Evaluation of an enzyme immunoassay kit for estrogen receptor measurements

Using an enzyme immunoassay (EIA) procedure, we evaluated the ability of the glass beads in the ER-EIA kit of Abbott Laboratories, which were coated with monoclonal anti-estrogen receptor (ER) antibodies, to bind hormone-free and hormone-filled Type I ER in cytosols, buffer washes, and 0.4 mol/L KCI extracts of ultracentrifugal pellets of breast cancer tissue homogenates. The unmodified ER-EIA technique yields higher values than does the dextran-coated charcoal (DCC) method for Type I ER. However, the antibody-coated beads fail to bind hormone-free ER and react with only a certain proportion of ER-[3H]estradiol complexes. The antigen saturation limit of the beads could not be determined because the quantity of antigen bound by the beads was disproportionate and unrelated to the total amount of the antigen available in the cytosols. In buffer extracts of tissue pellets that were ER-negative by the DCC assay, the EIA method detected high quantities of ER. We recommend checking the ability of the monoclonal antibodies to recognize proteins other than Type I ER in the extra-nuclear and nuclear compartments of target cells before using them for immunohistochemical detection of ER.     

Truncated forms of DNA-binding estrogen receptors in human breast cancer.

The likelihood a breast cancer will respond to antiestrogen therapy depends on the tumor content of immunoreactive or ligand-binding estrogen receptor (ER). To investigate the failure of many ER-positive breast cancers to respond to antiestrogen therapy, we examined by gel-shift assay the ability of tumor ER to bind its cognate estrogen response element (ERE). Analysis of 38 primary breast cancers showed that some tumors containing abundant immunoreactive ER failed to demonstrate DNA binding ER. In many other ER-positive tumors, the fraction of DNA binding ER was low and consisted primarily of truncated receptor forms, which on Western analysis were revealed to be 50 kD homodimers and 67-50 kD ER heterodimers. The use of protease inhibitors during tumor extraction and the demonstration of nuclear-localizing ER and ERE-binding COUP (chicken ovalbumin upstream promoter) protein in these tumors indicated that the truncated forms of ER were likely present in vivo. The presence of intact DNA binding ER correlated with higher tumor content of immunoreactive sex steroid receptors (ER and/or PR), standard predictors of tumor responsiveness to antiestrogen, suggesting that loss or truncation of DNA binding ER may be an important prognostic parameter accounting for some forms of clinical resistance to antiestrogen therapy.     

Estradiol receptors in human breast carcinomas assayed by use of monoclonal antibodies

Our aim here was to compare a new enzyme immunoassay for estradiol receptors (ER) in breast cancer with a standard assay involving radiolabeled estradiol. A significant correlation was obtained between the standard binding assay and immunoassay (rs = 0.945, p less than 0.001, n = 100). Immunoreactive ER were also found in endometrial carcinomas but not in lung carcinomas, malignant melanomas, or basal cell carcinomas. Patients with breast cancers containing immunoreactive ER survived significantly longer after operation than patients whose tumor lacked immunoreactive ER. This immunoassay for ER is simpler than the binding assay and requires less time and tissue.     

Characterization of estrogen response element binding proteins as biomarkers of breast cancer behavior

BackgroundWhile investigating estrogen response element (ERE) binding properties of human estrogen receptor-α (hERα) in breast cancer cytosols, other ERE-binding proteins (ERE-BP) were observed.Design and methodsRecognition properties of ERE-BP were evaluated by electrophoretic mobility shift assays (EMSA) with ERE sequences of the 5′-flanking region of the estrogen responsive gene vitellogenin A2 (VitA2). Cytosols were incubated 16 h, 4 °C with [32_P]ERE sequences and separated by EMSA. A method of estimating ERE-BP levels was developed by measuring band intensity from EMSA profiles, expressed in digital light units (DLU)/μg protein and normalized to total DLU. ERE-BP were purified by affinity chromatography and EMSA, and then identified by mass spectrometry.ResultsERE-BP in cytosols did not supershift in the presence of anti-hERα or anti-hERβ antibodies recognizing different ER epitopes suggesting that they are not fragments of either receptor isoform. ERE-BP competed with hERα for binding to VitA2–ERE. Increased levels of ERE-BP DNA-binding activities measured in 310 cytosols prepared from breast cancer biopsies correlated with decreased patient survival. Strikingly, breast cancer patients with ER negative status and high ERE-BP expression exhibited the poorest disease-free and overall survival. After purification, ERE-BP were identified as Ku70 (XRCC6) and Ku80 (XRCC5) using mass spectrometry. ERE-BP were confirmed to be Ku70/80 by supershift assay.ConclusionPresence of these novel ERE-binding proteins in a breast carcinoma is a strong predictor of poor prognosis. Our results suggest that ERE-BP, identified as Ku70/Ku80, in cytosols prepared from breast carcinoma biopsies are useful biomarkers for assessing risk of breast cancer recurrence.     

Changes in glucocorticoid and mineralocorticoid receptors of liver and kidney cytosols after pathologic stress and its regulation in rats

OBJECTIVE: As effectors, glucocorticoid and mineralocorticoid receptors play an important role in pathologic stress. This study was designed to observe the changes in glucocorticoid receptor of liver cytosols and mineralocorticoid receptor of kidney cytosols after pathologic stress in rats. DESIGN: Controlled laboratory study. SETTING: Medical university. SUBJECTS: Male Wistar rats (weight range, 180-200 g). INTERVENTIONS: Rats received a low-degree or heavy-degree immersion scald that covered 10% or 35% total body surface area and were randomly divided to receive either tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, KPV peptide (Ac-D-Lys-L-Pro-D-Val), or saline (control). The binding capacity and the apparent dissociation constant of the steroid-binding sites of normal, low-degree, and heavy-degree scalded rats were measured by radioligand-binding assay, with [3H]dexamethasone and aldosterone as the ligand, respectively. MEASUREMENTS AND MAIN RESULTS: The binding capacity of glucocorticoid receptor in hepatic cytosols in rats 12 hrs after heavy-degree scald (208.45 +/- 30.78 fmol/mg of protein) was lower than that of the control group (306.71 +/- 27.96 fmol/mg of protein; p < .01). The binding capacity of glucocorticoid receptor in hepatic cytosols in rats 12 hrs after low-degree scald (296.64 +/- 16.06 fmol/mg of protein) was not significantly different compared with the control group (p > .05). There were two types of mineralocorticoid receptor in kidney cytosols in rats, and their binding capacity and apparent dissociation constant were not identical. The binding capacity of mineralocorticoid receptor in rats 12 hrs after heavy-degree scald (binding capacity 1, 22.40 +/- 5.40 fmol/mg of protein; binding capacity 2, 196.30 +/- 32.50 fmol/mg of protein) was lower than that of the control group (binding capacity 1, 41.60 +/- 7.20 fmol/mg of protein; binding capacity 2, 317.60 +/- 70.00 fmol/mg of protein; p < .01). The binding capacity of mineralocorticoid receptor in kidney cytosols in rats 12 hrs after low-degree scald (binding capacity 1, 41.40 +/- 5.00 fmol/mg of protein; binding capacity 2, 314.80 +/- 45.70 fmol/mg of protein) was not significantly different compared with the control group (p > .05). The injections of anti-rat tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, and KPV peptide (Ac-D-Lys-L-Pro-D-Val) might prevent a reduction in the binding capacity of glucocorticoid receptor in hepatic cytosols and mineralocorticoid receptor in kidney cytosols in rats with heavy-degree scald in vivo. CONCLUSIONS: These studies suggest that the glucocorticoid receptor of hepatic cytosols and the mineralocorticoid receptor of renal cytosols decreased in rats with heavy-degree immersion scald and that the injections of anti-rat tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, and KPV peptide might increase the level of glucocorticoid receptor and mineralocorticoid receptor in vivo.     

Estradiol receptors in benign and malignant disease of the breast

Estrogen receptor in human breast carcinoma and other tissues was studied in the presence or absence of estrogen competitor by sucrose gradient centrifugation and a dextran-charcoal technique. Cytoplasmic estrogen receptor in tumor was demonstrated to be localized at approximately the 8 S region of a 10–30% sucrose gradient. In the dextran-charcoal method, by employing a binding index (BI), which compared the ratio of specific estradiol receptor binding (ER) to total binding (both specific and non-specific), tumors from 53 patients were classified into three groups: those with high BI, low BI and negative ER. The BI corresponded well with the cytoplasmic ER content in these groups and provided an excellent measurement of the relative binding capacity. Approximately two-thirds of the malignant tumors had ER. Only three of the 17 benign tumors had any measurable ER. Normal tissues from breast, ovary, adrenal, and skin did not contain receptor. There was no relationship between tumor histopathology and ER content in the tumors. The value of this simple dextran-charcoal technique, BI values and ER content for routine clinical purposes in predicting patient response to endocrine ablation is discussed.     

Evaluation of estradiol binding in human benign prostatic hyperplasia

We have systematically evaluated the effect of tris/phosphate buffers, ethylenediamine tetraacetate (EDTA), dithiothreitol (DTT), molybdate, and phenylmethylsulfonyl fluoride (PMSF) on the binding of 17β-estradiol to human BPH cytosol. Competition binding assays demonstrated that the estrogen receptor was highly specific for 17β-estradiol. There was statistically no difference in binding observed with tris or phosphate buffers. Two classes of binding (high affinity—type I, low affinity—type II) were observed. DTT (1 mM) showed maximum inhibition of type II binding. There was no significant difference in binding observed in the presence of 1.0 mM EDTA. However, 10 mM EDTA stimulated estradiol binding in both saturation analysis and glycerol gradients. Stimulation of estradiol binding by 10 mM EDTA was observed even in the presence of 1 mM DTT. Molybdate (20 mM), a reagent used to stabilize steroid receptors, significantly inhibited estradiol binding. PMSF inhibited estradiol binding at all concentrations studied and the nature of the inhibition appeared to be uncompetitive. A B_max (maximum number of binding sites) of 84.4 ± 68.5 fmoles/mg protein with a K_D (equilibrium dissociation constant) of 2.5 ± 1.7 mM was observed for type I estrogen receptor binding. Our B_max values are higher than those reported in other studies even after allowing for possible overestimation. In view of the potential importance of estrogen receptors in human prostate, careful reevaluation of estrogen receptors must be undertaken using proper assay conditions.     

Estrogen binding sites in peripheral blood mononuclear cells and thymocytes from 2 myasthenia gravis patients

In the present investigation the estrogen binding capacity of peripheral blood mononuclear cells (PBMC) and thymocytes from 2 young female Myasthenia Gravis (MG) patients has been studied. We found high levels of estrogen binding sites in both pre-thymectomy PBMC and lymphoid cells from the hyperplastic thymuses of the MG patients. A sucrose density gradient analysis revealed that the estrogen was binding to macromolecular species similar to those characterized in cytosols from classical estrogen target organs. A post-thymectomy time-dependent decrease of the estrogen binding capacity was also observed in PBMC, reaching levels similar to that of sex- and age-matched controls.     

Vitamin D and gonadal steroid-resistant New World primate cells express an intracellular protein which competes with the estrogen receptor for binding to the estrogen response element.

New World primates (NWP) exhibit a form of compensated resistance to vitamin D and other steroid hormones, including 17beta-estradiol. One postulated cause of resistance is that NWP cells overexpress one or more proteins which block hormone action by competing with hormone for its cognate hormone response element. Here we report that both nuclear and postnuclear extracts from NWP, but not Old World primate, cells contained a protein(s) capable of binding directly to the estrogen response element (ERE). This ERE binding protein(s) (ERE-BP) was dissociated from the ERE by excess of either unlabeled ERE or excess of the ERE half-site motif AGGTCAcag. DNA affinity chromatography using concatamers of the latter resulted in > 20,000-fold purification of the ERE-BP. The intensity of the ERE-BP-ERE complex in electromobility shift assay was indirectly related to the amount of wild-type Old World primate estrogen receptor (ER) but not affected when potential ligands, including 17beta-estradiol (up to 100 nM), or anti-ER antibody was added to the binding reaction. We conclude that vitamin D-resistant and gonadal steroid-resistant NWP cells contain a protein(s) that may "silence" ER action by interacting directly with the ERE and interfering with ER binding.     

Simplified Scatchard-plot assay for estrogen receptor in human breast tumor.

In this two-point Scatchard-plot assay, with which a test of competitive inhibition is combined, the sample is mechanically homogenized in a buffer containing dithiothreitol, ultracentrifuged to obtain a fat-free cytosol, the protein content of which is then adjusted, and free and bound labeled estradiol are separated with dextran-coated charcoal after overnight incubation. We tested the method for precision and reliability by assaying such cytosols from pregnant rabbit uteri before and after dilution with kidney cytosol, and by assaying several other target and nontarget animal and human tissues. The Scatchard plot data were more reliable than tests for percent inhibition of binding by a competitor (diethylstilbestrol). For a tumor tissue to be judged positive it must bind at least 8 fmol of estradiol per milligram of protein and have a Kd of 0.1 to 5 X 10(-10) mol/liter. Some non-target tissues showed less than 70% inhibition by 10(4)-fold concentrations (over labeled estradiol) of inhibitor. Of 19 breast-tumor specimens, seven were found to be positive.     

Expression of steroid receptors in intact rat uterus, mammary gland, and liver treated with selective estrogen receptor modulators and conjugated equine estrogens

The aim of the present study was to determine the effects of conjugated equine estrogens (CEE) and selective estrogen receptor modulators (SERM) (tamoxifen [TAM] and raloxifene [RAL]) on the expression of steroid receptors—estrogen receptor (ER) and progesterone receptor (PR)—in intact rat uterus, mammary gland, and liver. A total of 24 female rats weighing 250 to 300 g were randomized into 4 groups. Groups 1, 2, 3, and 4 were respectively given conjugated equine estrogen, tamoxifen, raloxifene, and vehicle for a 28-day period. ER and PR expression was detected in tissues of the uterus, mammary gland, and liver. Uterine wet weight and serum estradiol levels were established for all groups. No statistical difference was observed between groups in the ER expression of mammary gland and liver and in the PR expression of uterus, mammary gland, and liver, but differences were noted in serum estradiol levels and uterine ER expression. Serum estradiol levels were lower in the TAM-treated group; differences between the TAM-treated group and the other groups were statistically important (P > .05). Uterine ER expression was greater in the CEE-treated group; differences between the CEE-treated group and theTAM and RAL-treated groups were statistically important (P > .05). CEE or SERM versus vehicle treatment in controls did not seem to result in statistically important differences in ER and PR expression in intact rat uterus, mammary gland, and liver. Only ER expression in the uterus was found to be greater in the CEE-treated group than in SERM-treated groups.     

Measurement of estradiol receptors in human breast tumors by polyacrylamide gel electrophoresis.

1. Specific estradiol receptors in cytosols from human breast tumors were measured by discontinuous polyacrylamide gel electrophoresis. 2. Binding of estradiol to contaminating sex hormone binding globulin was clearly separated from that due to the specific receptor. 3. The amount of estradiol bound by the receptor was linear with respect to the amount of protein added to gel. 4. The estradiol receptor peak of radioactivity showed saturability and specificity characteristic of an intracellular receptor. 5. The results show that polyacrylamide gel electrophoresis can be used to quantitate specific estradiol receptors in the presence of other high affinity binding components.     

An enzyme immunoassay compared with a ligand-binding assay for measuring progesterone receptors in cytosols from breast cancers

To assay progesterone receptor (PR), we compared Abbott's enzyme immunoassay (PR-EIA) with a ligand-binding assay involving dextran-coated charcoal (PR-DCC), using cytosols prepared from 109 breast-cancer biopsies. Results by the two PR methods agreed well. Least-squares analysis produced a line of best fit having a slope of 0.88, an intercept on the PR-EIA axis of 16 fmol per milligram of protein, and a correlation coefficient (r2) of 0.87. To evaluate whether accurate PR-EIA measurements could be obtained on stored cytosols, we compared PR-EIA values for fresh cytosols with values for cytosols stored for various lengths of time up to 13 weeks. Agreement was excellent, especially when the samples showing very high binding (greater than 600 fmol per milligram of protein) were excluded. The lines of best fit after least-squares analyses of the remaining values had slopes between 1.0 and 1.1, intercepts less than 3 fmol/mg, and r2 all greater than 0.91.     

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.     

Comparison of sex steroid receptor determinations in human breast cancer by enzyme immunoassay and radioligand binding

Most investigators comparing ligand-binding procedures for quantifying estrogen and progestin receptors in human breast cancer with procedures employing monoclonal antibody-based methods have utilized an inappropriate variety of reaction conditions, including the elimination of sodium molybdate in the steroid-binding assays. We studied 197 biopsies of human breast cancer, comparing the results of simultaneous measurements of both estrogen and progestin receptors in identical cytosols by enzyme immunoassay and by radioligand binding using the commercially available kits developed by Abbott Laboratories and by DuPont/NEN Products, respectively. Regression analyses comparing the results from the two procedures indicated a linear relationship, with correlation coefficients ranging from 0.79 to 0.93 for both types of receptors over a wide range of data. Using the widely established cutoff value of 10 fmol/mg of cytosol protein for the ligand-binding method, and calculating sensitivity and specificity limits according to McNeil et al. (1975), an equivalent cutoff value of 15 fmol/mg of cytosol protein was determined for the enzyme immunoassay of these receptors. Endocrine status of the patient did not appear to alter the cutoff values of either estrogen or progestin receptors when determined by enzyme immunoassay. We recommend that these cutoff values be considered until the results of clinical correlations are completed.     

Assay of oestrogen and progestin receptors in human meningioma cytosols using immunological methods

Oestrogen (ER) and progestin receptors (PR) were assayed in human meningioma cytosol by radioligand binding assay with Scatchard plot analysis and by monoclonal antibody based enzyme immunoassays. For comparison, human breast cancer tissues were used. Results of both assays agreed very well. For human breast cancer, receptor levels assayed with both methods showed a highly significant (ER: r = 0.96; n = 74 and PR: r = 0.95; n = 19). Also for meningioma cytosols a good agreement was observed between the result of both assays. Thus, most meningiomas were devoid of ER but contained significant concentrations of PR. PR levels in meningioma determined with the enzyme immunoassay correlated well with those found by Scatchard plot analysis. After logarithmic transformation of the data the regression line was; PR(EIA) = 0.83 X PR(Scatchard) + 0.36 (r = 0.917; n = 24). The recognition of the progestin binder in meningioma by a monoclonal antibody against the progestin receptor is a further indication that progestin binding in meningioma occurs to a true receptor.     

Cardiovascular pharmacology of estradiol metabolites

A discussion of the role of endogenous estradiol metabolites in mediating important biological actions of estradiol is essentially nonexistent in standard textbooks of pharmacology and endocrinology. Indeed, the prevailing view is that all biological effects of estradiol are initiated by binding of estradiol per se to estrogen receptors and that estradiol metabolites are more or less irrelevant. This orthodox view, which is most likely incorrect, is the fundamental premise (an estrogen is an estrogen is an estrogen) underlying the design of important clinical trials such as the Heart and Estrogen/Progestin Replacement Study and the Women's Health Initiative Study. Accumulating data provide convincing evidence that some metabolites of estradiol, the major estrogen secreted by human ovaries, are biologically active and mediate multiple effects on the cardiovascular and renal systems that are largely independent of estrogen receptors. More specifically, metabolites of estradiol, particularly catecholestradiols and methoxyestradiols, induce multiple estrogen receptor-independent actions that protect the heart, blood vessels, and kidneys from disease. These protective effects are mediated in part by the inhibition of the ability of vascular smooth muscle cells, cardiac fibroblasts, and glomerular mesangial cells to migrate, proliferate, and secrete extracellular matrix proteins, as well as by an improvement in vascular endothelial cell function. The purpose of this review is to highlight the cardiovascular and renal pharmacology of catecholestradiols and methoxyestradiols. The take home message is simple: that when it comes to cardiovascular and renal protection, the concept that all estrogenic compounds are created equal may not be true.     

Estradiol enhances thiazide-sensitive NaCl cotransporter density in the apical plasma membrane of the distal convoluted tubule in ovariectomized rats.

Recent data suggest that sex hormones affect the thiazide-sensitive NaCl cotransporter (TSC) density or binding capacity (Chen, Z., D.A. Vaughn, and D.D. Fanestil. 1994. J. Am. Soc. Nephrol. 5:1112-1119). Thus, we determined the effect of ovariectomy (OVX) and estrogen replacement on the ultrastructural localization of TSC in rat kidney using immunocytochemistry. Kidneys of intact female (CON) and OVX rats fed ad libitum for 6 and 9 wk or pair-fed for 9 wk were processed for transmission electron microscopy. Immunogold localization of rat TSC (rTSC1) demonstrated intense label in the apical plasma membrane of CON distal convoluted tubule (DCT). In OVX DCT, rTSC1 label and apical plasma membrane microprojections were decreased. Western blots of renal membrane protein from pair-fed CON and OVX revealed bands at 129-135 kD, but the OVX signal was reduced. Morphometric analyses demonstrated that injecting 10 microg/ kg body weight 17beta-estradiol subcutaneously 4x/wk in OVX rats restored DCT apical microprojections and label density for rTSC1. Thus, in OVX rats (a) rTSC1 immunoreactive renal membrane protein is reduced; (b) apical plasma membrane complexity and immunogold label for rTSC1 in DCT is decreased; and (c) estradiol replacement restores DCT ultrastructure and rTSC1 label to normal. We conclude that estrogen enhances the density of rTSC1 in the DCT, and may alter renal Na transport by this mechanism.     

Correlation between tyrosine hydroxylase activity,melanogenesis, and estradiol binding in human melanoma cells

Cytosols fractions from human mammary carcinoma, or malignant melanoma cells, and tyrosine hydroxylase (TH) bind 3H-estradiol (3H.E) with strong affinity. Monoclonal antibodies secreted by cloned cell hybrids which were formed by the fusion of murine myeloma cells with spleen cells from BALB/c mice pre-immunized with purified TH were used to differentiate between 3H.E-binding protein in the cytosols of various neoplastic cells. These monoclonal TH-antibodies inhibited the enzymic activity and the binding of 3H.e to TH or to cytosol fractions of melanotic melanoma cells, but had no effect on 3H.E binding to cytosol fractions from various mammary epithelial or carcinoma cell lines. Cultivation of estradiol-responsive melanotic melanoma cells in media supplemented with the TH-antibodies, caused loss of pigmentation, the cells became amelanotic and non-responsive to 17 beta-estradiol. The results indicate that the estrogen does not bind to melanoma cells via a receptor as for mammary carcinoma cells but to tyrosine hydroxylase.