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PubMed1986

An immunochemical study of delta-aminolevulinate synthase and delta-aminolevulinate dehydratase in liver and erythroid cells of rat.

The relationship between erythroid delta-aminolevulinate (ALA) synthase and hepatic ALA synthase in rat was analyzed immunochemically, using antibodies directed against rat liver ALA synthase and against chicken liver ALA synthase. Rat erythroid ALA synthase showed no cross-reactivity with anti-liver ALA synthase antibodies, but hepatic ALA synthases from rat, mouse, and chicken share substantial cross-reactivity with one another. These results clearly distinguish the isozyme relationship between erythroid ALA synthase and hepatic ALA synthase in rat and suggest that there may be at least two different ALA synthase genes in rat. ALA dehydratase in rat liver, on the other hand, could not be immunochemically distinguished from ALA dehydratase in rat erythroid cells when antibody against rat erythroid ALA dehydratase was used. The finding that erythroid ALA synthase is an entity different from hepatic ALA synthase may provide a clue to understanding the different features in hepatic and erythropoietic porphyrias.

5-Aminolevulinate Synthetase↗
PubMed2002

Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

Acetylation↗
PubMed2001

Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses.

Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) by eIF2alpha kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2alpha kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2alpha kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2alpha phosphorylation upon arsenite treatment. HRI is also the major eIF2alpha kinase responsible for the increased eIF2alpha phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency.

Animals↗
PubMed1987

H5 gene specific trans-activation by nuclear extracts from avian erythroid cells.

Nuclear extracts from chicken erythroid cells selectively stimulate transcription of the chicken histone H5 gene (and not of other chicken histone genes) after coinjection into frog oocytes. This effect is shown to involve an enhancerlike activity, and a region of the H5 gene sufficient to mediate trans-activation is defined.

Animals↗
PubMed2000

Correlation between the oscillatory and adhesion activities in erythroid cells.

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.

Biological Clocks↗
PubMed1987

Identification of erythropoietin receptors on fetal liver erythroid cells.

Erythropoietin (EPO) has a central role in the growth and development of erythroid cells. Using a biologically active radioiodinated derivative, EPO receptors were identified on fetal mouse liver cells mostly consisting of erythroid cells. 125I-EPO was cross-linked to two receptors forms with apparent molecular masses of 110 and 95 kilodaltons, respectively and both having similar affinity toward EPO.

Animals↗
PubMed2006

Ikaros increases normal apoptosis in adult erythroid cells.

Ikaros is a critical transcriptional regulator of hematopoietic cell differentiation. In addition to its effects on the lymphoid system and hematopoietic stem-cell compartment, we have previously shown that Ikaros is also required for normal erythroid development. In this report, we compare Ikaros-dependent gene expression in erythroid cells of mice lacking the Ikaros protein with that of normal mice in purified adult bone-marrow erythroid cells (BMRC). Gene expression, measured by Affymetrix microarray analysis, indicates that in the BMRC of Ikaros-null mice, there is significant up-regulation of SMADs 6 and 7, serine protease inhibitor 3, and immediate-early protein 3 (IER3), all proteins that play a modulating role in apoptosis. We investigate the role of Ikaros in oxidative stress-induced apoptosis using Annexin-V staining and FACS analysis. We find a decrease in apoptosis in the BMRC of Ikaros-null mice compared to normal mice. This effect is also seen in nonerythroid cells but is stronger in BMRC. We conclude that normal Ikaros function increases normal apoptosis in erythroid cells. The data also suggest that Ikaros plays a role in apoptosis-mediated events in other normal hematopoietic cell lineages.

Animals↗
PubMed1991

Metabolic adaptation during erythropoietin-mediated terminal differentiation of mouse erythroid cells.

Metabolic development was examined in erythroid precursor cells, which were isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells). FVA cells undergo differentiation in vitro from the proerythroblast stage through the reticulocyte stage over a 48-hour period in the presence of erythropoietin. Concomitant with marked decreases in cellular size and energy demand, metabolic capacities of both glycolysis and oxygen consumption diminish after 48 hours in culture by 7- and 18-fold, respectively. Because the oxidative capacity decreases more than glycolytic ability does, the metabolic machinery increasingly shifts toward anaerobic metabolism. During the 48-hour period of differentiation, the 2,3-diphosphoglyceric acid (DPG) content per cell and 2,3-DPG mutase activity per cell increased eightfold and threefold, respectively. Freshly harvested FVA cells have adenosine triphosphate (ATP) levels of 7.23 +/- 2.52 mumol/10(10) cells or 3.76 +/- 1.31 mumol/mL cell water which are 12- or 2.3-fold higher, respectively, than the ATP levels of mature red blood cells. In the course of FVA cell differentiation, ATP content per cell decreases by fourfold, but ATP concentration in cell water remains unchanged because of a corresponding decrease in cellular size and water content during differentiation. These studies show that in the face of dramatic decreases in cell size and cellular energy demand, terminally differentiating erythroid cells maintain a constant ATP level by undergoing an involution of their glycolytic machinery as well as by losing their aerobic metabolic capacity.

2,3-Diphosphoglycerate↗
PubMed1999

Erythropoietin induces the expansion of c-kit+ progenitors for myeloid and erythroid cells, but not for lymphoid cells, in the bone marrow and liver.

In humans, the numbers of erythrocytes and granulocytes, but not that of lymphocytes, tend to increase in parallel. To determine the mechanism, we investigated how the administration of erythropoietin induces the expansion of erythroid cells and other lineage cells in the bone marrow, liver, and other organs of mice. When mice were injected twice (days 1 and 2) with erythropoietin at a dose of 20 or 200 IU/day/ mouse, a prominent expansion of TER 19+ (erythroid cells) and Gr-1high cells (granulocytes) occurred in the liver, spleen, and bone marrow day 3 after the initial injection. On the other hand, lymphoid cells, including NK cells, extrathymic T cells, and conventional T cells, did not expand. In parallel with the expansion of erythroid cells and granulocytes, the levels of c-kit(+)Lin- cells increased in the liver and bone marrow. Despite the increase in the proportion of c-kit(+) Lin(-) cells, the generation of lymphocytes (e.g., T cells) decreased when such bone marrow cells were injected to scid mice. These results suggest that erythropoietin has the ability to induce the expansion of not only erythroid cells but also granulocytes in the liver, spleen, and bone marrow. Furthermore, c-kit+ progenitors which may commit themselves to erythroid and myeloid cells, but not to lymphoid cells, were also activated in the liver and bone marrow of mice treated with erythropoietin.

Adult↗
PubMed2005

Induction of fetal haemoglobin expression in erythroid cells--a model based on iron availability signalling.

The K562 cell line has erythroid origin and is used for the study of fetal haemoglobin (HbF) production after treatment with several drugs, such as hydroxyurea, cisplatin and cytosine arabinoside (Ara C). It represents an important tool for the study of cancer differentiation therapy and treatment of thalassaemia and sickle cell disease. Although subject to intense research, the mechanisms involved in the induction of HbF are not fully established, and the regulation of several genes and signalling pathways has been proposed. Using the methodology of differential display, we investigated the changes in gene expression in K562 cells treated with doxorubicin and aclarubicin, which induce HbF expression and cell cycle arrest. Several genes were shown to present differential expression patterns, many of them related to the iron signalling pathway. Particular attention was given to Ndrg1, expressed as early as 24 h after treatment, which can be regulated by iron and is involved with blocking of the cell cycle. A review of the literature shows that, similar to doxorubicin and aclarubicin, most of the drugs used to induce HbF present some kind of effect on the iron signalling pathway, activating in the cells the machinery necessary for the incorporation of extracellular iron. Considering these results, as well as the fact that in erythroid cells the synthesis of haemoglobin is of vital importance, we propose that the production of fetal haemoglobin in erythroid cells is highly dependent on the iron signalling pathway.

Aclarubicin↗
PubMed2003

Haemoglobin biosynthesis site in rabbit embryo erythroid cells.

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.

Animals↗
PubMed1986

Fluorescence probe measurement of the pH of the transferrin microenvironment during iron uptake by rat bone marrow erythroid cells.

Fluorescence probe measurements of the transferrin micro-environment during iron uptake by rat erythroid cells revealed that part of the transferrin is taken up in an acidic environment. The pH of this intracellular transferrin environment is 5.7. When rat erythroid cell precursors are incubated with diferric transferrin then in the incubation medium monoferric transferrins TfNFe and TfFeC appear. In view of the known instability of TfNFe at acidic pH, TfNFe cannot arise after endocytosis of Tf2Fe in acid vesicles at pH below 6.0. The results support the existence of a mechanism other than endocytosis in the iron uptake process in rat erythroid cells.

Animals↗
PubMed1997

Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells.

Methylation of cytosines in the dinucleotide CpG has been shown to suppress transcription of a number of tissue-specific genes, yet the precise mechanism is not fully understood. The vertebrate globin genes were among the first examples in which an inverse correlation was shown between CpG methylation and transcription. We studied the methylation pattern of the 235-bp rho-globin gene promoter in genomic DNA from primary chicken erythroid cells using the sodium bisulfite conversion technique and found all CpGs in the promoter to be methylated in erythroid cells from adult chickens in which the rho-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the gene is transcribed. To elucidate further the mechanism of methylation-induced silencing, an expression construct consisting of 235 bp of 5' promoter sequence of the rho-globin gene along with a strong 5' erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, rho-CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp rho-globin gene promoter fragment at every CpG resulted in a 20- to 30-fold inhibition of transcription, and this effect was not overridden by the presence of potent erythroid-specific enhancers. The ability of the 235-bp rho-globin gene promoter to bind to a DNA Methyl Cytosine binding Protein Complex (MeCPC) was tested in electrophoretic mobility shift assays utilizing primary avian erythroid cell nuclear extract. The results were that fully methylated but not unmethylated 235-bp rho-globin gene promoter fragment could compete efficiently for MeCPC binding. These results are a direct demonstration that site-specific methylation of a globin gene promoter at the exact CpGs that are methylated in vivo can silence transcription in homologous primary erythroid cells. Further, these data implicate binding of MeCPC to the promoter in the mechanism of silencing.

Animals↗
PubMed2000

Differential effects of interleukin-3 on fetal and adult erythroid cells in culture: implications for the isolation of fetal cells from maternal blood.

Fetal clonogenic erythroid cells may be present in maternal blood and serve as a source of fetal DNA for prenatal genetic diagnosis. Proliferating nucleated red cells in cultures from first and second trimester fetal blood contain only fetal haemoglobin (HbF; F+A- cells), whereas nucleated red cells from adult blood contain also adult haemoglobin (HbA; F+A+ or F-A+ cells). Thus, fetal red cells can be identified and flow sorted. However, a few adult cells are also F+A-, which reduces the purity of fetal cell isolation. Culture media optimized for erythropoiesis contain interleukin-3 (IL3). We show here that IL3 strongly stimulates the growth of F+ cells (both F+A- and F+A+) in cultures from adult blood but has only a comparatively small effect on F+A- cells in cultures from fetal blood. This difference is maintained in the unified conditions of co-cultures of adult and fetal cells, so that the purity of fetal cell sorts can be increased by omitting IL3 from the culture medium. We further show that IL3 accelerates the exhaustion of the long-term division potential of adult cells, allowing fetal secondary colonies to be identified by their size following a two-stage culture scheme. Thus, the choice to omit or include IL3 in the growth medium of maternal blood cultures should depend on whether fetal nucleated red cells are to be isolated by flow sorting after one week, or by picking secondary colonies after a later secondary culture stage.

Adult↗
PubMed1981

Intracellular hemoglobin-a specific marker for erythroid cells in paraffin sections. An immunoperoxidase study of normal, megaloblastic, and dysplastic erythropoiesis, including erythroleukemia and other myeloproliferative disorders.

Intracellular hemoglobin represents an excellent marker for specific characterization of normal, megaloblastic, or dysplastic erythroid cells in paraffin sections. Using an immunoperoxidase indirect sandwich technique for detection of intracellular hemoglobin, erythroid cells at all stages of maturation were readily identified in bone marrow biopsies (58 specimens total) with a) normal erythropoiesis, b) megalobastic erythropoiesis, and c) various myeloproliferative disorders, including erythroleukemia. In other tissues (6 spleens, 2 lymph nodes, 1 liver) with extramedullary hematopoiesis, erythroid cells were similarly defined on the basis of this immunohistochemical method. Initial fixation in Zenker's-acetic acid solution (employed for bone marrow biopsies), B5 solution, or formalin, appeared equally effective in preserving the antigenicity of intracellular human hemoglobin. This sensitive and specific immunoperoxidase technique for erythroid cell characterization is particularly applicable to tissues with abnormal erythropoiesis, in which precise cell identification generally presents a diagnostic problem.

Bone Marrow↗
PubMed1976

The initiation of globin synthesis in differentiating rabbit-bone-marrow erythroid cells.

At a particular stage of erythropoiesis, the immature erythroid cell commences synthesis of the messenger RNA for globin. Experiments were performed to determine whether this mRNA is immediately translated into globin or whether there is any substantial delay and accumulation of mRNA awaiting a further stage of differentiation. Rabbit bone marrow erythroid cells were separated according to their degree of maturity by velocity sedimentation on a sucrose gradient. Their relative abilities to synthesise globin were measured by radioactive labelling in culture and the relative proportions of globin mRNA in cellular RNA were determined by hybridisation to DNA complementary to globin mRNA. The increasing ability to synthesise globin closely parallelled the concentration of globin mRNA at all stages of differentiation, thus suggesting that there is no significant delay between transcription and translation.

Animals↗
PubMed2005

The hydroxyurea-induced small GTP-binding protein SAR modulates gamma-globin gene expression in human erythroid cells.

Hydroxyurea (HU), a drug effective in the treatment of sickle cell disease, is thought to indirectly promote fetal hemoglobin (Hb F) production by perturbing the maturation of erythroid precursors. The molecular mechanisms involved in HU-mediated regulation of gamma-globin expression are currently unclear. We identified an HU-induced small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR) protein, in adult erythroid cells using differential display. Stable SAR expression in K562 cells increased gamma-globin mRNA expression and resulted in macrocytosis. The cells appeared immature. SAR-mediated induction of gamma-globin also inhibited K562 cell growth by causing arrest in G1/S, apoptosis, and delay of maturation, cellular changes consistent with the previously known effects of HU on erythroid cells. SAR also enhanced both gamma- and beta-globin transcription in primary bone marrow CD34+ cells, with a greater effect on gamma-globin than on beta-globin. Although up-regulation of GATA-2 and p21 was observed both in SAR-expressing cells and HU-treated K562 cells, phosphatidylinositol 3 (PI3) kinase and phosphorylated ERK were inhibited specifically in SAR-expressing cells. These data reveal a novel role of SAR distinct from its previously known protein-trafficking function. We suggest that SAR may participate in both erythroid cell growth and gamma-globin production by regulating PI3 kinase/extracellular protein-related kinase (ERK) and GATA-2/p21-dependent signal transduction pathways.

Antigens, CD34↗
PubMed1990

Isolation of a phospholipase-A2-like protein from human fetal intestine. Lysis of erythroid cells from bovine liver by this protein and porcine phospholipase A2.

Extracts of human fetal intestine contain factors that can stimulate or inhibit thymidine incorporation into fetal bovine erythroid cells. An inhibitory factor was purified to homogeneity by gel-permeation and reversed-phase high performance liquid chromatography. The inhibitory action was due to cell lysis. The first 25 amino acids of the N-terminal segment were identical to the human lung and pancreatic phospholipase A2. The isolated protein released arachidonic acid from 2-arachidonyl phosphatidylcholine. Porcine phospholipase A2 had the same effects as the intestinal protein, including its tissue-specific lysis of fetal bovine liver erythroid cells. No decrease of thymidine incorporation was seen in fetal bovine intestinal cells, 3T3 cells, or K562 cells incubated with the porcine enzyme. No release of hemoglobin or cell lysis was observed with human erythrocytes or fetal bovine erythrocytes. Porcine and bee phospholipases, which have low sequence homology, are nearly equipotent in inhibiting thymidine incorporation, whereas melittin and beta-bungarotoxin were less active than the pancreatic enzyme. These results support the tissue-specific effects observed with other phospholipases A2. The high sensitivity of liver erythroid cells towards some phospholipases A2 suggest that these enzymes may be involved in the elimination of hepatic erythroid cells at the end of gestation.

Amino Acid Sequence↗