Cytosolic Estrogen Receptor Research Articles

Effects of anterior roof deafferentation on lordosis behavior and estrogen receptors in various brain regions of female rats

This study investigated whether the estrogen receptors (ER), located at different brain areas and anterior pituitary (AP), changed after anterior roof deafferentation (ARD), and on the effects of facilitating the lordosis reflex in female rats. Female rats were ovariectomized and implanted with estradiol capsules. ARD or sham operation was performed with a Halász knife. All animals were tested for lordosis both before and after surgery. One day after the last test they were sacrificed. Cytosol and nuclear ER in the AP, medial preoptic area (MPOA), basal medial hypothalamus (BMH), amygdala (AMYG), septum (SEP), hippocampus (HPC), and cortex (CTX) were measured using an in vitro exchange assay. Rats with ARD showed significantly higher mean levels of lordosis quotient than the control and the sham groups before ARD surgery. An increase of both cytosol and nuclear ER in the BMH area compared to the control was observed, whereas the ER, in the SEP was reduced. ER in other areas were not affected by ARD. Serum estradiol and progesterone levels were not altered by the operation. These data suggest that the dorsal inhibitory pathway from the extrahypothalamus to the preoptic area and hypothalamus may modulate the estrogen receptor and the display of lordosis in female rats. Change of ER level in the BMH area may influence the hormonal sensitivity of lordosis in female rats.

Aromatase inhibitors regenerate the thymus in aging male rats

The thymus can be regenerated in aging rats by surgical or chemical castration and regeneration is inhibited by testosterone, which may exert this effect, at least in part, through its conversion to estradiol. An attempt has been made to regenerate the thymus in intact aging rats using inhibitors of the aromatase system, in the hope that this maneuver could lead to the use of such chemical intervention in the treatment of immunodeficiency syndromes. Young adult and aging (18-month-old) male rats were orchidectomized under ether anesthesia and 7 days later given s.c. implants of testosterone in silicone elastomer (SILASTIC) tubing. Some rats received testosterone together with a five-fold excess of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). One group of young intact rats received implants containing 25 mg ATD and group of 18-month-old intact rats received 125 mg ATD or 25 mg of another, more powerful aromatase inhibitor 4-hydroxyandrostenedione (4-OH). On the 28th day after implanting, rats were killed and the thymus, spleen, prostate gland and seminal vesicles removed for weighing and histology. In addition, estrogen receptors were measured in the thymus was enlarged after orchidectomy and greatly restored in aging rats. In aging rats, both aromatase inhibitors restored the thymus, which appeared normal histologically. In addition, ATD enlarged the thymus in young intact animals. Doses of testosterone which restored the accessory sex organs to weights measured in intact rats prevented the effects of orchidectomy on the thymus, and in old rats the effects of testosterone were blocked by ATD in both thymus and spleen. Available cytosolic estrogen receptors were reduced in thymus of testosterone-treated orchidectomized rats, and this effect blocked by ATD, which itself was apparently able to induce estrogen receptors. Receptors could not be detected in thymus from aging rats, but were measureable in cytosols from thymus of orchidectomized or ATD-treated old rats. It is therefore possible to restore the thymus in intact aging rats without recourse to surgical or chemical castration, and such a maneuver may possibly be of use to enhance an immune system weakened by aging or disease.

Influence of estrogen receptor variants in mammary carcinomas on the prognostic reliability of the receptor assay

We have used GR mouse mammary carcinomas as a model for evaluating the effect of estrogen receptor (ER) variants, present in the tumor cytosols, on the prognostic reliability of the receptor assay. Rapid, high resolution procedures were used for the assessment of wild-type and variant estrogen receptors in the cytosols of hormone-responsive and nonresponsive mammary tumors of GR mice. Two main ER types were resolved by high performance ion-exchange and size-exclusion chromatography: the wild-type receptor (II), and a fraction representing low molecular weight ER (I). ER types I and II both bound estradiol and both reacted with the anti-ER monoclonal antibodies H222 and D547 whose epitopes are in the C-terminal part of ER. The level of ER type II was higher in hormone-responsive than in hormone-nonresponsive tumors, whereas ER type I was about equally low in both types of tumor groups. ER types I and II both influence the standard ligand binding and antibody binding (Abbott EIA) test, but only the wild-type ER causes hormone-responsive growth of the tumor. These data suggest that prognostic clinical tests, currently in use for estimating ER in tumors of breast cancer patients, may be impaired by the presence of low molecular weight estrogen receptor variants in the tumor samples.

Effects of in vivo estrogen treatment on adipose tissue metabolism and nuclear estrogen receptor binding in isolated rat adipocytes

We have previously demonstrated the existence of nuclear estrogen receptors in isolated adipocytes (Pedersen et al. (1991) Biochim. Biophys. Acta 1093, 80–86). In the present study we have investigated the regulatory properties of these nuclear estrogen receptors, in addition to the metabolic effects of estrogen on adipose tissue metabolism. Estrogen treatment (20 μg 17β-estradiol in NaCl for 7 days) decreased lipoprotein lipase activity (LPL) in the adipose tissue by 62% ( p < 0.05), decreased adipocyte size by 27% ( p < 0.01) and diminished the normal postovariectomy weight gain. Furthermore, estrogen treatment increased the nuclear estrogen receptor binding in adipocytes; in addition, there was a tendency for increased cytosolic estrogen receptor content as well. Time course studies revealed that already 6 h after a single estrogen injection the B max increased from 3.82 ± 0.3 fmol/10 6 cells to 9.8 ± 3.6 fmol/10 6 cells ( p < 0.1) and 24 h after a single injection the B max was maximally increased to 12.7 ± 5.5 fmol/10 6 cells ( p < 0.05). The K d was similar at all time points (about 3–5 nM). Furthermore, the specific insulin receptor binding was increased in adipocytes from estrogen treated rats. The specific insulin binding was maximally increased by 149 ± 6% ( p < 0.001) after 4 days of daily estrogen injections. The increased binding seemed to be due to an increased number of insulin receptors on adipocytes from estrogen treated rats with no alteration of the ED 50 value. In conclusion it was found that estrogen treatment has a positive feedback effect on its own nuclear receptor. In addition, estrogen treatment was found to have several metabolic effects in the adipose tissue. Estrogen treatment increased the insulin receptor number, decreased LPL activity, increased the lipolytic response in adipocytes and finally decreased body weight and adipocyte cell size.

The effects of diethylstilbestrol, tamoxifen, and toremifene on estrogen-inducible hepatic proteins and estrogen receptor proteins in female rats

Alterations of estrogen-inducible hepatic proteins and estrogen receptor proteins were studied in female rats after daily oral administration of vehicle control (0.5% carboxymethylcellulose), the nonsteroidal antiestrogens tamoxifen (TAM; 3, 11, or 45 mg/kg body weight), toremifene (TOR; 3, 12, or 48 mg/kg body weight), or the potent synthetic estrogen diethylstilbestrol (DES; 10 mg/kg body weight) for 1 and 3 months. Serum corticosterone levels were significantly reduced by TAM 11, TOR 12, and DES at 1 month and by TOR 48 and DES at 3 months. Serum ceruloplasmin levels were unchanged at 1 month except for a significant reduction with TOR 48 and a significant increase in the DES-treated group. After 3 months, all doses of TAM and TOR had significant reductions whereas DES had elevations even greater than those at 1 month. The activity of the enzyme alanine aminotransferase in the liver was increased by TAM, TOR, and DES at 1 and 3 months of treatment. Both TAM and TOR caused a slight reduction in cytosolic estrogen receptor protein after 1 month and significant reductions at 3 months. The nuclear estrogen receptor (nER) protein levels were significantly increased at 1 and 3 months for TAM and TOR; whereas DES treatment resulted in nER levels no different than controls. In summary, chronic (up to 3 months) administration of TAM and TOR results in qualitatively and quantitatively similar hormone-related effects on the female rat liver. Thus, other mechanisms must be investigated to ascertain the hepatoproliferative differences seen with TAM administration but not with TOR.

Antiestrogenic action of 2,3,7,8-tetrachlorodibenzo- p-dioxin: Tissue-specific regulation of estrogen receptor in CD1 mice

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is a polychlorinated aromatic hydrocarbon with teratogenic and carcinogenic properties. Previous studies in our and other laboratories have demonstrated that TCDD has antiestrogenic properties. In order to elucidate the mechanism of action of TCDD on estrogen sensitive tissues, we studied its effects on serum estradiol and estrogen receptor (ER) levels in liver and uteri of CD1 mice. Treatment with TCDD did not result in alterations of serum estradiol levels at any of the doses tested (1.0–30 μg/kg). In contrast, TCDD treatment induced a dose-dependent decrease in hepatic and uterine ER protein as determined by an enzyme immunoassay and equilibrium binding assays. A decrease in cytosolic and nuclear ER levels in uteri occurred as early as 24 hr after initial treatment with 30 μg/kg TCDD and recovery occurred by 14 days. Hepatic cytosolic and nuclear ER also decreased at a dose of 30 μg/kg TCDD at 24 hr after treatment, but recovery occurred only by 21 days. Studies in ovariectomized mice indicate that the regulation of hepatic ER by TCDD is independent of ovarian factors, but ovariectomy inhibited the downregulation of uterine ER by TCDD. Furthermore, determination of TCDD-induced cytochrome P-450 levels indicates that the downregulation of uterine ER is uncoupled from induction of hepatic cytochrome P-450. This study indicates that the antiestrogenic effects of low doses of TCDD are mediated through its ability to decrease hepatic and uterine ER and are not due to alterations in serum estradiol levels. Our results on ovariectomized mice indicate that TCDD-induced downregulation of ER is tissue specific and may involve different mechanisms at transcriptional or posttranscriptional levels.

Sex differences in estrogen receptor binding in the rat hypothalamus: effects of subsaturating pulses of estradiol

The effects of subsaturating pulses of estradiol on cell nuclear retention of estrogen receptors in brain regions of male and female rats was determined. In the first experiment, age-matched adrenalectomized/gonadectomized (ADX/GDX) rats were killed 1 h after i.v. injection of a subsaturating dose of free estradiol (1.0 μg/kg b. wt.) and cell nuclear estrogen receptor binding was measured in microdissected brain areas by an in vitro exchange assay. As we have previously reported with saturating doses of estradiol (3.6–36.0 μg/kg b. wt.), a greater concentration of receptors was measured in the ventromedial nucleus (VMN), periventricular preoptic area (PVPOA), and medial preoptic area (mPOA) of the female than in the same regions of the male brain. Binding levels did not differ between the sexes in the bed nucleus of the stria terminalis, arcuate nucleus, or the corticomedial amygdala. In the second experiment, GDX/ADX male and female rats received either a single or double pulse of 0.5 μg estradiol/kg b. wt. administered 6 h apart. Rats were killed 1 h after the second pulse. In animals treated with a single pulse of estradiol 1 h before sacrifice, higher concentrations of estrogen binding were measured in the female VMN and PVPOA than in the male. Except in the mPOA, the concentration of estrogen receptor binding measured was not different from that measured in animals that had received an additional, earlier pulse of estradiol. In this area, receptor concentrations were lower in male rats treated with two pulses than in males treated with one pulse. Thus, receptor dynamics in the mPOA of the male may differ from that of the female and from that of other brain regions. In the third experiment, cytosol estrogen receptor concentrations measured in untreated male and female GDX/ADX rats were compared. Consistent with our data on cell nuclear binding after exposure to estradiol, cytosol receptor concentrations in the female VMN, PVPOA and mPOA were greater than those in the male. These results extend our previous findings of regional sex differences in maximal cell nuclear estrogen binding capacity by demonstrating that these differences exist in the presence of subsaturating pulses of estradiol and that they reflect differences in cytosol estrogen receptors.

Up-regulation of the uterine estrogen receptor and its messenger ribonucleic acid during the mouse estrous cycle: the role of estradiol.

Uterine estrogen receptor (ER) and ER mRNA were measured in cycling and ovariectomized (OVX) estrogen-treated mice to probe the physiological regulation of the intracellular distribution and biosynthesis of ER. On proestrus, when plasma estradiol (E2) levels are highest, the cell nuclear ER concentration was 2.4-fold greater than on metestrus. This increase was primarily attributable to an increase in total cellular ER (cytosolic plus nuclear ER) and only secondarily to an activation of ER, as measured by its redistribution from the cytosolic (i.e. nuclear-extractable) to the nuclear (nonextractable) fraction. Total cellular ER concentration was 1.8-fold higher on proestrus than on metestrus, whereas the fraction of total ER in the nuclear compartment (i.e. the percentage activated) was only 1.3-fold higher. The concentration of cellular ER mRNA was 3-fold greater on proestrus than on the other days of the estrous cycle, suggesting that the increased concentration of ER on proestrus was a consequence of increased ER gene expression. In OVX mice, physiological and, to a lesser extent, supraphysiological levels of E2 increased cell nuclear ER. As in proestrous mice, the increased ER content contributed more than ER activation to the increased cell nuclear ER concentration. Physiological, but not supraphysiological, concentrations of E2 increased ER mRNA in OVX mice. Together, these results suggest that up-regulation by E2 of ER mRNA and ER accounts for most of the increased nuclear binding of ER on proestrus. E2-dependent activation and consequent DNA binding of ER presumably initiate this process, but quantitatively account for only a small fraction of the increased nuclear binding of ER.

The effect of 6-nitro-1,3,8-trichlorodibenzofuran as a partial estrogen in the female rat uterus

Administration of 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF) caused a dose- and time-dependent increase in uterine wet weight and cytosolic and nuclear estrogen receptor (ER) and progesterone receptor (PR) levels in immature female Sprague-Dawley rats. These estrogenic effects persisted for up to 96 or 144 hr after initial administration of 6-NCDF and could be observed at a dose as low as 2 μmol/kg. In contrast, 6-NCDF (25 μmol/kg) did not increase rat uterine peroxidase activity or epidermal growth factor (EGF) receptor binding activity. 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), which exhibits a broad spectrum of antiestrogenic effects in the female rat uterus, inhibited the 17β-estradiol-induced increase in uterine wet weights, cytosolic and nuclear ER and PR levels, peroxidase activity, and EGF receptor binding activity. In contrast, 2,3,7,8-TCDD inhibited the uterotropic effects caused by 6-NCDF but did not affect the 6-NCDF-induced uterine ER and PR levels. 6-NCDF is a weak inducer of hepatic microsomal ethoxyresorufin O-deethylase activity and competitively binds to the aryl hydrocarbon (Ah) receptor but not the PR or ER. Thus both 6-NCDF and 2,3,7,8-TCDD, two ligands which bind to the Ah receptor, exhibit both partial estrogenic and antiestrogenic properties and serve as useful models for delineating the complex biochemical interactions between the ER and Ah receptor signal transduction pathways.

Multivariate analysis by the minimum spanning tree method of the structural determinants of diphenylethylenes and triphenylacrylonitriles implicated in estrogen receptor binding, protein kinase C activity, and MCF7 cell proliferation

The response profiles of 36 para-substituted diphenylethylenes (DPEs) and triphenylacrylonitriles (TPEs) have been compared by multivariate analysis. The responses measured were (a) relative binding affinity (RBA) for the cytosol estrogen receptor (ER), (b) ability to promote the growth of the human MCF7 breast cancer cell-line, (c) cytotoxicity in MCF7 cells, and (d) ability to stimulate or inhibit protein kinase C (PKC) III activity under three different conditions of enzyme activation. The prime object of the analysis was to observe the simultaneous influence of diverse combinations of substituents on all these in vitro responses. To do this, the minimum spanning tree (MST) method was used to organize the molecules into a network in which proximate molecules are closely related with regard to their responses whereas remote molecules are distinct. The MST of this population of molecules had four main branches. E2 and its TPE mime were located in a central position within the trunk whereas the tips of the branches tended toward molecules of different specificity, i.e., cytotoxic molecules that bind to ER and interfere with PKC, noncytotoxic molecules that also bind to ER and interfere with PKC but promote cell growth, molecules only active on PKC, and molecules active on all parameters except PKC stimulation. A parallel MST analysis of the relationships among the response parameters themselves confirmed previous conclusions: For this population of molecules, RBAs for ER are fairly closely related to ability to promote MCF7 cell growth and only little to cytotoxicity (Bignon et al. J. Med. Chem. 1989, 32, 2092). Cytotoxicity is much more clearly correlated with inhibition of diacylglycerol-stimulated PKC activity than with RBAs for ER. PKC inhibition differs substantially depending upon whether the substrate is H1 histone or protamine sulfate.

Increase in estrogen receptor levels in MNU-induced thyroid tumors in LE rats.

Estrogen receptor (ER) levels were evaluated in thyroid tumors induced by N-methyl-N-nitrosourea (MNU) and low iodine diet (LID) or propylthiouracil (PTU) in intact and estrogen (E2) loaded Long-Evans (LE) rats. MNU at 40 mg/kg body wt was injected in 50 day-old LE rats of both sexes. The animals were killed 17-22 weeks later and the thyroid tissues were subjected to ER assay. In LID-treated groups, cytosolic ER (cER) levels were 6.7 +/- 5.8 (fmol/mg protein, mean +/- SE) in females and 0.7 +/- 1.4 in males, E2 increased the ER levels. In E2-loaded LID groups, cER levels were 12.9 +/- 3.7 in females and 1.7 +/- 1.7 in males. PTU treatment produced almost comparable ER levels as LID treatment. PTU treatment as well as LID treatment increased the serum TSH levels with E2 treatment producing additional elevation. In evaluating ER levels by histological type of thyroid tumors, the level in cER plus nER showed the lowest value of 6 +/- 6.4 (fmol/mg DNA, mean +/- SE) in hyperplasia, followed by 129 +/- 52.3 in adenoma and 289 +/- 51.7 in carcinoma. The rates of BrdU incorporation in thyroid follicles indicated higher proliferation activity in the area of adenoma and carcinoma rather than in the hyperplastic area. These data suggested that E2 treatment increases the ER levels in MNU and LID/PTU-induced thyroid tumors. The level of ER was correlated to the histological type of thyroid tumors.

Two-site in-house quality control for estrogen receptor (ER) assay

As no external Quality Control Program has existed for ER assay since 1988, and commercial preparations are either unsuitable or expensive, a local QC program was set up between our hospitals. An ovarian tumour with high levels of ER was obtained. Cytosol was prepared, aliquoted into Nunc cryotubes and stored at -70°C. This material has been used in each ER assay (Abbott ER-EIA) at both the hospitals since January 1989. Mean cytosol ER values have not changed significantly over a 2.5 year period (see table). The difference in results between the laboratories was primarily due to different methods of proteins estimation. Mean intra-assay coefficients of variation (CV) were 2.1% and 4.8% for RWH and RMH respectively.RWHRMHNMEANSDCVNMEANSDCV1989 RESULTS378513154011728241990 RESULTS409918173810820181991 RESULTS19105131324140139.4 The cytosol preparation, although stable when frozen, was quite heat labile. It showed partial loss of activity after 10 minutes at room temperature, and total loss of activity after 30 minutes at 37°C. If stored at -70°C, it remains stable over at least two years, and provides a suitable cheap source of QC material. It, however, does not address the issue of inter-hospital error introduced in the homogenisation process.

Estrogen receptor levels and tumor growth in a series of pituitary clonal cell lines in rats.

Four kinds of in vitro clonal pituitary tumor cell lines named MtT/Se, MtT/SM, MtT/S and MtT/ E, each of which shows different sensitivity to estrogen on proliferation, were inoculated into fat pad of ovariectomized rats and estrogen‐loaded ovariectomized rats at 105 and 106 cells/site. They formed tumor with average latency ranging from 30 to 71 in ovariectomized rats and 13 to 63 days in estrogenized rats inoculated with 106 cells. MtT/Se was highly sensitive to estrogen for growth, and MtT/SM also grew well in estrogenized rats. With MtT/S and MtT/E, there was no significant shortening of average tumor latency in estrogenized rats. In vivo, the cytosolic estrogen receptor (ER) levels of MtT/Se, SM, S and E were measured to be 452 ±66, 370 ±115, 260 ±16 and 83 ±8 fmol/ mg protein, respectively. In vitro, however, the lowest ER level was noted in MtT/Se. Histologically, all four tumors grown in rats were composed of homogeneous round cells, and MtT/Se contained particularly large nucleated cells. In MtT/E, the cells appeared to be changing into ftbromatous cells. Three cell lines except MtT/E maintained the function of hormonal secretion in vivo as well as in vitro. Serum GH level was increased in rats with MtT/Se and MtT/S. Increased levels of both prolactin and growth hormone were measured in sera of rats with MtT/SM. Increases of hormones as well as tumor sizes were promoted by the estrogen.

Bovine uterine, cervical and ovarian estrogen and progesterone receptor concentrations

Bovine uterine cytosol estrogen (ERC) and progesterone receptors (PRC) were measured simultaneously in the endometrium and myometrium of cows with various serum progesterone concentrations. When serum progesterone concentrations were low (less than 2.7 nmol 1 −1), PRC concentrations in the endometrium (1162 fmol mg −1 cytosol protein) and in the myometrium (1315 fmol mg −1 cytosol protein) were relatively high. In animals with serum progesterone concentrations between 2.7 and 6.4 nmol l −1, endometrial and myometrial PRC concentrations were 695 fmol mg −1 protein and 694 fmol mg −1 protein, respectively. When the progesterone concentration was above 6.4 nmol 1 −1, PRC contents further decreased in endometrium and myometrium (491 fmol mg −1 protein and 485 fmol mg −1 protein, respectively). ERC concentrations seemed to follow those of PRC, but mostly the changes were not statistically significant. No differences were observed in PRC or ERC concentrations between the endometrium and myometrium at specified serum progesterone levels. Cervical PRC and ERC concentrations seemed to be lower than the corresponding uterine receptor levels and in the cervix, too, reduced PRC concentrations were associated with endogenous progesterone production. The latter finding strongly suggests that bovine uterine cervix tissue is under regulation by sex steroids. No differences in ERC or PRC contents were found between the two uterine horns of individual animals. Ovarian ERC and PRC concentrations were low compared to those detected in the uterus. Some synthetic C21-progestins were bound more avidly by the bovine PRC than by that of human, sheep, rabbit and guinea pig.

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors

The binding of [ 3H]estradiol and [ 3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [ 3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol ( K d = 0.1−0.6 nM) similar to that of the cytosolic ER. The displacement of [ 3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC 50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [ 3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.

Methyl and bromo derivatives of estradiol are agonistic ligands for the estrogen receptor of MCF-7 breast cancer cells

The binding affinity and relative estrogenic potency of 2-bromo-, 4-bromo-, 2-methyl- and 4-methylestradiol was evaluated in MCF-7 breast cancer cells. The relative binding affinities compared to estradiol were 47% for 2-methyl-, 25% for 4-methyl-, 37% for 4-bromo- and 17% for 2-bromoestradiol. However, both 2- and 4-methyl- as well as 2- and 4-bromoestradiol were able (a) to translocate the cytosolic estrogen receptor into the nucleus and (b) to induce the progesterone receptor in a concentration dependent manner. Finally, all ring-A substituted estrogens used in this study induced the pS2 mRNA as demonstrated by Northern-blotting. From these findings we conclude that 2-bromo-, 4-bromo-, 2-methyl- and 4-methylestradiol are agonistic ligands for the estrogen receptor in MCF-7 breast cancer cells.

Preclinical evaluation of a positron emitting progestin ([ 18F]fluoro-16α-methyl-19-norprogesterone) for imaging progesterone receptor positive tumours with positron emission tomography

Three 21-fluoro-progestins were investigated as potential imaging agents for the in vivo assessment of human progesterone receptor positive neoplasms with positron emission tomography. In competitive binding assays these compounds demonstrated high specificity, competing only for progesterone receptors. Binding to other steroid receptor types was negligible. Based on its high affinity binding, 21-fluoro-16α-methyl-19-norprogesterone was selected for further evaluation in vivo. Tissue distribution studies in immature estrogen primed female rats revealed high uterine uptake of 21-[ 18F]fluoro-16α-methyl-19-norprogesterone ([ 18F]FMNP). At 60 min after injection the ratio of uptake of radioactivity by uterine tissue to that of blood was 7. This ratio increased to 24 at 180 min. A selective decrease in uterine uptake was observed after administration of [ 18F]FMNP with excess unlabelled progestin. Rats bearing hormone responsive MT-W9A mammary adenocarcinomas were used to examine [ 18F]FMNP for tumour uptake. Animals were used irrespective of the phase of the estrous cycle. At 180 min the uterus to blood ratio and the tumour to blood ratio ranged from 3 to 20 and 3 to 17, respectively. Uterine and tumour tissue was assayed for cytosolic estrogen and progesterone receptors using a dextran-coated charcoal method and Scatchard plot analysis. The results indicate that the in vivo uptake of [ 18F]FMNP by uterine and mammary tumour tissue correlated well with the progesterone receptor concentration (r s = 0.98 and r s = 0.88, respectively). It is concluded that the uptake of [ 18F]FMNP by progesterone receptor positive tissue in vivo is primarily receptor related and that this uptake is attributable to the progesterone receptor. The study demonstrates the potential applicability of [ 18F]FMNP and positron emission tomography for imaging progesterone receptor positive neoplasms.

Inhibition of the Estradiol-Mediated Endometrial Gland Formation by the Antigestagen Onapristone in Rabbits: Relationship to Uterine Estrogen Receptors

There is evidence from previous studies that progesterone antagonists (antigestagens) modify estrogen responses at endometrial and myometrial levels without having affinity to the estrogen receptor (ER). The purpose of the present study was to investigate the influence of the antigestagen onapristone (ZK 98 299) on the uterus in ovariectomized (OVX) estradiol (E2)-substituted rabbits (3.0 micrograms/animal.day). The animals were treated for 8 days with different doses of onapristone (3.0, 10.0, and 30.0 mg/animal.day, sc). Uterine growth was not influenced by onapristone compared to that in OVX E2-substituted controls. However, morphological (light microscopy, transmission electron microscopy) and morphometric criteria indicated that there was a significant dose-dependent inhibition of the estrogen-induced gland formation within the endometrium and degenerative changes in glandular epithelial cells. By contrast, there were morphological signs of activation of the endometrial stroma (proliferation, increased capillarization, and vascularization, edema) above the level of E2-treated animals. A dose-dependent increase in the concentration of uterine cytosolic ER, nuclear ER, and ER mRNA (ER mRNA) was measured in uterine homogenates after onapristone treatment compared to values in OVX E2-substituted controls. Immunocytochemical analysis of ER in uterine sections suggests that the increase in ER after onapristone treatment took place predominantly in the myometrium and surface epithelium. To examine whether the observed interference was mediated via the progesterone receptor (PR), E2-substituted rabbits were treated, in a separate experiment, with onapristone (10.0 mg/animal.day, sc) and various doses of progesterone (1.0, 3.0, and 10.0 mg/animal.day, sc). Progesterone reversed all onapristone-induced changes, indicating that the observed effects were mediated via the PR. The data indicate that the antigestagen onapristone interacts with estrogen action in the absence of the natural PR ligand. The increase in ER and ER mRNA concentrations after onapristone treatment in OVX E2-treated animals suggests that this antigestagen abolished an inhibitory action of the unoccupied PR on ER biosynthesis.

Estrogen receptor stereochemistry: ligand binding and hormonal responsiveness

Estrogen stimulation of the uterus produces a spectrum of biochemical responses that are customarily linked together. This report is an overview of a series of studies by our laboratory investigating the role of different ligand structures in eliciting hormonal responses. Diethylstilbestrol (DES) and certain structural analogs, indenestrol A (IA), indenestrol B (IB), and pseudo-DES, were used as probes to segregate various genomic responses previously considered interrelated, most notably the events of specific protein synthesis and DNA synthesis. These compounds have weak uterotrophic activity; however, they interact with high affinity specifically with mouse uterine estrogen receptors (ERs). All of them produce stoichiometrically similar amounts of ER complex in the nucleus. Indenestrol A and IB possess a single chiral carbon atom and exist as a mixture of enantiomers (ENTs). Competitive binding assays of pure ENTs and cytosolic ERs demonstrated a stereochemical chiral preference for the IA isomer but not IB. This preference was also evident from nuclear ER occupancy experiments. Biologic activity of the IA ENTs also demonstrated differences as seen by receptor binding. Ornithine decarboxylase (ODC) activity was stimulated 600% by DES and partially by IA (rac). All of the ODC activity produced by IA (rac) was due to the IA(C3)-S ENT. Uterine DNA synthesis was measured after treatment with the IA compounds. Indenestrol A (rac) increased DNA synthesis to 40% of the level seen with DES. The weak ENTs showed no activity and the active ENTs were weaker than the IA racemic. These compounds should be useful probes for studying the individual responses involved in estrogen-induced uterine growth. The data also indicate that induction of some biologic responses, such as ODC, progesterone receptor, and DNA synthesis are not coupled. Therefore, stimulation of a certain uterine response may depend on the structure of the particular ligand receptor complex formed, and its interaction may be regulated by specificity at the genomic acceptor site.

Role of prolactin in growth of the rat mammary tumor MTW9

The growth of MTW9 mammary tumors exhibits different degrees of responsiveness to ovariectomy, ranging from sensitivity to resistance. This range of response is a function of time elapsing from tumor inoculation until performance of ovariectomy provided that prolactin (PRL) level is kept continuously high. In vivo studies showed that the MTW9 tumors developed by chronic administration of spiramide were sensitive to ovariectomy by 60 days, but they became resistant to ovariectomy by 100 days. However, when spiramide treatment was discontinued after tumor appearance, the tumors were still sensitive to ovariectomy by 100 days. Chromatofocusing (CF) profile of cytosolic estrogen receptors (ER) correlated with the responsiveness to ovariectomy. A 2-peak profile for tumors sensitive to ovariectomy, and only a one-peak profile for tumors resistant to ovariectomy, were seen. Although the prolactin level in rats bearing the tumors was higher than in the normal rats, no correlation between the PRL level and the change in CF profile of ER over time was seen. Also, these changes could not be correlated with the tumor size. In vitro studies showed that incubation of cytosolic ER from a sensitive tumor (2 peaks) with PRL led to a CF profile with only one peak, characteristic of a resistant tumor. Leupeptin, molybdate and phenylmethylsulfonylfluoride (PMSF) could not prevent this transition. The effect was not reproduced by incubation with growth hormone or progesterone. Our data suggest that PRL, either directly or through intermediates, may play a role in changing the response to hormonal therapy of the mammary tumor MTW9.