Michael F. Press

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.

Immunochemical Evaluation of Estrogen Receptor and Progesterone Receptor in Breast Cancer

Recent advances in the preparation of monoclonal antibodies to human estrogen receptor (ER) and progesterone receptor (PR) have led to the development of new quantitative and histochemical immunoassays for these proteins in reproductive tissues and related cancers [1–6] . Stich assays make use of very specific and sensitive probes that do not depend upon the ability of receptor protein to bind its cognate radiolabeled hormone in tissue extracts. In addition, these antibodies can be used to detect and quantify ER and PR directly in fixed tissue sections and cell dispersions, thereby permitting an evaluation of the type, proportion and distribution of cells that contain immunoreactive ER and/or PR. For metastatic breast cancer, a major potential clinical application of receptor immnnoassays is the prediction of response to endocrine therapy and assessment of prognosis (disease-free interval and survival). The clinical utility of ER and PR steroid-binding assays for evaluating these parameters in patients with advanced breast cancer has been well documented [7–9]. However, major limitations of current receptor assays are 1) their inability to predict response to endocrine therapy in 30–40% of the breast cancers defined as ER-or PR-rich and in 5–10% of those that are receptor-poor, and 2) the difficulty of performing such assays on small tumors, metastasies and needle biopsies. In addition, assays performed on extracts of breast tumors cannot provide information about the identity, proportion and distribution of receptor-containing cells.

Neuronal development in the cerebellum of lead poisoned neonatal rats

SummaryDevelopmental changes in the cerebellum of neonatal Long-Evans rats with lead encephalopathy were studied by Golgi technique and by light and electron microscopy. Lead encephalopathy was produced by administering daily doses of lead acetate (600 mg of lead acetate/kg of body weight) through an esophageal catheter. Histopathologic changes were observed at 3, 5, 8, 10, 13, and 15 days of age.Matrix cells and neuroblasts of the external granular layer were minimally altered prior to day 8 but thereafter the number of mitotic figures per folium decreased moderately and the number of pyknotic cells increased markedly. The thickness of the molecular layer in lead poisoned animals persistently lagged behind that of control animals after 3 days. Purkinje cells survived the cerebellar alterations of lead encephalopathy very well for the first 5–8 days. However, the rate of Purkinje cell maturation dropped off and at 10 days many still possessed the “mitre” of reticular cytoplasm and the perisomatic processes that are so characteristic of 5–8-day old Purkinje cells. This was particularly true for Purkinje cells located superficial to the “edematous cavities” produced by lead intoxication. The perisomatic processes of these Purkinje cells maintained synaptic contact with climbing fibers, whereas the climbing fibers of control animals had formed asymmetric synapses in the lower dendritic field and the cell soma had symmetric basket fiber synapses. A great variation existed in dendritic growth.Morphologically the neurons were minimally altered by lead intoxication until late in the disease process. The observed neuronal damage supports the suggestion that it is secondary to vascular disruption.

The effect of the α-emitting radionuclide lead-212 on human ovarian carcinoma: A potential new form of therapy

To improve response and survival of patients with ovarian carcinoma noncross-resistant forms of therapy must be developed. alpha-emitting radionuclides may be therapeutically useful since they can directly ionize with energies of 5 to 9 MeV, penetrate only a few cell diameters, and transfer a high amount of energy. The purpose of this study was to determine the effect of the alpha-emitter, lead-212 (212Pb), complexed to sulfur in a nude athymic mouse model (NIH:OVCAR-3) containing human ascites and solid epithelial ovarian carcinoma. Thirty-six nude mice 28 to 32 days old were injected with 10(7) to 10(8) carcinoma cells from donor mice. After 4 weeks, six groups of six nu/nu athymic BALB-C mice were intraperitoneally injected with 70, 50, 20, 5 microCi of 212Pb sulfur colloid, sulfur colloid, or saline. Tumor necrosis with a decrease in ascites and a dose-related survival were noted with doses of 50, 20, and 5 microCi. With 70 microCi acute gastrointestinal toxicity developed. These experiments form the basis for further investigations and the development of alpha-emitting radiocolloids which may be of therapeutic efficacy in the treatment of intraperitoneal ovarian carcinoma.

Comparison of the quantity of estrogen receptors in human endometrium and myometrium by steroid-binding assay and enzyme immunoassay based on monoclonal antibodies to human estrophilin

The recently developed enzyme immunoassay for estrogen receptors is more simple to perform with quality assurance than conventional steroid-binding assays with radioactive labeled estrogen. However, it is not known how well the results of the two assays agree for normal human uterine samples. We compared enzyme immunoassay (Abbott estrogen receptor enzyme immunoassay) and steroid-binding assay of normal human endometrium and myometrium. Low-salt, cytosol estrogen receptor determinations were performed by dextran-coated charcoal assay, and high-salt, nuclear estrogen receptors were measured by controlled pore glass bead assay. Results showed excellent correlation (p less than 0.0001) for cytosol estrogen receptor of endometrium (r = 0.95) and myometrium (r = 0.79) and also for a smaller number of nuclear estrogen receptors of myometrium (p less than 0.01, r = 0.69). Good agreement between steroid-binding assay and enzyme immunoassay was seen for estrogen receptors of both proliferative and secretory phase samples. Thus the data indicate that the simpler estrogen receptor enzyme immunoassay is useful to measure estrogen receptor in the normal uterus. Furthermore, with this sandwich assay, there is no evidence for the existence of significant quantities of receptor fragments that do not bind estrogen.

Quantitative ultrastructural analysis of coronary atherosclerotic involvement in two macaque species

Ultrastructural analyses were employed to observe and to compare in detail lesions of the coronary artery of cynomolgus and rhesus monkeys. Animals were fed individually with the same atherogenic ration under identical conditions for 4, 8, and 12 months, and controls of each species were fed with a low fat, cholesterol-free ration. Transmission electron microscopic studies of coronary arteries from these animals led to the following conclusions: (1) Synthetic smooth muscle cells (SMC) without lipid and macrophages without lipid appeared more frequently in the cynomolgus lesions than in the rhesus lesions. Furthermore, phenotypic expression of synthetic SMCs in the cynomolgus was more active with greater diversity, while the rhesus showed less phenotypic modulation. Macrophages without lipid appeared frequently in the cynomolgus media. (2) Increased percentages of both synthetic SMCs with lipid and macrophages with lipid were demonstrated in the cynomolgus lesions as compared to those in the rhesus. This indicates that foam cells, including SMC- and macrophage-derived foam cells, are more prevalent in cynomolgus than in rhesus. They are considered to play an important role in atherogenesis. (3) Medial disruption, synthetic SMCs, and macrophages containing lipid appeared more often in cynomolgus media than in rhesus media. (4) There were greater percentages of both synthetic SMCs and macrophages in the intima of the myocardial side of coronary arteries in both species. (5) Approximately 42% of all foam cells in the cynomolgus lesions were derived from SMCs. There were fewer macrophages in rhesus lesions. (6) The difference in expression between the two macaque species reflects different responses of macrophages to medial smooth muscle cell (SMC) components. The configuration of the artery wall could be one of the important indicators of these different expressions.

Golgi-electron microscopic study of sprouting endothelial cells in the neonatal rat cerebellar cortex

Intraparenchymal postnatal development of the CNS vasculature proceeds by a process of vascular budding or sprouting from existing endothelial cells and elongation of these immature vessels. A modified Golgi technique combined with gold toning and deimpregnation was used to identify and characterize sprouting endothelial cells in the neonatal rat cerebellar cortex with light and electron microscopy. Sprouting endothelial cells were the terminal endothelial cells of immature, developing blood vessels and they had characteristic morphologic features. The most distinctive feature was an array of tentacular processes (0.1-0.2 micron in diameter and ca. 20 microns in length) which radiated from the apex of these cells in the presumed direction of vessel growth. Cytoskeletal microtubules and microfilaments were the characteristic organelles of these tentacles. Sprouting endothelial cells were thin (1-3 microns in diameter). lacked a vessel lumen and basement membrane, had abundant cytoplasmic organelles and a prominent nucleus and were closely associated with the subterminal endothelial cell without interendothelial gaps. The developing blood vessels had a blood-brain barrier which excluded intravenously injected colloidal carbon from the neuropil.

Lead-induced permeability changes in immature vessels of the developing cerebellar microcirculation

SummaryThe ultrastructure and microcirculatory permeability changes of lead encephalopathy were studied in an animal model using horseradish peroxidase as an intravascular tracer. The fine structure of capillary sprouts in the developing cerebellar microcirculation of lead-poisoned rats were described. Immature vessels, characterized by the presence of endothelial sprouts, were found to have focal areas of endothelial injury with degenerating endothelial cells. These disruptions of the microcirculatory endothelium had tracer extending from the vessel lumen to the surrounding neuropil. The degenerating endothelial cells were found as early as 24–28 h after the first administration of lead acetate by gastric lavage (2–3-day-old rats). The early injury to endothelial cells of immature vessels in the developing microcirculation is suggested as an important component of the vascular permeability changes which characterize lead encephalopathy. Older animals (5–10 days old) had microaneurysmal vascular dilatations which had a complex internal structure formed by endothelial cells. These microaneurysmally dilated vessels may represent an endothelial response to preceding endothelial injury of immature vessels.

Immunocytochemical Localization of Estrogen Receptors with Monoclonal Antibodies

Monoclonal rat antibodies to MCF-7 human breast cancer estrogen receptor (estrophilin) have been used to study the structure and cellular location of receptor in reproductive tissues and cancers as well as to develop assays for receptor that do not depend on the binding of hormone to the receptor protein. Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from MCF-7 cell cytosol, with two different mouse myeloma lines [1–3], provided 13 cloned hybridoma cell lines; each of these (with one possible exception) secretes a unique idiotype of antibody that recognizes a distinct region of the receptor molecule. These antibodies have high affinity (Kd=10-9 -10-10 M) for both steroid-occupied and unoccupied estrogen receptor and recognize nuclear as well as cytosol forms of the receptor molecule. Although they vary in their cross reactivity with estrogen receptors from various animal species, each antibody appears to be completely specific for the 65,000-dalton steroid-binding subunit of the estrogen receptor complex, as judged by extensive sucrose gradient and immunoblot analyses of cytosol and nuclear extracts from a variety of tissues and cell lines. Cross reactivity patterns indicate both sequence homology and heterogeneity among mammalian and nonmammalian estrophilins. Some determinants (eg. H222 and H226) are common to all tested estrogen receptors, including those from hen oviduct, whereas others (eg. D547 and D58) are present only in mammalian receptors and one (D75) appears to be restricted to primate estrophilin.