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PubMed1998

The Raf-1 protein mediates insulin-like growth factor-induced proliferation of erythroid progenitor cells.

Previous studies from this and other laboratories have shown that insulin-like growth factor-1 (IGF-I) and insulin-like growth factor-2 (IGF-II) support erythroid colony formation in cultures supplemented with serum substitute and recombinant erythropoietin. Subpopulations of IGF-I- and IGF-II-dependent, erythropoietin-independent colony-forming unit-erythroid (CFU-E)-derived colonies and BFU-E-derived colonies were identified under serum-substituted conditions for adult bone-marrow-derived erythroid progenitors which proliferate in the absence and presence of exogenous anti-erythropoietin receptor monoclonal antibody and in serum-substituted medium that was preadsorbed with anti-erythropoietin IgG. To assess whether Raf-1 is required for the formation of IGF-dependent, erythropoietin-independent human erythroid colonies, 5-15 microM sense or antisense oligomer to raf-1 were added to serum-substituted cultures containing either 2 U/ml recombinant human erythropoietin (rHuEpo) alone or 0-1,000 ng/ml IGF-I or IGF-II with/without 2 U/ml rHuEpo. Both erythropoietin-induced and IGF-induced erythroid colony formation were completely blocked by antisense (but not sense) oligomers to raf-1. Purified human CFU-Es were examined for Raf-1 message and protein. Total RNA was extracted, and raf-1 mRNA was detected on Northern blots. Furthermore, a 74 kD protein, corresponding to Raf-1, was also detected in CFU-Es purified from human adult sources. Together, these studies support the hypothesis that the Raf-1 protein mediates both erythropoietin-induced and IGF-induced signal transduction in human erythroid progenitor cells.

Adult↗
PubMed1991

Growth of human normal erythroid progenitors in liquid culture: a comparison with colony growth in semisolid culture.

Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.

Cell Division↗
PubMed1993

Successful treatment of Diamond-Blackfan anemia with interleukin 3.

This report describes the response of 18 Diamond-Blackfan anemia (DBA) patients to recombinant human interleukin 3 (rhIL-3). rhIL-3 was administered s.c. once daily on an escalating dose schedule (0.5-10 micrograms/kg/day). The rhIL-3 dose was escalated every 21 days until erythroid response was attained, grade III or IV nonhematologic toxicity was observed, or the maximal rhIL-3 dose was reached. Four patients experienced clinically significant erythroid responses. Two of the responders were steroid-dependent and transfusion-independent, while two were steroid-independent and transfusion-dependent. Baseline clinical or laboratory parameters, in particular in vitro bone marrow erythroid progenitor assays, were not useful in predicting rhIL-3 response. Two of the responding patients remain on maintenance rhIL-3 without diminution of effect at 490 and 855+ days. rhIL-3 was discontinued in the other two responders because of the development of deep venous thrombi.

Animals↗
PubMed1993

Newer approaches in treating chronic myelogenous leukemia.

Allogeneic bone marrow transplantation (BMT) is a useful approach for treating patients with chronic myelogenous leukemia (CML). In patients who lack a suitable donor, various approaches to utilize the patient's own hematopoietic stem cells obtained from bone marrow and/or peripheral blood have been developed. Preferential recovery of normal human hematopoietic progenitors over CML clone in long-term bone marrow culture (LTBMC) has been observed for a long time. LTBMC initiated in tissue culture (TC) flasks to that in "Lifecell" bags, which are gas-permeable plastic bags in which feeder-layer cells cannot adhere, has been investigated for several years. In our laboratory, cells were incubated in the presence of various growth factors to develop improved methods for stem cell expansion. Sustained hematopoietic stem cell growth in the absence of a feeder layer in plastic gas-permeable bags has been observed. Growth of marrow from patients with CML is being investigated extensively. Combining effective drugs to decrease the Philadelphia (Ph) clone prior to bone marrow harvest and the use of cultured bone marrow may provide a useful method for treating patients with CML. Results of various international studies are also discussed below.

Blood Cell Count↗
PubMed1998

Trimidox-mediated morphological changes during erythroid differentiation is associated with the stimulation of hemoglobin and F-cell production in human K562 cells.

Trimidox (3,4,5-trihdroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase with accompanied growth inhibition and differentiation of mammalian cells. Hydroxyurea (HU) is the only ribonucleotide reductase inhibitor in clinical use for the treatment and management of sickle cell anemia, since this compound increases fetal hemoglobin (Hb F) production: a potent inhibitor of sickle hemoglobin (Hb SS) polymerization. However, the main limitations of HU is its lack of potency, myelosuppression and short half life. These studies investigated the effects of trimidox on the induction of hemoglobin and F-cells production in K562 erythroleukemia cells. Our study reveals that trimidox exhibits concentration dependent inhibitory effect on K562 cells with increase in benzidine positive normoblasts and F-cells production as well as morphological changes typical of erythroid differentiation. These findings provide the first evidence that the growth inhibitory differentiation of cells induced by trimidox enhance hemoglobin and F-cells production.

Benzamidines↗
PubMed1999

Differential expression of protein kinase C isoform transcripts in human hematopoietic progenitors undergoing differentiation.

Protein kinase C (PKC), a key component of the signaling pathways leading to proliferation and differentiation, consists of a family closely related serine/threonine protein kinases. The mRNA expression of these PKC isoforms has been characterized during hematopoietic differentiation. Using the reverse-transcriptase polymerase chain reaction technique, we have analyzed the levels of isoform transcripts in bone marrow CD34(+) hematopoietic progenitors and their progeny differentiated along erythroid, megakaryocyte, or granulocyte/monocyte lineages, upon exposure to growth factors. In contrast with isoforms alpha, beta(I), beta(II), delta, and epsilon, ubiquitously expressed, isoforms theta, eta/L, zeta, and iota/lambda exhibited a lineage-restricted expression. These qualitative changes, which allow to distinguish the erythroid and megakaryocyte phenotypes from the granulocyte/monocyte phenotype, include zeta exclusively upregulated in granulocytes/monocytes and theta, eta/L, and iota/lambda exclusively expressed in megakaryocytes and erythroblasts. In contrast, erythroblasts and megakaryocytes, which supposedly share a common bipotential progenitor, displayed only quantitative changes. These results evidence the selective expression of PKC isoforms at transcriptional and/or posttranscriptional levels in hematopoietic progenitors induced to differentiate, which may suggest a differential contribution of individual isoforms to cellular signaling.

Antigens, CD↗
PubMed2001

Phenotypic identification and development of distinct microvascular compartments in the postnatal mouse spleen.

In this paper we report the development of the sinus network of mouse spleen during the first postnatal month as studied with a set of new rat monoclonal antibodies (mAbs) against mouse splenic endothelial cell subpopulations. One of the new mAbs (IBL-7/1) also stained B-cell lineage cells in the spleen shortly after the birth as confirmed by three-color flow cytometry. This B-cell staining in the primordial follicles vanished by the fourth postnatal week, so that the expression of IBL-7/1 antigen was restricted to the marginal sinus endothelium and some red pulp sinuses and a minor B-cell subset in the spleen, presumably distinct from the follicular B-cell compartment. The other mAb (IBL-9/2) selectively labeled the sinusoids of the deeper part of the red pulp, without any reactivity against hemopoietic cells. The IBL-9/2-reactive cells in newborns appeared as isolated elements throughout spleen, and during the segregation of white and red pulps they formed an extensive network in the red pulp outside the marginal zone. Double-labeling immunofluorescence revealed that most of these sinusoids also stained weakly with IBL-7/1 mAb, whereas the strongly IBL-7/1-positive vessels of this region were IBL-9/2 negative. Neither of these mAbs reacted with the central artery. The comparative phenotypic analysis of the various vascular segments indicates that the splenic sinusoids of the marginal zone and red pulp, respectively, are lined with a heterogeneous array of endothelium. For the precise identification, isolation, and characterization of the possible homing function of these endothelium subsets these region-specific mAbs may be of potential value.

Age Factors↗
PubMed1993

The close relationship between DNA replication and the selection of differentiation lineages of human erythroleukemia cell lines K562, HEL, and TF1 into either erythroid or megakaryocytic lineages.

The selection of differentiation lineages into either erythroid or megakaryocytic series was analyzed with human erythroleukemia cell lines K562, HEL, and cytokine-dependent TF1. A tumor promoter, TPA, induced a megakaryocyte marker, glycoprotein IIb/IIIa (GP IIb/IIIa) or IIIa (GP IIIa), but suppressed erythroid differentiation. On the other hand, aphidicolin, which is a potent inhibitor of DNA replication, inhibited GP IIb/IIIa or IIIa expression, but induced the expression of erythroid phenotypes. These phenomena were observed in all erythroleukemia cell lines tested. The bromodeoxyuridine labeling experiments indicated that de novo DNA synthesis was completely suppressed by aphidicolin treatment but was well preserved in TPA-treated cells. Among these three cell lines, erythropoietin (EPO) treatment induced erythroid differentiation of TF1 cells, which was dependent on GM-CSF or IL-3. In this case, EPO functioned as the survival factor and mild stimulator for cell proliferation as well as the inducer of erythroid differentiation. However, when either GM-CSF or IL-3 was depleted from the culture medium, TF1 ceased cell growth; concomitantly, hemoglobin-positive cells appeared, which is consistent with the results obtained with aphidicolin. The incubation of K562 cells for 48 h with either TPA or aphidicolin induced the irreversible commitment of cells to megakaryocytic and erythroid lineages, respectively. Our results using three different erythroleukemia cell lines suggest that a possible linkage between the DNA replication system and the selection of a differentiation lineage is the common feature of human erythroleukemia cell lines, and that these culture systems provide a suitable model for the analysis of the signal transduction system for differentiation lineage selection.

Aphidicolin↗
PubMed2002

Mechanisms leading to sustained reversion of beta-thalassemia in mice by doxycycline-controlled Epo delivery from muscles.

Erythropoiesis has been considered as a potential treatment for beta-thalassemia. Although Epo secretion from genetically engineered muscles allowed long-term correction of the disease in the mouse, repeated injections of rHuEpo were disappointing in human patients. Whether different mechanisms operate in humans and mice or whether Epo exhibits different biological activity depending on the administration route is currently unknown. We provide evidence that mechanisms recruited over a 36-week follow-up in beta-thalassemic mice were similar to those acting during stress-induced erythropoiesis in humans. beta-Thalassemic mice were rendered steadily normocythemic by the intramuscular injection of a tetracycline-inducible AAV vector encoding mouse Epo. Doxycycline dosage was adapted to hematocrit. Circulating red blood cells essentially synthesized beta-minor globin, the mouse equivalent to human gamma-globin. Quantification of erythroid progenitors indicated a steady-state expansion of erythroid burst-forming units programmed for beta-minor globin synthesis and a hastening of their maturation to hemoglobin-synthesizing cells. We discuss hypotheses that could account for the failure to recruit this mechanism over the long term in beta-thalassemic patients and raise the possibility of Epo gene therapy trials to treat beta-thalassemia.

Animals↗
PubMed2000

Different patterns of restriction to B19 parvovirus replication in human blast cell lines.

B19 parvovirus can replicate in erythroid progenitor cells and in a small number of human blast cell lines. To better understand and analyze the B19 virus replicative cycle, we performed and compared the infection of bone marrow cells and of different blast cell lines with erythroblastoid and megakaryoblastoid phenotypic characteristics (UT-7, TF-1, M-07, and B1647). Following in vitro infection, B19-specific nucleic acids were characterized with regard to the genome-replicative intermediates, the transcription pattern, and the localization of virus-specific nucleic acids inside infected cells. While all cell lines tested proved to be susceptible to B19 virus infection, two different patterns of restriction to replication of B19 virus were observed. In the first restriction pattern, observed in UT-7 cells, the single-stranded viral DNA was converted to double-stranded replicative intermediates, identical to those found in bone marrow cells, and a full set of viral transcripts were observed. However, replication and transcription were restricted to a small subset of cells, and production of capsid proteins was not detected. In the second restriction pattern, observed in TF-1, M-07, and B1647 cells, the single-stranded viral DNA was not converted to double-stranded replicative intermediates.

Bone Marrow Cells↗
PubMed1995

Crosstalk between thrombin and adenylyl cyclase-stimulating agonists in proliferating human erythroid progenitor cells.

Human erythroid progenitor cells grown in a suspension culture system were used to study possible interactions between different guanine nucleotide-binding protein (G-protein)-coupled receptor-effector systems during normal cell differentiation. Agonist-stimulated adenylyl cyclase was not inhibited by any one of a panel of ligands (ADP, UTP, platelet-activating factor, thrombin, alpha2-adrenoceptor agonists, interleukin 8, lysophosphatidic acid) most of which are known, in other cells, to reduce cAMP formation by a Gi-mediated, pertussis toxin-sensitive mechanism. The first four of these ligands are also known to cause transient changes in intracellular [Ca2+] in erythroid cells. Rather than inhibiting, thrombin (but not ADP, UTP or PAF) specifically caused a fivefold increase in the maximum adenosine- or prostaglandin E1-stimulated cAMP formation, without any shift of the concentration/response curves. Thrombin did not enhance forskolin- and AlF4-stimulated cyclase activity and had only a marginal effect on isoprenaline-dependent stimulation. The effect of thrombin seemed to be unrelated to intracellular Ca2+ release but could be partially mimicked by phorbol ester (PMA)-induced stimulation of protein kinase C (PKC) and was inhibited by staurosporin or by inactivation of PKC after long-term incubation with PMA. The activity of thrombin was restricted to proliferating, colony-forming progenitor cells while proerythroblasts were completely unresponsive. Our results suggest that the interaction of thrombin with Gs-linked receptors requires phosphorylation of a target protein that is different from adenylyl cyclase, Gs or Gi but may be involved in the regulation of receptor desensitization.

Adenosine↗
PubMed1994

Development of nucleolar apparatus in the chick primitive erythroid cells.

The primitive erythroid line cells of chick embryos were studied during embryonic days 2-14 by means of a cytochemical method to investigate the appearance and frequency of the main nucleolar types. The populations of erythroblasts and erythrocytes were classified according to the presence of functionally dominant nucleoli in their nuclei. In the course of primitive erythroid cell differentiation and maturation, compact nucleoli and nucleoli with nucleolonemas (both supposed to be RNA biosynthetically active) were gradually replaced by ring-shaped nucleoli and finally by micronucleoli reflecting the reversible and irreversible inhibition of RNA synthesis, respectively. The occurrence of the main nucleolar types and their values in primitive erythroid cells of the developing chick depend not only on the maturation stage of the blood cells, but also on the developmental stage of the chick embryo. In comparison with the definitive erythroid line of the post-hatching chick and hen, the cells of the chick embryonic primitive erythroid line possess relatively high values of "active" nucleolar types. These are still present in advanced maturation stages, and occur also as definitive erythroid lines of lower vertebrates.

Animals↗
PubMed1993

Isolation of a new cDNA clone encoding an Rh polypeptide associated with the Rh blood group system.

The polymerase chain reaction (PCR) was used to amplify Rh-related cDNAs from erythroid cells cultured by the selective two-phase liquid culture system for human erythroid progenitors in peripheral blood. Direct sequencing based on PCR presents heterozygous bands. Two Rh polypeptide cDNAs have been isolated from the PCR products and tentatively designated RhPI cDNA and RhPII cDNA. Both cDNA clones have an open reading frame composed of 1251 nucleotides. The RhPI cDNA clone shows a single nucleotide substitution with no amino acid substitution compared with the published sequence. The RhPII cDNA clone, on the other hand, differs from the above by 41 nucleotide substitutions with the open reading frame, resulting in 31 amino acid substitutions.

Amino Acid Sequence↗
PubMed1993

Interleukin-9 stimulates the proliferation of enriched human erythroid progenitor cells: additive effect with GM-CSF.

In the study we report here we investigated the colony-stimulating activities of interleukin-9 (IL-9). In the presence of erythropoietin, IL-9 was found to stimulate the proliferation of relatively early erythroid progenitor cells (BFU-E) from normal human bone marrow cells depleted of mononuclear phagocytes and T lymphocytes. Neutralization experiments demonstrated that the observed BFU-E-stimulating effect was not the result of intermediate production of IL-3 or GM-CSF by residual accessory cells in response to IL-9. Accordingly, the effects of IL-9 were preserved when cell suspensions were further depleted of accessory cells using CD34 enrichment of progenitor cells. Furthermore, IL-9 did not stimulate bone marrow mononuclear cells to express mRNA for IL-3, GM-CSF, EPA (erythroid-promoting activity), or IL-4, as determined by a cDNA-PCR method. IL-9 is likely to act on a subpopulation of IL-3-responsive erythroid progenitor cells that are not stimulated by GM-CSF, since plateau concentration of IL-9 and GM-CSF had additive effects on BFU-E formation, whereas a combination of IL-9 and IL-3 did not. In addition to its burst-promoting activity, IL-9 was found to have a modest stimulatory activity on myeloid progenitor cells (CFU-GM) in some experiments, suggesting that this effect may be donor related.

Animals↗
PubMed1996

Molecular evidence for increased hematopoietic proliferation in the spleen of the b/b laboratory rat.

The splenomegaly and the appearance of a significant number of CFU-E (erythroid colony-forming units) and BFU-E1 (erythroid burst-forming units) in the Belgrade laboratory rat (b/b) spleen prompted us to analyse further the molecular evidence for increased hematopoietic proliferation in the b/b spleen. Messenger RNAs (mRNAs) specific for globins, proteins for iron transport and deposition and the band 3 protein were used in rat erythropoietic tissues as markers for proliferation and erythroid differentiation. In the b/b spleen, all mRNAs analysed display an erythroid-specific pattern of expression. This analysis also revealed an enhanced level of mRNA for ferritin in the +/b spleen, whereas erythrocyte-specific mRNA production was normal.

Animals↗
PubMed2002

The effect of thrombopoietin on erythroid progenitors in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare congenital red blood cell aplasia. Usually, erythropoiesis in vitro is defective, and decreased numbers of erythroid progenitors are found in colony assays performed with bone marrow cells. Recently, some investigators showed that thrombopoietin (TPO) also works on common multipotent progenitor cells and stimulates erythropoiesis. We examined the effect of TPO together with erythropoietin (EPO), stem cell factor (SCF), and/or interleukin-3 (IL-3) on erythroid burst-forming units (BFU-E) of bone marrow nonadherent mononuclear cells from 2 patients with DBA using a serum-free culture system. Very few BFU-E appeared in cultures containing EPO alone. Adding IL-3, SCF, or both to cultures containing EPO induced an increase in the number of BFU-E. When TPO was added to cultures containing EPO and IL-3, SCF, or both, the number of BFU-E further increased. Especially in patient 2, BFU-E formation was grossly equal to that in normal controls. We conclude that TPO enhances bone marrow erythropoiesis in patients with DBA in the presence of EPO and SCF and/or IL-3. The data raise the possibility of the combination of TPO and SCF as a therapeutic agent in DBA.

Anemia, Diamond-Blackfan↗
PubMed2001

Requirement of thrombopoietin-induced activation of ERK for megakaryocyte differentiation and of p38 for erythroid differentiation.

Thrombopoietin (TPO) plays a critical role not only in proliferation and differentiation of megakaryocytes but also in erythroid differentiation. We have investigated whether the different pathway of mitogen-activated protein kinase (MAPK) after TPO stimulation may discriminate megakaryocyte and erythroid differentiation. In this study, we have used human CD34+ hematopoietic progenitor cells (HPCs) from cord blood (CB) in serum-free liquid culture supplemented with TPO, to compare the respective effects of specific inhibitors of MAPK kinase (MEK) (PD98059) and p38 MAP kinase (p38) (SB203580) on megakaryocyte and erythroid development. PD98059, but not SB203580, significantly suppressed TPO-induced megakaryocyte differentiation when examined by the expression of CD41 and polyploidy assay. In the presence of SB203580, CD34+/CD36+ erythroid progenitors clearly decreased, whereas they increased when cultured with PD98059. These results indicate that activation of extracellular-signal-regulated kinase (ERK) is required for TPO-induced megakaryocyte differentiation and that p38 is required for TPO-induced erythroid differentiation.

Antigens, CD34↗