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At least 415 records · Page 23Linked to original sources
PubMed1998

Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors.

Tamoxifen (TAM), the only antiestrogen currently available for the endocrine therapy of breast cancer behaves as a mixed agonist/antagonist of estrogen action, thus limiting its therapeutic potential. We report the binding characteristics of a novel series of nonsteroidal antiestrogens to the rat uterine estrogen receptor. As measured by competition studies, the affinity of EM-652, the active metabolite of the prodrug EM-800, for the estrogen receptor is 7-11 times higher than that of 17beta-estradiol (E2), ICI 182780, and hydroxy-tamoxifen (OH-TAM), the active metabolite of Tamoxifen. EM-652 is 20x more potent than ICI 164384 and Droloxifene while it is 400 times more potent than Toremifene in displacing [3H]E2 from the rat uterine estrogen receptor. On the other hand, the prodrug EM-800 and Tamoxifen have respectively 150-fold and 410-fold less affinity for the estrogen receptor than the pure antiestrogen EM-652. No significant binding of EM-652, EM-800, TAM or OH-TAM was observed to the rat uterine progesterone receptor at concentrations up to 10,000 nM except for TAM that caused a 50% displacement of labeled R5020 at 4000 nM. No significant binding of EM-652 or EM-800 was observed on the rat ventral prostate androgen receptor or the rat uterine progesterone receptor. The present data demonstrate the high affinity and specificity of the new antiestrogen, EM-652, for the rat uterine estrogen receptor. The antiestrogen EM-652 thus becomes the compound having the highest known affinity for the estrogen receptor. Due to its unique potency and its pure antiestrogenic activity already demonstrated in many systems, this antiestrogen could well offer an important advance for the endocrine therapy of breast cancer, uterine cancer, and other estrogen-sensitive diseases in women.

Androgens↗
PubMed1999

Microbial models of drug metabolism: microbial transformations of Trimegestone (RU27987), a 3-keto-delta(4,9(10))-19-norsteroid drug.

Screening microorganisms for the biotransformation of the 3-keto-delta(4,9(10))-19-norsteroid RU27987 (Trimegestone) resulted in the isolation of nine identified metabolites, some of them being selectively produced by different strains. Eight metabolites were found to be hydroxylated on various positions of the rings, and one was additionally epoxidized. These microbial metabolites could be used as chromatographic standards and two of them were found identical to the unknown major human metabolites. Moreover, most microbial metabolites were produced in sufficient amounts to be tested for their biological activities. All these features demonstrate the usefulness and versatility of microbial biotransformation systems as a tool for early identification and convenient production of potentially active mammalian and non-mammalian metabolites.

Biotransformation↗
PubMed2001

Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases.

Steroid hormones have rapid nongenomic effects on cell-signaling pathways, but the receptor mechanisms responsible for this are not understood. We have identified a specific polyproline motif in the amino-terminal domain of conventional progesterone receptor (PR) that mediates direct progestin-dependent interaction of PR with SH3 domains of various cytoplasmic signaling molecules, including c-Src tyrosine kinases. Through this interaction, PR is a potent activator of Src kinases working by an SH3 domain displacement mechanism. By mutagenesis, we also show that rapid progestin-induced activation of Src and downstream MAP kinase in mammalian cells is dependent on PR-SH3 domain interaction, but not on the transcriptional activity of PR. Preliminary evidence for the biological significance of this PR signaling pathway through regulatory SH3 domains was shown with respect to an influence on progestin-induced growth arrest of breast epithelial cells and induction of Xenopus oocyte maturation.

Amino Acid Motifs↗
PubMed1991

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor.

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with [3H]progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020 (p less than 0.001). This indicated that a difference in the binding mechanism of RU486 and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable (p = 0.915). These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.

Animals↗
PubMed1992

Heat shock alters the composition of heteromeric steroid receptor complexes and enhances receptor activity in vivo.

Under normal cellular conditions, human progesterone receptors (PR), immune-isolated from cytosols of T47D breast cancer cells, associate with two heat shock proteins (hsps), hsp 90 and hsp 70. Receptors activated by hormone binding in vivo and extracted from nuclei with 0.5 M NaCl no longer associate with hsp 90 but retain association with hsp 70. We have examined the effect of heat shock treatment of cells on hsp-receptor interactions and on receptor function. Heat shock resulted in a partial reduction in cellular levels of PR, but receptors that remained were functional for both steroid and DNA binding activities. By steady-state [35S]methionine labeling prior to heat shock treatment, it was determined that heat shock did not affect the composition or maintenance of preexisting cytosolic PR.hsp 90.hsp 70 complexes. By contrast, immune isolation of PR complexes from cells pulse-labeled with [35S]methionine showed that heat shock altered the composition of newly synthesized hsps associated with PR. After heat shock, both the highly inducible form of hsp 70 (72K hsp) and a 100K hsp were bound to cytosol PR, and inducible 72K hsp remained bound with the nuclear-activated PR. Neither of these hsps were associated in detectable amounts with PR under normal cellular conditions. With respect to receptor function, heat shock treatment substantially enhanced the activity of PR in vivo as determined by measuring hormone-dependent PR-mediated transcription of a target reporter gene (MMTV-CAT) that was stably transfected into T47D cells. Heat shock treatment alone, in the absence of hormone, did not stimulate MMTV-CAT expression nor did it affect transcription from a control reporter gene, pSV2-CAT, suggesting that enhanced receptor activity was due to an effect on PR-mediated processes and not to a general effect on transcription. Induction of the heat shock response by a related chemical stress (sodium arsenite) also enhanced PR activity in vivo. Interestingly, sodium arsenite produced both a greater induction of hsp 90 and hsp 70 synthesis and a greater fold enhancement of PR-mediated gene transcription than did heat shock. This suggests that enhancement of PR activity is related not only to induction of hsp synthesis but also to the severity of the stress response. The present results provide an indication that in certain cells there may exist an interrelationship between the activation pathways by which cells respond to stress and to steroid hormones. Possible mechanisms responsible for heat shock effects on PR activity are discussed.

Arsenic↗
PubMed1992

Hormone-induced dissociation of the androgen receptor-heat-shock protein complex: use of a new monoclonal antibody to distinguish transformed from nontransformed receptors.

The hormone-induced transformation process of the androgen receptor in the androgen-responsive human prostatic carcinoma cell line LNCaP was studied. Immunoprecipitation of the nontransformed cytosolic receptor (8S on sucrose gradients) with a specific monoclonal antibody (F39.4.1) resulted in coprecipitation of three heat-shock proteins (hsp90, hsp70, and hsp56). Upon incubation of the cells with the synthetic androgen R1881, the sedimentation value of the receptor complex decreased to an intermediate form of 6S, and an almost complete loss of coprecipitating heat-shock proteins was observed. After a 2-h incubation, the receptor was recovered in considerable part from the nuclear fraction (extraction with high salt; 4.6S form). By use of the bifunctional cross-linker dimethyl pimelimidate, dissociation of the 8S complex, but not of the 6S complex, was blocked. A newly developed monoclonal antibody (F52.24.4), directed against the C-terminal part of the DNA-binding domain of the androgen receptor, specifically recognized both the 4.6S and the 6S forms of the receptor but did not react with the nontransformed 8S form. It is concluded that the unoccupied androgen receptor is associated with several heat-shock proteins and that transformation of the receptor to the tight nuclear-binding form is a multistep process that involves the dissociation of heat-shock proteins from the receptor.

Amino Acid Sequence↗
PubMed1992

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.

Baculoviridae↗
PubMed1994

Effects of molecular crowding on protein self-association: a potential source of error in sedimentation coefficients obtained by zonal ultracentrifugation in a sucrose gradient.

Theoretical and experimental studies have illustrated a potential source of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-association. The error stems from the excluded volume (molecular crowding) effect of the sucrose on the activity coefficients of monomeric and polymeric states. The consequent displacement of the equilibrium position in favor of polymeric state(s) is a function of sucrose concentration, and can therefore result in failure to detect the equilibrium coexistence of monomer if 5% sucrose suffices to displace the equilibrium completely toward dimer. In less extreme situations, it may result in the evaluation of an average sedimentation coefficient whose magnitude is a function of sucrose concentration and hence of the distance migrated into the sucrose gradient. These features are illustrated by the results of computer-simulated sedimentation of reversibly dimerizing systems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.

Centrifugation, Zonal↗
PubMed1994

Phosphorylation sites in ligand-induced and ligand-independent activation of the progesterone receptor.

Steroid hormone receptors are phosphoproteins that undergo hyperphosphorylation upon binding of hormone. The mechanism and the role of this reaction remain poorly understood. Two-dimensional analysis of ligand-free progesterone receptor (PR) tryptic digests showed the existence of seven main phosphopeptides. Incubation of the cells with the progestin R5020 led to a global increase in the levels of PR phosphorylation. However, the same phosphopeptides were seen, and their levels of labeling relative to each other were unchanged. A similar result was observed after incubation of cells with the antiprogestin RU486. The antiprogestin ZK98299 demonstrated only half of the activity of RU486 in terms of receptor hyperphosphorylation, but the same phosphopeptides, proportionally labeled to the same extent, were observed by chromatography electrophoresis. Ligand-induced DNA binding did not play a role in receptor hyperphosphorylation since the mutant delta 547-592, which is devoid of the first zinc finger region, exhibited the same phosphopeptides, labeled to the same extent, as did wild-type receptor after incubation of cells with hormone. These results suggest that the same kinase(s) act in vivo on ligand-free and on agonist or antagonist-bound progesterone receptor. Binding of different ligands produces different conformational changes in the ligand binding domain of the receptor which enhance, to varying extents, affinity of the receptor for the kinase(s). The DNA binding region also plays a role in the interaction with the kinase(s), although binding to DNA per se is not necessary for the hyperphosphorylation of the receptor to take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit.

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Synthesis and evaluation of 7 alpha-iodo-5 alpha-dihydrotestosterone as a potential radioligand for androgen receptor.

7 alpha-Iodo-17 beta-hydroxy-5 alpha-androstan-3-one (7 alpha-iodo-5 alpha-dihydrotestosterone, 7 alpha-IDHT) has been synthesized as a potential radioligand for the detection and measurement of androgen receptor and for imaging of androgen-receptor-containing tissues when labeled with the gamma-emitting radionuclides 125I and 123I, respectively. In vitro binding studies show that 7 alpha-IDHT binds with high affinity to the rat and human androgen receptor (RBA = 74) compared to R1881 (RBA = 100). Further, this compound showed high specificity for the androgen receptor. 7 alpha-IDHT showed only a marginal affinity for the progestin receptor and even less affinity for the estrogen receptor. No binding was detected to the glucocorticoid receptor. These characteristics make 7 alpha-IDHT a potentially ideal agent for imaging and evaluation of androgen-receptor-containing tissues.

Animals↗
PubMed1985

Synthesis and evaluation of potential radioligands for the progesterone receptor.

Several steroidal analogues were synthesized as potential gamma-emitting radioligands for the progesterone receptor. Each of these compounds was tested as an inhibitor of the specific binding of [3H]-17 alpha,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to the progesterone receptor in rabbit uterine cytosol. R5020 is a well-known progestin with high affinity for the receptor. Of the compounds synthesized, aromatic N-substituted C-17 steroidal carboxamides inhibited the binding only poorly. Three compounds, 16 alpha-iodo-4-estren-17 beta-ol-3-one, 17 alpha-[2(E)-iodovinyl]-4-estren-17 beta-ol-3-one, and 17 alpha-[2(Z)-iodovinyl]-4-estren-17 beta-ol-3-one were excellent competitors, each having a Ki less than or equal to that of the natural progestin, progesterone. Since similar iodinated analogues of estrogens have been shown to be extremely stable both in vivo and in vitro, these compounds are potentially useful ligands for the progesterone receptor.

Animals↗
PubMed1988

21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone: synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography.

We have synthesized 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone (FENP), a high affinity ligand for the progesterone receptor, labeled with the positron-emitting radionuclide fluorine-18 (t1/2 = 110 min). The synthesis proceeds in two steps from 21-hydroxy-16 alpha-ethyl-19-norprogesterone and involves [18F]fluoride ion displacement of the 21-trifluoromethanesulfonate (21-triflate). This material is purified by HPLC and is obtained in 4-30% overall yield (decay corrected) within 40 min after the end of bombardment to produce [18F]fluoride ion. The effective specific activity, determined by competitive radioreceptor binding assays, is 700-1400 Ci/mmol. In vivo, [18F]FENP demonstrates highly selective, receptor-mediated uptake by the uterus of estrogen-primed rats; the uterus to blood and uterus to muscle ratios were respectively 26 and 16 at 1 h and 71 and 41 at 3 h after injection. The high target tissue selectivity of this uptake suggests that this compound may be useful for the in vivo imaging of progestin target tissues and receptor-rich tumors (such as human breast tumors) by positron emission tomography.

Animals↗
PubMed1997

Characterization of ligand binding, DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands.

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect properties of PR from T47D cells that are similar to RU486.

Binding, Competitive↗
PubMed1997

Inhibition of proliferation of T47D human breast cancer cells: alterations in progesterone receptor and p53 tumor suppressor protein.

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effective inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24-2.4 micrograms/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels-a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR.

Antineoplastic Agents↗
PubMed1999

Agonist and antagonist-induced qualitative and quantitative alterations of progesterone receptor from breast cancer cells.

T47D cells, cultured in medium containing serum stripped of endogenous steroids, proliferate in response to treatment with the progesterone receptor (PR) agonist, R5020 or the PR agonist/antagonist, RU486, whereas the full PR antagonist, ZK98299 has no proliferative effects. Under estrogenized conditions, all of the PR ligands tested inhibit cell growth [23]. In order to determine whether the levels or phosphorylation state of PR are reflected in the growth patterns of T47D cells, we monitored the effects of these PR ligands on the immunoblotted PR band intensities, the relative intensities of PR-A and PR-B, and their phosphorylation states that are reflected in their altered mobility during SDS-PAGE. Under conditions where the PR ligands inhibit cell proliferation, each ligand had distinctively different qualitative and quantitative effects on PR. Short term treatment of the cells with R5020 or RU486 induced a characteristic phosphorylation-dependent upshift of both PR-A and PR-B. The phosphorylated PR was stable for up to 4 days after treatment of the cells with RU486, but was down regulated between 6-24 h after treatment with R5020. No replenishment of PR in cells treated with R5020 was detected. ZK98299, at concentrations tested, had no qualitative or quantitative effects on PR. Culturing cells for 8 days in medium containing steroid-depleted serum caused a significant reduction in the PR band intensity without causing a change in the ratio of PR-A and PR-B or their phosphorylation states. This decrease in the PR band intensity was reversed by maintaining the cells in 1 nM estrogen, but was potentiated by RU486 or ZK98299. These observations support the view that decreased PR levels may play a role in the stimulatory effects of R5020 and RU486 when cells are cultured under non-estrogenized conditions.

Blotting, Western↗
PubMed2001

Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells.

The contribution of estrogen (and progesterone) in driving cell cycle progression of hormone dependent breast cancer cells is well documented, however, the roles of the various relevant signal transduction pathways remain unclear. The immunosuppressant rapamycin is a potent inhibitor of cell cycle progression and has been used to define signal transduction pathways. In this study we have determined rapamycin's effects on cell cycle progression in estrogen dependent breast cancer cells using a novel method of inducing S-phase. In this method estradiol-17-beta alone induced S-phase without mitogen support. In our studies the T47D cells were quite sensitive to estradiol-17-beta, with half-maximal induction in the picomolar range. indicating that the estrogen can induce S-phase in the absence of mitogens such as insulin. The estrogen response does not seem to be particularly specific because estriol estrone and estradiol-17-beta-BSA were about as effective as estradiol-17-beta. R5020, a progestin also induced S-Phase, while rapamycin blocked steroid driven transition of cells from G1 to S-phase.

Antibiotics, Antineoplastic↗