Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: Evidence for multiple estrogen binding sites

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Estrophilin Immunoreactivity versus Estrogen Receptor Binding Activity in Meningiomas" Evidence for Multiple Estrogen Binding Sites K. P. L e s c h , M . D . , W . S c h o t t , M . D . , a n d S. G r o s s , M . D . Neurosurgical Clinic, University of Erlangen-Nuernberg, Erlangen, Federal Republic of Germany

Lesch KP, Schott W, Gross S. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: evidence for multiple estrogen binding sites. Surg Neurol 1987;28:181-8.

The existence of estrogen receptors in human meningiomas has long been a controversial issue. This may be explained, in part, by apparent heterogeneity of estrogen binding sites in meningioma tissue. In this study, estrogen receptors were determined in 58 meningiomas with an enzyme immunoassay using monoclonal antibodies against human estrogen receptor protein (estrophilin) and with a sensitive radioligand binding assay using uSI-labeled estradiol (l~SI-estradiol) as radioligand. Low levels of estrophilin immunoreactivity were found in tumors from 62% of patients, whereas radioligand binding activity was demonstrated in about 46% of the meningiomas examined. In eight (14%) tissue samples multiple binding sites for estradiol were observed. The immunoreactive binding sites correspond to the classical, high affinity estrogen receptors: the Kd for *25I-estradiol binding to the receptor was approximately 0.2 nM and the binding was specific for estrogens. The second, low affinity class of binding sites considerably influenced measurement of the classical receptor even at low ligand concentrations. The epidemiological and clinical data from patients with meningiomas, and the existence of specific estrogen receptors confirmed by immunochemical detection, may be important factors in a theory of oncogenesis. KEY WORDS: Estrogen receptor; Monoclonal antibody; Meningioma

Until very recently, all data about the steroid-receptor interactions, the subcellular distribution of receptors, and quantitative assays for receptors in both normal and neoplastic tissues were entirely based on the binding of

Address reprint requests to: Dr. K. P. Lesch, Universit~.ts-Nervenklinik, Fiichsleinstrasse 15, 8700 Wiirzburg, Federal Republic of Germany. © 1987 by Elsevier Science Publishing Co., Inc.

radioactively labeled steroids [8,9,18,20]. The widely recognized procedure for assaying steroid hormone receptors is a biochemical saturation analysis. Tumor cytosol is incubated with increasing quantities of radioactive steroids and then free and receptor-bound steroids are separated by adsorption on dextran-coated charcoal (DCC). The computation of the binding data is carried out by the method of Scatchard [6,356]. However, this method has several inherent disadvantages including interference by endogenous or administered steroids and standardization problems. In addition, multiple sources of variability have been identified such as errors in counting efficiency, variation in tracer solutions, differences in estimation of nonspecific binding, selection of separation procedures, and methods of constructing Scatchard plots. The production of monoclonal antibodies against estrogen receptor (ER) protein (estrophilin) by Greene et al[ 15,16] has permitted the development of estrogen receptor assays based on direct antigenic recognition rather than steroid binding activity. These antibodies have high affinity (Kd = 10 9-10 m M) for both steroid-occupied and unoccupied ERs, and recognize nuclear as well as cytosol forms of the receptor [ 16,21 ]. In human mammary carcinomas statistical analysis revealed a strong correlation between the results of the radioligand binding assay (RBA) and enzyme immunoassay (EIA) techniques. When monoclonal antibodies to estrophilin were used to detect receptors in estrogen-sensitive tissues, specific binding was confined exclusively to the nucleus, suggesting that both cytosol and nuclear forms of the receptor protein may reside in the nuclear compartment [ 14,21]. The hormone modulation of the growth of mammary carcinomas has been documented frequently [8,18,19]. The determination of sex steroid receptor levels to predict the effectiveness of hormone therapy has become standard practice. Steroid hormone analysis is established as a routine method not only in cases of mammary carcinoma, but also for endometrial carcinoma; for ovarian carcinoma and prostate carcinoma this diagnostic method is gaining increasing significance [33,40]. 0090-3019/87/$3.50

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Epidemiological and clinical data suggest that the meninges and their tumors are target tissues for sex steroids as well [1,2,7,28,32,38]. Consequently, the presence of estrogen, progesterone, and androgen receptors in meningeal tissue has been extensively investigated [5,22,27,30]. Meningiomas appear to contain high concentrations of progesterone receptors and moderate concentrations of androgen receptors, whereas the presence of ERs remains controversial [3,10,13, 23,31,36,43-45]. Low concentrations of ERs as found in meningiomas may occur when exclusively unspecific, low affinity binding components are present; when nuclear receptors resist complete extraction; when small numbers of receptor-containing cells are present in otherwise receptor-negative tissue; or when estrogen binding is impaired by endogenous ligands. In order to investigate these possibilities the estrogen binding sites were determined by an EIA using monoclonal antibodies against human ER protein (estrophilin) [15,16], and by a conventional RBA using the DCC assay with 125I-estradiol as radioligand [17].

Materials and Methods

Clinical and Histological Data Fifty-eight samples of tumor tissue were obtained during operation from 55 patients with meningiomas. The epidemiological characteristics as well as the localization and histopathology of a typical statistical series of meningiomas were expected. This series included 39 (71%) women and 16 (29%) men, with a mean age of 56.3 -+ 1.0 years (mean -+ SEM; n = 55). Eight of the 39 women had regular menstrual cycles, 3 were perimenopausal, and 28 were postmenopausal. Of the 58 meningiomas, 50 were intracranial, 4 were extracranial, and 4 were intraspinal. Of the 50 intracranial meningiomas, 5 were infratentorial and 45 were supratentorial tumors. Of the 45 supratentorial tumors, 28 were excised from the convexity or from the parasagittal region; 17 were located basally and originated in the tentorium, the cribriform plate, the sphenoid bone, or the sella turcica. The histological assignment was carried out according to the W H O classification [46]. Fiftytwo tumors were benign meningiomas of the meningotheliomatous, transitional and fibrous type; of these, 8 revealed regressive changes with calcification and necrosis (psammomatous subtype). Two meningiomas were angiomatous and 4 were anaplastic. Eight tumors were recurrent. Forty-nine of the patients had been treated for 1-14 days before the operation with 3-24 mg/day dexamethasone orally and/or parenterally.

Lesch et al

Reagents 1251-16~-iodo-3,17~8-estradiol (125I-E2, 1 4 0 - 1 8 4 Ci/mmol), diethylstilbestrol (DES), estradiol, R 5020, dexamethasone, and lyophilized receptor control substance were purchased from New England Nuclear (Boston, MA). 5o~-dehydrotestosterone was obtained from Sigma (Munich, F.R.G.). PENG-buffer (10 mmol/liter KH2PO4/K2HPO4, 1.5 mmol/liter EDTA disodium, 3 mmol/liter NAN3, 5 mmol/liter monothioglycerine, 10% glycerol (vol/vol), pH 7.5) was obtained from Serva (Heidelberg, F.R.G.). Monoclonal antiestrophilin antibodies (ER-EIA technique) were obtained from Abbott Laboratories (N. Chicago, IL). Protein standard was obtained from Bio-Rad (Munich, F.R.G.).

Enzyme Immunoassay The ER-EIA technique is a solid phase enzyme immunoassay based on the "sandwich" principle. Tissue homogenization and cytosol preparation were conducted as previously described [22]. Cytosol extracts from meningiomas, controls, and appropriate standards were assayed for immunoreactive ER by a convenient immunocolorimetric procedure using two monoclonal antibodies that recognize human estrophilin [15,16]. Briefly, 100/*L cytosol (control or standard) was incubated for 18 hours at 4°C with a polystyrene bead coated with antiestrophilin immunoglobulin G (IgG). Unbound proteins were then removed by washing twice with 4-6 mL deionized water, and each bead was incubated for 60 minutes at 37°C with antiestrophilin IgG conjugated with horseradish peroxidase. After removal of unbound antibodies, beads were incubated with a solution of 0phenylenediamine for 30 minutes at room temperature and the absorbances of the supernatant fractions were measured at 450 nm in a spectrophotometer (Quantum I, Munich, F.R.G.). All assays were performed in duplicate. Parallel incubations containing known amounts of ERs (standards) were used to generate a standard curve. The estrophilin immunoreactivity was expressed in terms of the protein concentration of the cytosol (femtomoles per milligram). Calculations were carried out using a computer program. The assay sensitivity was approximately 2 fmol/mL (unpublished observations); the intraassay reproducibility was about 6% and the interassay reproducibility was approximately 10%.

Radioligand Binding Assay Because of its very high specific activity, the use of ~25IE2 as tracer [17] made it possible to determine ERs at low ligand concentrations. The determination of ERs was carried out by titration of cytosol [22] with five

Estrophilin lmmunoreactivity in Meningiomas

Surg Neurol 1987 ;28:181-8

different concentrations of the radioactively labeled steroid. All assays were carried out in duplicate. To determine total binding, 100 IzL of '25I-E2 dissolved in PENG-buffer at 0°C was placed in glass tubes (12 × 75 mm) and to each, 1 0 0 / , L cytosol was added. (The protein concentration in the cytosol had been previously adjusted to 1-4 mg protein/mL by dilution with P E N G buffer.) The mixture was incubated for 16 hours at 0-4°C. The tracer concentration was in the range 0. l - 1 0 nmol/liter. Nonspecific binding was determined in replicate mixtures in which, in addition to the radioactive ligands, DES (a relevant competitor) was added in a 200-fold excess (calculated with respect to the highest concentration of the labeled steroid). To estimate the ligand specificity of the estrogen binding component, cytosol from a meningioma was incubated for 16 hours at 0-4°C with 0.5 nmol/liter 1~5IE, in the presence or absence of a 100-fold excess of a different unlabeled competitor. At the end of the incubation time, the flee and receptor-bound steroids were separated with DCC. T o each tube 500 /,L DCC-suspension was added and the mixture was shaken gently for 10 minutes before centrifuging at 1,500 g at 4°C for 10 minutes. Radioactivity was determined in a 9,-counter (Packard, Downers Grove, IL) in 500/.LL of the supernatant. The binding data were evaluated exclusively by the Scatchard method [6,35]. The total amount of steroid bound specifically (B) and the ratio of specifically bound to free steroid (B/F) were calculated. For each assay, B on the abscissa was plotted against B/F on the ordinate and the straight line of best fit was determined. The intercept of the extrapolated straight line with the abscissa gave the total receptor concentration B ..... in moles per liter; the gradient of the line is the dissociation

constant K,I. The receptor concentration was expressed in terms of the protein concentration of the cytosol and given in femtomoles receptor per milligram cytosol protein. The data processing was carried out using a computer program.

Protein Determination Protein determinations in tumor cytosol were carried out according to the method of Bradford [4] using bovine albumin as standard.

Statistics The results are expressed as the mean -+ SEM. The data were analyzed using nonparametric statistical methods; mean comparison by the Mann-Whitney U-test or Kruskal-Wallis H-test; correlations by the Spearman rank order correlation.

Results

Saturation Analysis High affinity, low capacity binding for estradiol (Figure 1) was demonstrated in 26 (45%) of the 58 meningiomas (B ..... : m e a n -+ SEM = 8.3 -+ 1.2 fmol/mg protein; K,t: 1.0 _+ 0.2 nM; n -- 26). In eight tissue samples analysis of the binding data produced curvilinear Scatchard plots (Figure 2), indicating the presence of more than one class of binding sites. One component of the Scatchard plot (concentration range of radioactive ligand: 0.05-0.5 nM) had a slope characteristic of high affinity binding (K~: 0.04-0.8 nM). The second component of the Scatchard plot representing a lower affinity estrogen binding

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ing to multiple estrogen binding sites. High affinity estrogen receptor." Bmax = 4.2 fmol/ml cytosol; Kj = 0.06 riM. Lower affinity estrogen binding component: B l l . 4 fmol/ml cytosol; Ka - 0.5 nM. The maximum number of specific, high affinity estrogen receptors (B,,,~) was obtained by vectorial analysis. The curved line represents the sum of the two linear plots (for details, see the text). ....

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site was not due to interference from testosterone-estradiol binding globulin (TEBG), because [3H]moxestrol (R 2858) and IzsI-E2, neither of which binds to TEBG, displayed the same binding behavior [31]. The maximum number of specific binding sites (Bmax) obtained by extrapolating the steepest component of the Scatchard plot to the x axis was approximately 1.5 times the Bma~ data obtained by vectorial analysis. The Bmaxof the second component (Kd: 0.5-4.2 nM) was not affected significantly by vectorial analysis [12,34]. The ratio of the number of low to high affinity binding sites averaged 4:1. For studies of ligand specificity of the high affinity binding component, it was important to select low concentrations of 125I-estradiol in order to minimize the influence of the lower affinity binding site. To investigate the specificity of estrogen binding, parallel saturation analyses were carried out in the absence or presence of different unlabeled competitors. Neither unlabeled D H T , R 5020, nor dexamethasone influenced the specific binding of 125I-estradiol significantly. No significant difference between female and male patients (Figure 1) was detectable (7.4 _+ 1.4 versus 9.9 -+ 2.0; U = 114, NS). No correlation of ER binding activity with age of the patients, with application of preoperative glucocorticoid therapy, or with the histological subtype and location of the tumors has been demonstrated.

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F i g u r e 3. Correlation of enzyme immunoassay (EIA) and radioligand binding assay (RBA) for estrogen receptor (ER) determinations. (B) Meningiomas containing exclusively specific, high affinity estrogen binding components (linear Scatchard plots in the RBA technique). (&) Meningiomas showing multiple estrogen binding sites (cum,ilinear Scatchard plots in the RBA technique). * Linear regression between ER immunoreactit,ity and ER binding activity: rs = 0.140, NS; Spearman's rank order correlation. • * Linear regression between ER immunoreactivity and ER binding activity of tumors with linear Scatchard plots in the RBA technique: r~ = 0.884, p < 0.001.

Immunoassay Immunoreactive ERs (Figure 3) were present at measurable levels in 36 (62%) of the 58 meningiomas (B; 3.7 + 0.5 fmol/mg protein; n = 36). The ER immunoreactivity was significantly lower than ER radioligand binding activity (U = 93, p < 0.001) and no correlation emerged when corresponding values of the EIA and RBA technique were compared (rs = 0.140, NS). However, in 11 meningiomas with linear Scatchard plots a considerable correlation (rs = 0.884, p < 0.001) between ER immunoreactivity and ER binding activity was found (Figure 3). In eight tissue samples with curvilinear Scatchard plots, specific ER radioligand binding was clearly overestimated by conventional analysis; 14 samples revealed exclusively immunoreactive ERs and 7 samples showed only ER binding activity. The results demonstrate a significant difference (U = 56, p < 0.01) between female (4.3 +- 0.6, n = 24) and male patients (2.5 +_ 0.4, n = 12). O f the 8 female premenopausal women, only 1 had measurable ER immunoreactivity, but all 4 meningiomas of the perimenopausal women with irregular menstrual cycles contained comparatively higher ER levels (7.3 -+ 1.8, n = 4). In 3 recurrent meningiomas, including a pulmonary metastasis, the tissue samples contained considerably higher ER levels (4.1 _+ 1.8) than the primary tumors

Estrophilin Immunoreactivity in Meningiomas

(1.6 -+ 1.1). Because o f the size of the subsample, these differences were not significant. A definitive dependence of ER levels on the histological subdivision and topographical location of the tumors or on the preoperative administration of glucocorticoids was not demonstrated.

Discussion The existence of ERs in human meningiomas has long been a matter of debate, but the results of previously published series in which estrogen binding components were determined cannot be compared directly either with each other or with the results of this study. Blankenstein et al [3], Schnegg et al [36], Vaquero et al [44], and Markwalder et al [26] found no evidence for the presence of specific ERs in meningiomas; other groups [5,10,13,27,31,43,45] reported low but significant ER levels in 9 % - 9 4 % o f tumors. These discrepancies in the data may be accounted for in a number of ways. The data o f Cahill et al [5] and other groups [27,36,43,44], as well as those o f our series, are expressed in femtomoles per milligram protein; the other authors report their results in femtomoles per gram tumor tissue [10,13,31], or some use both systems [26]. The existence of a correlation between the two systems cannot always be assumed [26]. A second problem is associated with variations in the definition of significance for a positive receptor level. In the data of Magdelenat et al [23] and Poisson et al [31], which were published as data of the same group [30], receptor concentrations less than 100 fmol/g tissue were classified as negative. According to Tilzer et al [43], for a positive receptor value the level must be above 8 fmol/mg protein and the dissociation constant below 3 nM. In the studies of Cahill et al [5] and in our previous series [22], the internationally accepted minimum significance value of 10 fmol/mg protein was applied [8,20]. In a number of other series significance levels were not considered. This definition, which is derived from clinically based experience with mammary carcinomas, is supposed to be o f fundamental importance for the anticipation of response to antiestrogen therapy, and is therefore a prerequisite for interpretation of false-negative and false-positive receptor values. For example, Donnel et al [10] reported measurable ERs in five of six meningiomas. Only two of these tumors, however, had ER levels above I00 fmol/g tissue. Similar problems of interpretation occur in various data series. However, because the absolute level o f estrogen binding that can correlate with a treatment response can only be determined by a clinical study, it is premature to report hormone-binding assays to be "positive" as determined by some arbitrary level o f receptor reactivity, as the binding

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may not be that of a true receptor (vide infra) and the response of meningiomas to the therapies may not be the same as the response o f mammary carcinomas. Therefore, in this study, we have chosen to report the actual receptor concentrations measured. Another plausible reason for the wide spectrum o f data is the range o f different methods used. In most data series tritium-labeled estradiol was used as radioligand. The studies o f Pertuiset [30], Poisson [31], and Magdelenat et al [23 ] used R 2858 (1 l~e-methoxy-17/3-ethynylestradiol) as estradiol analog. The concurrent measurement of ERs by a sensitive RBA using ~25I-E2 as radioligand and a specific EIA using monoclonal antibodies has not been reported previously. Most frequently, the quantitative DCC assay has been used; however, in some of the studies, the assay used was a qualitative density gradient centrifugation [5,10,13,43]. Isoelectric focusing has been applied as well [3,45]. The iodine-labeled DCC assay is largely identical, as far as the methodology is concerned, with the conventional RBA using 3H-estradiol as radioligand. A statistical comparison revealed a high correlation between the results of the two modifications [22]. In a large number of the studies a single point saturation analysis was carried out [5,23,27,30,31], which has been shown to lead to false-positive and false-negative results in the region of borderline significance. Scatchard analyses [6,35] were carried out only in isolated cases. The use of iodine-labeled estradiol with high specific activity makes it possible to carry out Scatchard analyses of ERs consistently, even with relatively small amounts of tumor tissue. There were no differences among the various studies in biopsy technique, freezing of the tissue, storage times, and homogenization methods. The epidemiological data of the patients and the histological and topographical characteristics of the tumors were comparable as well. Furthermore, differences in the application of preoperative glucocorticoid therapy could offer a possible explanation for the wide range of published data. In the study of Magdelenat et al [23], 13 of the 42 patients (31%) received preoperative glucocorticoids; in the study of Cahill et al [5] and in our study, 9 1 % and 8 3 % of the patients, respectively, received glucocorticoid therapy (patients with extracranial and intraspinal meningiomas and peripheral metastasis were excluded). Most authors did not comment on this point. It may, however, be assumed that the frequency was within the range described above. Glucocorticoids in high molar concentrations compete with the sex steroids for the ER, and thus a reduction of receptor reactivity as a result of therapy does seem possible. Blocking the ERs with endogenous estrogens (and/or glucocorticoids) could account for receptor levels below the sensitivity of the

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RBA technique (vide infra). Studies investigating competition and specificity using a representative variety of ligands have been unable to clarify this point so far. Finally, the interference of a relatively unspecific low affinity estrogen binding component may account for the inconsistency and wide range of results. Heterogeneity of estrogen binding sites has been reported in different types of tissues [ 11,24,25,29,41,42]. In rat uterine cytosol Eriksson et al [11] described two types of estradiol binding components, which were referred to as type I and type II. The type I binding site had the properties of a classic high affinity receptor; the type II binding components had a 30-fold lower affinity for estradiol. The presence of the type II binding component markedly disturbs the accurate quantitation of the classical high affinity ER. The first and second class of binding sites reported in this study have many similarities with the high and low affinity binding components described above. The most important difference between rat uterine and human meningioma ERs appears to be the fourfold to eightfold higher affinity of *25I-estradiol for both binding sites in meningiomas. It is of interest, however, that the affinities of ~25I-estradiol for these sites in meningiomas appear to be similar to those present in human mammary carcinoma [29]. In individual meningiomas the low affinity binding will influence the determination of the high affinity estradiol binding when saturation analysis is performed with ligand concentrations in the range of 0.5-10 nM (a range often selected for Scatchard analysis); thus, quantitation of ER solely by a single point assay cannot be carried out accurately unless the low affinity binding component can be selectively blocked. Because of the variable shape of the curve of the second binding component these Scatchard plots are difficult to interpret. In order to accurately measure the high affinity class of ERs in the meningioma, an adequate number of ligand concentrations within the range of 0.01-1.0 nM should be applied. The recently developed alternative approach to the estimation of ERs in neoplastic tissues was used to decide whether meningiomas contain true ERs, and whether endogenous steroids do interfere with the estrogen binding sites. A number of results are indicative of complex relations between the two types of binding sites. The existence of several meningioma subpopulations-differing with respect to their receptor affinity and specificity-is suggested by the fact that, on the one hand, tissue samples exist that contain exclusively specific, high affinity estrogen binding components and, on the other hand, tissue samples can contain relatively unspecific, lower affinity binding sites that evaded detection by the EIA technique (as in several tumors of our study), or they can contain both types of binding sites at the same

Lesch et al

time. The low dissociation constant found in tissues that contain only high affinity ERs are also obtained for the high affinity binding component of samples with curvilinear Scatchard plots, and provide strong evidence for the existence of and the reliable measurement of a specific, high affinity ER. Furthermore, it may be presumed that blocking ERs by endogenous steroids can in fact play a significant role, as a large proportion of samples in the present study contained exclusively immunoreactive ERs. Thus, we may conclude that an exact determination--in particular, of the lower affinity binding components that are not recognized by the antibody--is hindered by interference from endogenous steroids (vide supra). Our results indicate a significant difference between female and male patients with regard to the levels of ER immunoreactivity. In the female subsample we observed a trend towards higher levels of ERs in meningiomas from perimenopausal patients compaired to postmenopausal patients; in most premenopausal women we failed to find any ERs. That these differences are not solely related to the prevalent hormone status, but rather to a common hormonal imbalance, is suggested by epidemiological and clinical data that emerge when the sex incidence is compared to the hypothetically estimated onset of tumor growth. The information provided in this study should help resolve some of the controversies in this field and stimulate generation of more uniform data on ERs in meningiomas. Because of its possible biological role in the oncogenesis of meningiomas, the ER should be further studied as a potential marker for hormone sensitivity of individual tumors. In performing these studies it is important to carry out the assays with the correct ligand concentration preferentially combined with immunochemical techniques. Results derived from assays based solely on the use of high ligand concentrations will be difficult to interpret as the importance of the second binding component is as yet not known. Certainly, data on the two binding sites need to be analyzed separately. Ultimately, the question can be raised as to whether detection of such low amounts of receptors is of practical importance. For human mammary carcinoma a distinction between ER-positive and ER-negative tumors is often selected arbitrarily in the range of 3-10 fmol/mg protein. However, very little is known concerning the amount of receptor necessary to mediate a physiological response in different tissues [19,25], and further in vitro studies using meningioma tissue cultures [27] to assess the biological activity of both types of estrogen binding sites should certainly be encouraged. Moreover, preliminary data from our laboratory indicate that the level of ER in meningioma tissue is as high as 8% of the progestin and 29% of the androgen receptor (unpublished

Estrophilin l m m u n o r e a c t i v i t y in Meningiornas

observations). In summary, we conclude that when performing estrogen binding studies in meningioma tissue it is necessary to be aware of the complexity of such binding. The classical high affinity, low capacity binding generally agreed to be of importance for estrogen sensitivity of a tissue is measurable only at ligand concentrations below 0.5-1 nM, or, alternatively, by a sensitive immunochemical method. At higher concentrations the data are markedly distorted by a second class of binding sites. The characteristics of the first binding component are typical of an ER. A detailed characterization of the second estrogen binding component is beyond the scope of the present investigation.

The authors wish to express their gratitude to Dr. P. Thierauf, Mrs. P. Roth, and Dr. A. J0rss for their assistance in this study.

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