Biomedical subjects

G L Greene

Publications and source records attributed to G L Greene.

At least 73 records · Page 4Linked to original sources

PubMed1988

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines.

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗

PubMed1988

Purification of T47D human progesterone receptor and immunochemical characterization with monoclonal antibodies.

In order to obtain steroid-independent probes for human progesterone receptor (PR), the A [88-93 kilodalton (kDa)] and B (109-119 kDa) forms of PR from T47D human breast cancer cells were partially purified and used to generate a series of 14 monoclonal antibodies. Initially, unoccupied PR was isolated from cytosol extracts by steroid affinity chromatography, followed by chromatography on diethylaminoethyl Bio-Gel. The partially pure (3-15%) PR consisted of two steroid-binding components that migrated at 89 kDa and 109 kDa in reducing sodium dodecyl sulfate gels after being photoaffinity labeled with the synthetic progestin [3H]R5020. Two unique monoclonal antibodies to PR were derived from a male Lewis rat immunized with this material. One of these antibodies (JU601) was coupled to Sepharose 4B and used to purify T47D nuclear PR for additional immunizations. Highly purified (30-70%) PR migrated as 93 kDa and 119 kDa progestin-binding proteins in sodium dodecyl sulfate gels. In all, thirteen monoclonal antibodies were obtained that recognized epitopes shared by both receptor forms. One mouse immunoglobulin G (KC146) was completely specific for the larger B form. Interestingly, the epitope for this antibody was present on all PRs tested, including the B form of PR from chicken oviduct, whereas nine other antibodies recognized only human PR and the remaining four cross reacted with rabbit PR. With the exception of the JU145 and JU601 rat immunoglobulin Ms, all antibodies appeared to be completely specific for the A or B forms of PR. Each recognized the cytosol and nuclear forms of occupied as well as unoccupied PR. Although the relationship between B and A was not established, it is clear that an amino-terminal region of B is not present in A, and that a significant portion of A and B are either identical or very similar in amino acid sequence.

Antibodies, Monoclonal↗

PubMed1988

Progesterone receptor distribution in the human endometrium. Analysis using monoclonal antibodies to the human progesterone receptor.

Two monoclonal antibodies to the human progesterone receptor (PR), JZB39 and KD68, were used in determining the immunohistochemical distribution of PR in the human endometrium throughout the menstrual cycle and after menopause. These antibodies recognized PR, as demonstrated by a downfield shift in the radiolabeled progestin binding peak when KD68 or JZB39 was added to high salt sucrose density gradients. The specificity of both antibodies for PR was confirmed with Western immunoblots and competition studies performed with purified receptor. Progesterone receptor was identified with these antibodies and the peroxidase-antiperoxidase technique in the nuclei of epithelial cells, stromal cells, and myometrial smooth muscle cells. The receptor content of endometrial epithelium and stroma varied with the menstrual cycle. The variation was most marked in the epithelium, which demonstrated very strong PR immunostaining during the proliferative phase and postovulation Days 1-3 of the early secretory phase, but PR immunostaining decreased sharply at postovulation Day 4 and remained relatively weak or absent during the mid and late secretory phase. In contrast, stromal cell nuclei were moderately to strongly immunostained even during the secretory phase. Progesterone receptor was not localized in vascular smooth muscle cells or endothelial cells. Specific cytoplasmic staining for PR was not identified in any of these cases, even prior to ovulation, when circulating levels of progesterone are low, indicating that both the steroid-occupied and -unoccupied forms of human progesterone receptor, like rabbit and guinea pig PR, and estrogen receptor, is a nuclear protein.

Antibodies, Monoclonal↗

PubMed1988

Progesterone receptors in the human heart and great vessels.

Progesterone receptors (PgR) were identified in 31 of 50 specimens of human (men and women) thoracic ascending aorta, internal carotid, coronary artery, and left atrial appendage. This was accomplished with a peroxidase-antiperoxidase immunocytochemical assay employing a highly specific monoclonal antibody to primate PgR. In the aorta, specific staining was seen in the nuclei of smooth muscle cells and endothelium of intima, media, and adventitia. In the myocardium, staining was localized to the nuclei of the myocardial fibers. In internal carotid and coronary arteries, PgR was localized to endothelial nuclei of intima, and in vascular channels within the atherosclerotic plaques. PgR was also visible in the smooth muscle cell nuclei of uninvolved media and intima and at the plaque periphery. In contrast, receptor was not identified in vessels of the human uterus, breast, prostate, kidney, or gastrointestinal tract. These findings suggest that the heart and great vessels are target organs for steroid hormones.

Adult↗

PubMed1987

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases.

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

Antibodies, Monoclonal↗

PubMed1987

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.

Animals↗

PubMed1987

Phosphorylation of the chicken progesterone receptor.

We have examined the phosphorylation of the chicken progesterone receptor in tissue slices and in vitro. The receptor is phosphorylated in tissue slices and this phosphorylation is stimulated by progesterone. As others have reported, partially purified receptor preparations contain a kinase activity which phosphorylates histones and receptor. We have shown that this activity can be separated from the receptor. The receptor is a substrate for several kinases, including the catalytic subunit of the cAMP-dependent protein kinase and PPdPK, a polypeptide-dependent protein kinase. Phosphorylation by the cAMP-dependent protein kinase results in an apparent increase in the molecular weight of the receptor when the receptor is analyzed by SDS-PAGE. These results are consistent with apparent changes in molecular weight observed for rabbit and human progesterone receptor upon treatment of tissue or cells with hormone.

Animals↗

PubMed1987

The progesterone receptor gene maps to human chromosome band 11q13, the site of the mammary oncogene int-2.

Progesterone is involved in the development and progression of breast cancers, and progesterone receptors (PR) are important markers of hormone dependence and disease prognosis. We have used a human PR cDNA probe, genomic DNA blotting of a series of Chinese hamster-human cell hybrids, and in situ hybridization to map the human PR gene to chromosome 11, band q13. This band also contains the human homolog of the mouse mammary tumor virus integration site, int-2, which surrounds a protooncogene thought to be involved in the development of murine mammary cancers. That these two genes share the same chromosomal location raises important questions about their possible linkage and about the relationship between the mammary-specific oncogene and the steroid hormone in the development, growth, and hormone dependence of human breast cancers.

Animals↗

PubMed1987

Immunostaining of estrogen receptor in paraffin sections of breast carcinomas using monoclonal antibody D75P3 gamma: effects of fixation.

Immunostaining of estrogen receptor was carried out on paraffin sections of breast carcinomas using an anti-estrophilin monoclonal antibody (D75P3 gamma) and the avidin-biotin technique. The tumors were fixed in Bouin's solution or in formalin for varying periods of time at room temperature or at 4 degrees C. Best results were obtained following fixation in Bouin's at room temperature or in formalin at 4 degrees C. The staining was localized in the nuclei of carcinoma cells and was heterogeneous in intensity and extent. Prolonged fixation resulted in decreased immunoreactivity and in the appearance of nonspecific cytoplasmic and background staining. The estrogen receptor immunostaining on paraffin sections was found to be in concordance with that on frozen sections (Abbott ER-ICA) and with the steroid-binding assay (dextran-coated charcoal) in over 90% of the cases. This method is of easy and rapid execution and yields reliable and reproducible results.

Aged↗

PubMed1987

Estrogen receptor localization in formalin-fixed, paraffin-embedded endometrium and endometriotic tissues.

Monoclonal estrogen receptor (ER) antibody and indirect immunoperoxidase techniques were used to study the immunohistochemical localization of ER in paraffin sections of 20 surgical specimens containing foci of endometriosis. The eutopic endometrium, available in 16 of 20 cases, was dated histologically and stained for ER. Specific nuclear staining was observed in glandular and stromal cells of the endometrium during the proliferative and early secretory phases. Mid and late secretory phase endometria showed only focal staining. Dating of the endometriotic tissues using morphologic criteria revealed that these were in phase with the endometrium in nine of 16 cases. In these cases the ER staining of both the endometriotic tissues and endometrium was similar. The endometriotic tissues could not be dated as either proliferative or secretory in seven cases and stained variably for ER. Diffuse nuclear staining for ER was present in the endometriotic glands of three of these cases in which the endometriotic glands morphologically resembled glands of the basalis contrasting with weak, focal staining in the corresponding eutopic secretory endometria. The majority of endometriotic tissues examined mimicked the cyclic ER expression of the eutopic endometrium and thus appeared to correspond to the morphologic appearance of the endometriotic glands. Immunohistochemical staining with monoclonal estrophilin antibodies may be helpful in supporting the histopathologic diagnosis of endometriosis.

Adult↗

PubMed1987

Estrogen receptors in the nuclear matrix: direct demonstration using monoclonal antireceptor antibody.

Estradiol-binding sites, as assayed by exchange with radiolabeled steroid, become associated with the nuclear matrix of estrogen-responsive tissues after treatment with estrogen in vivo. Using monoclonal estrogen receptor antibodies, we have now obtained direct evidence that these matrix-associated estradiol-binding sites are estrogen receptor proteins similar to those found in the cytosol before estrogen treatment. Proteins of the liver nuclear matrix from untreated or ethinyl estradiol-treated female rats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and probed with the monoclonal estrogen receptor antibody H222Sp gamma. A single prominent immunoreactive 67,000 mol wt band, indicating the presence of estrogen receptors, was found in the liver nuclear matrix of estrogen-treated animals. This band was detectable, but of much lower intensity, in the liver nuclear matrix of untreated animals. Liver cytosol estrogen receptor from untreated rats also migrated as a 67,000 mol wt band. These immunoreactivity data corroborated data obtained by [3H]estradiol-binding assays. Scatchard analysis of specific high affinity [3H]estradiol-binding sites showed high levels of these sites in the liver nuclear matrix of estrogen-treated rats and low levels in untreated rats. Therefore, both direct and indirect methods of receptor identification demonstrate the specific association of estrogen receptors with the nuclear matrix after estrogen treatment in vivo.

Animals↗

PubMed1987

Structural organization and regulation of the chicken estrogen receptor.

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.

Animals↗

PubMed1987

Immunochemical studies of estrogen receptors.

The preparation of specific antibodies, and especially monoclonal antibodies, to human estrophilin has furnished a means of detecting and studying receptor localization, structure and function, independent of its ability to bind labeled steroid. These reagents have been especially useful in providing improved methods for the determination of estrogen receptors in human breast cancers, as a guide to clinical prognosis and therapy, and in facilitating the cloning of estrophilin cDNA leading to an elucidation of the aminoacid sequence of the receptor molecule.

Animals↗

PubMed1986

Receptor interconversion model of hormone action. II. Nucleotide-mediated conversion of estrogen receptors from nonsteroid binding to the lower affinity binding state.

Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third nonestrogen binding form of the receptor, designated Rnb, is now described which exists in short-term estrogen withdrawn chick oviduct cytosol. A model is presented in which the receptor can be interconverted between the three states. The interconversion is monitored by Scatchard analysis, sucrose density gradient analysis, and affinity labeling using [3H]tamoxifen aziridine followed by receptor purification with estrogen receptor monoclonal antibody affinity chromatography and sodium dodecyl sulfate-gel electrophoresis. The results are consistent with each state existing in different conformations having a common molecular weight of approximately 66,000. This paper defines the conditions and nucleotide requirements for the Rnb to Ry conversion. The conversion to the steroid binding form is induced by ATP, ADP, and GTP. Cyclic nucleotides are ineffective. There is a specific requirement for Mg2+; neither Ca2+ nor Mn2+ will substitute. Nonhydrolyzable nucleotide analogues were tested for their relative efficiency to convert Rnb to Ry. Conversion occurred with alpha,beta-methylene adenosine triphosphate, but beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate were inert. Thus, activation of Rnb to form Ry appears to be catalyzed by an event requiring the loss of the terminal phosphoryl moiety from either ATP or ADP. Receptor derived from conversion of Rnb to Ry has the same physical properties as native Ry. Activation of Rnb is to Ry specifically; no increase in the Rx form of estrogen receptor was ever observed. The accompanying paper similarly describes the Rx to Ry conversion. Since these data also explain observations made with glucocorticoid and with epidermal growth factor receptors, it is speculated that the receptor interconversion model may have general application to hormone action.

Adenine Nucleotides↗

PubMed1986

Molecular cloning of the chicken progesterone receptor.

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Amino Acid Sequence↗

PubMed1986

Sequence and expression of human estrogen receptor complementary DNA.

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.

Amino Acid Sequence↗

PubMed1986

Structure and dynamics of the estrogen receptor.

To evaluate the structure and function of estrogen receptor (ER) in various mammalian systems, the cytosolic forms of receptor from calf uterus and from MCF-7 human breast cancer cells have been purified to virtual homogeneity by sequential selective adsorption to estradiol-Sepharose and heparin-Sepharose. In both cases, the purified steroid-receptor complex appears to exist as an activated 5S homo- or heterodimer of mol. wt 65,000 (4S) steroid-binding subunits. Purified ER has high affinity for DNA and serves as a substrate for phosphorylation by a purified rat brain kinase. Several monoclonal antibodies prepared against affinity-purified MCF-7 cytosol ER have been used to localize receptor by an indirect immunoperoxidase technique in fixed, frozen sections of human breast tumors, human uterus, rabbit uterus and in other mammalian reproductive tissues and cancers, as well as in fixed MCF-7 cell cultures and in paraffin-embedded sections of breast tumors and human endometrium. In all cases, we have observed only nuclear localization of immunoreactive receptor in tissues and whole cells, even under conditions in which virtually all of the receptor is found in a low-salt extract (cytosol) of the target cells. Treatment of cells or tissues in vivo or in vitro with estradiol alters the intensity but not the distribution of specific staining for ER. By immunoelectron microscopy, receptor was localized in the euchromatin, but not in the marginated heterochromatin or nucleoli of MCF-7 nuclei and epithelial and stromal nuclei of postmenopausal human endometrium. These observations suggest that the majority of the unoccupied receptor may actually reside in the nucleus, rather than in the cytoplasm as previously thought. Thus, hormone action may involve binding of the steroid directly to receptor loosely associated with nuclear components, followed by conversion of the steroid-receptor complex to an activated form which becomes more tightly associated with chromatin.

Animals↗

PubMed1986

Estrogen receptor localization in the female genital tract.

The authors have used monoclonal estrophilin antibodies and the peroxidase antiperoxidase technique to characterize the distribution of estrogen receptor in the human vagina, uterus, and fallopian tubes. Exclusively nuclear localization of estrogen receptor was observed in epithelial cells, stromal cells of the lamina propria, and smooth muscle cells with both immunohistologic studies and immunoelectron-microscopy studies. The endometrium in various regions of the uterus (uterine isthmus, corpus, and fundus) was stained for estrogen receptor, with similar staining intensities in each of the respective cell types. There was no systematic regional variation in the staining intensity or distribution of cells with stained nuclei. The functionalis of the endometrium showed distinctive variation in the intensity of the staining for estrogen receptor with the menstrual cycle. The staining intensity of both endometrial epithelium and stroma was greatly reduced in the functionalis during the secretory phase. The vaginal epithelium did show some variation with the menstrual cycle, but it was much less than that observed in the functionalis of the endometrium. The basal cells of the vaginal stratified squamous epithelium, strongly stained during the proliferative phase, were less strongly stained during the secretory phase. No variation in the staining intensity for estrogen receptor was observed in different regions of the fallopian tube (isthmus, ampulla, and infundibulum), and the staining intensity varied only minimally with the menstrual cycle. The serosa of the female reproductive tract, connective tissues in the muscularis and in the vicinity of blood vessels, as well as neutrophils, eosinophils, mast cells, and lymphoid cells in the female genital tract were not stained for estrogen receptor.

Antibodies, Monoclonal↗