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PubMed1989

Folate (pteroylglutamate) uptake in human red blood cells, erythroid precursors and KB cells at high extracellular folate concentrations. Evidence against a role for specific folate-binding and transport proteins.

Membrane-associated folate (pteroylglutamate, PteGlu)-binding proteins (FBPs) play an important role as PteGlu-transport proteins in malignant and normal human cells. Since high extracellular folate (PteGlu) concentrations (EFC) profoundly influenced uptake and toxicity of the anti-PteGlu methotrexate in malignant KB cells, we studied human cells to determine additional mechanisms for PteGlu uptake when the EFC was varied. At low EFC (less than 10 nM), the predominant mechanism for folate uptake in mature erythrocytes was through binding to externally oriented FBPs which were quantitatively insignificant (4-6 orders of magnitude lower) and of no apparent physiological relevance when compared with KB cells. However, the predominant mechanism of PteGlu accumulation at high EFC [10-250 nM] in intact erythrocytes and sealed right-side-out (RSO) ghosts was not FBP-mediated and non-specific. This conclusion was based on the findings that radiolabelled PteGlu uptake: (i) continued even in the presence of a 1000-fold excess of unlabelled PteGlu and was linear and not saturable up to 250 nM; (ii) was two-fold higher at pH 4.5 than 7.5; (iii) was less than 2-fold increased at 37 degrees C compared with 4 degrees C; and (iv) was unaffected after trypsin-mediated proteolysis of greater than 75% FBPs. The [3H]PteGlu and 125I-PteGlu (histamine derivative) accumulated intracellularly through the non-specific PteGlu-uptake mechanism was unaltered biochemically and in a soluble compartment. Raising the EFC 500-fold higher than controls during erythropoiesis in vitro resulted in reversal of the expected anti-(placental folate-receptor)-antiserum-induced megaloblastic changes in orthochromatic normoblasts derived from burst-forming unit-erythroid colonies. Furthermore, at EFC greater than 0.1 microM, KB-cell accumulation of [3H]PteGlu was also predominantly through a mechanism that did not involve specific FBPs. Thus, at high EFC, a major component of PteGlu transport in human cells is not mediated through FBPs and is likely to be a passive diffusion process.

Carrier Proteins↗
PubMed1980

Erythroid cell differentiation in unincubated chick blastoderm in culture.

Morphologically distinct erythroid cell types characteristic of the primitive and the definitive erythroid cell lines, and embryonic and adult haemoglobins are produced when the unincubated chick blastoderm is cultured ventral side down on a filter raft to inhibit morphogenetic movements and subsequent primitive-streak formation mechanically in serum-free minimal essential medium. The primitive and definitive erythroid cell populations appear consecutively in culture even though there is no axis formation nor apparent morphogenesis. The information to produce both the early and late haemoglobins and erythroid cell types is independent of axis formation and of specific extra-embryonic influences, such as progressive induction exerted by the yolk mass.

Animals↗
PubMed1989

Receptors for erythropoietin in mouse and human erythroid cells and placenta.

High and lower affinity receptors for erythropoietin (EP) were initially identified on a very pure population of EP-responsive erythroblasts obtained from the spleens of mice infected with anemia strain of Friend virus (FVA). The structure of the receptor for EP in these cells was determined to be proteins of 100 and 85 Kd by cross-linking 125I-EP. In this investigation, studies on the receptors for EP were extended to other mouse erythroid cells and human erythroid cells as well as to the placentas of mice and rats. Only lower affinity receptors for EP were detected on erythroblasts purified from the spleens of mice infected with the polycythemia strain of Friend virus and a murine erythroleukemia cell line, both of which are not responsive to EP in culture. Internalization of 125I-EP was observed in both groups of cells. The structure of the receptor determined by cross-linking 125I-EP was two equally labeled proteins of 100 Kd and 85 Kd molecular mass in all these mouse erythroid cells. The structure of the receptor was found to be very similar in human erythroid colony forming cells cultured from normal blood. These cells respond to EP with erythroid maturation and were previously shown to have high and lower affinity receptors. Placentas from mice and rats were found to have only lower affinity receptors for EP, and when placental membranes were cross-linked to 125I-EP, the same 100 Kd and 85 Kd bands were found as seen in mouse and human erythroid cells. The structure of the receptor was similar in cells that have high affinity receptors (FVA-infected and human erythroid colony-forming cells) and nonresponsive erythroid cells and placenta that have lower affinity receptors, but only the cells with the high affinity receptors respond to the addition of EP with erythroid maturation.

Animals↗
PubMed1987

Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells.

Erythropoietin (Epo) is a glycoprotein hormone that regulates erythroid development and interacts with surface receptors on developing erythroid cells. In this laboratory, a cell system with a relatively pure population of erythroid cells that respond to Epo has been developed. Immature erythroid cells are obtained from the spleens of mice infected with the anemia strain of Friend virus. The binding of 125I-labeled Epo (125I-Epo) to plasma membranes from these cells was studied in this investigation. 125I-Epo binding reached equilibrium within 20 min at 37 degrees C. Twenty percent of the receptors bound 125I-Epo with a Kd of 0.08 X 10(-9) M, while the remaining receptors bound the hormone with a Kd of 0.6 X 10(-9) M. In this study, a receptor for Epo was identified by cross-linking 125I-Epo to the receptor in intact cells and plasma membrane preparations using disuccinimidyl suberate. Polyacrylamide gel electrophoresis revealed two labeled bands of 100 and 85 kDa. The 85-kDa band was more heavily labeled (65%) than the 100-kDa band. Both bands were equally decreased when increasing amounts of unlabeled Epo were included in the binding mixture, indicating a specific interaction of 125I-Epo with the receptor.

Animals↗
PubMed1987

Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells.

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.

Ammonium Chloride↗
PubMed1977

Comparative ultrastructure of late rabbit-embryo erythroid cells in liver and peripheral blood.

A morphological study of hemoglobin biosynthesis activity in rabbit-embryo liver and peripheral blood was comparatively developed. Orthochromatic erythroblasts and reticulocytes of the same maturing degree were analysed through thin sections, as to their organellar constitution and behaviour regarding iron incorporation. It was found that peripheral blood erythroid cells contain mainly hemosomes, organelles taken as sites of final hemoglobin molecule biosynthesis. The ratio between the mean number of hemosomes in blood erythroid cell sections and that in liver erythroid cell sections reaches a little more than 6:1. Inversely, late liver erythroid cells are predominantly constituted by mitochondria that directly or indirectly participate in hemosome formation. The ratio between the mean number of mitochondria in blood erythroid cell sections and that in liver erythroid cell sections reaches 1:11. Besides this, the iron incorporation activity is higher in peripheral blood erythroid cells than in liver erythroblasts and reticulocytes. It is apparent that erythroid cells in the liver accumulate heme, given the ratio of the means of iron incorporation activity per cell to hemosome per cell (3.67), while blood erythroid cells, with a 1.08 ratio, synthesize heme, which is immediately afterwards integrated into the globin chains. The sudden increase in the formation of hemosomes when liver orthochromatic erythroblasts and reticulocytes enter the peripheral blood, reflecting an enhancement of hemoglobin synthesis, agrees with biochemical findings of other authors.

Animals↗
PubMed1969

RNase activity in erythroid cell lysates.

The characteristics of degradation of reticulocyte ribonucleic acid (RNA) and ribosomes were studied in a whole erythroid cell lysate system. The process followed Michaelis-Menten kinetics, and indicated that RNA degradation in the erythroid cell is mediated by an enzyme previously isolated from reticulocyte hemolysates. Erythroid cell RNase activity had a temperature optimum of 50 degrees C, a pH optimum of 7.0, was not energy dependent, was heat labile at physiologic pH, and was inhibited by Mg(++), Ca(++), and exposure to bentonite and deoxycholate. Free sulfhydryl groups were not essential for RNase activity. Of the substrates occurring naturally within the erythroid cell, isolated ribosomal RNA was most susceptible to the action of the enzyme, intact ribosomes least susceptible, and transfer RNA intermediate between them. Natural substrates were degraded completely to nucleotides in cell lysates. Competitive inhibition studies indicate that one enzyme system is capable of degrading both RNA and ribosomes, although the existence of more than one enzyme has not been excluded. Erythroid cell lysates quickly broke down polyribosomes into single ribosomes. The more rapid degradation of ribosomes, as compared with transfer RNA, which occurs in vivo, as opposed to findings in vitro, suggests that there is a special intracellular mechanism responsible for ribosome degradation in the maturing erythroid cell.

Animals↗
PubMed2000

Growth factor-independent proliferation of erythroid cells infected with Friend spleen focus-forming virus is protein kinase C dependent but does not require Ras-GTP.

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.

Adaptor Proteins, Signal Transducing↗
PubMed1979

Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.

Adult↗
PubMed2002

Stat3 beta inhibits gamma-globin gene expression in erythroid cells.

We demonstrated previously gamma-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated gamma gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5'-untranslated gamma-globin promoter region. Furthermore, co-transfection experiments with Stat3 beta demonstrate gamma promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5'-untranslated region was mutated. These studies establish a novel mechanism for gamma gene silencing through the STAT signal transduction pathway.

5' Untranslated Regions↗
PubMed2001

Cytokine-Synthesizing Activity of Erythroid Cells.

The presence of mRNA of cytokines IL-1alpha, IL-1beta, IL-4, IL-6, IFN-gamma, TGF-beta, GM-CSF and the absence of mRNA of IL-2, IL-3 and IL-5 were identified in erythroid cells isolated from spleen of mice subjected to erythropoiesis-stimulating actions (treated with phenylhydrazine, acute hypoxia) and from spleen of newborn mice. Gene expression of cytokines in erythroid cells therewith differed both qualitatively and quantitatively depending on erythropoiesis-stimulating action. Erythroid cells of newborn mice contained mRNA of GM-CSF, but did not contain mRNA of IFN-gamma, whereas erythroid cells of mice under acute hypoxia or treated with phenylhydrazine had mRNA of IFN-gamma, but no mRNA of GM-CSF. After cell cultivation with erythropoietin, qualitative and quantitative changes in gene expression of cytokines were observed including the appearance of mRNA of IL-2, IL-3 but not of IL-5. The fact of gene expression of cytokines was confirmed on cells from single erythroid colonies. The level of mRNA expression of cytokines in erythroid cells was comparable with, and for a number of cytokines even higher than in splenocytes of mice immunized with T-dependent antigen. Moreover, it was demonstrated that erythroid cells are capable of translating proteins themselves. The production of GM-CSF and IFN-gamma by erythroid cells extracted from spleen of newborn mice, as well as from mice after erythropoiesis-stimulating actions (treatment with phenylhydrazine, acute hypoxia, acute blood loss) was detected. The level of IFN-gamma production was comparable with the level of production of this cytokine by mitogen-stimulated splenocytes. Thus, erythroid cells, as well as the cells of practically all hemopoietic lineages are capable to express and produce a number of hemo- and immunoregulatory cytokines by means of which they can participate in the regulation of hemo- and immunopoiesis, as well as exert a self-regulatory lineage restricted effect on cell proliferation and differentiation.

Journal Article↗
PubMed1976

Non-histone chromatin proteins during maturation of avian erythroid cells.

1. In chicken erythroid cells (erythroblast, reticulocyte and erythrocyte) maturation was accompanied by a decrease in the content of non-histone chromatin proteins. 2. Phenol-soluble non-histone chromatin proteins (phosphoproteins) from the three cell populations studied, showed differences in the behavior on sodium dodecyl sulphate polyacrylamide-gel electrophoresis, and isoelectric focusing. Phosphoprotein of immature cells had a higher content of fractions of about 86 000 and 23 000 daltons than the phosphoproteins of erythrocytes.

Anemia, Hemolytic↗
PubMedDate not supplied

Accumulation of a heat shock-like protein during differentiation of human erythroid cell line K562.

The human erythroid cell line K562 provides a model system for studying erythroid differentiation and eukaryotic gene regulation. These cells express glycophorin A, spectrin and i antigen. They accumulate embryonic and fetal haemoglobins on induction of erythroid differentiation with haemin, sodium butyrate or hydroxyurea. In the present study, the protein composition of K562 cells during haemin-mediated induction of erythroid maturation was analysed by two-dimensional gel electrophoresis. Under conditions in which haemin did not effect cell viability and proliferation, a protein of approximately 70,000 molecular weight (MW) accumulated in the differentiated K562 cells. The accumulation appears to be due to an increase in the rate of RNA synthesis for this protein. The protein is related in sequence to a 70,000-MW heat shock protein. An antigenically related protein was also demonstrated in human bone marrow and accumulates at particular stages of human erythroid maturation.

Cell Differentiation↗
PubMedDate not supplied

[Insulin binding by erythroid cells of swines].

Insulin binding by erythroid cells of newborn and 1-, 10-, 30-days pigs was investigated. The process intensity in piglets was found to decline in the period from birth to 30 days of life. The obtained changes were determined by the reduced receptors number in high affinity site in early postnatal ontogenesis as well as by decrease of receptor affinity constants in 30-days animals. During investigated period the level of insulin binding by erythrocytes was conditioned by hormone binding ability of "young" erythroid cells, which was found to be high in newborn and decreased considerably in 10-30 days pigs. It was established that postnatal decrease of insulin binding by erythrocytes occurred simultaneously with sharp increase of insulin concentration in animal during the 10-days period after birth. So, it is supposed that insulin may be involved in regulation of its own receptor number in erythrocytes.

Aging↗
PubMed1995

Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer.

The 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors.

Animals↗
PubMed1986

Isolation and characterization of an erythroid cell line highly inducible to form erythroid burst-like colonies.

The study of induction of Friend erythroleukemic cell lines during the last decade has enriched our understanding of late erythroid differentiation. In comparison, little information is available on early erythroid differentiation. We describe here the isolation and characterization of a highly inducible clone from a murine erythroid cell line, which is capable of forming colonies that possess properties of the early erythroid burst progenitor. We found that a combination of erythropoietin (Epo), spleen conditioned medium (SCM), and plasma from a patient with aplastic anemia (Apa) induces over 95% of cells from this clone (clone 12) to form colonies with the properties of burst or mixed burst blast-like colonies. Examination of the culture conditions of these cells indicated that alpha medium was more efficient for colony induction than Iscove's medium, and that the addition of two-mercaptoethanol did not improve the induction process. These factors (EPo, SCM, and Apa) must be present for 4 days in order for induction to take place. It is hoped that the isolation of this highly inducible cell clone will enrich our understanding of early erythroid differentiation.

Animals↗