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Biomedical subjects

E Milgrom

Publications and source records attributed to E Milgrom.

At least 91 records · Page 5Linked to original sources
PubMed1993

Immunohistochemistry of pS2 in normal human breast and in various histological forms of breast tumours.

Expression of pS2 was studied by immunocytochemistry in normal breast tissue (n = 20), benign tumours (n = 9) and 145 breast cancers representative of the different histological types. pS2 immunostaining was scored as negative (D1 = 0-5% stained cells), positive (D2 = 5-75% stained cells) or highly positive (D3 > 75% stained cells). pS2 protein was evident in all normal breast samples examined. Six of nine benign lesions showed pS2 staining. In both cases, immunostaining was weaker than in breast cancers. Of breast cancers, 77/145 (53.1%) were pS2 positive, including 33.1% with intense staining. The presence of pS2 was not correlated with the age of patients, the size of the primary tumour, or lymph node status, but was correlated with histological grading and nuclear grading. pS2 expression was also correlated with menopausal status and oestrogen receptor status (59% of receptor-positive tumours were pS2 positive), but not to progesterone receptor status. pS2 expression in breast carcinomas is not a characteristic of specific histological types. Although this protein is predominantly expressed in oestrogen receptor-positive and differentiated tumours, it shows oestrogen-independent expression in about 30% of cases.

Aged↗
PubMed1992

Variant forms of the pig lutropin/choriogonadotropin receptor.

The cloning and sequencing of porcine lutropin/choriogonadotropin (LH/hCG) receptor messenger RNAs have shown the presence of a full-length receptor (pLHR-A) and of shorter variants lacking either the transmembrane and the intracellular domains (pLHR-B and pLHR-C) or only the transmembrane domain (pLHR-D). Moreover, immunoblotting of testicular membrane extracts has detected 85-, 68-, and 45-48-kDa proteins reacting with antireceptor antibodies. Transfection experiments were performed to assign the protein species to the various messenger RNAs and to study the function of the various receptor species. COS-7 and L-cells transfected with an expression vector encoding full-length receptor pLHR-A yielded a protein of apparent molecular mass of 105 kDa. This corresponded to the complete receptor which had undergone a different glycosylation pattern to that found in testis, since after digestion with peptide N-glycosidase F both the 105-kDa COS-7 protein and the 85-kDa testicular glycoprotein yielded a holoprotein of approximately 63 kDa. Transfection with pLHR-A also yielded a high proportion of the 68-kDa glycoprotein which was shown by digestion with endoglycosidase H to be a high-mannose precursor of the full-length receptor. The existence of a large pool of precursor species in both transfected cells and Leydig cells evokes possible physiological regulations at the level of receptor maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Two-subunit structure of the human thyrotropin receptor.

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular alpha subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning beta subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of beta subunits over alpha subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.

Amino Acid Sequence↗
PubMed1992

Enzyme-linked immunosorbent assay of pS2 in breast cancers, benign tumors, and normal breast tissues. Correlation with prognosis and adjuvant hormone therapy.

An enzyme-linked immunosorbent assay using monoclonal and polyclonal antibodies against recombinant pS2 was devised. It was used to measure pS2 concentration in the cytosol of 339 breast cancer, 15 fibroadenomas, 16 cases of benign breast disease, and 6 normal breast tissues. The mean value of pS2 concentration was higher in cancer, but the protein could be detected readily in benign tumors and even in normal breast. The concentration of pS2 was significantly lower in postmenopausal women and tumors of differentiation Grade 3. The pS2 concentration was correlated strongly with the presence of estrogen receptors (ER) and progesterone receptors (PR). No correlation was observed with the size, histologic type of the tumor, and lymph node status. The prognostic value of pS2 appeared relatively limited. It was clear cut only for a relatively small group of patients (approximately 15%), who had low concentrations of pS2 (less than or equal to 0.32 ng/mg of protein). These patients had a shorter disease-free interval and overall survival time. The most striking correlation was observed with the outcome of adjuvant hormone therapy. pS2 concentration was shown to be the most potent prognostic factor, preceding even ER.

Adult↗
PubMed1992

Prognostic value of progesterone receptor after long-term follow-up in primary breast cancer.

In a previous study of a series of 105 patients with primary breast cancer we found that the progesterone receptor (PgR) status was an important prognostic factor for early recurrences. 95 patients from the same series were followed-up for a median of 9.5 years and reassessed for the prognostic value of PgR status by univariate and multivariate statistical methods. In univariate analysis, the disease-free interval was only related to the lymph-node status. For overall survival, PgR and combined PgR-ER (oestradiol receptor) status had a prognostic value (P = 0.035 and 0.05, respectively). Moreover, PgR status was found to be discriminant for the survival of the node-negative patients (P = 0.017). In multivariate analysis, ER and PgR status were not significant, indicating that receptor status is not a powerful predictor of the course of breast cancer.

Adult↗
PubMed1992

Mechanisms of nuclear localization of the progesterone receptor.

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 was 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. A second putative nuclear localization signal is located in the DNA binding domain activated either through ligand binding or through production of constitutive receptor. 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↗
PubMed1992

Oestrogen receptor negative-progesterone receptor positive phenotype in 1,211 breast tumours.

From 1,211 breast cancers, 15 oestrogen receptor (ER) negative-progesterone receptor (PgR) positive breast cancers by conventional dextran coated charcoal steroid binding assays in cytosol were reassessed using Elisa techniques with monoclonal antireceptors antibodies in the cytosolic and nuclear fractions, and immunocytochemistry on cryostat sections. Three categories of results were found in this series. Two tumours were false negative ER due to receptor sites occupancy by hormonal contraceptive treatment. A second group of ten tumours, with high PgR concentrations and immunoreactive ER, corresponds to non ER-binding forms of receptors. One PgR positive tumour was found to be devoid of PgR by using monoclonal antiPgR antibodies might contain a progesterone binding cyst protein. Only two tumours were found to be true ER negative-PgR positive by all methods. This rare phenotype deserves further study of the regulation of the PgR gene.

Adult↗
PubMed1992

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Animals↗
PubMed1992

The cytoskeleton and the cellular traffic of the progesterone receptor.

Previous studies on glucocorticoid receptors have suggested the existence of interactions between the receptor and microtubule or actin networks. It was hypothesized that such interactions may contribute to the guidance of steroid hormone receptors towards the nucleus. We used a permanent L cell line expressing the delta 638-642 progesterone receptor. This mutant has all the characteristics of the wild type receptor except that the deletion of five amino acids inactivates the constitutive karyophilic signal. Consequently, the receptor is cytoplasmic in the absence of hormone but is shifted into the nucleus when administration of hormone activates the second karyophilic signal. Optical microscopy and confocal laser microscopy were used in intact cells or in cells depleted of soluble elements by permeabilization with detergents. By immunofluorescence, the receptor was found to be mainly concentrated in the perinuclear area. A small fraction of progesterone receptor (PR) persisted in this region after Triton X100 treatment. These observations suggested that the receptor could interact with some insoluble constituent(s) of the cytoplasm. However, careful colocalization studies showed that this heterogenous distribution was not due to interactions with microtubule, microfilament, or intermediate filament networks. Functional involvement of these networks in the translocation of the receptor into the nucleus was studied after cell treatment with cytoskeletal drugs such as nocodazole, demecolcine and cytochalasin. None of these compounds prevented or even delayed the hormone-dependent transfer of delta 638-642 PR into the nucleus. Similar conclusions were reached with the wild type receptor expressed by transfection in Cos-7 cells. PR was shifted from the nucleus into the cytoplasm by administration of energy-depleting drugs. After disruption of the various cytoskeletal networks normal nuclear reaccumulation of the receptor was observed when these drugs were removed. The results thus suggest that the progesterone receptor is not colocalized with the main cytoskeletal components. Disruption of the cytoskeletal networks does not prevent its nuclear translocation. Thus, karyophilic signals and interactions with the nuclear pore seem to be the primary determinants of the cellular traffic of the progesterone receptor.

Animals↗
PubMed1992

Control of biosynthesis and post-transcriptional modification of the progesterone receptor.

The rabbit progesterone receptor undergoes dual regulation at the level of transcription: positive by estrogens and negative by progestins. The two aspects of this regulation are mediated by a single intragenic estrogen-responsive element. Estrogen receptor binding to this element has been demonstrated but progestin down-regulation does not proceed through DNA binding of the progesterone receptor. This result suggests some kind of protein-protein interaction--direct or indirect--between estrogen and progesterone receptors. At the post-transcriptional level, the progesterone receptor undergoes a hormone-dependent hyperphosphorylation of serine residues localized in the N-terminal region. Studies of progesterone receptor mutants have determined the influence of the different receptor domains in the phosphorylation mechanism. A casein kinase copurifies with the receptor. The role of this phosphorylation remains to be determined.

Animals↗
PubMed1992

New functional zonation in the ovary as shown by immunohistochemistry of luteinizing hormone receptor.

Monoclonal anti-LH receptor antibodies were used to study receptor distribution in the various structures of porcine ovary. Primordial and primary follicles were not labeled. Immunoreactivity appeared on the thecal cells of secondary follicles of approximately 100-200 microns in diameter. In large antral follicles and preovulatory follicles granulosa cells were also labeled, whereas two zones were observed in the theca interna. The region close to the lamina basalis (approximately 1/3 of the thecal cells) appeared devoid of LH receptors, whereas a strong labeling was observed on the most external region. Thecal cells were also labeled in atretic follicles. There was no labeling of the oocyte. In cyclic corpora lutea only the most external cells, probably of thecal origin, were stained. The most abundant cells, of granulosa origin, were not labeled. In the interstitium few cells were immunolabeled; they proceeded either from remnants of atretic follicles or existed as groups of isolated cells. Thus both the theca interna of preovulatory follicles and the corpus luteum are heterogenous structures composed of two zones of which only one is directly responsive to LH. No LH immunoactivity could be detected in nontarget organs (liver, lung, thyroid gland, small intestine, fallopian tube, and uterus).

Animals↗
PubMed1992

Effects of luteal estradiol on the secretory transformation of human endometrium and plasma gonadotropins.

To study the role of luteal estradiol (E2), we interrupted the supply of E2 during the luteal phase of E2 and progesterone (P) replacement cycles. Thirty-one women, aged 26-37 yr, with absent or inactive ovaries received three different treatment regimens: group I (n = 11) received transdermal E2 and vaginal P according to a protocol designed to approximate levels of estrone (E1), E2, and P seen during the menstrual cycle. Groups II (n = 11) and III (n = 9) received identical treatments, except that in group II no E2, and in group III no E2 or P, was administered after day 15. Endometrial biopsies were obtained on days 20 and 24 in groups I and II, and on days 14 and 20 in group III. In group I, plasma E1 and E2 reached menstrual cycle levels, whereas in groups II and III, discontinuation of the E2 supply on day 15 resulted in a prompt decrease to castrate levels of plasma E1 and E2. In groups I and II, menopausal FSH and LH levels decreased to 26 +/- 6 and 30 +/- 7 IU/L, respectively, on day 13 (mean +/- SEM). In group I, administration of E2 and P starting on day 15 further lowered plasma gonadotropin levels. In group II, administration of P only failed to induce a similar decrease in plasma FSH and LH. No uterine bleeding occurred before day 25 in women of groups I or II, while women of group III bled within 2 days of E2 withdrawal. Endometrial biopsies were similar in groups I and II. Histological features were characteristic of early and late luteal phases on days 20 and 24, respectively. Endometrial maturation assessed by estrogen and progesterone receptors identified by immunocytochemistry showed the typical distribution seen on day 24 of the menstrual cycle with no difference between groups I and II. We conclude that in women deprived of ovarian function, administration of P only after 14 days of E2 priming prevented uterine bleeding and induced normal secretory transformations of the endometrium, but failed to suppress plasma gonadotropins.

Administration, Cutaneous↗
PubMed1992

Immunolocalization of steroid hormone receptors in normal and tumour cells: mechanisms of their cellular traffic.

Experimental conditions are described for the detection of steroid receptors in tissue sections or cells at the light microscope level. Current knowledge about the ultrastructural distribution of these receptors is summarized; the mechanisms of their nuclear localization are described. Karyophilic signals involved in nuclear translocation are characterized by means of in vitro mutagenesis of steroid receptor cDNAs. Studies analysing the subcellular distribution of various transfected receptor mutants in energy depleted cells together with fusion experiments provide evidence for nucleoplasmic shuttling of progesterone receptors. We conclude that the "nuclear" location of the wild type progesterone receptor reflects a dynamic equilibrium between active nuclear import and outward diffusion. We also describe the use of immunocytochemistry in pathology, especially for the detection of steroid receptors in hormone dependent tumours.

Animals↗
PubMed1992

[LH and TSH receptors. A new family of G protein-coupled receptors].

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. Cross hybridisation with the LH receptor clone allowed to isolate a clone corresponding to the human TSH receptor from thyroids. The structure of both receptors have been determined. They show similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor lacking transmembrane domains have been isolated. The obtention of monoclonal antibodies against both receptors allowed immunochemical and immunocytochemical studies to be performed. The human LH receptor gene have been localized to chromosome 2p21 and TSH receptor gene to chromosome 14q31. The complete organisation of the human TSH receptor gene has been determined.

Animals↗
PubMed1992

[Intracellular traffic of the progesterone receptor].

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone-dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.

Animals↗