tamoxifen-aziridine has been researched along with Breast-Neoplasms* in 23 studies
23 other study(ies) available for tamoxifen-aziridine and Breast-Neoplasms
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Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF).
Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression. Topics: Animals; Binding Sites; Breast Neoplasms; Cattle; Chickens; COUP Transcription Factor I; DNA-Binding Proteins; ERRalpha Estrogen-Related Receptor; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Gene Expression Regulation; Humans; Ligands; Luciferases; Nuclear Receptor Co-Repressor 2; Ovalbumin; Precipitin Tests; Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; Repressor Proteins; Response Elements; Tamoxifen; Transcription Factors; Transcription, Genetic; Transfection; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins | 1999 |
Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D.
[3H]Tamoxifen Aziridine ([3H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [3H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [3H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [3H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [3H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/ mg p.). The 4S peak of [3H]TAZ was partially inhibited by both estrogens and antiestrogens. When [3H]E2 was used instead of [3H]TAZ, only an 8S peak was detected. [3H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [3H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [3H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [3H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [3H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor. Topics: Biomarkers, Tumor; Breast Neoplasms; Cathepsin D; Electrophoresis, Polyacrylamide Gel; Estrogen Antagonists; Female; Humans; Neoplasms, Hormone-Dependent; Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Trefoil Factor-1; Tumor Suppressor Proteins | 1998 |
Estrogenic and antiestrogenic regulation of the half-life of covalently labeled estrogen receptor in MCF-7 breast cancer cells.
Effect of estrogens and antiestrogens (AEs) on estrogen receptor (ER) half-life was analyzed in MCF-7 cells by assessing its progressive disappearance after covalent labeling in situ with [3H]tamoxifen aziridine ([3H]TAZ). Cells were incubated for 1 h with 20 nM [3H]TAZ either in the absence or presence of a 500-fold excess of unlabeled estradiol (E2) (non-specific binding). The entire ER population was labeled by this method as established by subsequent incubation of the cells with [125I]E2. [3H]TAZ labeled cells were maintained in culture for additional 5 h in the absence (control) or presence of increasing amounts (0.1 nM - 1 microM) of either a given estrogen (E2, estrone, diethylstilbestrol, bisphenol), a pure AE (RU 58 668, ICI 164 384) or an AE with residual estrogenic activity (RU 39 411, 4-hydroxytamoxifen, keoxifene). The progressive disappearance of nuclear and cytosolic [3H]TAZ-ER complex during 5 h incubation were assessed by their immunoprecipitation with anti-ER monoclonal antibody (H 222) followed by scintillation counting or SDS-PAGE and fluorography. Fading of labeled receptors was extremely slow (approximately 10% loss after 6 h) in absence of any hormone/antihormone indicating a long half-life of the [3H]TAZ-ER complex. Addition of estrogens as well as pure AEs led to a dramatic reduction of the half-life while AEs with residual estrogenic activity were extremely less efficient in this regard providing an explanation for the ability of latter compounds to up-regulate the receptor since they do not affect ER mRNA synthesis and stability. Receptor disappearance induced by estrogens was closely related to their binding affinity for ER. Newly synthesized ER emerged during the treatment with hormones or antihormones seems to be implicated in the phenomenon since [3H]TAZ was covalently bound and could, therefore, not be displaced by these compounds. Induction of synthesis of a short half-life peptide(s) with degradative activity was demonstrated by addition of cycloheximide or puromycine (both at 50 microM) which completely blocked ER disappearance. The fact that no cleavage products of ER were detected by SDS-PAGE suggested a lysosomial hydrolysis. Hence, hormonal modulation of only a part of ERs may down-regulate their total population until it reaches the steady-state level. Topics: Breast Neoplasms; Cell Nucleus; Cycloheximide; Cytosol; Estradiol; Estrogen Antagonists; Estrogens; Half-Life; Humans; Indicators and Reagents; Molecular Weight; Protein Synthesis Inhibitors; Puromycin; Receptors, Estrogen; RNA, Messenger; Tamoxifen; Tumor Cells, Cultured; Up-Regulation | 1996 |
Tamoxifen aziridine labeling of the estrogen receptor-potential utility in detecting biologically aggressive breast tumors.
Expression of estrogen receptor (ER) is a helpful predictor of response to endocrine therapy and disease free survival in breast cancer patients. The presence of variant estrogen receptors has been demonstrated at the RNA/DNA level and might represent an escape of tumors from hormonal control mechanisms. However, the demonstration that the corresponding peptides do exist is a real challenge. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of covalently bound [3H]tamoxifen aziridine ([3H]TAZ) to ER demonstrates a specific, multiband peptide pattern recognized by anti-ER monoclonal antibodies (anti-ER Mo Abs). The native 66 kDa ER form identified through its hormone binding domain by the H-222 Mo Ab was the most prominent one followed by 50, 35, and 28 kDa forms on fluorography. Such patterns from early human breast tumors were compared to the ones of more advanced disease, namely large primary breast cancers, metastatic lymph nodes, and soft tissue relapses: in these cases, molecular forms of 43 and 35 kDa were identified with a remarkable consistency. The 43 kDa peptide was more frequently identified by the H-226 Mo Ab (which maps a region near the DNA binding domain)-albeit with low labeling intensity as compared to H-222 Mo Ab. In addition, the 43 kDa peptide was inversely correlated to ER levels. This altered ER or related peptide could potentially be a marker of biologically aggressive breast tumors. Topics: Antibodies, Monoclonal; Binding Sites; Blotting, Western; Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Epitopes; Humans; Lymph Nodes; Neoplasms, Hormone-Dependent; Polymorphism, Genetic; Receptors, Estrogen; Tamoxifen; Tritium | 1996 |
Functional estrogen receptors in a human preosteoclastic cell line.
The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis. Topics: Adenocarcinoma; Antibodies, Monoclonal; Base Sequence; Blotting, Western; Breast Neoplasms; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; DNA Primers; DNA Probes; DNA-Binding Proteins; Estradiol; Filaggrin Proteins; Humans; Kinetics; Molecular Sequence Data; Osteoclasts; Polymerase Chain Reaction; Progesterone; Receptors, Estrogen; Recombinant Proteins; RNA, Messenger; Tamoxifen; Transfection; Tumor Cells, Cultured | 1995 |
Subunit structure of the nonactivated human estrogen receptor.
The nonactivated estrogen receptor of human MCF-7 mammary carcinoma cells was investigated with respect to stoichiometry of protein subunits. The native receptor complex stabilized by molybdate had a molecular mass of approximately 300 kDa. Chemical cross-linking with several bifunctional reagents resulted in complete stabilization of the same receptor form of approximately 300 kDa and was achieved both in cell extracts and in intact cells. Incubation of the cross-linked receptor with a receptor-specific monoclonal IgG1 antibody increased the molecular mass by approximately 135 kDa--i.e., no more than one immunoglobulin molecule bound to the complex. Partial and progressive cross-linking of affinity-labeled receptors revealed patterns of labeled bands upon denaturing gel electrophoresis indicative of a heteromeric structure. The completely cross-linked receptor was purified to homogeneity and analyzed for protein components. In addition to the receptor polypeptide of approximately 65 kDa, we detected the heat shock proteins hsp90 and p59; the hsp90 band was roughly twice as intense as the p59 band. The heat shock protein hsp70 and the 40-kDa cyclophilin were not detected as components of the highly purified cross-linked receptor of approximately 300 kDa. We suggest a heterotetrameric structure consisting of one receptor polypeptide, two hsp90 molecules, and one p59 subunit, for which the molecular mass adds up to approximately 300 kDa. Thus, the nonactivated estrogen receptor has a molecular architecture homologous to those of glucocorticoid and progesterone receptors, even though phylogenetically the estrogen receptor gene forms a distinct subgroup within the gene family of nuclear hormone receptors. Topics: Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Chromatography, Gel; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Immunoglobulin G; Kinetics; Macromolecular Substances; Receptors, Estrogen; Succinimides; Tamoxifen; Tumor Cells, Cultured | 1995 |
Major molecular weight heterogeneity of estrogen receptor from breast cancer is not related to neoplasia.
Recent investigation from our laboratory revealed that the estrogen receptor (ER) from breast cancer is characterized by a high molecular weight polymorphism: SDS-polyacrylamide gel electrophoresis of [3H]-tamoxifen aziridine ([3H]-TAZ) labeled cytosols usually display several bands corresponding to the native receptor (67 KDa) and lower molecular cleavage products. High frequency of such altered receptors was confirmed here by size exclusion FPLC of [125I]-E2 labeled cytosols from a series of 98 breast cancers: on the average, 60% of the ER molecules were strongly degraded (Mr < or = 37 KDa). The absence of transcriptional activating domains (ABC domains) in such receptors was further demonstrated by assessing their ability to bind to hydroxylapatite (HAP). Thus, in presence of 500 mM KCI, 55% of ERs from another series of 54 cytosols failed to strongly adsorb to this phosphocalcic matrix, a characteristic property of receptors without exposed ABC domains. Finally, [3H]-TAZ labeled cytosols from normal uterine tissue and MCF-7 human breast cancer cells growing in nude mice displayed identical multibands electrophoretic patterns revealing in both cases native and cleaved receptors. Since latter receptor forms were never detected in MCF-7 cells growing in monolayer culture, we put forward the hypothesis that they were produced under the action of proteolytic enzymes acting at the time of tissue processing. Hence, most of the truncated receptors detected in human breast cancer cytosols should not be markers of malignancy. Topics: Animals; Breast Neoplasms; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Female; Humans; Mice; Mice, Nude; Molecular Weight; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Protein Processing, Post-Translational; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured; Uterus | 1995 |
The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours.
Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6.1, 6.3, 6.6 and 6.8. Of these the first (pI 6.1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6.3 and pI 6.8 were significantly correlated with the presence of progesterone receptors. To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6.3, 6.8 and 6.6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6.3, 6.8 and 6.6 isoforms, could form dimers. We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome. Topics: Blotting, Western; Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Estradiol; Female; Humans; Isoelectric Focusing; Molecular Weight; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen | 1993 |
Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.
In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Aziridines; Binding Sites; Breast Neoplasms; Escherichia coli; Estradiol; Humans; Ketones; Kinetics; Ligands; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Peptide Fragments; Protein Binding; Protein Conformation; Receptors, Estrogen; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Tamoxifen | 1992 |
Accumulation of a non-binding form of estrogen receptor in MCF-7 cells under hydroxytamoxifen treatment.
It is well known that MCF-7 cells, when incubated with hydroxytamoxifen (OH-Tam) loose their capacity to bind [3H]estradiol. By using Western blotting and [3H]tamoxifen aziridine labeling of KCl extracts from these cells we found that this loss in binding capacity was not associated with a disappearance of the estrogen receptor (ER) protein, an event known to occur after incubation with estradiol. Attempts to label under exchange conditions these ER molecules, which, on the basis of enzyme immunoassays appear to accumulate under OH-Tam treatment, were unsuccessful. Cell fractionation suggested that their origin is nuclear. Assessment of a few triphenylethylenic antiestrogens, as far as their inhibitory potency towards the in vitro MCF-7 cell growth is concerned, indicated a correlation between accumulation of these non-binding ER molecules and the antiestrogen antiproliferative action. However, we were unable to demonstrate absence of such an ER accumulation in two tamoxifen-resistant variants. Impaired folding of the ER protein or impaired phosphorylation of its hormone-binding domain are attractive hypotheses to account for these non-binding ER molecules. Whether these ER molecules have any physiological role, such as competition with the "normal" receptor molecules for the estrogen responsive elements on the DNA is unknown and deserves further study. Topics: Blotting, Western; Breast Neoplasms; Cell Line; Centrifugation, Density Gradient; Estradiol; Estrogen Antagonists; Female; Humans; Molecular Weight; Receptors, Estrogen; Receptors, Progesterone; Structure-Activity Relationship; Tamoxifen; Tumor Cells, Cultured | 1992 |
Comparison of tritiated estradiol and tamoxifen aziridine for measurement of estrogen receptors in human breast cancer cytosols.
We examined the estrogen receptor measurement in 265 human breast cancer cytosols by using a specific method based on [3H]tamoxifen aziridine labeling, sequential immunoadsorption with an antiestrogen receptor monoclonal antibody (H-222), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These new tools of molecular endocrinology revealed an impressive estrogen receptor molecular polymorphism. Given the recent finding of a similar estrogen receptor polymorphism at the messenger RNA level by several laboratories, it is tempting to speculate about its possible biological significance. To gain insight into the potential clinical relevance of this polymorphism in terms of breast cancer hormone dependence, we compared the 265 cytosols for their [3H]tamoxifen aziridine- and [3H]estradiol-binding capacities using the above-mentioned method and the conventional dextran-coated charcoal assay. We failed to identify a specific [3H]tamoxifen aziridine electrophoretic pattern with respect to the tumor estrogen receptor content as measured by the dextran-coated charcoal assay. However, an excellent correlation overall was found between the intensities of both labeling methods. Some tumors were positive for only one of these two ligands. It will be clinically important to see whether the tumors positive for [3H]tamoxifen aziridine only correspond to the small subset of tumors (10%) which respond to tamoxifen treatment despite very low estrogen receptor levels, as measured by the dextran-coated charcoal technique. Topics: Antibodies, Monoclonal; Autoradiography; Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Electrophoresis, Polyacrylamide Gel; Estradiol; False Negative Reactions; Humans; Receptors, Estrogen; Tamoxifen; Tritium | 1991 |
Effect of ATP on the regulation of the steroid binding activity of the oestradiol receptor.
We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed. Topics: Adenosine Triphosphate; Affinity Labels; Animals; Breast Neoplasms; Cell Nucleus; Cytosol; Estradiol; Female; Humans; Immunosorbent Techniques; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinases; Receptors, Estradiol; Sheep; Tamoxifen; Tumor Cells, Cultured; Uterus | 1990 |
Modulation of the estrogen receptor structure, evidence of a heterogeneity.
In order to analyse the molecular weight polymorphism of the estrogen receptor (ER) in MCF-7 cells, we have developed a procedure which allowed in situ linkage of ER by (3H) tamoxifen aziridine and provided labelled proteins in conditions which minimized protease activities. After labelling, cell lysis was performed in SDS buffer containing various concentrations of [symbol: see text]-mercaptoethanol. Proteins extracted with phenolic solution and precipitated by cold acetone were analysed by SDS PAGE. It appears that beside the form of 67 kDa already described, binding entities of tamoxifen aziridine were also present at a molecular mass of 110 kDa and 45 kDa. On the other hand, investigations on the effect of 12-0-Tetradecanoyl Phorbol 13-Acetate (TPA) showed that TPA induces a decrease of the 67 kDa entity. Topics: Breast Neoplasms; Endopeptidases; Humans; Intracellular Fluid; Molecular Weight; Receptors, Estrogen; Sodium Dodecyl Sulfate; Tamoxifen; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured | 1990 |
Target size analysis of estrogen receptor in cultured intact cells: change in receptor structure between subconfluency and superconfluency in culture.
MCF-7 human breast cancer cells were submitted to the tritiated antiestrogen tamoxifen aziridine, frozen at -170 degrees C, stored and irradiated at -78 degrees C in a calibrated Gammacell 60Co irradiator. A three-step protein extraction procedure provided protein samples for the determination of the target size (TS) of the covalently labelled estrogen receptor (ER). From the TS it is shown that ER bound to an antiestrogen was, in whole cells, part of a 265 kDa polypeptide structure if measured in MCF-7 cells at subconfluency, or of a 360 kDa species in superconfluent cells. Topics: Breast Neoplasms; Cobalt Radioisotopes; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Weight; Receptors, Estrogen; Tamoxifen; Tritium; Tumor Cells, Cultured | 1990 |
Phosphorylation of the estradiol receptor in MCF-7 human breast cancer cells in culture.
Double labelling and Western blot techniques were used to demonstrate phosphorylation of estradiol receptor. Cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine [( 3H]TA). Labelled receptor was precipitated with the monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or transferred to nitrocellulose paper. Receptor protein was detected on the Western blot with the monoclonal antibody H222 and rabbit anti-rat peroxidase conjugate. Phosphorylated receptor was visualized by autoradiography. Tritium and 32P activities were monitored in the gels. Two phosphorylated forms of the receptor (molecular weights 67 and 50 kDa) have been detected in MCF-7 cells. Estradiol treatment of the cells was found to increase phosphorylation of the receptor. In estradiol-treated cells both phosphorylated receptor forms were present mainly in the nuclear extract. Both forms bound [3H]TA as evidence by SDS-PAGE. [3H]TA binding was abolished by excess non-radioactive estradiol. In addition two phosphorylated proteins of approximately 120 and 90 kDa were regularly coprecipitated with receptor in cytosol. These proteins did not bind [3H]TA. The 90 kDa phosphorylated protein was identified as a heat shock protein (hsp-90). Topics: Blotting, Western; Breast Neoplasms; Cell Nucleus; Cytosol; Electrophoresis, Polyacrylamide Gel; Estradiol; Heat-Shock Proteins; Humans; Molecular Weight; Phosphorylation; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured | 1990 |
Electrophoretic analysis of large molecular weight forms of the estrogen receptor in MCF-7 human breast cancer cells.
The denatured and nondenatured forms of the estrogen receptor (ER) in MCF-7 human breast cancer cells have been characterized by gel electrophoresis using the covalently attaching ligand [3H]tamoxifen aziridine ([3H]TA). A comparison of the ER binding properties of the recently introduced commercial preparation (Amersham) of [3H]TA with the prototype showed no quantitative or qualitative differences, with both preparations binding efficiently and selectively at either 4 degrees C or 37 degrees C. The nuclear and cytosolic labeled ER, when analyzed by denaturing SDS-PAGE, migrate as 67,000 dalton proteins. [3H]TA labeled ER from cytosol prepared in low salt buffer without protease inhibitors migrates on 5%, 6%, or 7% nondenaturing PAGE as two discrete forms. The molecular weights of these two species, determined by Ferguson plot analysis with multiple standard protein markers, are 260,000 and 67,000 daltons, respectively. The 260 kDa form is dissociated by 0.4 M KCl into the 67 kDa form, suggesting that the larger form is composed of one or more noncovalently attached subunit proteins. When enzyme inhibitors leupeptin and molybdate are included in cytosol prepared in low salt, two high molecular weight [3H]TA labeled peaks are identified, the first 600,000 and the second 430,000 daltons. These two forms migrate together on 4%, 5%, and 6% non-SDS, non-denaturing gels, and are not dissociated when cytosol is made 0.4 M in KCl. These findings indicate that the ER of these human breast cancer cells exists in one or more large discrete forms of mol. wt 430,000-600,000 daltons. These forms are not dissociated by high salt but may be digested by proteases to the subunit 67,000 dalton form. These findings support the proposal that the ER is present as a large molecular weight "holoreceptor", and may be important for our understanding the metabolism and function of the ER in estrogen target tissues and human breast cancer. Topics: Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Weight; Radioligand Assay; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured | 1989 |
Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis.
Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule. Topics: Affinity Labels; Animals; Aziridines; Azirines; Breast Neoplasms; Cell Line; Cell Nucleus; Female; Humans; Ketones; Peptide Fragments; Peptide Hydrolases; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Receptors, Estrogen; Tamoxifen; Uterus | 1988 |
Estrogenic affinity labels: synthesis, irreversible receptor binding, and bioactivity of aziridine-substituted hexestrol derivatives.
To develop an affinity label for the estrogen receptor that would be an estrogen agonist, rather than antagonist, we prepared several aziridine derivatives of the potent nonsteroidal estrogen hexestrol [3R,4S)-3,4-bis(4-hydroxyphenyl)hexane) bearing an aziridine function on the side chain. Three functional groups link the hexestrol ligand and the aziridine: a carbonyl group (ketone or ester), a thioether, or a methylene chain. The apparent competitive binding affinity of these derivatives for the estrogen receptor ranges from 1.8% to 25% that of estradiol, and most of them bind in a time-dependent, irreversible manner with the receptor, although the rate and efficiency of this binding vary widely, often with relatively small changes in structure. This is consistent with the irreversible attachment requiring a precise alignment of activating and reacting residues in the binding site of the receptor. The estrogenic and antiestrogenic activity of these aziridine derivatives was investigated in MCF-7 human breast cancer cells. Most of the compounds are agonists, with one being an antagonist. The derivative (6R,7S)-1-N-aziridinyl-6,7-bis(4-hydroxyphenyl)-5-nonanone (keto-nonestrol aziridine 3) appears to have the most ideal behavior of the estrogenic affinity labeling agents prepared: It is an agonist, and it binds to receptor irreversibly, efficiently, and quite rapidly. Topics: Affinity Labels; Animals; Aziridines; Azirines; Binding, Competitive; Breast Neoplasms; Cell Division; Cell Line; Chemical Phenomena; Chemistry; Estrogen Antagonists; Female; Hexestrol; Humans; Receptors, Estrogen; Sheep; Structure-Activity Relationship; Uterus | 1987 |
Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity.
We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region. Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Cell Nucleus; Cytosol; Female; Humans; Molecular Weight; Peptide Fragments; Rats; Receptors, Estrogen; Tamoxifen; Trypsin; Uterus | 1987 |
Selection and characterization of a breast cancer cell line resistant to the antiestrogen LY 117018.
We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M. The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant. LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance. Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2. The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7. The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin. The nuclear estrogen receptor when covalently labeled with [3H]tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a competitive binding assay, LY 117018 competes for [3H]estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells. When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells. However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with [35S]methionine. The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer. In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study. Topics: Binding, Competitive; Breast Neoplasms; Cell Division; Cell Line; Drug Resistance; Estradiol; Female; Humans; Molecular Weight; Phenotype; Pyrrolidines; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Thiophenes | 1985 |
Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen.
We have used a covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])1,2-diphenyl-1-butene], to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens, and the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to [3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KCl extract of the nuclear fraction; this receptor has an apparent mol wt of 63,000 +/- 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The mol wt and pI of cytosol receptor labeled with [3H] TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10(-6) M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis of nuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 mol wt species; no significant quantities of lower mol wt TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen-binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar mol wt and isoelectric points for soluble cytosol and nuclear extracted receptors under strongly denaturing and disaggregating conditions reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells. Topics: Affinity Labels; Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Cell Nucleus; Cytosol; Estradiol; Female; Humans; Isoelectric Point; Kinetics; Molecular Weight; Receptors, Drug; Receptors, Estrogen; Tamoxifen; Tritium | 1984 |
Efficient and highly selective covalent labeling of the estrogen receptor with [3H]tamoxifen aziridine.
Topics: Animals; Breast Neoplasms; Cell Line; Cytosol; Estradiol; Female; Humans; Isotope Labeling; Kinetics; Molecular Weight; Rats; Receptors, Estrogen; Substrate Specificity; Tamoxifen; Tritium; Uterus | 1983 |
Tamoxifen aziridines: effective inactivators of the estrogen receptor.
Two analogs of the antiestrogen tamoxifen, which bear a chemically reactive aziridine function in place of the dimethylamino group, bind to the estrogen receptor from rat uterus and from MCF-7 human breast cancer cells and appear to react irreversibly with the receptor at the estrogen binding site, in a time-and concentration-dependent fashion. BEcause these compounds are effective receptor inactivators in uterus and breast cancer cells, they should prove to be useful probes for investigating the role of receptor in regulating cellular responses to estrogen and in studying the dynamics of estrogen receptor synthesis and turnover. Topics: Animals; Breast Neoplasms; Cell Line; Cell Nucleus; Cytosol; Female; Humans; Kinetics; Rats; Receptors, Estrogen; Structure-Activity Relationship; Tamoxifen; Uterus | 1981 |