Cytoplasmic Estrogen Receptor Research Articles

An update on the immunohistochemical localization of estrogen receptors in mammary carcinomas utilizing polyclonal anti-receptor antibodies

An immunohistochemical (IF) method utilizing polyclonal antibodies to cytoplasmic estrogen receptors (ER) and a tissue processing procedure which allows in situ precipitation of cytoplasmic ER without denaturation of antigenic sites were first described in this journal. In this report we present data on the immunohistochemical classification of 153 cases of human breat carcinomas (123 frozen sections, 28 cytological specimens) as ER-positive (IF+) or ER-negative (IF−). Results on correlation between the biochemical vs immunohistochemical assessment of the ER status and the relationship between quantity of ER and the proportion of IF+/IF− tumor cells are presented. In addition, a procedure which has been successfully applied to study in vitro translocation of cytoplasmic ER in cryostat sections of human breast carcinomas is described. A positive correlation between ER translocatability and status of progesterone receptors (PR) was obvious. All biochemical and immunohistochemical data are collectively reviewed and the question of whether the antibodies are detecting Type I ER is examined.

Cytoplasmic estrogen receptor concentrations in the endometrium of Finnish and Japanese women

Cytoplasmic estrogen receptor concentrations in the endometrium were determined in 13 Finnish and 20 Japanese women. All the women were premenopausal and aged between 40 and 50. The concentration of estrogen receptors in the proliferative endometrium for Finnish women was 246.9 ± 46.2 mol/mg protein (mean ± S.E.M.) and that for Japanese women 45.7 ± 17.1 mol/mg protein. The difference is significant ( P < 0.01). In the secretory endometrium the corresponding figures were 97.8 ± 33.7 mol/mg protein for Finnish and 20.0 ± 6.4 mol/mg protein for Japanese women ( P < 0.05). During the proliferative phase the serum estrone concentration was significantly higher in Finnish than in Japanese women ( P < 0.05).

Estrogenicity of coumestrol in the mouse: fluorescence detection of interaction with estrogen receptors.

The estrogenicity of coumestrol, a fluorescent phytoestrogen, has been examined in murine uteri. Coumestrol competed with 17 beta-[3H]estradiol for binding to cytoplasmic estrogen receptors, caused cytoplasmic estrogen receptors to associate with chromatin in the nucleus, and induced progesterone receptors. By use of size-exclusion high-performance liquid chromatography (SEHPLC), the interaction of coumestrol with estrogen receptors was examined directly by monitoring the fluorescence associated with macromolecules having properties characteristic of estrogen receptors. These analyses were made possible by the addition of dimethylformamide to the elution buffer, at a concentration (7.5%) which improved recoveries but did not interfere with estrogen receptor binding. It was possible to detect fluorescent coumestrol at approximately 0.5 nM. All determinations were performed with preparations in which estrogen receptor activity was 3-10 nM. Exposure of these preparations to coumestrol (50 nM) resulted in the elution of increased fluorescent activity in the regions where estrogen receptors eluted during SEHPLC. This fluorescent activity was reduced when diethylstilbestrol, 17 beta-estradiol, hexestrol, or tamoxifen was present as a competitor (2 microM) but was unaffected by testosterone or progesterone. Diethylstilbestrol reduced fluorescence below endogenous base lines and thereby displayed a fluorescence quench property which was not observed with other ligands. When hepatic and renal estrogen receptor preparations were used, the injected receptor activity was observed to be the major limiting factor in detecting the interaction of coumestrol with estrogen receptors. These observations are relevant to attempts to visualize estrogen receptors in tumor cells and demonstrate that accepted biochemical criteria for ligand-receptor interaction can be satisfied when fluorescent ligands are examined.

The effect of tamoxifen on the growth of human malignant melanoma in Vitro

The effect of tamoxifen on the growth of malignant melanoma was investigated using human cell lines and single-cell suspensions prepared from patients' tumours cultured in soft agar. Tamoxifen stimulated both [ 3 H]-thymidine incorporation and cell numbers in all of the cell lines tested. Cytoplasmic oestrogen receptor (ER) was detected in one of the responding lines and progesterone receptor (PR) in another. Tumour colony formation in soft agar culture was satisfactorily established from tumour cell suspensions from 13 of 21 patients, only one of which had detectable cytoplasmic ER. Greater than 50% reduction in colony formation with 5 × 10 −7 M tamoxifen occurred in two tumours, neither of which contained ER. These results indicate that tamoxifen has the potential to either retard or accelerate the growth of human malignant melanoma.

Action of calpain on the basic estrogen receptor molecule of porcine uterus.

Basic estrogen receptor (ER) molecule (vero-ER) of the cytosol of porcine uterus was purified 1,200-fold after successive chromatographies on phenyl-Sepharose, hydroxylapatite, and DEAE-cellulose, followed by Sephadex G-150 gel filtration. The purified vero-ER was completely free from endogenous protease and ER-binding factor. The action of Ca2+-dependent cysteine proteinase (calpain) on vero-ER was studied by utilizing the purified receptor and calpains from porcine uterus (endogenous calpain), porcine kidney, and human erythrocytes. Proteolysis of vero-ER was followed by monitoring the disappearance of the binding capability of vero-ER with "8S" ER-forming factor. Vero-ER was proteolyzed by both the endogenous and the exogenous calpains in the presence of Ca2+. The calpains did not attack vero-ER in the absence of Ca2+. The results indicated the absolute requirement by calpain for Ca2+ for the limited hydrolysis of vero-ER. Uterine cytosol was shown to contain, in parallel with calpain, a protease which does not require Ca2+ for the limited proteolysis of vero-ER. The strongly hydrophobic domain of vero-ER, recently shown to be indispensable for the nuclear translocation of vero-ER (Murayama, A. & Fukai, F. (1983) FEBS Lett. 158, 255), was preferentially destroyed by both the Ca2+-requiring and -nonrequiring enzymes. It was assumed that calpain might intervene in the estrogen action by diminishing irreversibly the amount of the cytoplasmic ER capable of translocating into the nucleus.

Development of steroid-receptor systems in guinea pig brain. I. Cytoplasmic estrogen receptors

The development of the cytoplasmic estrogen receptor (CER) system in male and female guinea pigs was examined to determine whether attainment of sensitivity to estrogen action related to lordosis behavior is correlated with increases in brain CER concentrations. Ovariectomized neonatal (7 days old) guinea pigs failed to display lordosis after priming with 10 μg estradiol benzoate (EB) and 0.5 mg progesterone (P). The percentage of ovariectomized steroid-treated females displaying lordosis increased at 29–33 days of age. At 50–65 days of age this percentage increased even further. Plasma levels of estrogen in 4–6 day old animals at 4, 8 and 12 h after 10 μg EB were at least equal to those seen in 50–65 day old animals. Neither the concentration nor the affinity of CER in hypothalamus (HYPO), preoptic area (POA), amygdala (AMYG) and cortex (CORT) were different in 4–6 day olds vs 50–65 day olds. The rank order for concentrations of CER in brain regions was: POA > HYPO > AMYG > CORT. A sex difference in CER was found in HYPO (females > males), but not POA, AMYG or CORT of 4–6 day olds.

Establishment of human endometrial adenocarcinoma cell line containing estradiol-17β and progesterone receptors

The cell line designated HHUA was established from a well-differentiated endometrial adenocarcinoma (grade 1) of a 54-year-old female on December 10, 1980. The cell line grew well without interruption for 22 months and was subcultivated more than 56 times. The cells were spindle or polygonal in shape, formed multilayers, and did not show contact inhibition. The chromosome number varied widely and showed aneuploidy. The modal number was 46 and no marker chromosome was identified. The cells were transplanted into the subcutis of BALB/C nude mice and produced tumors which were interpreted as adenocarcinoma. The cells contained estradiol-17β and progesterone receptors. Administration of estradiol-17β successfully depleted cytoplasmic estrogen receptors and brought about increased progesterone receptor levels.

Interactions of methylestrogens with cytoplasmic and nuclear estrogen receptors in rat pituitary gland, hypothalamus and uterus

The in vitro affinities to cytoplasmic estrogen receptors of the methylestrogens 2-methylestradiol-17β, 4-methylestradiol-17β and 4-hydroxy-2-methylestradiol-17β, which are useful probes to test the biological importance of 2- or 4-hydroxylation of estradiol-17β (catecholestrogen formation), have been determined in hypothalamic, pituitary and uterine tissue of the ovariectomized rat. Moreover, the in vivo capacity of these compounds to translocate estrogen receptors into the cell nucleus of pituitary and uterine tissue has been studied. Methylestrogens exhibited estrogen receptor affinities which were not significantly different from the binding affinity of estradiol-17β. When given at a high dose (100 μg/animal) their nuclear translocation capacity was equal (4-methylestradiol-17β) or even higher (2-methylestradiol-17β) than that of estradiol-17β. However, at a low dose (5 μg/animal) 4-methylestradiol was completely ineffective in both the pituitary gland and the uterus, and 2-methylestradiol-17β was less potent than estradiol-17β in the pituitary gland.

Effect of chemotherapy on cytoplasmic estrogen receptors (ER c) in advanced epithelial ovarian carcinoma

Effect of chemotherapy on cytoplasmic estrogen receptors (ER c) in advanced epithelial ovarian carcinoma

Effects of fasting on the distribution of cytoplasmic and nuclear oestrogen receptors in rat liver, uterus, pituitary and hypothalamus before and after exogenous oestrogen administration.

The accumulation of oestrogen receptors in the liver cell nuclei of intact female rats 45 min after administration of 100 micrograms 17 alpha-ethynyloestradiol-17 beta i.p., decreased progressively during a 72-h fast from 2550 +/- 860 to 257 +/- 67 fmol/mg DNA, a level not significantly different from that in uninjected animals. Cytoplasmic oestrogen receptor concentrations also decreased, but only to about 60% of the original level (from 84.1 +/- 27.5 to 50.3 +/- 2.09 fmol/mg protein during the fast). Similar differences were found when these parameters were examined in normally fed and 72-h-fasted ovariectomized rats. On the other hand these parameters were unaffected in uterus, pituitary and hypothalamus. Uterine cytoplasmic receptor concentrations remained at about 500 fmol/mg protein during the fasting period, those in the pituitary and hypothalamus at about 230 and 30 fmol/mg protein, respectively. Nor was in vivo translocation in these organs affected by fasting. Regardless of nutritional status, the nuclear oestrogen receptor concentrations in uterus rose from about 500 to 2000 fmol/mg DNA after ethynyloestradiol administration, those in the pituitary and hypothalamus from approximately 250 to 2000 and from 250 to 500 fmol/mg DNA respectively.

Effects of temperature, storage and sodium molybdate on the analysis of estrogen and progesterone receptors in rabbit uterine tissue and gynecologic tumor.

The cytoplasmic estrogen receptor (ERc) and progesterone receptor (PRc) in mammary tumors have been recognized as useful biochemical markers for predicting the objective response of patients with advanced breast cancers to endocrine therapy. These proteins are also useful in the prognosis of gynecologic carcinoma. This report presents data showing the effect of sodium molybdate in the stabilization of estrogen and progesterone receptors. In rabbit uterine tissue, molybdate (20 mM) increased the binding of progesterone and estrogen to the receptors in several ways: (a) the apparent loss of detectable receptors during lengthy sucrose gradient analysis and at elevated temperature (30 degrees C) was reduced; (b) the instability of receptors due to storage at -70 degrees C was lessened, and (c) the conversion of the 7S PRc to the 3.5S form was minimized. Similarly, molybdate caused a qualitative and/or a statistically significant quantitative difference in receptor values for some human gynecologic tumors presented herein; the molybdate-associated changes vary with tumor specimen. Of the 8 tumors for which receptor values in the presence of molybdate (M+) and its absence (M-) can be compared, detectable ERc of 6 and PRc of 7 tumors increased with molybdate, and ERc of 2 and PRc of 1 tumor showed no change. In addition to the increase in receptor values, a concomitant shift of the 3-4S molecules to the 7-8S moieties was noted for some tumors (1 of 6 for ERc and 3 of 7 for PRc). In 2 receptor-poor tumor samples, ERc was only detected in M+ cytosols. These results show that molybdate is effective in reducing receptor degradation and stabilizes the 7-8S molecules from converting to 4S moieties. The addition of molybdate may be helpful for better quantitation of steroid receptors in clinical specimens.

Activated oestrogen receptors in breast cancer and response to endocrine therapy

The status of oestrogen and progesterone receptors has been measured in 147 primary breast tumours. In addition to the measurement of cytoplasmic oestrogen receptors, the ability of these receptors to bind to oligo(dT)-cellulose has been assessed. This indicates the capability for activation of cytoplasmic receptors to a form able to bind in the nuclear compartment in vivo and thus be part of a functional receptor pathway. All the receptor concentrations measured were increased in the postmenopausal group of patients. All nuclear oestrogen receptors in this group were available for labelling at 4°C, in contrast to the premenopausal group. The apparent functionality of the oestrogen receptor pathway could be equally assessed either by the co-presence of cytosol progesterone receptor with nuclear oestrogen receptor (30 or 4°C) or with activated cytosol oestrogen receptor. The presence of activated cytosol oestrogen receptor was as reliable (80%) as the presence of either nuclear oestrogen receptor at 30 (83%) or 4°C (81%) in predicting the response of breast tumours to endocrine therapy.

Identification of cytoplasmic estrogen receptors in the accessory sex organs of the rabbit and their comparison to the cytoplasmic estrogen receptor in the epididymis

Estrogen receptors are present in cytosol prepared from the accessory sex organs (vesicular gland, proprostate, prostate, bulbourethral gland) of sexually immature and of sexually mature rabbits. The receptor in these organs from animals of both age groups has a sedimentation coefficient of 8–10S on low ionic strength (0.01 M KCl) sucrose gradients. Under high ionic strength (0.4 M KCl) conditions, the receptor sediments at approximately 4S. The cytoplasmic estrogen receptor from the epididymis shows age-dependent changes in its sedimentation coefficient. It is 8S under low ionic strength conditions when prepared from immature rabbits and 4S under identical conditions when prepared from sexually mature animals. Although the dissociation constant of the cytoplasmic estrogen receptor in the immature and mature epididymis and accessory sex organs remains constant during development ( $ ̃ 0.1 nM), the number of available cytoplasmic estrogen binding sites declines from about 160 fmoles/mg cytosol protein in the-immature rabbit to about 40 fmoles/mg cytosol protein in the adult animal. The estrogen receptor in the accessory sex organs is highly specific, the relative affinities of various potential competitors being: estradiol and estrone = 1, diethylstilbestrol = 0.3, estriol = 0.2, tamoxifen = 0.08, testosterone = 0.0004 and 5α-DHT = 0.00005. Changes with age in the physicochemical characteristics of the estrogen receptor and in the concentration of binding sites suggest that the estrogen receptor may be involved in the development and physiological regulation of the male reproductive tract.

Estrogen receptor levels in the oviducts and endometria of cynomolgus macaques during the menstrual cycle.

We sampled oviducts and endometria of 27 cynomolgus macaques during the menstrual cycle and measured the concentration of nuclear and cytoplasmic estrogen receptors in these tissues by exchange assay. We assessed the stage of the cycle by correlating serum estradiol (E2) and progesterone (P), as measured by radioimmunoassay, with the morphological condition of the ovaries, oviducts and endometrium of each animal. We have previously identified a series of oviductal stages that reflected the orderly sequence of cytological changes in the oviduct during the cycle, and we normalized receptor measurements to these stages. The amounts of nuclear and cytoplasmic estrogen receptor in both the oviduct and the endometrium were approximately twofold greater in the follicular phase than in the luteal phase. In the follicular phase, elevated receptor levels were associated with oviductal proliferation and differentiation, as well as with endometrial proliferation. During the luteal phase, lowered levels were correlated with atrophy and dedifferentiation in the oviduct, but with hypertrophy and progestational development in the endometrium. When the luteal phase of one cycle ends and the follicular phase of the next begins, it is a decline in serum P, not a rise in serum E2, that precedes the elevation in estrogen receptor level and the onset of proliferation in the oviduct and endometrium. Proliferation of the reproductive tract and elevations in nuclear estrogen receptor levels during the early follicular phase can therefore be viewed as consequences of the release of the system from antagonism by P.

Progesterone receptors and human breast cancer; ‘wassink lecture’ presented at the 3rd EORTC Breast Cancer Working Conference

INTRODUCTION THE DEVELOPMENT and widespread use of estrogen receptor (ER) assays to predict the endocrine dependence of a patient’s tumor has changed the clinician’s approach to the treatment of breast cancer. When it was discovered that cytoplasmic ER is responsible for the uptake of estrogen into target cells and is necessary for estrogen action, it was reasoned that ER might be useful in determining the endocrine responsiveness of breast cancers in patients with metastatic disease. This hypothesis has been confirmed, and it is now generally accepted that tumors lacking ER rarely respond to endocrine therapy whereas ER+ tumors frequently regress with hormonal manipulations. Determination of the ER status of the tumor is now standard practice in the United States. The ER assay is most helpful when the tumor is ER-. These patients rarely respond to endocrine therapy. On the other hand, a positive ER does not guarantee a response to endocrine therapy, since about 40% of patients fail to responddespite a positive assay. Horwitz et al. [l] suggested that certain ER+ tumors do not regress with endocrine manipulation because of a defect in the estrogen response pathway distal to the binding step, leading to autonomous growth. What was needed was a way of testing the integrity of the entire metabolic sequence that should be triggered by the binding of estrogen to receptor. Based on the observation that progesterone receptor (PgR) is induced by estrogen in normal reproductive tissues and in human breast cancer cells in culture, they hypothesized that PgR might be a better marker than ER for an intact estrogen

Estrogen agonistic and antagonistic action of 8 non-steroidal antiestrogens on progestin receptor induction in rat pituitary gland and uterus

All 8 non-steroidal antiestrogens tested considerably increased progestin receptor concentration in the uterus and, to a lesser extent, in the pituitary of ovariectomized rats. However, the pituitary was more sensitive than the uterus to the estrogen antagonistic action of these compound, in that monohydroxytamoxifen, LY 117, 018, enclomiphene, nitromifen, nafoxidine and trans-tamoxifen completely blocked progestin receptor induction by estradiol benzoate. In these tissues the order of the in vitro binding affinity of antiestrogens to cytoplasmic estrogen receptors was not correlated with either their in vivo estrogen agonistic or antagonistic potency.

Estrogen and antiestrogen effects on different lymphoid cell populations in the developing fetal thymus of guinea pig.

Estrogen responsiveness in the developing fetal thymus of guinea pig has been investigated. One-day estradiol (E2) treatment (1 mg/kg BW) of pregnant animals selectively decreases the number of larger lymphoid cells of the outer cortex of the fetal thymus, without affecting the global number of cortical lymphocytes. E2 treatments for longer periods (from 2 until 6 days) provoke a further impairment in the pattern of outer cortical lymphocytes, selectively reducing larger lymphoid cells, and also decrease the global number of cortical lymphocytes without affecting medullary lymphocytes. Treatment with the antiestrogen tamoxifen (2 mg/kg BW . day for 6 days) results in a transfer of cytoplasmic estrogen receptor to the nuclei of fetal thymus and also affects the histology of the fetal organ, although to a lesser extent than E2. However, when tamoxifen is administered with E2 (1 mg/kg BW . day during the last 3 days of tamoxifen treatment), it antagonizes all of the effects induced by E2 alone on the fetal thymus. The 6-day E2 treatment decreases the weight of the fetal thymus in an age-dependent manner; this effect is progressively more intense from 42 days of gestation at the time of treatment (mean +/- SE, 25 +/- 5% decrease) up to the end of gestation (63 +/- 7% decrease). The effect of E2 on large lymphoid cells also increases in an age-dependent manner. The extent of the estrogen responses observed is correlated with the levels of cytoplasmic estrogen receptors, which increase from 38 days to the end of gestation. The intracellular compartmentalization of estrogen receptor shows that cytoplasmic estrogen receptors are transferred to the nuclei by E2 at both younger and older ages. It is concluded that E2 has differential actions on the lymphoid cell population of the fetal thymus of guinea pig by events antagonized by the antiestrogen tamoxifen and related to the availability and development of cytoplasmic estrogen receptors.

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure.

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.

Characterization and physiological variation of estrogen receptors in rabbit corpora lutea throughout pregnancy and pseudopregnancy: the effect of hysterectomy and sustained estradiol treatment.

Previous studies suggest that regression of the rabbit corpus luteum is associated with a uterine-induced loss of responsiveness to estradiol. To determine if this is due to loss of estrogen receptor, cytoplasmic and nuclear estrogen receptors were measured in pseudopregnant, hysterectomized-pseudopregnant and pregnant rabbits throughout luteal life. Estrogen receptor levels were higher in corpora lutea than in nonluteal tissue and were generally higher in nuclei compared to cytosol. Estrogen receptor levels were low on Day 3, increased 2- to 3-fold by Day 6-8, reached peak levels by Days 8-10, and then gradually decreased in a pattern similar to the pattern of serum progesterone typical of each group. Hysterectomy was not associated with elevated cytoplasmic or nuclear estradiol receptor levels. When hysterectomized rabbits were treated with estradiol-filled Silastic implant on Day 1, nuclear estradiol receptor levels fell by Day 20 to levels seen in untreated hysterectomized rabbits. Despite substantial losses in nuclear estrogen receptor, serum progesterone remained elevated on Days 16 and 20. Thus, the ability of estradiol to maintain serum progesterone in hysterectomized rabbits did not correlate directly with the level of estrogen receptor.

Intrauterine injection of cholera toxin induces estrogen-like uterine growth.

Uterine growth in response to estrogen involves nuclear interaction of cytoplasmic estrogen receptors. The magnitude of the uterine growth response, however, and other observations have led to the suggestion that an additional mechanism or mechanisms of estrogen action may be at work in the uterus. This was investigated indirectly by examining estrogen-like growth responses of the uterus to the intrauterine (i.u.) injection of cholera toxin and water. Ovariectomized rats received either a subcutaneous injection of estradiol or vehicle and an i.u. injection of cholera toxin, water, or nothing. At 4 h after treatment of uterine wet weight was determined, and at 24 h the uterine protein/DNA ratio, [3H] leucine incorporation into protein, and [3H] thymidine incorporation into DNA were measured. Estradiol caused a 1.5-fold increase in the protein/DNA ratio and cholera toxin caused a 2.0-fold increase. Similarly, estradiol increased uterine wet weight 2.1-fold and cholera toxin increased it 1.9-fold. Estradiol and cholera toxin caused similar increases in the incorporation of [3H]leucine into protein: 3.3-fold and 2.3-fold, respectively. The effects of the treatments on the incorporation of [3H] thymidine into DNA were even greater, with estradiol producing a 10.3-fold increase, cholera toxin a 12.0-fold increase, and water a 3.6-fold increase. Since vascular permeability is known to be increased in the uterus by estrogen and in other systems by cholera toxin, these results support the idea of a relationship between uterine growth and increased vascular permeability.