Methyl p-hydroxyphenyllactate. An inhibitor of cell growth and proliferation and an endogenous ligand for nuclear type-II binding sites

We previously described and partially characterized endogenous ligands for nuclear type II sites in normal and malignant tissues. Chromatography of these ligands on Sephadex LH-20 revealed that two peaks with binding activity (alpha and beta) could be resolved. The beta-peak component was present in all normal tissues that we examined, but not in malignant tissues, and it inhibited the growth of MCF-7 human breast cancer cells in vitro. Conversely, the alpha-peak component was found to be present in both normal and malignant tissues, and did not inhibit MCF-7 cell growth. The present studies describe the purification and identification of the alpha-peak and beta-peak components in bovine serum and an assessment of the effects of these compounds on normal and malignant cell growth. Gas chromatography-mass spectroscopy analysis of the purified beta-peak component demonstrated that the compound was methyl p-hydroxyphenyllactate (MeHPLA). Competition analysis revealed that MeHPLA binds to nuclear type II sites with a high binding affinity, while physiological levels of this compound blocked estradiol stimulation of uterine growth in vivo and inhibited the growth of MCF-7 human breast cancer cells in vitro. The alpha-peak component was found to be the corresponding acid, p-hydroxyphenyllactic acid (HPLA). This compound interacted with nuclear type II sites with a relatively low affinity and did not block uterotropic response to estradiol or inhibit MCF-7 cell growth. These studies demonstrate that HPLA and MeHPLA are ligands for nuclear type II sites and that MeHPLA may be a very important regulator of normal and malignant cell growth.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.     

Uterine type II estrogen-binding sites are not of eosinophil origin

A recent report by Lyttle et al. (Lyttle, C. R., Medlock, R. L., and Sheehan, D. M. (1984) J. Biol. Chem. 259, 2697-2700) suggested that nuclear type II sites in the rat uterus are of eosinophil origin and may represent [3H]estradiol binding to eosinophil peroxidase. To further evaluate this hypothesis we examined the response of nuclear type II sites to estrogen under conditions where eosinophils are not present. Results of our experiments show that physiological levels of estradiol-17 beta (10 nM for 72 h) will stimulate nuclear type II sites in highly purified cultures (21-25 days; 4 passages) of rat uterine stromal and myometrial cells. The magnitude of the response of type II sites to estradiol in these stromal (4-fold) and myometrial (80-fold) cell cultures was essentially identical to that observed in the uterine cell types following in vivo estrogen treatment. Since these highly purified cultures of uterine cells were prepared from the uterus of a 21-day ovariectomized rat which is devoid of eosinophils, we conclude that estradiol stimulation of nuclear type II sites is a direct intracellular response to estrogen which occurs independent of eosinophil accumulation. Furthermore, we have found that type II sites in the rat uterus are not peroxidase. This was demonstrated by experiments which show type II sites are present in the 39,000 X g supernatant fraction of uterine cytosol, whereas peroxidase activity is quantitatively recovered in the crude mitochondrial (39,000 X g) pellet. Likewise, the small amount of peroxidase activity (approximately 10%) in the total homogenate which contaminates our nuclear pellet preparations was extracted (98-100%) with 0.5 M CaCl2. Type II estrogen-binding sites (95-100%) remained associated with the nuclear pellet fraction after peroxidase extraction. Therefore, stimulation of cytosol and nuclear type II sites by estrogen in the rat uterus is a direct intracellular response to the hormone unrelated to eosinophil accumulation and/or peroxidase activity.     

Reconstitution of the type II [3H]estradiol binding site with recombinant histone H4

Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd approximately 20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient approximately 4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.     

Multiple species of estrogen binding sites in the nuclear fraction of the rat prostate

Specific estrogen-binding sites have been demonstrated in purified nuclear fractions of prostates from intact rats. Saturation analysis of nuclei over a wide range of [³H]-estradiol concentrations (0.15 to 90 nM) has shown two different types of binding sites: a) one with high affinity (Kd of 0.5–0.8 nM) and low capacity for estradiol (approximately 162 fmole/mg DNA); b) a second with a lower affinity (Kd of 30–40 nM), which shows a higher capacity (approximately 860 fmole/mg DNA), and displays a saturation curve that is sigmoidal and that appears to be similar to those for Type II estrogen-binding sites in rat uterus. These results suggest that the actions of estradiol in the prostate are mediated by specific nuclear binding sites.     

Heterogeneity of [3H]estradiol binding sites in the rat prostate: properties and distribution of type I and type II sites

In order to assess the rat prostate as a target tissue for receptor-mediated estrogen action, we have studied the properties and distributions of estrogen binding sites in the dorsolateral (DLP) and ventral (VP) prostate. Saturation analyses over a wide range of [3H]estradiol ([3H]E2) concentrations (0.5-100 nM) revealed two distinct types of binding sites in the cytosol and nuclear fractions of DLP of intact rats. The high affinity (type I) estrogen binding sites saturated at 2-4 nM of [3H]E2 and had a capacity of 170 fmol/mg DNA in the cytosol and 400 fmol/mg DNA in the nuclei. DLP type I sites had ligand specificity similar to that described for the classical estrogen receptors (ERs) found in female target tissues. The moderate affinity (type II) estrogen binding sites saturated at 15-30 nM of [3H]E2 and had a capacity of 850 fmol/mg DNA in the cytosol and 1600 fmol/mg DNA in the nuclei. DLP type II sites shared some characteristics of the type II ERs described for the rat uterus; they were estrogen specific, heat labile, and sensitive to reducing agents such as dithiothreitol. Saturation analyses on VP cytosols and nuclear fractions revealed only high affinity sites but no moderate affinity sites in the tissue preparations. Our finding that prostatic type II estrogen binding sites are present exclusively in the DLP supports the concept that basic biological differences exist between the two major prostatic lobes of the rat. Furthermore, our findings may help elucidate the observed differences in susceptibility between these two lobes to the hormonal induction of proliferative prostatic lesions.     

Effects of Salt Extraction on the Quantitation of Nuclear Estrogen Receptors: Interference by Secondary Estrogen Binding Sites

Abstract

The effects of salt-extraction on type I and type II estrogen binding sites were examined in uterine nuclei. Injection (10 ug) of estradiol or estriol in adult ovariectomized rats induced maximum numbers (80–100%, ~ 1 pmole/uterus) of 0.4 M KCL resistant type I estrogen complexes at 1 hour. Only estradiol, which sustained these levels for long periods of time (4–24 hours) stimulated true uterine growth.

Likewise, a single injection of estradiol, but not estriol, also elevated nuclear type II sites throughout the entire uterine growth period (1–48 hours). However extraction of these nuclei from estradiol injected rats with 0.4 M KCL increased the numbers of type II sites from ~ 1 pmole/uterus (non-extracted nuclei) to ~ 8 pmoles/uterus (salt resistant plus salt-extractable fractions). Sixty percent of these sites were resistant to salt-extraction. Continuous exposure to either estradiol or estriol by beeswax implants stimulated nuclear type II sites which were highly resistant (80%) to KCL-extraction, and additional sites were not exposed by high salt. Thus chronic treatment with both estrogens “locked in” nuclear type II sites such that they were resistant to KCL-extraction. This resistance of type II sites to salt-extraction correlated with the ability of estradiol and estriol implants to stimulate true uterine growth. The procedures presented here for nuclear preparation and assay have reduced non-specific binding considerably in the uterine system, and may eliminate the need to perform exchange assays on salt-extracted nuclei in other systems.     

Type II estrogen binding site agonist: synthesis and biological evaluation of the enantiomers of methyl-para-hydroxyphenyllactate (MeHPLA)

A high-affinity ligand for the type II estrogen binding site (EBS) was identified. Methyl para-hydroxyphenyllactate (MeHPLA) was observed to suppress the cellular proliferation of estrogen-sensitive MCF-7 breast cancer cells in vitro and to suppress the growth of rat uteri in vivo. The high affinity of MeHPLA for the type II EBS suggests that this interaction is responsible for the observed suppression of cell growth. In this study, the enantiomers of MeHPLA were synthesized and separated by three methods and evaluated for biological activity. When the methods were compared, it was found that the method using an optically pure amine to form the diastereomers of the enantiomers gave the superior yield. Binding studies for the enantiomers to the type II EBS showed that the S-MeHPLA had a higher affinity for the binding site. However, higher binding affinity did not translate into superior cell growth suppression. Both enantiomers suppressed cell growth equally.     

Cytosol type II sites in the rat uterus: interaction with an endogenous ligand

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.     

Interaction of DP-TAT-59, an active metabolite of new triphenylethylene-derivative (TAT-59), with estrogen receptors.

DP-TAT-59, (Z)-2-(4-(1-(4-hydroxyphenyl)-2-(4-isopropylphenyl)-1-butenyl) phenoxy)-N, N-dimethylethylamine, has been reported to inhibit estrogen-stimulated growth of MCF-7 cells as well as rat uterus at lower concentrations than the hydroxymetabolite of tamoxifen (4-OH-TAM). In the present study, the growth of mouse Leydig cell tumor, B-1F cells were also more effectively inhibited by DP-TAT-59 than 4-OH-TAM. Additionally, the expression of estrogen responsive element ligated CAT gene transfected into B-1F cells was also suppressed by DP-TAT-59. Thus, the interaction of DP-TAT-59 with estrogen receptor (ER) was characterized and compared with that of 4-OH-TAM using immature rat and bovine uteri. The dissociation constant of DP-TAT-59 to ER of immature rat uterus was 0.24 nM and was similar to that of 4-OH-TAM (Kd = 0.20 nM) and estradiol (Kd = 0.29 nM). Using sucrose density gradients, the sedimentation constant of DP-TAT-59 with bovine uterus was 4.9S, which was similar to that of estradiol (5.1S) and 4-OH-TAM (5.3S). However, the elution profile of the DP-TAT-59-ER complex from a DEAE-Sephadex column was different for both estradiol-and 4-OH-TAM-ER complexes. These results suggest that ER forms different complexes with DP-TAT-59 than estradiol or 4-OH-TAM, while the ER binding affinity of these compounds are similar to each other.     

Cytosolic Type II Estrogen Binding Site in Rat Uterus: Specific Photolabeling with Estrone

Abstract

A low affinity (Kd = 30 nM), large capacity (Bmax = 2.6 pmol/g tissue) estrogen binding site was photolabeled from estradiol-stimulated rat uterus cytosol. To maximize levels of this binding site and reduce those of the type I binding site, ovariectomized rats were injected with high doses of estradiol (10μg per day) for four days with the last injection two hours before sacrifice. This treatment depleted type I estrogen receptors from the cytosol (by 90%) and raised levels of type II sites in the nucleus without affecting cytosolic type II levels. The type II estradiol binding sites were distinguished from the type I sites on the basis of their dissociation kinetics, pH-sensitivity and their behavior towards potassium chloride, somatostatin, sodium thiocyanate, sulfhydryl reagents and ammonium sulfate precipitation. These type II binding sites could be covalently photolabeled with tritiated estrone. A molecular weight of 43 kDa was found on SDS PAGE.     

Estrogen dependent induction of low affinity binding sites in the nuclear fraction of rat uterus

Type II estradiol binding sites characterized by lower affinity and higher capacity than type I receptor sites have been described in rat uterine nuclei. These sites appeared to be dependent on estrogen stimulation. Reducing agents prevented estradiol binding to these sites. In the present study, the situation prevailing in adult rats (Ad) was studied and compared to ovariectomized (Ox) and ovariectomized estrogen prestimulated rats (OxPS). Nuclear precipitate from Ad, Ox and OxPS rats were incubated with tritiated estradiol (E2(3)H) in the presence and in the absence of mercaptoethanol as reducing agent. In the presence of mercaptoethanol, saturation was attained at E2(3)H concentrations above 16 nM. In the absence of reducing agents, a secondary binding was observed in Ad and OxPS which was not saturated at E2(3)H levels up to 80 nM. Non-specific binding obtained with paired aliquots containing 100-fold excess of DES as competitor was not linear but showed a saturation profile, distorting the saturation curve of the specific sites, obtained by subtracting non-specific from total E2(3)H binding. Increasing DES concentrations up to 10,000 nM did not allow to reach complete exchange with E3(3)H ligand bound to specific sites, preventing measurement of binding sites concentration. Incubation of nuclear fractions with increasing concentrations of E2(3)H (up to 6,000 nM) gave a saturation curve with a linear kinetics above 1-2,000 nM, which represented saturation concentration of the specific sites. From this, non-specific and specific moieties could be estimated. Binding capacity of specific sites was of the order of 50-80 pmol uterus. Half saturation was attained between 300 and 600 nM E2(3)H, which approximated the Kdiss of these sites, at variance with the Kdiss of 15-30 nM originally reported for type II binding sites. In conclusion, these results show that secondary binding sites were present in uterine nuclei of Ad and OxPS rats. Binding capacity was about 30-fold higher than that of type I sites. Affinity was however very low, and casts some doubt on the role of these sites as active estradiol binders in physiological situations. Their increase under the influence of estrogen may however be related to some as yet undetermined role.     

Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo.

The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.     

Ligand-binding properties of estrogen receptor proteins after interaction with surface-immobilized Zn(II) ions: evidence for localized surface interactions and minimal conformational changes

The site- or domain-specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification and purification of receptor-associated regulatory components and nucleic acids. We have demonstrated previously that restricted surface regions of the estrogen receptor protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in receptor at the stationary phase immobilized metal ion interface were evaluated by monitoring alterations in the equilibrium dissociation constant (Kd) for [3H]estradiol. Soluble estrogen receptor proteins (unliganded) present in immature calf uterine cytosol were immobilized via surface-exposed Zn(II)-binding sites to beads of agarose derivatized with iminodiacetate (IDA)-Zn(II) ions. The IDA-Zn(II) bound receptor was incubated with increasing concentrations of [3H]estradiol (0.01-20 nM) in the presence and absence of unlabeled competitor (diethylstilbestrol) to determine the level of specific hormone binding. Steroid-binding experiments were performed in parallel with identical aliquots of soluble receptor. Analyses of the equilibrium binding data revealed the presence of a single class of high-affinity (Kd = 2.44 +/- 1.5 nM, n = 10) steroid-binding sites which were only marginally affected by receptor immobilization via surface-exposed Zn(II) bindings sites (Kd = 2.58 +/- 0.56 nM, n = 4). These data are consistent with the location of surface accessible Zn(II) binding site(s) on the receptor at or near the DNA binding domain which, upon occupancy, do not influence the steroid binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear estradiol binding in rat adipocytes. Regional variations and regulatory influences of hormones.

The nuclear estrogen receptor was characterised in isolated rat adipocytes. The binding reaction with [3H]estradiol was performed with intact isolated rat adipocytes and the radioactivity associated with the nucleus was subsequently determined after cell lysis. The nuclear uptake of [3H]estrogen in rat adipocytes was temperature dependent and steroid specific. The steady-state binding was achieved after 30 min at 37 degrees C and was constant for several hours. Estradiol was found to bind to a homogeneous class of nuclear receptors in epididymal adipocytes with an apparent Kd of 3.1 +/- 0.76 nM and a Bmax of 7.98 +/- 1.11 fmol/10(6) cells corresponding to about 4800 receptors per nucleus. The estradiol binding exhibited regional variations in isolated adipocytes. In lean rats the highest receptor number was found in epididymal adipocytes, whereas there was a significantly lower number of nuclear binding sites in perirenal and subcutaneous adipocytes (P less than 0.05), unlike in older and more obese rats where the nuclear estradiol binding was greatest in adipocytes from the perirenal fat depot. Incubations with isoproterenol (10 microM) and dibutyryl-cAMP (2.5 mM) both reduced estradiol binding by 56% (P less than 0.005), while insulin (1 nM) enhanced the estradiol binding by 37% (P less than 0.01). In conclusion, a specific and high affinity nuclear estradiol receptor was demonstrated in rat adipocytes and regional differences in nuclear estradiol binding were detected. Furthermore, it was demonstrated that nuclear estradiol binding could be modulated by other agents known to affect adipocyte metabolism.     

Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.