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Query: UNIPROT:P06889 (Mol)
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Rapid segregation and purification of haemopoietic progenitor cells by simple methods is necessary for a proper analysis of the control of early haemopoiesis. In this paper we describe the use of a rat monoclonal antibody, YW 13.1.1, for that purpose. This reagent reacts with more than 90% of foetal liver cells but spares stem cells. A single-step lysis with antibody and complement achieves a tenfold enrichment for early progenitor cells. The marker also shows an increasing level of expression on the three defined subsets of erythroid progenitor cells. This parallels their developmental pathway and erythropoietin responsiveness. Simple quantitative considerations therefore permit separation of cells at different stages of erythropoiesis.
Mol Biol Med 1984 Oct
PMID:Haemopoietic progenitor cell heterogeneity revealed by a single monoclonal antibody, YW 13.1.1. 654 83

Erythropoietin-responsive progenitor cells (CFU-E) from normal mouse foetal liver have been substantially purified on the basis of their differential binding to two monoclonal antibodies in conjunction with flow cytometry. This has allowed the formal identification of the foetal liver CFU-E as an early erythroid blast cell with a highly basophilic cytoplasm. It has also been possible to show a quantitative association of a membrane marker with proliferative potential within a single differentiation lineage. This is the first demonstration of such an association. Progenitor cells enriched in this way should allow study of the molecular mechanisms of erythropoietin action and also serve as a target for determining the cellular specificity of erythroid-transforming viruses.
Mol Biol Med 1983 Jul
PMID:Selective isolation of murine erythropoietin-responsive progenitor cells (CFU-E) with monoclonal antibodies. 667 74

Plethoric mice treated with pharmacological doses of estradiol have decreased concentration of erythropoietin-responsive cells (ERC) in the marrow. We used the methylcellulose-culture system for growth of erythroid stem cells (CFU-E and BFU-E) to define more accurately these estrogen-induced changes. As an animal model we utilized plethoric mice given repeated injections of estradiol cypionate and found that at 14 days after the onset of treatment there was no significant change in the concentration of femoral CFU-E whereas there was a significant decrease of the BFU-E content. Both CFU-E and BFU-E increased progressively in the spleen over a 42-day period. Addition of estradiol directly to the cell-culture system showed no effect on CFU-E growth but induced a significant depression of BFU-E growth. This depression seemed to require the presence of adherent cells. It is our hypothesis that estrogens suppress only the early stages of erythroid proliferation and/or differentiation by a mechanism involving possibly the stromal (adherent) cells of the marrow microenvironment.
Mol Cell Endocrinol 1981 Oct
PMID:The effect of estrogens on erythroid stem cells in polycythemic mice. 732 99

Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH- and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.
Mol Cell Biol 1994 Mar
PMID:Growth hormone and erythropoietin differentially activate DNA-binding proteins by tyrosine phosphorylation. 750 51

We recently reported that interleukin-3, Steel factor, and erythropoietin all induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cell lines. We have now further characterized the proteins that become associated with Shc following stimulation with these cytokines and found that, in response to all three, the tyrosine-phosphorylated form of Shc binds to common 145- and 52-kDa proteins which also become tyrosine phosphorylated in response to these growth factors. The 145-kDa protein, which appears, from antiphosphotyrosine blots of two-dimensional O'Farrell gels, to exist in four different phosphorylation states following cytokine stimulation (with isoelectric points ranging from 7.2 to 7.8), does not appear to be immunologically related to the beta subunit of the interleukin-3 receptor, c-Kit, BCR, ABL, JAK1, JAK2, Sos1, eps15, or insulin receptor substrate 1 protein. Silver-stained sodium dodecyl sulfate gels indicate that the association of the 145-kDa protein with Shc occurs only after cytokine stimulation and that it can bind to the tyrosine-phosphorylated form of Shc in its non-tyrosine-phosphorylated state. The latter finding, in conjunction with the observations that p145 does not bind, in vitro, to the Src homology 2 (SH2) domain of Shc, that it is not present in anti-Grb2 immunoprecipitates, and that a phosphopeptide which blocks the binding of Shc to the SH2 domain of Grb2 also blocks the binding of Shc to p145, suggests that p145 contains an SH2 domain and competes with Grb2 for the same tyrosine-phosphorylated site on Shc. This implicates p145 as a potential regulator of Ras activity and, perhaps, of other as yet unidentified functions of Shc.
Mol Cell Biol 1994 Oct
PMID:Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc. 752 59

Vascular endothelial growth factor (VEGF) expression is highly stimulated by hypoxia, both in vitro and in vivo. Recent findings suggest that the VEGF gene utilizes an oxygen sensing mechanism similar to the one used by the erythropoietin gene. The genomic sequences that control the VEGF response to hypoxia are, however, largely unknown. In utilizing transient transfection assays in HeLa cells we determined that hypoxia/cobalt responsive enhancer elements are present at the 5' and 3' flanking regions of the human VEGF gene. The 3' enhancer is contained in a 160 bp fragment located about 60 bp downstream of the polyadenylation site. It contains a sequence stretch of about 12 bp which are highly homologous to sequences in the erythropoietin hypoxia-responsive enhancer. The 5' flanking enhancer is contained in a 100 bp fragment located about 800 bp upstream of the start site. This fragment does not contain significant homologies with the erythropoietin enhancer. Thus, it appears that the response to hypoxia of the VEGF gene is controlled by two regulatory elements; one which may be related to the erythropoietin enhancer and a second, which appears to be a completely unrelated sequence.
Cell Mol Biol Res 1994
PMID:Hypoxia regulatory elements of the human vascular endothelial growth factor gene. 752 97

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.
Mol Biol Cell 1995 May
PMID:A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF. 754 29

Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperature-sensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32 degrees C by undergoing G1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G1. The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G1 arrest. These observations indicate that apoptosis and G1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32 degrees C by undergoing G1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.
Mol Cell Biol 1995 Nov
PMID:Cytokines inhibit p53-mediated apoptosis but not p53-mediated G1 arrest. 756 57

1. To investigate the possibility that cholinesterase inhibitors may cause adverse hematopoietic effects, we employed antisense oligodeoxynucleotides selectively inhibiting butyrylcholinesterase gene expression (AS-BCHE). Complementary sense (S) oligonucleotides served as controls. 2. In primary bone marrow cell cultures grown with interleukin 3 (IL-3), AS-BCHE but not S-BCHE reduced growth of megakaryocyte colony-forming units (CFU-MK) in a dose-dependent manner at the micromolar range. 3. In cultures grown with IL-3, transferrin, and erythropoietin (Epo), cell counts increased up to twofold, yet colony counts (CFU-GEMM) remained unchanged under AS-BCHE treatment. 4. Electrophoretic measurements of DNA ladder as an apoptotic index revealed that the above oligonucleotide effects were not due to nonspecific induction of programmed cell death. 5. Differential cell counts demonstrated increased myeloidogenesis and reduced levels of early megakaryocytes in CFU-GEMM under AS-BCHE, suggesting requirement of the BuChE protein for megakaryopoiesis. 6. In vivo injection of AS-BCHE reduced BCHE mRNA levels in both young and mature megakaryocytes for as long as 20 days, as shown by in situ hybridization. 7. Ex vivo growth of primary bone marrow cells revealed a twofold reduction in CFU-MK colonies grown from the AS-BCHE- but not the S-BCHE-injected mice, 15 days posttreatment. 8. These findings demonstrate that deficient butyrylcholinesterase expression, and hence interference with this enzyme's activity through treatment with or exposure to cholinesterase inhibitors, may cause hematopoietic differences in treated patients.
Cell Mol Neurobiol 1994 Oct
PMID:Antisense inhibition of butyrylcholinesterase gene expression predicts adverse hematopoietic consequences to cholinesterase inhibitors. 762 7

Ping-pong amplification is an efficient process by which helper-free retrovirions replicate in cocultures of cell lines that package retroviruses into distinct host-range envelopes [11]. Transfection of a retroviral vector DNA into these cocultures results in massive virus production, with potentially endless cross-infection between different types of packaging cells. Because the helper-free virus spreads efficiently throughout the coculture, it is unnecessary to use dominant selectable marker genes, and the retroviral vectors can be simplified and optimized for expressing a single gene of interest. The most efficient ping-pong vector, pSFF, derived from the Friend erythroleukemia virus, has been used for high-level expression of several genes that could not be expressed with commonly employed two-gene retroviral vectors. Contrary to previous claims, problems of vector recombination are not inherent to ping-pong methods. Indeed, the pSFF vector has not formed replication-competent recombinants as shown by stringent assays. Here we review these methods, characterize the ping-pong process using the human erythropoietin gene as a model, and describe a new vector (pSFY) designed for enhanced expression in T lymphocytes. Factors that limit tissue-specific expression are reviewed.
J Mol Med (Berl) 1995 Mar
PMID:Amplified and tissue-directed expression of retroviral vectors using ping-pong techniques. 763 47


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