, prostaglandin D2 did not show any effect. Among phospholipids tested the inhibitory effect of phosphatidylinositol on the [3H]R5020 binding was evident, but no significant effect was found with phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine or sphingomyelin. The phosphatidylinositol inhibition was dose dependent. Analysis on kinetics and Scatchard plot have revealed the noncompetitive type of inhibition by arachidonic acid and phosphatidylinositol. From these results it is suggested that the unsaturated nonestrified fatty acid, arachidonic acid, and phosphoinositides modulate the brain progestin and, possibly, glucocorticoid receptors through their binding at sites different from steroid binding sites on the respective receptor molecules.     

Progestin binding in mammary tissue of prepartum,nonlactating and postpartum,lactating cows

Binding of [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [³H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [³H]R5020 binding to progestin binding sites, while estradiol-17β, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), dexamethasone (9-fluoro-11β, 17, 21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for specifically bound [³H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 ( ± 0.3) and 0.8( ± 0.2) × 10?⁹M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 ± 53) than postpartum, lactating (41 ± 15) cows. Specific binding components in cytosol from lactating cows sedimented in the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.     

Antiprogestin-receptor complexes: differences in the interaction of the antiprogestin RU38,486 and the progestin R5020 with the progesterone receptor of human breast cancer cells

In order to understand the molecular basis for antiprogestin action, we have compared the interaction of the antiprogestin [3H]RU38, 486 (RU486) and the progestin [3H]R5020 with the progesterone receptor (PR). In both MCF-7 and T47D human breast cancer cells, we have observed marked differences in the sedimentation properties of the PR on high salt sucrose gradients: while the R5020-receptor complexes sediment at approximately 4 S (4.4 +/- 0.1 S), the RU486-receptor sediments as a prominent 6 S species as well as a 4 S species. This binding is abolished by excess unlabelled R5020, RU486 or progesterone, but is unaffected by excess unlabelled hydrocortisone or dexamethasone, indicating that both the 4 S and 6 S species represent the PR and not glucocorticoid receptor. Although the relative distribution of 4 S and 6 S forms is not altered by treatment with DNAse or RNAse, exposure to 10 mM thioglycerol or to 3 M urea results in conversion of the 6 S to the 4 S form, suggesting that disulfide bonds and hydrophobic interactions are important in maintaining the integrity of the 6 S form. These findings suggest that the 6 S antiprogestin complex is formed as a result of the interaction of PR units with each other or with a different protein. This change in receptor association state may be an important aspect of the antiprogestin activity of RU486.     

Characterization of R5020 and RU486 binding to progesterone receptor from calf uterus

We have examined and compared the binding characteristics of the progesterone agonist R5020 [promegestone, 17,21-dimethylpregna-4,9(10)-diene-3,20-dione] and the progesterone antagonist RU486 [mifepristone, 17 beta-hydroxy-11 beta-[4-(dimethylamino) phenyl]-17 alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] in calf uterine cytosol. Both steroids bound cytosol macromolecule(s) with high affinity, exhibiting Kd values of 5.6 and 3.6 nM for R5020 and RU486 binding, respectively. The binding of the steroids to the macromolecule(s) was rapid at 4 degrees C, showing saturation of binding sites at 1-2 h for [3H]progesterone and 2-4 h for both [3H]R5020 and [3H]RU486. Addition of molybdate and glycerol to cytosol increased the extent of [3H]R5020 binding. The extent of [3H]RU486 binding remained unchanged in the presence of molybdate, whereas glycerol had an inhibitory effect. Molybdate alone or in combination with glycerol stabilized the [3H]R5020- and [3H]RU486-receptor complexes at 37 degrees C. Although the rate of association of [3H]RU486 with the cytosolic macromolecule was slower than that of [3H]R5020, its dissociation from the ligand-macromolecule complex was significantly slower than [3H]R5020. Competitive steroid binding analysis revealed that [3H]progesterone, [3H]R5020, and [3H]RU486 compete for the same site(s) in the uterine cytosol, suggesting that all three bind to the progesterone receptor (PR). Sedimentation rate analysis showed that both steroids were bound to a molecule that sediments in the 8S region. The 8S [3H]R5020 and [3H]RU486 peaks were abolished by excess radioinert progesterone, RU486, or R5020.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]DU41165: a high affinity ligand and novel photoaffinity labeling reagent for the progesterone receptor

17 alpha-Acetoxy-6-fluoro-16-methylene-(9 beta, 10 alpha)pregna-4,6-dien- 3,20-dione (DU41165), a retroprogestin (9 beta, 10 alpha) embodying a fluorine-substituted dienone system, has been prepared in high specific activity tritium-labeled form (4 Ci/mmol) and shown to be a high affinity ligand for the progesterone receptor (PgR) and a highly selective photoaffinity labeling reagent for PgR. The radiosynthesis involved conversion of DU41231 (the 17 alpha-hydroxy analog of DU41165) to DU41165 by treatment with tritium-labeled acetic anhydride. The binding affinity of DU41165 for PgR was determined by both a competitive binding assay and a direct binding assay (Scatchard analysis) to be 1.6-2.2-times higher than that of the high affinity synthetic progestin promegestone (R5020). In unlabeled form, DU41165 demonstrates photoinactivation of PgR to the extent of 60% at 60 min. In radiolabeled form [3H]DU41165 demonstrates specific covalent attachment with an efficiency of 5-7%. SDS-polyacrylamide gel electrophoresis of photoattached [3H]DU41165 confirms that there is covalent labeling of both the B subunit (Mr = 118,000), and the A subunit (Mr = 88,000) of PgR in a molar ratio of approximately 1:3.     

Mammalian progesterone receptor shows differential sensitivity to sulfhydryl group modifying agents when bound to agonist and antagonist ligands

Modulation of calf uterine progesterone receptor (PR), in relation to its binding to synthetic steroids with known agonist (R5020) and antagonist (RU486) properties, was studied in the presence of iodoacetamide (IA), N-ethylmaleimide (NEM), beta-mercaptoethanol (MER), and dithiothreitol (DTT). Pretreatment of uterine cytosol at 4 degrees C with NEM (4-10 mM) reduced the binding of [3H]RU486 to PR by 40%, but [3H] R5020 binding was completely abolished. Whereas IA (2-10 mM) treatment did not affect [3H]RU486 binding, [3H]R5020 binding was totally eliminated. DTT or MER increased the binding of both steroids slightly (15%). [3H]R5020- or [3H]RU486-receptor complexes (Rc) migrated in the 8 S region and were eliminated upon pretreatment with NEM. At 23 degrees C, DTT increased the amount of 4 S [3H]R5020-Rc, but had no effect on the [3H]RU486-Rc. In the control, [3H]RU486 binding to the 8 S PR could be competed with radioinert R5020 or RU486, but R5020 failed to compete in the presence of IA. The heat-treated [3H]R5020- and [3H]RU486-Rc showed reduced binding to DNA-cellulose in the presence of NEM and IA. The results of our study suggest that SH group modifications differentially influence the properties of mammalian PR complexed with either R5020 or RU486. In the presence of IA, the [3H]RU486-Rc remained in the 8 S form when incubated at 23 degrees C, indicating that RU486 binding causes conformational changes in PR which are distinct from those that result upon R5020 binding.