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Results for “Erythroid Precursor Cells”

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PubMed1977

An assay for serum cytotoxicity against erythroid precursor cells in pure red cell aplasia.

Several reports have indicated that a circulating serum inhibitor (antibody) is involved in the pathogenesis of acquired pure red cell aplasia (PRCA). In the present study, the pathophysiologic significance of this inhibitor was assessed according to the status of erythroid progenitor cells in the bone marrow. So far, direct proof for the antibody acting against erythroid stemcells was lacking. Employing an "in vitro" assay, erythroid colony forming cell (CFU-e) numbers in PRCA marrow were quantified and the cytotoxic effect of PRCA serum on CFU-e was investigated. It was revealed that the CFU-e population size in the marrow of PRCA patients was severely reduced; at the same time the relative number of myeloid colony forming cells was normal. The serum was demonstrated to contain a factor cell which was cytotoxic to CFU-e, in the presence of complement. The results indicate that inhibition of erythropoiesis in PRCA is achieved by a complement dependent plasma factor which eliminates or inactivates CFU-e and which constitutes an effective block at the precursor cell level in the differentiation pathway of the erythroid line. The data present a practical assay for measuring cytotoxic factors affecting erythroid stem cells.

Adult↗

PubMed2005

Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells.

Polycythemia vera (PV) is a clonal myeloproliferative disorder characterized by excessive erythrocyte production. Most patients with PV harbor an activating JAK2 mutation, but the molecular links between this mutation and erythrocyte overproduction are unknown. The interaction between death receptors and their ligands contributes to the physiological regulation of erythropoiesis through the inhibition of erythroblast proliferation and differentiation. With the use of an in vitro culture system to generate differentiating erythroid cells, we found that erythroblasts derived from patients with PV harboring the JAK2 V617F mutation were able to proliferate and generate higher numbers of mature erythroid cells in the presence of inhibitory signals delivered by CD95 (Fas/Apo-1) and TRAIL receptor stimulation. JAK2-mutated PV erythroblasts showed lower levels of CD95-induced caspase activation and incomplete caspase-mediated cleavage of the erythroid transcription factor GATA-1, which was entirely degraded in normal erythroblasts on CD95 stimulation. JAK2 mutation was associated in PV erythroblasts with cytokine-independent activation of the JAK2 effectors Akt/PKB and ERK/MAP and with a deregulated expression of c-FLIP(short), a potent cellular inhibitor of death receptor-induced apoptosis. These results show the presence in PV erythroblasts of proliferative and antiapoptotic signals that may link the JAK2 V617F mutation with the inhibition of death receptor signaling, possibly contributing to a deregulation of erythropoiesis.

Adult↗

PubMed1987

Induction of globin mRNA transcription by erythropoietin in differentiating erythroid precursor cells.

The effects of erythropoietin (epo) on the proliferation of late erythroid progenitor cells (CFU-E) and on the formation of hemoglobin and of globin mRNA in these cells are described. CFU-E were isolated from thiamphenicol-pretreated anemic mice by elutriation and Percoll density gradient methods. These CFU-E are restricted in their capacity to proliferate in vitro without added epo. The epo dependence in vitro was not absolute. With no epo in the culture medium the first cell division was unimpaired, whereas the third division was only 1%-2% of the control. In the absence of epo the synthesis of hemoglobin is very low in CFU-E, but is increased significantly after about 5 h of incubation with epo present. In epo deprived cells there was considerable hemoglobin formed at about 14 h, but not earlier. The presence as detected by the Northern blot technique of globin mRNA, isolated from CFU-E, was variable, probably depending on the presence of some more mature erythroid cells. By an extrapolation method we show evidence that pure CFU-E would have virtually no detectable globin mRNA. The production of globin mRNA is rapidly (2 h) induced in cells incubated with epo. We conclude that epo, besides having a mitogenic effect on CFU-E, induces the rapid expression of the globin genes.

Animals↗

PubMed1980

Quantification of spectrin-containing erythroid precursor cells in normal and perturbed erythropoiesis.

The presence of spectrin in erythroid cells of female BALB/c mice has been detected by specific antisera to spectrin, using an immunofluorescent sandwich technique. Spectrin-containing cells are more numerous in bone marrow (18.9%) than in spleen (3.7%). In marrow, spectrin-containing cells exceed benzidine positive cells by about 6.2% whereas in spleen, numbers are almost equivalent. This suggests that normal proerythroblasts and perhaps more primitive erythroid cells contain spectrin, which is consistent with cytological characteristics of some spectrin-positive cells. Proerythroblast cells from spleens of Rauscher virus induced erythroleukemic mice contain spectrin although markers for hemoglobin synthesis are absent. Changes in CFUE number are closely correlated with those of spectrin-positive erythroid cells when erythropoiesis is perturbed. In red cell transfused, erythropoietically suppressed mice undergoing hemopoietic regeneration the administration of erythropoietin results in appearance of CFUE and spectrin-positive cells within 24-72 h. Due to different methods of assaying for CFUE and presence of spectrin, numerical data cannot be compared quantitatively because of uncertainty in the level of detection. Although it is not possible to demonstrate the presence or absence of spectrin in CFUE, changes in spectrin-containing cells and CFUE are closely correlated in time. Spectrin provides a convenient marker for both normal and leukemic nucleated erythroid cells and particular provides a functional marker for proerythroblasts which have previously been identified only by morphology.

Animals↗

PubMed1985

A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro.

A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.

Animals↗

PubMed1982

A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells.

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O-tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.

Animals↗

PubMed1987

Expression of the 5'-flanking region of the beta major-globin gene in cultured murine erythroid precursor cells.

The amounts of mouse beta major-globin mRNA and globin gene upstream RNA polymerase III transcripts were compared during the differentiation of purified erythroid colony-forming progenitor cells (CFU-E) in vitro. The accumulation of each RNA was determined relative to the 100% levels in fetal liver erythroid cells. The mRNA level was low in freshly isolated CFU-E and began to accumulate only after a lag period of approximately 2-4 h. It continued to accumulate for approximately 40 h thereafter, at which point it was comparable to that in the fetal liver. In contrast, in three of four CFU-E preparations, the relative level of upstream RNAs was high in freshly isolated CFU-E and reached the maximal level (defined as 100% of the fetal liver level) more rapidly than did the mRNA. The early induction and accelerated accumulation of upstream RNAs in immature erythroid cells suggest some role for these RNAs at an early stage of globin gene activation.

Animals↗

PubMed1988

Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.

Erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells)-are erythropoietin (EP)-sensitive cells at the late colony forming unit-erythroid (CFU-E) and cluster forming unit stages of differentiation (Koury et al., J. Cell. Physiol. 121:526-532, 1984). We investigate here the EP requirements of FVA cells in vitro for viability, proliferation, and maturation. By delaying the addition of EP to FVA cell cultures or by withdrawing EP at early times of culture, the subsequent viability, cell numbers, and maturation were diminished. The longer the delay in EP addition or the earlier the EP withdrawal, the more diminished these parameters were when compared to cultures which contained EP throughout the 48 h of differentiation. FVA cells had a period of EP requirement in vitro that lasted for only 24 h or less after the initiation of culture. During these crucial first 24 h, EP induced an increase in the synthesis of all size classes of RNA. Protein synthesis was maintained at a stable level in cells cultured with EP, but it declined in cells cultured without it. In contrast, the synthesis rate of DNA and the content of DNA per cell were not affected by the presence of EP in the culture. However, FVA cells cultured without EP had progressive accumulation of small sized DNA due to breakage of higher molecular weight DNA. The rate of DNA breakdown was sufficient to prevent DNA accumulation and thus it probably plays a role in the abortion of cell proliferation. No such breakage was found in cells cultured with EP. Our results indicate that EP exerts an effect on FVA cells in culture which is reflected in their viability, cell number, and maturation. This effect is not mediated by a stimulation of the rate of DNA synthesis, but is accompanied by stimulation of overall RNA synthesis and maintenance of protein synthesis.

Animals↗

PubMed1979

Particle-induced erythropoietin-independent effects of erythroid precursor cells in murine bone marrow.

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.

Animals↗

PubMed1995

[Large volume, isovolemic erythrocytapheresis in treatment of polycythemia vera. Effect of massive iron depletion of proliferation behavior of erythroid precursor cells (BFU-E)].

BACKGROUND: Isovolemic large volume erythrocyte-apheresis (EA) is a rapid, effective and well-tolerated treatment modality for red blood cell (RBC) depletion in patients with polycythaemia vera (PV). According to clinical observations its long lasting effect (median interval from EA to EA about 6 months) may, at least in part, be due to the associated loss of iron. METHODS: Therefore we investigated the influence of EA on the proliferative capacity of erythroid (BFU-E) and granulocyte-macrophage (GM-CFU) progenitor cells and on the erythropoietin-(EPO-)independent, spontaneous in vitro growth of BFU-E in particular. In six patients RBC and iron parameters as well as the proliferative capacity of hematopoietic progenitor cells were determined before and after EA. RESULTS: RBC parameters (in median) were before/after EA: RBC 7.64/5.93 x 10(6)/microliter; Hct 53/40%; Hb 15.5/12.0 g/dl and remained at reduced levels for several months; serum iron and ferritin levels decreased, while transferrin levels and transferrin receptor expression on peripheral mononuclear cells were enhanced. Serum EPO-levels were temporarily but only slightly increased. In all patients there was a significant inhibition of the growth of BFU-E detectable after EA while the GM-CFU were less affected. Within 3 to 6 weeks, the inhibition of endogenous BFU-E ranged from 53% to 100% and of EPO-dependent BFU-E from 31% to 74%. The inhibition of EA associated BFU-E growth could be reduced by in vitro addition of FeCl3. On the other hand, in vitro exposure of progenitor cells to an equivalent concentration of the iron chelator DFO (deferoxamine mesylate) resulted in a total suppression of progenitor cell growth. CONCLUSION: Our data suggest that the long lasting effect of EA is not only due to the high volume removal of RBC itself, but also to the growth inhibition of EPO-independent and -dependent BFU-E which is obviously mediated by the considerable loss of iron by EA.

Blood Component Removal↗

PubMed1986

The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation.

The plasma membrane of murine erythro-leukemia (MEL) cells contains a 140-kD protein that binds specifically to fibronectin. A 125I-labeled 140-kD protein from surface-labeled uninduced MEL cells was specifically bound by an affinity matrix that contained the 115-kD cell binding fragment of fibronectin, and specifically eluted by a synthetic peptide that has cell attachment-promoting activity. The loss of this protein during erythroid differentiation was correlated with loss of cellular adhesion to fibronectin. Both MEL cells and reticulocytes attached to the same site on fibronectin as do fibroblasts since adhesion of erythroid cells to fibronectin was specifically blocked by a monoclonal antibody directed against the cell-binding fragment of fibronectin and by a synthetic peptide containing the Arg-Gly-Asp-Ser sequence found in the cell-binding fragment of fibronectin. Erythroid cells attached specifically to surfaces coated either with the 115-kD cell-binding fragment of fibronectin or with the synthetic peptide-albumin complex. Thus, the erythroid 140-kD protein exhibits several properties in common with those described for the fibronectin receptor of fibroblasts. We propose that loss or modification of this protein at the cell surface is responsible for the loss of cellular adhesion to fibronectin during erythroid differentiation.

Animals↗

PubMed1999

Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34(+) cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 micromol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V-binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

Androstadienes↗

PubMed1997

Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly-devised quantitative flow-cytometric assay.

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine-RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by 125I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34+CD38 cells (approximately 1600 sites/cell), and decreased in the following order: CD34+CD38- cells > CD34+CD38+ cells > CD34-CD38+ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34+ fraction expressed more EpoR than their CD34- fraction. However, the expression levels of EpoR of these fractions were lower than CD34+CD38- cells. These results indicated that EpoR was highly expressed on CD34+ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.

ADP-ribosyl Cyclase↗

PubMed1994