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Biomedical subjects

G L Greene

Publications and source records attributed to G L Greene.

At least 109 records · Page 6Linked to original sources
PubMed1983

Estrogen receptors in human prostate: evidence for multiple binding sites.

The existence of estrogen receptors in the human prostate has long been a controversial issue. This may be explained partly by the apparent heterogeneity of estrogen-binding sites in prostatic tissue. We herein report on multiple binding sites for estrogens in cytosol as well as nuclear preparations of human prostatic tissues. One class of binding sites corresponds to the classical, high affinity estrogen receptor; the Kd for [3H]estradiol binding to the receptor was approximately 0.10 nM and the binding was specific for estrogens. The second class of binding sites appeared to have a Kd for [3H]estradiol in the range of 5-10 nM. This second, lower affinity class of binding sites markedly influenced studies of the classical receptor even at low ligand concentrations. Saturation analysis should be performed over a wide range of ligand concentrations (0.05-10 nM) to allow separation of the two binding components. Quantitation of estrogen receptor by a single point assay cannot be carried out accurately unless the low affinity binding component can be blocked. Multiple binding sites for estradiol were observed in the cytosol as well as in the nuclear salt extractable and salt-resistant compartments of normal, benign hyperplastic, and cancerous human prostates. Normal peripheral and cancerous prostates contained significantly (P less than 0.01) higher amounts of cytosol estrogen receptor compared to benign hyperplastic tissue.

Binding, Competitive↗
PubMed1982

Monoclonal antibodies as probes for estrogen receptor detection and characterization.

Splenic lymphocytes from a Lewis rat, immunized with purified estradiol-receptor complex of calf uterine nuclei, were fused with cells of three mouse myeloma lines to yield several monoclonal lines of hybridoma cells that secrete antiestrophilin antibodies, which, like the antiserum of the immunized rat, react specifically with estrophilin of calf tissues. In contrast, a Lewis rat, immunized with cytosol estradiol-receptor complex from MCF-7 human breast cancer cells after purification by a novel affinity chromatography technique, gave antiserum that crossreacts with receptor from mammalian as well as avian tissues. Monoclonal antibodies secreted by three hybridoma cell lines derived from this immunized rat showed interesting differences in cross reactivity. All recognized receptor from primate sources, two of the three monoclonal preparations recognized receptor from calf and rat uterus as well, but none reacted with estrophilin from hen oviduct. Thus, in addition to similarities, there appear to be immunocytochemical differences between estrogen receptors from mammalian and avian sources and between receptors from primate and non-primate tissues. These monoclonal antibody preparations, recognizing different determinants on the estrophilin molecule, provide a novel approach to the study of receptor structure and function as well as the basis for a simple immunoradiometric determination of estrogen receptors in human breast cancers. Preliminary studies indicate that they also may prove useful for the immunocytochemical detection of estrophilin in tissue sections.

Animals↗
PubMed1980

Monoclonal antibodies to estrophilin: probes for the study of estrogen receptors.

Splenic lymphocytes from a Lewis rat, immunized with purified estradiol-receptor complex of calf uterine nuclei, were fused with cells of three different mouse myeloma lines (P3-X63-Ag8, P3-NSI/I-Ag4-1, and Sp2/0-Ag14) to yield hybridoma cultures, 9% of which produced antibodies to the receptor protein (estrophilin). When cloned by limiting dilution, approximately 70% of the viable cultures secreted antiestrophilin antibody. When expanded in suspension culture, three clones derived from Sp2/0-Ag14 were found to secrete rat IgG (gamma 2a class), whereas seven other clones (from all three myeloma lines) secreted IgM. Monoclonal IgG shows comparable affinity for nuclear and extranuclear receptors, whereas IgM reacts preferentially with the nuclear form. Both classes of antibody react with unoccupied as well as with occupied receptor and do not interfere with its ability to bind to estradiol. By growing IgG-secreting clones in the presence of [35S]methionine, radiolabeled monoclonal antiestrophilin has been prepared. Unlike antiestrophilin antibody previously generated in the rabbit or the goat, which crossreacts with estrogen receptors from every animal species tested, antibodies produced by the Lewis rat and by hybridomas derived from its spleen cells react specifically with estrophilin from calf tissues. These monoclonal antibodies provide reagents for the application of immunochemical techniques to study estrogen receptors in calf target tissues.

Animals↗
PubMed1980

Monoclonal antibodies to human estrogen receptor.

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.

Animals↗
PubMed1979

The immunoendocrinology of estrophilin.

Immunoglobulin from the serum of rabbits immunized with highly purified estradiol-receptor complex from calf uterine nuclei has been shown to contain specific antibodies to estrophilin by five criteria. Antibodies to calf nuclear estrophilin cross react with nuclear estradiol-receptor complexes of rat, rabbit and sheep uterus, rat endometrial and pituitary tumor, and MCF-7 human breast cancer cell line. They also react with extranuclear receptor of calf, rat, mouse, rabbit, guinea pig, monkey and sheep uterus, rat mammary, endometrial and pituitary tumor, and human breast cancer. There is no interaction of the antibody with estradiol itself. The nuclear form of estrophilin appears to bind more immunoglobulin molecules than does the cytosol form. The antibodies do not react with either the nuclear or extranuclear dihydrotestosterone-receptor complexes of rat prostate, with the extranuclear progesterone-receptor complexes of rabbit uterus, chick oviduct or rat endometrial tumor, or with rat and mouse alpha-fetoprotein. These findings indicate an immunochemical similarity among estrophilins from several mammalian species, as well as between nuclear and extranuclear forms of the receptor, but not among receptor proteins for different steroid hormones. Immunoglobulin from the serum of a goat immunized with similar antigen shows a considerably higher titer of antibodies to estrophilin. These react with nuclear and extranuclear estradiol-receptor complexes of calf uterus to produce somewhat larger entities than those formed with the rabbit antibody. Unlike the rabbit antibody, interaction with the goat antibody causes a noticeable decrease in estradiol-binding affinity of the extranuclear estrophilin as well as an apparent decrease in the total hormone-binding capacity. Specific antibodies to estrophilin offer promise as valuable reagents for receptor analysis and purification, as well as for the elucidation of many still unresolved questions concerning receptor synthesis, localization and function.

Animals↗
PubMed1977

Antibodies to estrogen receptor: immunochemical similarity of estrophilin from various mammalian species.

Immunoglobulin obtained from the serum of rabbits immunized with a highly purified preparation of estradiol-receptor complex from calf uterine nuclei has been shown to contain specific antibodies to the receptor protein (estrophilin) by four criteria: (a) precipitation of the radioactive steroid upon addition of goat antibody against rabbit gamma globulin to a mixture of the tritiated estradiol-receptor complex and the immunoglobulin, (b) adsorption of the estradiol-receptor complex by the immunoglobulin linked to Sepharose, (c) adsorption of the estradiol-receptor complex in the presence of the immunoglobulin by Staphylococcus aureus protein-A linked to Sepharose, and (d) the ability of the immunoglobulin to increase the sedimentation rate of the estradiol-receptor complex. Antibodies to calf nuclear estrophilin were shown to crossreact with the nuclear receptor of rat uterus, as well as with the extranuclear receptor of calf, rat, mouse, and guinea pig uterus and of human breast cancer. The antibodies do not react with either the nuclear or extranuclear dihydrotestosterone-receptor complexes of rat prostate or with the extranuclear progesterone-receptor complex of chick oviduct. These findings indicate an immunochemical similarity among estrophilins from several mammalian species, as well as between extranuclear and nuclear forms of the receptor, but not among receptor proteins for different steroid hormones.

Animals↗
PubMed1974

Use of phosphorus oxychloride in synthesizing nucleotides and oligonucleotides.

Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3'-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; beta-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact. Application of the technique is illustrated by the synthesis of dpT, dTp, d(CF(3)CONH)Tp, dTp(N)Tp, and dTp(N)Tp(N)Tp. In addition, the utilization of phosphorus oxychloride in joining thymidine derivatives and dinucleoside phosphotriester blocks via phosphodiester links is described.

Deoxyribonucleotides↗
PubMed1968

Endogenous gibberellins of a radiation induced single gene dwarf mutant of bean.

The distribution of endogenous gibberellins in Dwarf-1, a single gene dwarf mutant of Mexico 80-R red beans, was studied. Parallel extraction and fractionation of seeds of this mutant and those of a normal homozygous line followed by thin layer chromatography and bioassays using Rumex obtusifolius, wheat seed endosperm and dwarf bean plants revealed that a stem elongation control factor was contained in the non-acidic fraction from normal, but not from Dwarf-1, seeds. It was concluded that the single gene mutation causes a block either in gibberellin precursor formation or in production of a non-acidic fraction gibberellin-like substance.

Journal Article↗
PubMedDate not supplied

Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis-acting EREs.

Animals↗
PubMedDate not supplied

Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells.

Although it is widely accepted that specific intracellular receptor proteins are involved in the oestrogenic regulation of gene expression and growth in reproductive tissues, the precise nature of the regulation is poorly understood. Among the unresolved issues are the distribution and dynamics of the oestrogen receptor protein (oestrophilin) in target tissues in the presence and absence of oestrogens and antioestrogens. The use of radiolabelled and unlabelled receptor ligands to detect and measure oestrogen receptors in tissues has been complicated by the presence of other intracellular steroid-binding proteins and by the low concentration of receptors in responsive tissues. We report here the development of an immunocytochemical procedure that is suitable for localizing oestrophilin directly in frozen tissue sections or cells from human and several non-human sources. When monoclonal antibodies to oestrophilin were used to detect receptor in various oestrogen-sensitive tissues, specific staining was confined to the nucleus of all stained cells, suggesting that both cytosol and nuclear forms of the receptor protein may reside in the nuclear compartment.

Antibodies, Monoclonal↗