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PubMed1984

Polypeptide components of two 8 S forms of chicken oviduct progesterone receptor.

Two 8 S forms of progesterone receptor from the chicken oviduct were purified to near homogeneity and analyzed for peptide composition by gel electrophoresis. Form I contains two major peptides with molecular weights of 90,000 and 75,000. Form II also contains two major peptides with molecular weights of 90,000 and 110,000. In glycerol gradients containing molybdate, the 90,000, 110,000, and 75,000 molecular weight peptides co-sediment with the [3H]progesterone peak at 8 S. In high salt gradients lacking molybdate, the [3H]progesterone peak co-sediments with the 110,000 and 75,000 molecular weight peptides at 4 S, while the 90,000 molecular weight peptide sediments at 6-7 S. On photoactivation, the synthetic progestin R5020 binds covalently to the 110,000 and 75,000 molecular weight peptides. Thus, both 8 S forms of progesterone receptor contain 90,000 molecular weight peptides which do not bind progesterone, and each 8 S form contains a separate form of progesterone-binding peptide. When receptor is purified from oviduct minces incubated with [32P]orthophosphate, autoradiography indicates the presence of 32P in the 90,000 and 110,000 molecular weight peptides and in a peptide which appears to be slightly larger than 75,000 and may be a more highly phosphorylated fraction of the 75,000 molecular weight peptide. Thus, three separate peptides have been identified as components of the 8 S form of progesterone receptor, and all three appear to exist as phosphoproteins.

Animals↗
PubMed1984

Interaction of ionizing irradiation with steroid receptors in human breast cancer cells.

Human mammary tumor cells in continuous culture (MCF-7 cells) are hormone- and radiosensitive. The interaction of both factors is analyzed. Ionizing irradiation lowers the concentration of both the estradiol and progesterone receptors per cell. The reduction is dose dependent. However, the effects on the cytoplasmic and nuclear forms of the receptors are not similar. For the estradiol receptor, an accumulation in the nuclear fraction is observed 48 hr after irradiation when no appreciable amounts of estrogens are present. After administration of 10(-8) M estradiol, the cytoplasmic clearance is comparable to the unirradiated controls. However, nuclear accumulation is impaired. The processing of the nuclear estrogen receptor remains identical. Nuclear progesterone receptor is not significantly increased due to irradiation in the absence of progestins. Cytoplasmic decrease after incubation with progestins is unaffected. Again, nuclear accumulation is impaired in contrast to the unchanged processing of the nuclear form of the progesterone receptor. A decrease in "nuclear acceptor sites" for both receptors after irradiation may be an explanation for these observations. No significant effects of ionizing irradiation are observed in the initial steps of steroid hormone action.

Breast Neoplasms↗
PubMed1984

Biological activity and receptor binding of a strongly interacting estrogen in human breast cancer cells.

A substantial proportion of human breast cancers contain estrogen receptors, and it is believed that the growth of some of these tumors and their synthesis of specific proteins are stimulated by estrogens. Since natural estrogens, such as estradiol, react reversibly with estrogen receptors, it was of interest to determine the biological consequences that would result from very strong, possibly irreversible interaction of an estradiol-based ligand with the estrogen receptor of breast cancer cells. For these studies, we have examined the receptor interactions and biological character of 11 beta- chloromethylestradiol (CME) and 11 beta- bromomethylestradiol (BME) as potential estradiol-based affinity labeling ligands in MCF-7 human breast cancer cells which contain high levels of estrogen receptors. The apparent relative binding affinities of CME and BME for MCF-7 estrogen receptor measured by competitive binding assay are 230 and 15%, respectively, whereas the affinity of estradiol is considered 100%. Incubation of receptor preparations from MCF-7 cells or rat uteri with CME at 21 degrees results in a concentration- and time-dependent decrease in receptor content measured by exchange assays with [3H]estradiol. This may be due to covalent attachment of CME to receptor and is termed "inactivation." Inactivation of 80 to 85% of the receptors occurs within 30 min at 21 degrees by exposure to 5 or 20 nM CME, with 2 nM giving 20 to 40% inactivation. This receptor inactivation is prevented by preincubation with 2000 nM estradiol, indicating that the interaction of CME is occurring at the estradiol binding site on the receptor. MCF-7 cells incubated with 20 nM CME show a rapid loss of cytosol receptor sites and no accumulation of receptors detectable by exchange assay in the nucleus, while 20 nM estradiol shows nuclear localization of receptor. BME, in contrast, inactivates only a portion (approximately 40%) of estrogen receptors. CME and BME both behave as estrogen agonists. They stimulate the proliferation of MCF-7 cells and increase cellular progesterone receptor content and plasminogen activator activity. CME is at least as potent as estradiol on a molar basis in increasing all of these activities, while BME shows a biopotency of only 1% of that of estradiol or CME. Hence, although CME reacts very strongly and apparently irreversibly with estrogen receptors in MCF-7 cells, it still behaves as a potent estrogen agonist.

Animals↗
PubMed1983

Photoaffinity labeling of the chick progesterone receptor proteins. Similar hormone binding domains detected after removal of proteolytic interference.

Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.

Affinity Labels↗
PubMed1983

Progesterone receptor replenishment in T47D human breast cancer cells. Roles of protein synthesis and hormone metabolism.

T47D are unusual human breast cancer cells that do not require estrogen to synthesize high levels of progesterone receptors. These cells can, therefore, be used to study the mechanisms by which progesterone, freed of estrogen interference, controls the synthesis of its receptors. In a recent paper we described progesterone receptor translocation and a subsequent very rapid nuclear processing step that results in an apparent loss of 60 to 80% of cellular progesterone receptors, 30 to 60 min after progesterone treatment. This paper deals with the replenishment of cellular receptors following processing. If progesterone is removed from cells after 60 min of treatment, cytoplasmic progesterone receptors replenish in 16 to 20 h. However, replenishment occurs even during chronic progesterone treatment; this is an artifact created by the extremely rapid (t1/2 approximately 2 h) metabolism of progesterone in media exposed to cells. If progesterone metabolism is blocked, then replenishment is not seen, probably because the hormone continuously retranslocates the newly replenished sites. There is an early protein synthesis-dependent step; cycloheximide in the first 4 h inhibits replenishment 24 h later, but if cycloheximide is slightly delayed (beyond 4 h), replenishment proceeds normally. In contrast to progesterone, the synthetic progestin R5020 completely suppresses progesterone receptor replenishment even 96 h after its removal from the medium. This compound can bind covalently to receptors and may be very difficult to remove from cells. Clearly, progestin treatment, and by analogy, circulating progesterone, will have profound effects on cytoplasmic and nuclear progesterone receptor levels when these are measured in biopsied human tumors as an adjunct to endocrine therapy.

Breast Neoplasms↗
PubMed1983

Nuclear estradiol-binding sites in human breast cancer.

The binding of estradiol to nuclear fractions extracted from human breast carcinomatous tissue was demonstrated. The material, which was extracted with KCl, had an approximate molecular weight of 37,000 and bound estradiol with both high and low affinity (Kd congruent to 1 nM, type A receptors; Kd congruent to 30 nM, type B receptors) as calculated according to the method of Scatchard. Competition studies indicated that both components were specific for estradiol, and among the 134 tumors studied the receptors were found to be linked in almost all cases. Thirty-six % of the tumors were nuclear receptor positive. Cytoplasmic estradiol and progesterone receptors were also measured. Among the cytoplasmic tumors positive for cytoplasmic and progesterone receptors, 37% were devoid of both types of nuclear receptors; this may explain the failure of endocrine therapy in some cases. The determination of nuclear binding sites in human breast tumors appeared to be an interesting criterion for the assessment of estradiol-dependent cell growth.

Adenocarcinoma↗
PubMed1982

Progestin receptor levels in rat hypothalamic and limbic nuclei.

We have utilized a method to minimize cytosol progestin receptor loss during freezing in order to localize the quantify estrogen-inducible progestin receptors in individual nuclei of the female rat brain. Ovariectomized females received estradiol benzoate (20 micrograms for 3 days) or vehicle prior to sacrifice. All animals were perfused with cold distilled H2O containing the cryoprotective compound, dimethyl sulfoxide (DMSO; 10% (v/v)). Thirty-one nuclei or brain regions were removed from frozen sections (300 micrometers) according to the method of Palkovits (Palkovits, M. (1973) Brain Res. 59: 449-450) and were assayed in vitro using a synthetic radioligand, [3H]R5020. In ovariectomized animals perfused with DMSO, a basal level (1 to 8 fmol/mg of protein) of progestin receptors was observed in a variety of preoptic, hypothalamic, and limbic structures. Moreover, estrogen treatment induced high levels (24 to 49 fmol/mg of protein) of progestin receptors in regions of the preoptic area of hypothalamus which contain high levels of estrogen receptors. These regions included the medial, periventricular, and superchiasmatic nuclei of the preoptic area, the periventricular anterior hypothalamus, the ventromedial nucleus, and the arcuate-median eminence. Moderate levels (2 to 8 fmol/mg of protein) of progestin receptors were induced by estrogen in other hypothalamic and limbic structures, including the anterior and lateral hypothalamus, the bed nucleus of the stria terminalis, the cingulate cortex, the medial amygdaloid nucleus, and the CA subfield of the hippocampus. By contrast, some areas, such as the caudate-putamen and the supraoptic nucleus, were devoid of both estrogen-inducible and uninduced progestin receptors. These results support the hypothesis that progesterone action in the central nervous system is mediated by cytosol receptors in discrete brain regions and provide the first quantitative map of progestin binding in a vertebrate brain.

Animals↗
PubMed1981

Absence of high-affinity binding of progesterone (R 5020) in human placenta and fetal membranes.

Increased levels of maternal serum progesterone occur during the last stages of human gestation. The function of the high level of this steroid is unknown. The presence of a progesterone receptor in the placenta was investigated to determine whether progesterone action on the placenta might serve as one function of the high level of this steroid. Cytosol and nuclear fractions, derived from human placentae and fetal membranes, were examined for the presence of progesterone receptors by conducting exchange assays, using tritiated R 5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) as the radiolabelled ligand. High-affinity, low-capacity binding, characteristic of steroid receptors, was estimated as the difference between binding of radiolabelled ligand in the presence of no unlabelled ligand and that in the presence of a 100-fold excess of unlabelled ligand. These exchange assays were conducted during a 24-hour period at 0 degrees C, to allow maximal stability of the receptor, and during a 3-hour period at 20 degrees C, to allow the rapid exchange of radiolabelled ligand for any bound endogenous progesterone. The assays of all fractions showed no specific binding of the R 5020, thus indicating the absence of progesterone receptors in the cytosol and nuclei of the human placenta and the fetal membranes.

Extraembryonic Membranes↗
PubMed1980

Steroid receptors in osteoblasts.

Using the whole-cell incubation system at 37 degrees C, the specific bindings for 3H-dexamethasone, 3H-estradiol-17 beta, 3H-dihydrotestosterone and 3H-R5020 were measured in the purified, putative osteoblasts isolated from fetal rat calvaria by collagenase digestion. More than 90% of the purified cells contained intense alkaline phosphatase activity. The specific binding for 3H-dexamethasone with high affinity and low capacity was demonstrated in the isolated osteoblasts. Most of the binding was found in the nuclear fraction, indicating nucler binding of the 3H-dexamethasone-receptor complex. The apparent dissociation constant (Kd) for 3H-dexamethasone was estimated to be 3.3 x 10(-9)M and the number of binding sites was calculated to be 65 fmol/ml (4 x 10(6) cells) or 9,750 binding sites per cell. High salt: sucrose gradient analysis of nuclear extracts revealed a radioactive 4.0 S peak. These results indicate that the purified osteoblasts are among the target cells for glucocorticoids. On the other hand, the specific bindings for 3H-estradiol-17 beta and 3H-dihydrotestosterone were not detectable in the isolated osteoblasts, which suggests that estrogens and androgens act on osteoblasts only indirectly.

Animals↗
PubMed1980

Stabilization of 8S progesterone receptor from human prostate in the presence of molybdate ion.

In the presence of 10 mM molybdate ion, we are able to detect the appearance of a [6,7-3H]-17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione-([3H]R5020) binding moiety in human prostatic cytosol which sedimented at approximately 8S in a glycerol density gradient. The specifically bound [3H]-R5020 was displaced by progesterone, tiramcinolone acetonide, and R5020 but not by cortisol, dihydrotestosterone, 17 beta-estradiol, or diethylstilbestrol. Specific binding of [3H]R5020 in the presence of molybdate ion was shown to saturate at 7 x 10(-10) M (n = 64 fmol/mg of protein). The optimum concentration of molybdate ion for enhancing specific binding of [3H]-R5020 was determined to be between 7 and 20 mM. Molybdate ion was also effective in stabilizing the 8S R5020-binding moiety in a preincubation for 16 hr at 0 degrees in the absence of added steroid.

Binding, Competitive↗
PubMed1980

Steroid receptors in human renal carcinoma.

Glucocorticoid- and androgen-binding receptors were demonstrated in cytosol of human renal cell carcinomas by the dextran-coated charcoal technique. Sedimentation studies using preparative ultracentrifugation indicated a 7 to 8 S binding component. None of the renal carcinoma cells tested contained detectable estrogen receptors and a few samples had a very low but demonstrable progestin receptor level. In the normal kidneys tested, cytoplasmic components that bound only androgen could be detected. Competition studies showed that progestin competed for the glucocorticoid receptor sites in all the renal tumors tested, whereas diethylstilbestrol and testosterone were weak or not competitive. It is suggested that the response of some patients with renal carcinoma to high doses of progesterone or androgens could be explained by the ability of these hormones to block the activity of glucocorticoid receptors, which might be involved in tumor growth.

Adenocarcinoma↗
PubMed1981

A comparison of progesterone and R5020 binding in endometrium, ovary, pituitary, and hypothalamus.

A cytoplasmic receptor is necessary for target tissues to respond to steroid hormones. Since the hypothalamic-pituitary unit is responsive to progestins, it might be expected to have a progesterone receptor. The clinical evaluation of receptors located in the central nervous system is not possible, so the clinical investigator must extrapolate from accessible tissues. To determine whether such extrapolation is valid, binding affinities and specificity of progesterone receptor sites in the cytoplasm of bovine uterus, ovary, hypothalamus, and pituitary were measured. In each particular tissue the receptors showed similar binding characteristics with progesterone and R5020. It is concluded that in a given species the receptor for progesterone is similar in all target tissues and probably results from the expression of the same gene.

Animals↗
PubMed1981

Simultaneous measurement of estrogen and progesterone receptors in tumor cytosols with use of 125I-labeled estradiol and of 3H-R5020.

We describe a dual-isotope assay for measuring the concentration of estrogen receptors and progesterone receptors in tumor cytosols. The concentrations of these receptors are derived from separate Scatchard-plot analyses of a single dextran-coated charcoal assay that incorporates both radioiodinated estradiol and tritiated R5020 as the labeled ligands. The two isotopes, radioiodine and tritium, are easily measured with a liquid-scintillation counter. The concentrations of estrogen and progesterone receptors derived from the dual-label assay are identical to values derived from the respective single-label assays. Moreover, values obtained from an assay with dextran-coated charcoal that incorporates radioiodinated estradiol are identical to those obtained from an assay in which tritiated estradiol is used as the labeled ligand. The dual-isotope assay requires both less time and less tissue.

Breast Neoplasms↗
PubMed1981

Photoaffinity labeling of glucocorticoid receptors.

The cross-reactivity of progestins for glucocorticoid receptors was exploited to photoaffinity label glucocorticoid receptors from cultured rat hepatoma (HTC) and mouse lymphoma (S49) cell cytosol. The synthetic progestin, 17 alpha, 21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020), rapidly forms covalent bonds with protein upon irradiation of either cytosol with 350 nm light. Polyacrylamide gel electrophoresis under denaturing conditions reveals a single band photolabeled by R5020 that is not observed when excess nonradioactive dexamethasone is included in the incubation. This protein band corresponds to a molecular weight of about 87,000 in both HTC and S49 cell cytosol; it is entirely absent in cytosol from glucocorticoid-resistant S49(r-) cells which lack receptor-binding activity. Another steroid-resistant mutant, S49 (nti), which exhibits normal levels of steroid-binding activity but increased binding of receptor-steroid complexes by the nucleus, yields a receptor which, when photolabeled, has an apparent molecular weight of only 39,000. These results demonstrate that glucocorticoid receptors can be photoaffinity-labeled; the data are consistent with the notion that the binding form of the receptor consists of a single polypeptide chain, Mr = 87,000, in two different species, rat and mouse, and in cells of either hepatic or lymphoid origin. The data also suggest that the lesion in the steroid-resistant S49 (nti) lymphoma cell line is a mutation of the structural gene for the glucocorticoid receptor which results in the synthesis of a truncated protein.

Affinity Labels↗
PubMed1982

Identification of a steroid receptor on the surface of Xenopus oocytes by photoaffinity labeling.

The synthetic progestin, 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020), was used to photoaffinity label a steroid-binding protein on the Xenopus oocyte plasma membrane. R5020 effectively induced oocyte maturation with half-maximal response at a concentration of 1.4 X 10(-6) M. Polyacrylamide gel electrophoretic analysis of plasma membranes following photolysis with tritiated R5020 resulted in the identification of a single labeled protein with a Mr = 110,000. The specificity of this steroid receptor interaction for R5020 was demonstrated by the competitive inhibition of R5020 photolabeling with nonradioactive R5020 and the lack of inhibition by 17 beta-estradiol. Covalent labeling of the 110,000-dalton protein was saturable with both time of photolysis and concentration of R5020 with the maximum number of photolabeled binding sites equal to 0.7 pmol/oocyte, and kinetic analysis of the photolabeling of the 110,000-dalton receptor protein yielded an apparent KD of 1 X 10(-6) M R5020. Progesterone had a biphasic effect on the kinetics of photolabeling with concentrations of progesterone below 5 microM increasing photolabeling by elevating Vmax up to 5-fold and higher concentrations of progesterone reducing the rate of photolabeling. Membrane-associated adenylate cyclase measured in the presence of guanyl-5'-yl imidodiphosphate was inhibited up to 70% after photolysis with [3H]R5020. Inhibition was proportional to the level of [3H]R5020 covalently bound to the 110,000-dalton protein, and significant inhibition was observed at 1 X 10(-6) M R5020.

Adenylyl Cyclases↗
PubMed1981

Progestin receptors in testes from various animal species.

Using a synthetic progestin, R5020 (17 alpha, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione), we investigated the presence of progestin receptor(s) in testicular cytosol from various animal species including humans. After incubation of cytosol from adult chickens with [3H]R5020 in the presence or absence of various unlabeled steroids, specific progestin binding was found in the 8 S region with an equilibrium dissociation constant of 2 nM and a binding capacity of 75 fmoles/mg protein. Specific progestin binding was also found in cytosol from immature chickens sedimenting in the 4 S region with a dissociation constant of 61 nM and a binding capacity of 1 pmole/mg protein. Although 5 S binding was found in the cytosols from humans, a monkey and rats, the binding specificity was low. These results clearly indicate that the testicular cytosols from chickens contain progestin receptor(s), whereas those from the other species do not.

Adult↗