Human Erythroid Progenitor Cells Research Articles

Proliferation of human erythroid progenitor cells is stimulated by protein kinase cα and inhibited by thrombin

We have used erythropoietin(Epo)- and stem cell factor(SCF)-dependent cultures of early erythroid progenitors in serum-free medium to study the effect of PKC- and of G protein-coupled receptor-linked signals on cell proliferation. DNA-synthesis was measured by 3H-thymidine incorporation. PKC activity was quantified after subtype-specific immunoprecipitation. The phorbol ester PMA, an activator of Ca2+-dependent and -independent PKC subtypes stimulated DNA synthesis to the same extent as did growth factors. Epo-, SCF- and PMA-dependent proliferation was strongly (80-90%) reduced by an inhibitor of Ca2+-dependent PKC isoforms (Gö 6976) or by thrombin (2 U/ml). Unlike PMA, neither thrombin nor Epo or SCF induced membrane translocation of individual PKC subtypes. Epo and SCF specifically stimulated cytosolic PKCα but no other PMA-sensitive PKC subtypes. This activation was blocked by Gö 6976 but not by thrombin. The inhibitory effect of thrombin on DNA synthesis required the activation of the small GTPase Rho and could be completely eliminated by the C3 exotoxin from C. botulinum, a specific blocker of Rho-mediated effects. Ceramides, possible mediators of Rho-induced apoptosis in erythroleukemia cells, inhibited Epo and SCF-dependent DNA synthesis and enhanced the effect of thrombin. We conclude from these observations that (1) PKCα plays a central role in cytokine-controlled development of erythroid progenitors and (2) that thrombin, via a Rho-dependent but PKC-independent pathway, acts as an endogenous inhibitor of human erythroid progenitor proliferation.

Stem cell factor and erythropoietin inhibit apoptosis of human erythroid progenitor cells through different signalling pathways.

Erythropoietin (EPO) and stem cell factor (SCF) are two important factors in human erythropoiesis. We have recently demonstrated that SCF and EPO synergistically activate mitogen-activated protein (MAP) kinase, thereby promoting growth of human erythroid colony-forming cells (ECFCs). In the present study, we have examined the intracellular mechanisms by which SCF and EPO maintain survival of these cells. In the absence of SCF and EPO, human ECFCs underwent rapid apoptosis. The process was significantly inhibited by addition of a single factor and was totally prevented in the presence of both factors. Treatment of ECFCs with wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI3K), inhibited the antiapoptotic effect of SCF but had no effect on that of EPO, indicating that SCF but not EPO inhibits apoptosis through the PI3K pathway. In contrast, treatment of ECFCs with PD98059, a specific inhibitor of MAP kinase/ERK kinase (MEK), inhibited cell growth but had no effect on the antiapoptotic activity of either SCF or EPO, suggesting that SCF and EPO prevent apoptosis of human ECFCs independent of the extracellular signal-regulated kinase (ERK) pathway. Interestingly, both EPO and SCF induced activation of PI3K. However, through PI3K, SCF caused activation of protein kinase B (PKB), an anti-apoptosis signal, whereas EPO led to activation of ERKs. Furthermore, the SCF- and EPO-maintained expression of antiapoptotic protein Bcl-XL was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl-XL may not have a major role in preventing apoptosis of human ECFCs. Phosphorylated BAD was not affected by SCF, EPO or wortmannin. Taken together with our previous results, the present study indicates that SCF and EPO support survival and growth of human ECFCs through different signalling pathways and that they transduce distinctly different signals through activation of PI3K.

Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown

·ivikova K. , J . Dianovsk : Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown. Acta Vet. Brno 2000, 69: 45–50. The in vitro effect of the herbicide Modown (with active component bifenox) was tested for the ability to influence cell proliferation of PHA-stimulated bovine peripheral lymphocytes. Mitotic (MI) and proliferation (PI) indices were determined as an alternative for the screening of the cytostatic activity. The herbicide Modown exerted a clear effect on the inhibition of MI and PI over a concentration-tested range of 25 μg/ml to 1000 μg/ml. An expressive proliferation delays was found after the treatment with herbicide at a dose of 250 μg/ml (P < 0.001), while the higher doses of 500 and 1000 μg/ml had caused nearly complete mitotic inhibition in each donor (P < 0.05 and P < 0.001, respectively). A correlation between the PI and MI inhibition refers rather to cytostatic than cytotoxic effects of the herbicide. The results support the possibility of immunosuppression by herbicide exposure. Herbicide Modown, bovine peripheral lymphocytes, cell proliferation, cytostatic effect Nitrodiphenylether herbicides are persistent and lipophilic environmental contaminants may contribute to the impairment of animal health and production. The presence of pesticide residues has been demonstrated in raw bovine milk and in different domestic animal tissues (Lioi et al. 1998). All nitrodiphenylether herbicides exert their phytotoxic activity in chlorophyll of plants by the reduction itself to radical species, which initiate destructive reactions in membrane lipids, leading to the cell leakage (Corbet t et al. 1984). Members, as acifluorfen and oxyfluorfen, e.g. are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane–bound enzyme involved in the heme and chlorophyll biosynthesis pathways (Camadro et al. 1995). The later was studied for the induction of haematological diseases in human erythroid progenitor cells but a cytotoxic effect had been seen only at very high concentrations (Rio et al. 1997). Bifenox is a selective herbicide used in control of annual broad-leaved weeds and some grasses, e. g. in cereals, maize, soya beans, rice and other crops. J inno et al. (1999) described the cytotoxic and porphyrogenic effects of bifenox in rat hepatocytes. They found the maximum porphyrin accumulation at 0.25 mM of the herbicide and an inhibion of protoporphyrogen oxidase, resulting in the accumulation of protoporphyrin IX. Francis et al. (1999) evaluated maternal and developmental toxicity of ten diphenylether herbicides (including bifenox) in mice. They found a correlation among the position of chlorine substituents and the potential for inducing prenatal and postnatal syndroms. But no prenatal and postnatal embryotoxicity was shown. ACTA VET. BRNO 2000, 69: 45–50 Address for correspondence: RNDr. Katarina ·ivikova, CSc. Department of Veterinary Genetics, University of Veterinary Medicine Komenskeho 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 95 63 321 11, (ext. 482) Fax: +421 95 63 236 66 E-mail: sivikova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm

Inhibition of β-globin gene expression by 3′-azido-3′-deoxythymidine in human erythroid progenitor cells

3′-Azido-3′-deoxythymidine (AZT) treatment in HIV-infected patients is limited by bone marrow suppression including neutropenia and anemia. Previous studies had shown a direct effect of high concentrations of this drug on globin gene expression in K-562 erythroleukemia cells. To better define the mechanism(s) of AZT-induced bone marrow toxicity, the present study evaluates these effects in more relevant human erythroid progenitor liquid cultures, because AZT is 100 times more toxic to human bone marrow cells than K-562 cells. At a clinically relevant concentration of 1 μM, AZT inhibited specifically erythroid cell growth by ∼58% as compared with untreated cells. The percentage of cells synthesizing hemoglobin was decreased also by 47% in AZT-treated cells with β-globin mRNA levels accounting for 0.27 pmol in treated cells as compared with 1.44 under control conditions while β-actin levels remained unchanged. Under the same conditions, AZT inhibited the β-globin chain synthesis by ∼60% as compared with the control. Consistent with the data described above was the finding that a concentration as low as 0.1 μM of AZT decreased by almost 40% the binding level of the erythroid-specific transcription factor GATA-1. These findings demonstrate that AZT, at clinical relevant concentrations, specifically inhibits β-globin gene expression in human erythroid progenitor liquid cell culture.

Possible interactions between the NS-1 protein and tumor necrosis factor alpha pathways in erythroid cell apoptosis induced by human parvovirus B19.

Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G(2) phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-alpha and NS-1 in human erythroid cells.

Haemopoietic Lineage Sensitivity and Individual Susceptibility to PCB126: Relation to Glutathione S-transferase μ

The effect of PCB126 on the in vitro growth potential of human myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells derived from human cord blood haemopoietic cells was examined. The possible link between individual variability in susceptibility and the presence of the xenobiotic metabolizing enzyme glutathione S-transferase class mu (GST μ) was studied. After density centrifugation separation, mononuclear cord blood cells were cultured in the presence of PCB126 (10 −6 m and 10 −8 m). Several cord blood samples were simultaneously tested for: (1) colony formation and (2) the presence of the GST μ gene measured by PCR. An interindividual variability in the response to PCB126 was present. At 10 −8 m and 10 −6 m PCB126, respectively 8 (40%) and 10 (50%) out of 20 cord blood samples showed a significant dose-dependent decrease in CFU-GM numbers. Erythroid progenitors were less affected by PCB126. At 10 −8 m and 10 −6 m PCB126, respectively only two (12%) and three (18%) out of 17 cord blood samples showed a significant decrease in BFU-E numbers. The presence or absence of the GST μ gene was determined using PCR. The GST μ gene was present in 52% (14 out of 27 samples tested) of the cord blood samples. Damage to the myeloid and erythroid haemopoietic progenitor cells at either PCB126 concentrations was not correlated with the presence of the GST μ gene.

Erythropoietin– and Stem Cell Factor–Induced DNA Synthesis in Normal Human Erythroid Progenitor Cells Requires Activation of Protein Kinase Cα and Is Strongly Inhibited by Thrombin

Proliferation, differentiation, and survival of erythroid progenitor cells are mainly regulated by stem cell factor (SCF) and erythropoietin (Epo). Using normal human progenitors, we analyzed the role of Ca2+-sensitive protein kinase C (PKC) subtypes and of G-protein-coupled receptor ligands on growth factor-dependent DNA synthesis. We show that stimulation of DNA synthesis by the two growth factors requires activation of PKCalpha. Inhibitors of Ca2+-activated PKC subtypes blocked the growth factor-induced 3H-thymidine incorporation. SCF and Epo caused no significant translocation of PKCalpha into the membrane, but treatment of intact cells with either of the two cytokines resulted in enhanced activity of immunoprecipitated cytosolic PKCalpha. Stimulation of PKC with the phorbol ester PMA mimicked the cytokine effect on DNA synthesis. Epo-, SCF-, and PMA-induced thymidine incorporation was potently inhibited by thrombin (half-maximal inhibition with 0.1 U/mL). This effect was mediated via the G-protein-coupled thrombin receptor and the Rho guanosine triphosphatase. Adenosine diphosphate caused a modest Ca2+-dependent stimulation of DNA synthesis in the absence of cytokines and specifically enhanced the effect of SCF. Cyclic 3', 5'-adenosine monophosphate exerted a selective inhibitory effect on Epo-stimulated thymidine incorporation. Our results define PKCalpha as major intermediate effector of cytokine signaling and suggest a role for thrombin in controlling erythroid progenitor proliferation.

Interferon γ Induces Upregulation and Activation of Caspases 1, 3, and 8 to Produce Apoptosis in Human Erythroid Progenitor Cells

Interferon gamma (IFNgamma) induces apoptosis in purified human erythroid colony-forming cells (ECFC) and inhibits cell growth. Fas (APO-1; CD95) and Fas ligand (FasL) mediate apoptosis induced by IFNgamma, because Fas is significantly upregulated by IFNgamma, whereas Fas ligand is constitutively present in the ECFC and neutralization of FasL greatly reduces the apoptosis. Because conversion of caspases from their dormant proenzyme forms to active enzymes has a critical role in transducing a cascade leading to apoptosis, we performed further studies of the expression and activation of caspases in normal human and IFNgamma-treated day-6 ECFC to better understand the mechanism of IFNgamma action in producing this cell death. RNase protection assays showed that the caspase-1, -2, -6, -8, and -9 mRNAs were upregulated by IFNgamma, whereas the caspase-5 and -7 mRNAs were not increased. Western blots showed that FLICE/caspase-8 was upregulated and activated by 24 hours of incubation with IFNgamma. FADD was not similarly altered by incubation with IFNgamma. Western blots of ICE/caspase-1, which might be required for amplification of the initial FLICE activation signal, showed that pro-ICE expression significantly increased after treatment with IFNgamma for 24 hours and cleavage of pro-ICE also increased. CPP32/apopain/caspase-3, responsible for the proteolytic cleavage of poly (ADP) ribose polymerase (PARP), was also studied and treatment of ECFC with IFNgamma resulted in an increased concentration of caspase-3 by 24 hours and a clear induction of enzyme activation by 48 hours, which was identified by the appearance of its p17-kD peptide fragment. The cleavage of PARP was demonstrated by an obvious increase of the 89-kD PARP cleavage product, which was observed at almost the same time as caspase-3 activation in the IFNgamma-treated cells, whereas untreated ECFC showed little change. Peptide inhibitors of the caspase proteins, DEVD-fmk, DEVD-cho, YVAD-cho, and IETD-fmk, were incubated with the ECFC to obtain further evidence for the involvement of caspases in IFNgamma-induced apoptosis. The activation of FLICE/caspase-8 and CPP32/caspase-3 and cleavage of PARP clearly were inhibited, but the reduction of cell growth due to apoptosis, induced by IFNgamma, was only partially blocked by the presence of the inhibitors. These results indicate that IFNgamma acts on ECFC not only to upregulate Fas, but also to selectively upregulate caspases-1, -3, and -8, which are activated and produce apoptosis, whereas the concentrations of FasL and FADD are not demonstrably changed.

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.

Characterization of CKβ8 and CKβ8-1: Two Alternatively Spliced Forms of Human β-Chemokine, Chemoattractants for Neutrophils, Monocytes, and Lymphocytes, and Potent Agonists at CC Chemokine Receptor 1

Two new members of human beta-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human beta-chemokine, CKbeta8, whereas the other is a splicing variant of CKbeta8, therefore named CKbeta8-1. CKbeta8 was short in 51 nucleotides (17 amino acids) compared with CKbeta8-1. The mature proteins of CKbeta8-1 and CKbeta8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKbeta8-1 and CKbeta8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active beta-chemokines from a single gene.

Characterization of CKβ8 and CKβ8-1: Two Alternatively Spliced Forms of Human β-Chemokine, Chemoattractants for Neutrophils, Monocytes, and Lymphocytes, and Potent Agonists at CC Chemokine Receptor 1

AbstractTwo new members of human β-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human β-chemokine, CKβ8, whereas the other is a splicing variant of CKβ8, therefore named CKβ8-1. CKβ8 was short in 51 nucleotides (17 amino acids) compared with CKβ8-1. The mature proteins of CKβ8-1 and CKβ8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKβ8-1 and CKβ8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active β-chemokines from a single gene.

Molecular cloning of leukotactin-1: a novel human beta-chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3.

A new member of human beta-chemokine cDNA was isolated and named leukotactin-1 (Lkn-1). Lkn-1, along with murine macrophage inflammatory protein-related protein-1 and -2, defines a subgroup of beta-chemokines based on two conserved cysteines in addition to the four others conserved in all beta-chemokines. The putative mature Lkn-1 is composed of 92 amino acids with a calculated m.w. of 10,162. The Lkn-1 gene was mapped to human chromosome 17, region q12. Recombinant Lkn-1 was a potent chemoattractant for neutrophils, monocytes, and lymphocytes and induced calcium flux in these cells. Lkn-1 specifically induced calcium flux in CCR1- and CCR3-expressing HOS cell lines. Lkn-1 suppressed colony formation by human granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. Hence, we have isolated and characterized a human C6 beta-chemokine that is a potent agonist at CCR1 and CCR3 and shows broad biologic activities, including leukocyte chemoattraction.

Interferon γ Downregulates Stem Cell Factor and Erythropoietin Receptors But Not Insulin-Like Growth Factor-I Receptors in Human Erythroid Colony-Forming Cells

AbstractInterferon γ (IFNγ) has been shown to inhibit proliferation and differentiation of erythroid progenitor cells and to produce apoptosis of erythroid cells, whereas stem cell factor (SCF ), erythropoietin (EP), and insulin-like growth factor-I (IGF-I) have distinct roles in enhancing erythroid cell production and preventing apoptosis. The mechanism by which IFNγ exerts an inhibitory effect on the positive roles of these growth factors is unknown. Although some inhibitory cytokines including IFNγ have been shown to downregulate growth factor receptors, the effect of IFNγ on SCF, EP, and IGF-I receptors of human erythroid progenitor cells has not been defined. We obtained highly purified day-5 or day-6 erythroid colony-forming cells (ECFCs) from human blood in sufficient quantity and purity for radiolabeled cytokine binding studies and analysis of mRNA. When day-5 ECFCs were incubated with increasing concentrations of recombinant human (rh) IFNγ for 24 hours at 37°C, specific binding of 125I-rhSCF to SCF receptors was significantly decreased by 25% to 40% in a dose-dependent fashion, with the maximum effect at 2,500 to 5,000 U/mL of IFNγ. The decrease was apparent by 12 hours of incubation and was only slightly lower by 24 hours. The numbers of SCF and EP receptors, but not of IGF-I receptors, per ECFC, calculated by Scatchard analysis, were significantly decreased by 30% and 23% to 25%, respectively, after incubation with 2,500 U/mL rhIFNγ for 24 hours at 37°C, whereas the binding affinities were not affected. This decrease in SCF receptors was confirmed by flow cytometry using an anti–c-kit mouse monoclonal antibody. Northern blot analysis showed that the mRNAs for the SCF and EP receptors, but not for the IGF-I receptors, were decreased by 50% to 60% after 3 hours of incubation at 37°C with 2,500 U/mL of rhIFNγ. This persisted for 24 hours without alteration of the stability of the SCF and EP receptor mRNAs. These observations suggest that one means by which IFNγ inhibits erythroid cell proliferation and differentiation and produces apoptosis may be through the reduction of the number of target receptors for SCF and EP and that this occurs through transcriptional inhibition of the corresponding mRNAs.

Interferon γ Downregulates Stem Cell Factor and Erythropoietin Receptors But Not Insulin-Like Growth Factor-I Receptors in Human Erythroid Colony-Forming Cells

Interferon γ (IFNγ) has been shown to inhibit proliferation and differentiation of erythroid progenitor cells and to produce apoptosis of erythroid cells, whereas stem cell factor (SCF ), erythropoietin (EP), and insulin-like growth factor-I (IGF-I) have distinct roles in enhancing erythroid cell production and preventing apoptosis. The mechanism by which IFNγ exerts an inhibitory effect on the positive roles of these growth factors is unknown. Although some inhibitory cytokines including IFNγ have been shown to downregulate growth factor receptors, the effect of IFNγ on SCF, EP, and IGF-I receptors of human erythroid progenitor cells has not been defined. We obtained highly purified day-5 or day-6 erythroid colony-forming cells (ECFCs) from human blood in sufficient quantity and purity for radiolabeled cytokine binding studies and analysis of mRNA. When day-5 ECFCs were incubated with increasing concentrations of recombinant human (rh) IFNγ for 24 hours at 37°C, specific binding of 125I-rhSCF to SCF receptors was significantly decreased by 25% to 40% in a dose-dependent fashion, with the maximum effect at 2,500 to 5,000 U/mL of IFNγ. The decrease was apparent by 12 hours of incubation and was only slightly lower by 24 hours. The numbers of SCF and EP receptors, but not of IGF-I receptors, per ECFC, calculated by Scatchard analysis, were significantly decreased by 30% and 23% to 25%, respectively, after incubation with 2,500 U/mL rhIFNγ for 24 hours at 37°C, whereas the binding affinities were not affected. This decrease in SCF receptors was confirmed by flow cytometry using an anti–c-kit mouse monoclonal antibody. Northern blot analysis showed that the mRNAs for the SCF and EP receptors, but not for the IGF-I receptors, were decreased by 50% to 60% after 3 hours of incubation at 37°C with 2,500 U/mL of rhIFNγ. This persisted for 24 hours without alteration of the stability of the SCF and EP receptor mRNAs. These observations suggest that one means by which IFNγ inhibits erythroid cell proliferation and differentiation and produces apoptosis may be through the reduction of the number of target receptors for SCF and EP and that this occurs through transcriptional inhibition of the corresponding mRNAs.

Selective Expression of the Receptor Tyrosine Kinase, HTK, on Human Erythroid Progenitor Cells

AbstractHTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34low, and glycophorin A−/low. Assays of progenitors showed that HTK+c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK−c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

Human parvovirus B19 infection.

Human parvovirus B19 shows remarkable tropism for human erythroid progenitor cells. The pathogenesis of disease resulting from infection is a dynamic between the viral suppression of erythropoiesis and the host's ability to mount an effective immune response. Understanding this process has led to strategies for treating chronic anemia in persistently infected patients and may result in the development of a vaccine to prevent infection.

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.

Adeno-associated virus 2-mediated transduction and erythroid lineage-specific expression in human hematopoietic progenitor cells.

Parvoviruses are among the smallest of the DNA-containing viruses that infect a wide variety of vertebrates (Siegl et al. 1985). Two parvoviruses of human origin, the nonpathogenic adeno-associated virus 2 (AAV) and the parvovirus B19, a common human pathogen, have been studied extensively (Berns and Bohenzky 1987; Brown et al. 1994). AAV requires coinfection with a helper virus, such as adenovirus or herpesvirus, for its optimal replication (Berns 1990), but in the absence of a helper virus, the AAV genome establishes a latent infection in a site-specific manner (Kotin and Berns 1989; Kotin et al. 1990, 1991, 1992; Samulski et al. 1991). B19, by contrast, is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells (Ozawa et al. 1986, 1987; Yaegashi et al. 1989; Srivastava and Lu 1988; Takahashi et al. 1990). We have described the construction of a recombinant AAV-B19 hybrid genome, in which we combined the remarkable features of these two parvoviruses, and speculated that such a hybrid vector may prove useful for high efficiency transduction of primary human hematopoietic progenitor cells (Srivastava et al. 1989). Indeed, it has become increasingly clear that the AAV-based vector system may prove to be a useful alternative to the more commonly used retroviral and adenoviral vectors for its potential use in human gene therapy (Muzyczka 1992; Carter 1993; Srivastava 1994). Despite these advances, a number of fundamental questions related to AAV remain unanswered. For example, the molecular details of viral assembly and the mechanism of viral entry into the host cell have not been rigorously analyzed. Furthermore, the feasibility of obtaining tissue-specific expression of an AAV-transduced gene has not been adequately addressed. Here, we provide experimental evidence to suggest that the vector assembly requires a precise signaling mechanism and that AAV infection of human cells is receptor-mediated. We also document erythroid lineage restricted expression following AAV-B19 hybrid vector-mediated transduction of primary human hematopoietic progenitor cells. Elucidation of the molecular details of these aspects of AAV biology will have important implications in the potential use of AAV as a vector in human gene therapy.

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.