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PubMed2003

Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity.

Studying the effects of microgravity on cell differentiation will enhance our understanding of fundamental biology and is indispensable for a sustained space program. Rauscher murine erythroleukemic cells were chosen as a model system to study erythroid cell differentiation aboard the International Space Station because these cells undergo differentiation in response to the natural inducer, erythropoietin, as well as various chemical-inducers. We have now developed a method to quantify hemoglobin in Rauscher cells after weeks of fixation and storage required for such space biology experiments. By exploiting the pseudoperoxidase activity of hemoglobin and by using reagents that yield a soluble chromophore that freely passes out of fixed cells, we developed a highly specific and sensitive assay applicable to cells fixed as long as 4 months.

Animals↗
PubMed1981

Effect of heme on globin messenger RNA synthesis in spleen erythroid cells.

Synthesis of globin mRNA in erythroid spleen cells from anemic mice was measured after in vitro incubation under conditions in which the level of intracellular heme was manipulated. This newly synthesized globin mRNA was isolated by hybridization with globin cDNA covalently bound to cellulose. Isonicotinic acid hydrazide (INH) and penicillamine were used as specific inhibitors of heme synthesis. It has been found that a 120-min incubation of spleen erythroid cells with 5mM INH or 5mM penicillamine reduced [3H] uridine incorporation into globin mRNA by 24% or 36%, respectively. The addition of heme to INH- or penicillamine-treated cells almost completely restored [3H] uridine incorporation into globin mRNA. These results indicate that heme stimulates transcription of processing of globin mRNA.

Animals↗
PubMed2002

Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides.

Correct human beta-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with beta-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/beta(E) genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in beta-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human beta-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.

Animals↗
PubMed2003

An endonuclease activity similar to Xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human beta-globin mRNA in erythroid cells.

beta-globin mRNA bearing a nonsense codon is degraded in the cytoplasm of erythroid cells by endonuclease cleavage, preferentially at UG dinucleotides. An endonuclease activity in polysomes of MEL cells cleaved beta-globin and albumin mRNA in vitro at many of the same sites as PMR1, an mRNA endonuclease purified from Xenopus liver. Stable transfection of MEL cells expressing normal human beta-globin mRNA with a plasmid vector expressing the catalytically active form of PMR1 reduced the half-life of beta-globin mRNA from 12 to 1-2 h without altering GAPDH mRNA decay. The reduced stability of beta-globin mRNA in these cells was accompanied by an increase in the production of mRNA decay products corresponding to those seen in the degradation of nonsense-containing beta-globin mRNA. Therefore, beta-globin mRNA is cleaved in vivo by an endonuclease with properties similar to PMR1. Inhibiting translation with cycloheximide stabilized nonsense-containing beta-globin mRNA, resulting in a fivefold increase in its steady-state level. Taken together, our results indicate that the surveillance of nonsense-containing beta-globin mRNA in erythroid cells is a cytoplasmic process that functions on translating mRNA, and endonucleolytic cleavage constitutes one step in the process of beta-globin mRNA decay.

Animals↗
PubMed1990

Isolation from fetal bovine serum of an apolipoprotein-H-like protein which inhibits thymidine incorporation in fetal calf erythroid cells.

A 46 kDa heparin-binding protein which inhibits thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on heparin-Sepharose, ion-exchange chromatography, gel filtration and reversed-phase h.p.l.c. The N-terminal sequence of the first 22 amino acids showed 81% identity with the published sequence of human apolipoprotein H. The isolated protein inhibited thymidine incorporation with an ED50 (concn. producing 50% of maximal effect) of 36 nM. A 100% inhibition of thymidine incorporation and a 40% decrease in cell numbers in cultures of fetal calf erythroid cells were observed at a protein concentration of 840 nM. No effects could be seen in cultures of 3T3 cells used as controls. Human apolipoprotein H had no inhibitory activity in any of the cell cultures tested, suggesting a species-specificity or a different structure or function for the bovine heparin-binding protein.

Amino Acid Sequence↗
PubMed1975

Polyribosomal structures inactive for protein synthesis present in erythroid cells during terminal stages of differentiation.

During the terminal stages of differentiation nucleated erythroid cells from the fetal mouse synthesize hemoglobin at a lower rate because after the last cycle of cell division about half of their polyribosomal structures are rendered inactive for protien synthesis though they maintain their aggregated shape. Partially inactive polyribosomes are tested in comparison with normal polyribosomes for the capacity to support polypeptide chain synthesis in cell-free conditions. The following observations are made: a) no difference is found for the profile on sucrose density gradients; b) partially inactive polyribosomes carry growing polypeptide chains in reduced amounts in comparison with active polyribosomes; c) partially inactive polyribosomes are not capable to release "run off" 80 S ribosomal monomers and to dissociate to active ribosomal subunits. These data are interpreted as the evidence for a block of chain termination producing inactivation of polyribosomes during the late maturation of nucleated erythroid cells.

Animals↗
PubMed1977

Transferrin receptors in developing murine erythroid cells.

Techniques of cell separation were used to isolate murine erythroid cells at different stages of maturation. The number of transferrin receptors in these cell populations was assayed by measuring binding of 125I-labelled transferrin. Nearly 23 times as many receptors were found in the least mature cells, chiefly pronormoblasts, as in reticulocytes. Iron transport, determined by measurement of the rate of 59Fe uptake from 59Fe-labelled transferrin, was proportional to the number of receptors at all stages of differentiation. Electron microscope radioautographic studies of the interaction of 125I-labelled transferrin with erythroid precursor cells demonstrated that 15-33% of cell associated transferrin was intracellular in erythroid precursors.

Animals↗
PubMed1980

[Enzymes of NAD+ catabolism in differentiating rabbit erythroid cells].

A study was made of the hydrolysis of NAD+ in the process of rabbit erythroid cell differentiation. Activities of NAD-hydrolase and NAD-pyrophosphatase, examined during maturation of erythroid cells, demonstrated a more significant decrease in the course of erythroblast-reticulocyte transformation, than at the next step of erythropoesis (reticulocyte-erythrocyte transformation). Both enzymes are strongly associated with the stroma. Under discussion is a question of the possible role of NAD+ catabolism.

Animals↗
PubMed1986

Transferrin receptor number, synthesis, and endocytosis during erythropoietin-induced maturation of Friend virus-infected erythroid cells.

Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 37 degrees C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I-transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these cells.

Animals↗
PubMed2006

Evidence for downregulation of erythropoietin receptor in bone marrow erythroid cells of patients with chronic idiopathic neutropenia.

OBJECTIVE: The aim of this study is to probe the mechanisms underlying anemia in patients with chronic idiopathic neutropenia (CIN) by evaluating parameters of bone marrow (BM) erythropoiesis. PATIENTS AND METHODS: Ten CIN patients fulfilling the criteria of anemia of chronic disease, 27 nonanemic CIN patients, and 30 healthy volunteers were enrolled in the study. Reserves and survival characteristics of BM erythroid cells were evaluated using flow cytometry and clonogenic assays. Serum erythropoietin (EPO) was measured with ELISA. Expression of EPO receptors (EPORs) on BM erythroid cells was evaluated by flow cytometry and reverse-transcription polymerase chain reaction. RESULTS: CIN patients display defective erythropoiesis in addition to previously reported impaired granulopoiesis. Patients have low number of CD34(+)/CD71(+) progenitor and CD36(-)/ Glycophorin A(+) (GlycoA(+)) precursor BM cells, and increased proportion of apoptotic cells within the CD34(+)/CD71(+) and CD36(+)/GlycoA(+) compartments. Burst-forming units erythroid (BFU-Es) in BM mononuclear or purified CD34(+) cells were significantly reduced in the patients. Patient BFU-Es increased significantly following in vitro treatment with tumor necrosis factor-alpha (TNF-alpha) and/or interferon gamma (IFN-gamma)-neutralizing antibodies. Local TNF-alpha and IFN-gamma production was higher in anemic than in nonanemic patients. EPO production was appropriate in the patients, but EPOR expression was significantly reduced in patient GlycoA(+) cells, especially in anemic patients. CONCLUSION: Impaired BM erythropoiesis in CIN patients is probably the result of increased local production of TNF-alpha and IFN-gamma that induce apoptosis, cell growth inhibition, and downregulation of EPOR expression on erythroid cells. We suggest that anemia in CIN patients displays overlapping pathophysiologic features with anemia of chronic disease.

Adult↗
PubMed1985

In vitro differentiation of a human erythroid cell line (KMOE) induced by some metabolic inhibitors.

KMOE-2/05, a continuous human erythroid cell line derived from a patient with acute erythremia was capable of differentiating into benzidine positive cells following exposure to cytosine arabinoside (CA), mitomycin C, or daunorubicin. Among the three substances, CA was the most effective inducer. Other compounds, reported as effective inducers on human and murine erythroid cells, were also tested but they were ineffective. Benzidine positive cells were counted to be approximately 500/1 X 10(5) cells after 10 days incubation with CA at its optimal concentration of 1 X 10(-5) M. Under the same conditions, the hemoglobin (Hb) concentration quantitated by radioimmunoassay (RIA) was more than 500 ng/1 X 10(6) cells. Quantitative kinetics of synthesized Hb and of benzidine-positive cell counts, after exposure to CA were closely correlated.

Antimetabolites↗
PubMed1997

[Changes in the kinetics of the hemoglobin, total protein and DNA content in rat erythroid cells in experimental anemia. II. The bone marrow].

Cytophotometry and population analysis of rat bone marrow erythroid cells, during anaemia induced by phenylhydrazin (4-8 days after the beginning of infections), revealed that in the period of restoration of erythron after the rats acute anaemia in the bone marrow blood formation the reciprocally complementary processes of microcytosis, macrocytosis and normal erythropoiesis occur. These processes are based on the normally functioning regulation mechanisms and on the reserve mechanisms, including DNA hyper-replication, which are turned on in the extreme conditions. The various paths of the bone marrow erythroid cells development during anaemia, the means and rates of the micro- and macrocytes formation and part of each line of the erythrocyte development in the restoration of blood cells quantity and haemoglobin homeostasis are analysed. It is suggested that mechanisms of the reserve erythropoiesis, activated during the acute anaemia of adult animals, could function also during the process of normal primary erythropoiesis of embryos.

Acute Disease↗
PubMed2003

HS5 of the human beta-globin locus control region: a developmental stage-specific border in erythroid cells.

Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken beta-globin locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human beta-globin locus. Here, we have characterized HS5 of the human beta-globin locus control region. We have examined its tissue-specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full beta-globin locus. Our results show that HS5 is erythroid-specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Surprisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage-specific border in erythroid cells.

Animals↗
PubMed1978

Regulation of differentiation in normal and transformed erythroid cells.

Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.

Cell Cycle↗
PubMed1991

Expression of embryonic globins by erythroid cells in juvenile chronic myelocytic leukemia.

Juvenile chronic myelocytic leukemia (JCML) is a rare hematopoietic neoplasia of early childhood with distinct hematologic and biochemical features. We studied the biologic properties and the globin synthetic profiles of JCML erythroid cells both in vivo and in vitro from a total of 24 patients. In these cases we observed the exuberant colony-forming unit-macrophage (CFU-M) colony growth, as reported previously. Furthermore, in contrast to previous reports, we found significant erythroid colony growth in most of our cases (average: 1,182 burst-forming unit-erythroid [BFUe] per 10(5) plated cells, range: 40 to 6,927). This growth was by and large erythropoietin-dependent and was not greatly influenced by other added cytokines. By several criteria all erythroid colony growth detected in vitro was derived from JCML progenitors. The globin synthetic profile of JCML erythroid cells showed high levels of fetal hemoglobin both in vivo and in vitro (gamma/gamma + beta: 53% to 94% in reticulocytes, 62% to 98% in BFUe-derived cells). In addition (in seven cases studied) we detected embryonic globins (epsilon and zeta) at the protein and messenger RNA level, a novel finding for primary leukemic cells. We speculate that the transformed erythroid cells in JCML harbor a trans environment supporting expression of developmentally earlier genes (fetal, embryonic). However, in contrast to other acute or subacute leukemias, JCML erythroid cells also have the ability to reach full maturation to the red cell level, thus allowing detection of this primitive program in vivo.

Bone Marrow↗
PubMed1999

Protein kinase B (c-Akt), phosphatidylinositol 3-kinase, and STAT5 are activated by erythropoietin (EPO) in HCD57 erythroid cells but are constitutively active in an EPO-independent, apoptosis-resistant subclone (HCD57-SREI cells).

We found that erythropoietin (EPO) and stem cell factor (SCF) activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid cells. To better understand signals controlling proliferation and viability, erythroid cells that resist apoptosis in the absence of EPO were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of PKB/Akt because the PI3-kinase inhibitor LY294002 blocked both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.

Apoptosis↗
PubMed1977

Translation of endogenous message in embryonic chick erythroid cell polysomes in a cell-free protein-synthesizing system.

Polysomes from 5.5-day and 6.5-day embryonic chick erythroid cells contain messenger RNA (mRNA) which can be translated into products in a cell-free protein-synthesizing system. The products of the cell-free system coelectrophoresed with carrier globin chains from the cells from which the polysomal pellet was isolated. Wheat germ S30 fraction increases by 1.5--2.0 fold [3H] leucine incorporation into trichloroacetic acid-precipitable material directed by the endogenous mRNA on chick erythroid cell polysomes. The wheat germ probably provides a specific factor or factors absent in a shortage in the incubation medium.

Animals↗
PubMed2000

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells.

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.

Animals↗