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E Milgrom

Publications and source records attributed to E Milgrom.

At least 127 records · Page 7Linked to original sources
PubMed1989

Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers.

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone); it was almost fully active. The second mechanism consisted of the activation of the DNA binding domain. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand, the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.

Animals↗
PubMed1989

Novel monoclonal antibodies against human uterine progesterone receptor. Mapping of receptor immunogenic domains.

Progesterone receptor was purified in a single step from human uteri using immunoaffinity chromatography with monoclonal antibodies raised against the rabbit receptor. A total of 39 monoclonal antibodies were prepared against the human receptor and characterized. Immunoblot experiments using crude uterine cytosol or purified receptor showed that the antibodies belonged to three groups: they recognized either a single receptor species (apparent molecular mass 110 kDa), two species (110 and 79 kDa) or three species (110, 79 and 65 kDa). The species specificity of the antibodies was very variable; some recognized only the human receptor, others interacted with several mammalian receptors (rabbit, guinea pig and rat), while a single one also cross-reacted with the chick receptor. The epitopes recognized by 15 of the antibodies showing the strongest affinity for the human receptor were mapped using a method recently described [Lorenzo, Jolivet & Milgrom (1988) Eur. J. Biochem. 176, 53-60] which involves immunoprecipitation of C-terminally truncated proteins obtained by transcription and translation of cloned cDNA in vitro. The antibodies recognized five different regions of the receptor, all localized on the N-terminal half of the protein. None of the antibodies interacted with an epitope present in the DNA-binding or steroid-binding regions of the receptor. Comparison of the pattern of receptor species recognized by the antibodies and the localization of their epitopes showed that the 79 and 65 kDa receptor species were derived from the 110 kDa form by deletion of its N-terminal part. The N-terminus of the 79 kDa species was found to lie between amino acids 121 and 208, and that of the 65 kDa species between amino acids 208 and 296.

Antibodies, Monoclonal↗
PubMed1989

Immunohistochemical evidence of the presence of estrogen and progesterone receptors in the same neurons of the guinea pig hypothalamus and preoptic area.

Estrogen and progesterone interact in the regulation of various brain functions including mechanisms controlling gonadotropin secretion and female sexual behavior. Various methods have been used to map the regions where these hormones act and where specific receptors can be detected. However, it remains unknown if both steroids act on the same neuron or if there are neural populations responding to either one or the other of these hormones To answer this question, we used various immunocytochemical procedures to detect estrogen and progesterone receptors (ER-IR and PR-IR) on the same histological section, taking advantage of the fact that anti-ER monoclonal antibodies were raised from rats whereas anti-PR monoclonal antibodies were raised from mice. Initial experiments showed that the number of cells displaying ER and PR immunoreactivity changed with hormonal treatment. Prolonged treatment of ovariectomized guinea pigs with high doses of estradiol benzoate (EB) (10 or 15 micrograms/day for 4 or 6 days) increased the number of PR-positive cells in the preoptic area and the hypothalamus. Inversely, with this estrogen priming regimen the number of ER-positive cells decreased, as did the immunostaining intensity in their nuclei. With a lower dose of estrogen administered for a shorter period (2-microgram dose of EB for 2 days) the two receptor populations could be easily observed. Under these hormonal conditions, the sequential procedure revealed that the vast majority of cells containing ER-IR in the preoptic area (nucleus preopticus periventricularis, medialis) and the mediobasal hypothalamus (nucleus periventricularis, arcuatus, ventrolateral part of nucleus ventromedialis and premamillaris) showed PR immunoreactivity. This was true regardless of the order in which dual immunocytochemical staining was performed. There were, however, a few ER-positive/PR-negative cells in these regions. However, only ER-positive neurons were found in the n. interstitialis stria terminalis, the n. amygdaloideus medialis and the n. supraopticus. Thus, in guinea pigs receiving moderate doses of estrogen, all PR-positive cells contain ER-IR whereas there is a population of ER-positive cells which are devoid of PR-IR.

Animals↗
PubMed1989

Immunocytochemical staining of progesterone receptor in paraffin sections of human breast cancers.

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.

Antibodies, Monoclonal↗
PubMed1988

Receptors bound to antiprogestin from abortive complexes with hormone responsive elements.

The mechanism of action of antisteroids is not understood and explanations of their antagonistic activity have been sought at all levels of hormone action. It has been proposed that antisteroids, after binding to receptor, trap it into a non-activated (non DNA-binding) form possibly through interaction with a heat-shock protein of relative molecular mass (Mr) 90,000 (90 K), or that the antisteroids provoke binding of receptor to nonspecific DNA sites but not to hormone responsive elements (HREs), or that the antisteroid-receptor complexes can bind to HREs but form abortive complexes that fail to regulate transcription. We have constructed a deleted cDNA encoding a mutant form of rabbit progesterone receptor which exhibits constitutive activity, that is, binds to HREs in the absence of hormone and thus bypasses the first two steps discussed above. Co-transfection experiments allowed the expression of both constitutive and wild-type receptors in the same recipient cells. Antiprogestin RU486-wild-type receptor complexes completely suppressed the activity of the constitutive receptor on a reporter gene, showing that the inhibition is at the level of their common responsive elements.

Animals↗
PubMed1988

A rapid method of epitope mapping. Application to the study of immunogenic domains and to the characterization of various forms of rabbit progesterone receptor.

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C-terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C-terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action. This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1-60, 101-110, 295-325 and 370-396. There were no antibodies directed against the DNA-binding or the steroid-binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross-reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1-60). The 79-kDa form ('subunit A') of the receptor was shown to lack the two more N-terminally localized immunogenic domains (amino acids 1-60 and 101-110). The 65-kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N-terminal part of the protein.

Animals↗
PubMed1988

Organisation of the entire rabbit progesterone receptor mRNA and of the promoter and 5' flanking region of the gene.

cDNA clones corresponding to the 3' and 5' non coding regions of the rabbit progesterone receptor (rPR) mRNA and genomic clones corresponding to the promoter and 5' flanking region of this gene were isolated and sequenced up to nucleotide -2761. The 3' non coding region is very long (3058-3553 nucleotides) and contains three different polyadenylation sites. Primer extension experiments and S1 mapping showed the existence of 2 transcription initiation sites 699 and 712 bp upstream from the initiator ATG. The promoter region contains two modified TATA boxes: TAGAAA at -17 and TAGA at -37bp. A CAACT sequence is present at position -100 and one consensus binding site for the transcription factor Sp1 is found at position -51. A 317 bp sequence was observed (positions -2590 to -2273) which belongs to the C family of the short interspersed repeats of the rabbit. Sequences resembling the consensus for estrogen and progesterone responsive elements are observed at several locations in the 5' flanking region. The progesterone receptor is present in tissue extracts mainly as a mixture of two molecular species (110 and 79 kDa) whose origin remains currently debated. By Northern blot analysis we have shown, using rabbit and human mRNAs, that these receptor species are not derived from separate mRNAs. Transcription-translation experiments also showed that, at least in vitro, they are not derived by use of different translation initiation sites on the same messenger RNA.

Amino Acid Sequence↗
PubMed1988

Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action.

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.

Animals↗
PubMed1988

Immunocytochemical demonstration of estrogen and progesterone receptors in muscle cells of uterine arteries in rabbits and humans.

Modifications of uterine blood flow are implicated in many important aspects of reproductive physiology and in several of their pathological disorders. These modifications are hormonally regulated but remain poorly understood, and various complex mechanisms have been proposed. The aim of this study was to investigate the presence and some characteristics of estrogen receptors (ER) and progesterone receptors (PR) in uterine blood vessels. Using monoclonal antibodies and immunocytochemistry we observed the presence of ER and PR in muscle cells (tunica media) of uterine arteries of rabbits and women. ER or PR immunoreactivity was not detected in the endothelium of uterine arteries nor in uterine capillaries or veins. Staining for both receptors was also present in arterial walls from the fallopian tube (isthmus and ampulla) and vagina but not in arteries of nonreproductive tissues (intestinal, renal, hepatic, femoral, and pulmonary arteries, aorta). PR immuno-staining was increased by estrogen in all cell types of the rabbit uterus, but the doses necessary to provoke an intense nuclear staining in uterine arteries were higher than those required for observing strong labeling in glandular, stromal, or myometrial cells. These results suggest that, contrary to many hypotheses previously put forward, sex steroid hormones may regulate uterine blood flow through a direct effect on uterine arterial walls.

Animals↗
PubMed1988

Use of immunocytochemistry of progesterone and estrogen receptors for endometrial dating.

Endometrial progesterone and estrogen receptors were studied by immunocytochemistry using monoclonal antibodies during the menstrual cycle in normal women. We initially compared immunocytochemical staining of progesterone and estradiol receptors on endometrial fragments obtained by either aspiration or endometrial biopsy and found that immunocytochemistry could be performed easily on tissue obtained in either way. The immunocytochemical studies showed that the concentration and distribution of receptors changed markedly during the normal menstrual cycle. These changes were distributed in three characteristic phases. During phase I, corresponding to the midfollicular period (days 7-8), a small proportion (25%) of stromal and glandular cells stained positively for the progesterone receptor, whereas estrogen receptor staining was more intense and more frequent (50% of cells). Phase II, which included both the late follicular and early luteal periods (days 9-19), was characterized by a marked staining of progesterone receptors in the majority of glandular cells (75%) and somewhat less abundant and less frequent staining in stromal cells (50%). Estrogen receptor staining was present in about half of the glandular and stromal cells. Phase III, the mid- and late luteal period (days 21-27), was characterized by the disappearance of estrogen and progesterone receptor staining in glandular cells, although faint staining for both receptors was found in stromal cells. These variations in progesterone receptor staining are potentially useful for determining the effect of progesterone on endometrial maturation.

Biopsy↗
PubMed1988

Radioimmunoassay of progesterone receptor in human tissues: application to breast cancer.

A radioimmunoassay method to measure progesterone receptor in rabbit and human tissues was devised and applied to human breast cancer. A specific progesterone receptor antibody was prepared by purifying rabbit receptor by immunoaffinity chromatography, sodium dodecylsulphate-polyacrylamide gel electrophoresis and injection of the isolated 110,000 dalton receptor band into a goat. Immunoblot studies of progesterone target and non-target tissues showed the specificity of the antibody, which was used at a dilution of 1/45,000. The tracer consisted of 125I-labelled electroeluted 110,000 dalton receptor. The sensitivity of the method was 1 fmol/tube for the rabbit receptor and 3 fmol/tube for the human receptor. The intra-assay coefficient of variation was 11% for tumours positive for the progesterone receptor and 9.9% for those on the borderline (10-30 fmol receptor/mg protein). The interassay coefficients of variation were 20 and 19% respectively. The correlation between the radioimmunoassay and a steroid-binding assay was studied in 40 tumour biopsies. In 39 cases, very good correlation was found (r = 0.99); in a single case an immunoreactive protein was detected which apparently bound steroid poorly. One important feature of this method was that receptor immunoreactivity remained unchanged when either the tissue or the cytosol was exposed to a temperature of 20 degrees C for relatively long periods of time. Under the same conditions the steroid-binding capacity declined rapidly. This characteristic of the radioimmunoassay may prevent errors due to improper handling of tissue samples. Such stability was not observed for oestrogen receptors when measured by a sandwich immunoenzymatic method after incubation of tissue at 20 degrees C.

Animals↗
PubMed1987

Characterization of a casein kinase which interacts with the rabbit progesterone receptor. Differences with the in vivo hormone-dependent phosphorylation.

Previous in vivo studies have shown that the rabbit progesterone receptor undergoes two phosphorylation reactions: one basal and a second one which is hormone-dependent. We report here on the presence and characteristics of a kinase activity found in receptor preparations highly purified by immunoaffinity chromatography. 1. This kinase activity is not due to the receptor molecule itself since the two proteins may be separated by several chromatographic and immunological methods. 2. The presence of the kinase in receptor preparations is not an artefact of the purification procedure. The kinase binds to the receptor as shown by coelution in immunoaffinity experiments and during various chromatographies. This interaction probably takes place in vivo and is not artefactually formed during solubilization of the receptor since the kinase also copurifies with receptors isolated from the uterine nuclei of progestin-treated rabbits. 3. This enzyme may be classified as a casein kinase since it readily phosphorylates the latter substrate (Km approximately equal to 0.15 mg/ml) and is not regulated by cyclic nucleotides, Ca2+ and calmodulin or phospholipids. Its classification as a casein kinase I or II is difficult since on the one hand it is inhibited by heparin, activated by polyamines and may use both ATP and GTP, but on the other hand it modifies only serine residues, and is not inhibited by heparin when the receptor itself is employed as a substrate. 4. The kinase which copurifies with the receptor does not mimic in vitro the effects of the hormone-dependent phosphorylation of the receptor observed in vivo: there is no enhancement of kinase activity by the hormone, and the phosphorylated receptor does not exhibit the characteristic "upshift" in its electrophoretic mobility. Thus either this kinase is not the enzyme responsible for the hormone-dependent receptor phosphorylation or, during purification, a factor has been lost which is necessary for retaining the hormone dependency of the reaction.

Animals↗
PubMed1987

A novel progesterone-induced messenger RNA in rabbit and human endometria. Cloning and sequence analysis of the complementary DNA.

Complementary DNAs (cDNAs) prepared from messenger RNAs (mRNAs) isolated from endometria of 5 day pregnant rabbits were inserted into the plasmid pBR322. A library of 2400 recombinant plasmid clones was prepared and screened by differential in situ hybridization with cDNAs prepared from mRNAs of rabbits either injected with progesterone or untreated by the hormone. Clones encoding uteroglobin were identified and discarded. Several progesterone-induced and progesterone-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.2% of clones in the library) of 2300 nucleotides. The induction of this messenger RNA by progesterone was totally suppressed by the antagonist RU486. This compound displayed a limited agonistic activity when administered alone. A very small increase in mRNA concentration was observed after estradiol administration. The messenger RNA was also found in the liver (where it was constitutively expressed), the ovaries, and the Fallopian tubes of rabbits. A cross-hybridizing messenger RNA was detected in human endometrium during the luteal phase. Sequence analysis showed that the messenger RNA encoded a protein of 370 amino acids with a calculated molecular weight of 40,800. A search in Genbank and National Biomedical Research Foundation data banks showed no identity or marked similarity with previously published DNA or protein sequences.

Animals↗
PubMed1987

Immunocytochemical study with monoclonal antibodies to progesterone receptor in human breast tumors.

Mouse hybridomas secreting monoclonal antibodies against rabbit uterine progesterone receptor (PR) have been prepared. Several of these immunoglobulins exhibited high affinity towards human progesterone receptor and two (LET 126 and LET 64) were selected as giving the best immunoperoxidase staining of human progesterone target organs. Using the indirect peroxidase-antiperoxidase method of Sternberger, optimal conditions for demonstrating PR involved brief fixation of frozen sections with formaldehyde-containing fixatives, among them picric acid-paraformaldehyde. This method allowed us to detect the receptor in breast carcinoma epithelial cells, T47D cell line, and uterine endometrium and myometrium. No staining was observed in intestine and muscle. Specific staining for PR was confined to the nucleus, irrespective of the concentration of progesterone in the blood of the patient. In a preliminary study of 27 human breast cancers by the immunocytochemical method, the presence or absence of nuclear staining for PR correlated well with the concentration of cytosolic progesterone receptor determined by a steroid-binding assay on tumor extracts. Differences in the intensity and distribution of staining within a section were observed, suggesting heterogeneity of the PR content of breast cancer cells. In 19 tumors, the immunocytochemical method for PR localization was also used in combination with a slightly modified Abbott ER-ICA staining for estrogen receptor to compare the distribution of both receptors within the same biopsy on adjacent frozen sections. Various combinations of estrogen receptor and PR contents that have been determined by steroid-binding assay have also been detected by the double immunocytochemical assay.

Antibodies, Monoclonal↗
PubMed1987

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.

Amino Acid Sequence↗
PubMed1987

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA.

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).

Amino Acid Sequence↗