Primary Human Erythroid Cells Research Articles

HIF–prolyl hydroxylase inhibition results in endogenous erythropoietin induction, erythrocytosis, and modest fetal hemoglobin expression in rhesus macaques

AbstractThe hypoxia-inducible factor (HIF) pathway is crucial in mitigating the deleterious effects of oxygen deprivation. HIF-α is an essential component of the oxygen-sensing mechanisms and under normoxic conditions is targeted for degradation via hydroxylation by HIF–prolyl hydroxylases. Several HIF–prolyl hydroxylase inhibitors (PHIs) induced erythropoietin (epo) expression in vitro and in mice, with peak epo expression ranging from 5.6- to 207-fold above control animals. Furthermore, several PHIs induced fetal hemoglobin (HbF) expression in primary human erythroid cells in vitro, as determined by flow cytometry. One PHI, FG-2216, was further tested in a nonhuman primate model without and with chronic phlebotomy. FG-2216 was orally bioavailable and induced significant and reversible Epo induction in vivo (82- to 309-fold at 60 mg/kg). Chronic oral dosing in male rhesus macaques was well tolerated, significantly increased erythropoiesis, and prevented anemia induced by weekly phlebotomy. Furthermore, modest increases in HbF-containing red cells and reticulocytes were demonstrated by flow cytometry, though significant increases in HbF were not demonstrated by high-pressure liquid chromatography (HPLC). HIF PHIs represent a novel class of molecules with broad potential clinical application for congenital and acquired anemias.

Developmental and Differentiation-Specific Patterns of γ- and β-Globin Promoter DNA Methylation in Primary Human Erythroid Cells.

Despite years of investigation in a variety of experimental systems, the mechanisms underlying human β-globin locus developmental gene switching remain elusive. Several lines of evidence implicate DNA methylation in this process. As an initial step in studying the role of epigenetic modifications in the human switching process and in determining the mechanisms by which DNA methyltransferase inhibitors reverse the switch, we have characterized the DNA methylation patterns of the individual CpGs in the γ- and β-globin promoters in fetal liver (FL) and adult bone marrow (BM) primary erythroid cells and during in vitro differentiation of adult erythroid cells. Using the bisulfite conversion method we evaluated all CpGs in the ~500 bp regions centered on the γ- and β-globin promoter start sites. Fetal liver (FL) and adult bone marrow (BM) samples were obtained using IRB approved protocols and informed consent procedures. Erythroid cells were purified using anti-glycophorin A (glyA) magnetic beads. Purity was confirmed to be greater than 95%. Samples from five independent BM and FL samples were analyzed and 8–20 bisulfite converted sequences were determined for each promoter in each sample. Our results show that all 8 CpGs between −249 and +210 of the Gγ and Aγ-globin promoters are less than 20% methylated in FL and greater than 80% methylated in BM except for the −158 CpG which is only 40% methylated in BM(p<0.002). The 6 CpGs between −415 and +110 of the β-globin promoter show an inverse pattern with lower levels of DNA methylation in BM. Histone H3 acetylation of the γ-globin promoter, as determined by ChIP analysis, showed a complimentary pattern with higher levels in FL than BM. We next evaluated γ-globin promoter methylation patterns during in vitro erythroid differentiation from CD34+ BM cells. In this experiment, cells were grown with SCF, Flt3 ligand and IL-3 for 7 days and then in EPO for 14 days producing erythroid cells which express 99%HbA and 1% HbF. The initial day 0 CD34+ cells showed 90–100% methylation of all γ promoter sites. By day 3 in culture, before the initiation of erythroid differentiation, methylation at all sites upstream of the promoter had decreased to less than 60% and the CpG at −53 (the site of Stage Selector Protein complex binding) had decreased to less than 20%. The three CpG sites down-stream of the promoter (+6, +17 and +50) remained highly methylated. The pattern was unchanged at day 10, early in erythroid differentiation, when γ-globin mRNA expression was beginning. By day 14, when β-globin expression was peaking, methylation of the upstream promoter had increased back to the 70–100% level at all CpGs. These experiments provide a comprehensive picture of γ- and β-globin promoter methylation during the fetal and adult stages of erythroid development and of the γ-globin promoter during adult erythroid differentiation. The finding of transient γ-promoter hypomethylation during differentiation offers a potential mechanism to explain the transient γ-globin gene expression seen during normal adult erythropoiesis. Our results also raise the possibility that, just as domains of altered histone modification exist in β-globin gene loci, there may also be developmentally-specific domains of DNA methylation.

High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.

Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.

Role of Atypical p50:p50:Bcl-3 NFκlB Complex in Differentiation of Erythroid Cells.

We recently reported that mouse and human primary erythroid progenitor cells and erythroid cell lines synthesize and respond to Tumor Necrosis Factor-alpha (TNF-α). The nuclear transcriptional control complex, NFκlB is central in signaling downstream from TNF-α; so we began to study the function of NFκlB in erythroid cells. We made three very interesting initial findings: 1) first we found that NFκlB binding to DNA increased very slowly in HCD57 erythroid cells treated with erythropoietin (EPO, the hormone required for red blood cell development). An inhibitory effect of adding a neutralizing antibody to TNF-α on EPO-stimulated NFκlB DNA suggested this increase in NFκlB was due to TNF-α rather than direct EPO signaling. 2) We also found that NFκlB binding to DNA increased 10-fold or greater during erythroid differentiation. We found greatly increased NFκlB DNA binding in HCD57 cells that differentiated due to over-expression of JunB, F-MEL induced by DMSO, or human UT7-EPO or murine HCD57 cells induced to differentiate with hemin. 3) Surprisingly, we found that the NFκlB DNA binding complex in mouse primary erythroid cells and the erythroid cells lines tested was almost exclusively composed of the atypical p50/p50/Bcl3 NFκlB rather than the canonical p65/p50 or the non-canonical p65/p52 NFκlB. When we begin to study the biological significance of this atypical NFκlB in EPO-mediated erythroid differentiation in vivo using genetic tools, we found marked deficiencies in the development of erythroid cells in either the nfkb1−/− mice (p50−/−) or the bcl3 −/− mice. The nfkb1−/− mice were mildly anemic. The number of red blood cells in the circulation of these mice was statistically lower than in control mice. The number of CFU-e was also reduced in nfkb1−/− mice. Using the Ter-119 and CD71 staining method, we noted that proerythroblasts and immature erythroid cells increased and mature erythroblasts decreased in either non-anemic bone marrow or anemic spleens of nfkb1−/− mice. Forward scatter of Ter-119+ cells also showed an increased size of the average immature erythroid cell in the bone a marrow of nfkb1 −/− mice, suggesting a block in differentiation and continued cell cycling of the immature erythroblasts. Similar erythroid defects were observed in the spleens of anemic bcl-3−/− mice. nfkb1−/− mice and bcl-3−/− mice are also apparently unable to produce new reticulocytes as effectively as wild type mice after induction of anemia. Our working hypothesis is without expression of either p50 or Bcl-3 NFκlB proteins, immature erythroid cells continue to proliferate and ineffectively differentiate. In summary, the atypical p50/p50/Bcl-3 NFκlB complex appears necessary for maximal differentiation of immature erythroid cells.

Characterization of a Newly Discovered Human Alpha-Globin Gene.

It is well known that the globin genes are organized within the human genome as two gene clusters on chromosomes 16 and 11 (α cluster: ζ2-ζ1-α2-α1-# and β cluster: ε-Gγ-Aγ-δ-β). Switching in the expression patterns of those genes during human ontogeny generally follows a pattern determined by their genomic arrangement, but the genetic mechanism is not defined. In an effort to better understand the γ-to-β globin switching phenomenon, gene expression patterns from reticulocyte RNA were studied. cDNA were generated from the reticulocytes of 28 separate donors (14 cord blood; 14 adult blood) and analyzed using Affymetrix HG-U133 arrays. Quantitative PCR was used to confirm the array expression patterns. Among those cDNA identified as having expression patterns that were relatively decreased among adult reticulocytes, we were surprised by expression associated with a cluster of expressed sequence tags (EST) located within the α-globin locus in the region between the ζ1 and α2 genes. In the context of the 44,229 arrayed probes, high-level expression from that EST cluster was detected (ranked < 25th). Informatic analysis revealed alignment with the second and third exons previously described for the Ψα2 globin gene, but no significant similarity with the first exon of Ψα2. Based upon these comparisons, we used adult human reticulocyte RNA to amplify a cDNA clone for further study (GenBank: AY698022; named μ-globin). Sequencing of that clone revealed an insert that encodes a predicted 423 nt. open reading frame that is one amino acid residue shorter than the α2-globin gene. While the mature μ-globin mRNA results from splicing of two introns, it lacks a canonical Kozak sequence. BLASTP and CLUSTALW analyses revealed the highest level of homology with the avian αD-globin protein with a sequence identity of 55% (78/141 amino acids). In addition, the predicted heme- and globin-binding amino acids of μ-globin and avian αD-globin are largely conserved. Experimentally, we analyzed the expression level of the μ-globin transcripts using quantitative real-time PCR. Non-erythroid tissues including whole brain, white blood cells, and the Jurkat cell line demonstrated only background levels. The expression level of μ-globin (copies per ng cDNA) was 3.04x10(4) ± 1.68x10(3) in fetal liver, 1.71x10(5) ± 9.51x10(4) in cord reticulocytes, 1.15x10(4) ± 1.19x10(3) in bone marrow, and 2.17x10(4) ± 6.84x10(3) in adult reticulocytes. Based upon the consistent decrease in μ-globin expression between fetal and adult erythroid tissues, we measured the expression of γ-globin in those tissues for comparison. The expression level of γ-globin (copies per ng cDNA) was 2.49x10(6) ± 1.48x10(5) in fetal liver, 1.49x10(8) ± 7.88x10(7) in cord reticulocytes, 3.84x10(3) ± 3.83x10(2) in bone marrow, and 5.70x10(5) ± 5.80x10(5) in adult reticulocytes. To determine if μ-globin expression is regulated during erythropoiesis, we assayed primary human erythroid progenitor cells cultured in erythropoietin. The expression pattern of μ-globin was similar to the other globin genes increasing from background levels on day 0 to maximum levels in committed erythroblasts (3.56x10(5) ± 1.99x10(4) copies per ng cDNA). These results suggest that the human genome encodes a previously unrecognized α-globin that most closely resembles avian αD-globin. Expression of that gene is decreased during the fetal-to-adult transition of human ontogeny and regulated during erythropoiesis.

Hypoxia enhances human B19 erythrovirus gene expression in primary erythroid cells

Human B19 erythrovirus replicates in erythroid progenitors present in bone marrow and fetal tissues where partial oxygen tension is low. Here we show that infected human primary erythroid progenitor cells exposed to hypoxia (1% O 2) in vitro increase viral capsid protein synthesis, virus replication, and virus production. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor involved in the cellular response to reduced oxygenation, is shown to bind an HIF binding site (HBS) located in the distal part of the B19 promoter region, but the precise mechanism involved in the oxygen-sensitive upregulation of viral gene expression remains to be elucidated.

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells.

p38alpha, p38beta, p38gamma, and p38delta are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38alpha and p38gamma transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38delta mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38beta is minimally expressed in early CD34(+) hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38alpha, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38alpha, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38alpha does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38alpha and p38delta isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor alpha, an inflammatory cytokine that is modulated by p38alpha, is expressed by differentiating erythroblasts and inhibition of p38alpha or tumor necrosis factor alpha results in reduction in differentiation. Taken together, our data demonstrate that both p38alpha and delta isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

α5β1 integrin as a cellular coreceptor for human parvovirus B19: requirement of functional activation of β1 integrin for viral entry

AbstractReplication of the pathogenic human parvovirus B19 is restricted to erythroid progenitor cells. Although blood group P antigen has been reported to be the cell surface receptor for parvovirus B19, a number of nonerythroid cells, which express P antigen, are not permissive for parvovirus B19 infection. We have documented that P antigen is necessary for parvovirus B19 binding but not sufficient for virus entry into cells. To test whether parvovirus B19 utilizes a cell surface coreceptor for entry, we used human erythroleukemia cells (K562), which allow parvovirus B19 binding but not entry. We report here that upon treatment with phorbol esters, K562 cells become adherent and permissive for parvovirus B19 entry, which is mediated by α5β1 integrins, but only in their high-affinity conformation. Mature human red blood cells (RBCs), which express high levels of P antigen, but not α5β1 integrins, bind parvovirus B19 but do not allow viral entry. In contrast, primary human erythroid progenitor cells express high levels of both P antigen and α5β1 integrins and allow β1 integrin–mediated entry of parvovirus B19. Thus, in a natural course of infection, RBCs are likely exploited for a highly efficient systemic dissemination of parvovirus B19.

Identification and characterization of mechanistically distinct inducers of γ-globin transcription

Inhibition of HbS polymerization is a major target for therapeutic approaches in sickle cell anemia. Toward this goal, initial efforts at pharmacological elevation of fetal hemoglobin (HbF) has shown therapeutic efficacy. In order to identify well-tolerated, novel agents that induce HbF in patients, we developed a high-throughput screening approach based on induction of γ-globin gene expression in erythroid cells. We measured γ-globin transcription in K562 cells transfected with either γ promoter elements fused with the locus control region hypersensitivity site 2 and luciferase reporter gene (HS2γ) or a β-yeast artificial chromosome in which the luciferase reporter gene was recombined into the γ-globin coding sequences (γYAC). Corresponding pharmacological increases in HbF protein were confirmed in both K562 cells and in human primary erythroid progenitor cells. Approximately 186,000 defined chemicals and fungal extracts were evaluated for their ability to increase γ gene transcription in either HS2γ or γYAC models. Eleven distinct classes of compounds were identified, the majority of which were active within 24–48 hr. The short chain hydroxamate-containing class generally exhibited delayed maximal activity, which continued to increase transcription up to 120 hr. The cyclic tetrapeptide OSI-2040 and the hydroxamates were shown to have histone deacetylase inhibitory activity. In primary hematopoietic progenitor cell cultures, OSI-2040 increased HbF by 4.5-fold at a concentration of only 40 nM, comparable to the effects of hydroxyurea at 100 μM. This screening methodology successfully identifies active compounds for further mechanistic and preclinical evaluation as potential therapeutic agents for sickle cell anemia.

The proapoptotic factor Nix is coexpressed with Bcl-xL during terminal erythroid differentiation

AbstractTranscriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)–based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

The AML1-ETO fusion gene promotes extensive self-renewal of human primary erythroid cells

The t(8;21) translocation, which encodes the AML1-ETO fusion protein (now known as RUNX1-CBF2T1), is one of the most frequent translocations in acute myeloid leukemia, although its role in leukemogenesis is unclear. Here, we report that exogenous expression of AML1-ETO in human CD34(+) cells severely disrupts normal erythropoiesis, resulting in virtual abrogation of erythroid colony formation. In contrast, in bulk liquid culture of purified erythroid cells, we found that while AML1-ETO initially inhibited proliferation during early (erythropoietin [EPO]-independent) erythropoiesis, growth inhibition gave way to a sustained EPO-independent expansion of early erythroid cells that continued for more than 60 days, whereas control cultures became growth arrested after 10 to 13 days (at the EPO-dependent stage of development). Phenotypic analysis showed that although these cells were CD13(-) and CD34(-), unlike control cultures, these cells failed to up-regulate CD36 or to down-regulate CD33, suggesting that expression of AML1-ETO suppressed the differentiation of these cells and allowed extensive self-renewal to occur. In the early stages of this expansion, addition of EPO was able to promote both phenotypic (CD36(+), CD33(-), glycophorin A(+)) and morphologic differentiation of these cells, almost as effectively as in control cultures. However, with extended culture, cells expressing AML1-ETO became refractory to addition of this cytokine, suggesting that a block in differentiation had been established. These data demonstrate the capacity of AML1-ETO to promote the self-renewal of human hematopoietic cells and therefore support a causal role for t(8;21) translocations in leukemogenesis.

Role of JunB in Erythroid Differentiation

The role of junB as a regulator of erythroid cell survival, proliferation, and differentiation was tested by controlled expression of JunB in the erythropoietin (EPO)-dependent erythroleukemia cell line HCD57. JunB induced erythroid differentiation as evidenced by increased expression of the erythroid-specific proteins beta-globin, spectrin-alpha, and TER-119. Expression of JunB for at least 48 h was required for the differentiated phenotype to emerge. Differentiation was accompanied by a slower rate of proliferation and an increase in the expression of the cell cycle inhibitory protein p27. p27 protein expression increased due to reduced turnover without changes in transcription, indicating global changes in cell physiology following JunB induction. JunB expression was also studied in mouse and human primary erythroid cells. JunB expression increased immediately in both primary mouse cells and HCD57 cells treated with EPO and quickly returned to base-line levels, followed by a secondary rise in JunB in primary erythroid cells, but not in HCD57 cells, 36-48 h later. This result suggested that the initial EPO-dependent JunB induction was not sufficient to induce differentiation, but that the late EPO-independent JunB expression in primary erythroid cells was necessary for differentiation. This study suggests that JunB is an important regulator of erythroid differentiation.

Stem cell factor protects c-kit+ human primary erythroid cells from apoptosis.

It has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC). Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit+ ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells. Glycophorin A+ (GPA+) c-kit+ cells were purified from primary cultured ECFC derived from purified human CD34+ cells. The GPA+c-kit- and nonerythroid cells were generated from the same CD34+ cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry. For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting. In GPA+c-kit+ cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO. In contrast, no significant DNA fragmentation was seen in GPA+c-kit- and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF. PP2 also inhibited SCF-induced phosphorylation of Akt. These data indicate that SCF protects purified human GPA+c-kit+ cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival.

Recombinant human parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient for successful transduction of human hematopoietic cells.

The blood group P antigen, known to be abundantly expressed on erythroid cells, has been reported to be the cellular receptor for parvovirus B19. We have described the development of recombinant parvovirus B19 vectors with which high-efficiency, erythroid lineage-restricted transduction can be achieved (S. Ponnazhagan, K. A. Weigel, S. P. Raikwar, P. Mukherjee, M. C. Yoder, and A. Srivastava, J. Virol. 72:5224-5230, 1998). However, since a low-level transduction of nonerythroid cells could also be detected and since P antigen is expressed in nonerythroid cells, we reevaluated the role of P antigen in the viral binding and entry into cells. Cell surface expression analyses revealed that approximately 75% of primary human bone marrow mononuclear erythroid cells and approximately 31% of cells in the nonerythroid population were positive for P antigen. Two human erythroleukemia cell lines, HEL and K562, and a human promyelocytic leukemia cell line, HL-60, were also examined for P antigen expression and binding and entry of the vector. HEL and K562 cells showed intermediate levels, whereas HL-60 cells demonstrated high levels of expression of P antigen. However, the efficiency of vector binding to these cells did not correlate with P antigen expression. Moreover, despite P antigen positivity and efficient viral binding, HEL, K562, and HL-60 cells could not be transduced with the vector. Low levels of P antigen expression could also be detected in two primary cell types, human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblasts (NHLF). In addition, vector binding occurred in both cell types and was inhibited by globoside, indicating the involvement of P antigen in virus binding to these cells. These primary cells could be efficiently transduced with the recombinant vector. These data suggest that (i) P antigen is expressed on a variety of cell types and is involved in binding of parvovirus B19 to human cells, (ii) the level of P antigen expression does not correlate with the efficiency of viral binding, (iii) P antigen is necessary but not sufficient for parvovirus B19 entry into cells, and (iv) parvovirus B19 vectors can be used to transduce HUVEC and NHLF. These studies further suggest the existence of a putative cellular coreceptor for efficient entry of parvovirus B19 into human cells.

Signal transduction in primary cultured human erythroid cells.

Development of erythrocytes is a complex process governed by multiple cytokines. Colony assays have revealed the physiologic importance of these cytokines, although biochemical studies of highly purified human colony-forming unit-erythroid (CFU-E) generated in vitro from CD34+ cells have only recently begun. Studies from our groups and others suggested that signal transduction in primary erythroid cells differs considerably from that in cell lines or primary cells from other species. In this review, we summarize results of these studies with emphasis on possible implications for hematotherapy.

Quantitative PCR analysis of HbF inducers in primary human adult erythroid cells

The development and evaluation of drugs to elevate fetal hemoglobin in the treatment of the genetic diseases of hemoglobin would be facilitated by the availability of reliable cell assays. We have used real-time, quantitative polymerase chain reaction (PCR) analyses of globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to modulate levels of adult (beta) and fetal (gamma) globin mRNA. Complementary DNA synthesized from total RNA extracted at timed intervals from aliquots of cells were assayed throughout the period that the culture was studied. gamma-globin mRNA levels were found to be much lower (less than 1%) than beta-globin mRNA levels. At concentrations of agents chosen for minimal effect on cell division, we find that the 3 drugs studied, 5-azacytidine (5 micromol/L), hydroxyurea (40 micromol/L), and butyric acid (0.5 mmol/L), significantly increase gamma-globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on beta-globin mRNA levels, while butyric acid caused a twofold inhibition of beta-globin mRNA levels, and 5-azacytidine had little effect on beta-globin mRNA levels. The net result of all 3 drugs was to increase the gamma/(gamma + beta) mRNA ratios by threefold to fivefold. These data suggest that the mechanism is distinct for each drug. The profile of butyric-acid-induced changes on globin gene expression is also quite distinct from changes produced by trichostatin A, a known histone deacetylase inhibitor. Quantitative PCR analyses of human erythroid cells should prove useful for studying the mechanism(s) of action of known inducers of gamma-globin and identifying new drug candidates.

Distinct Roles of JNKs/p38 MAP Kinase and ERKs in Apoptosis and Survival of HCD-57 Cells Induced by Withdrawal or Addition of Erythropoietin

Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

Distinct Roles of JNKs/p38 MAP Kinase and ERKs in Apoptosis and Survival of HCD-57 Cells Induced by Withdrawal or Addition of Erythropoietin

AbstractErythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

The Glucocorticoid Receptor Cooperates With the Erythropoietin Receptor and c-Kit to Enhance and Sustain Proliferation of Erythroid Progenitors In Vitro

AbstractAlthough erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid progenitors. We examined the role of corticosteroids in the in vitro expansion of primary human erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone, cooperated with Epo and stem cell factor to induce erythroid progenitors to undergo 15 to 22 cell divisions, corresponding to a 105- to 106-fold amplification of erythroid cells. Dex acted directly on erythroid progenitors and maintained the colony-forming capacity of the progenitor cells expanded in liquid cultures. The hormone delayed terminal differentiation into erythrocytes, which was assayed by morphology, hemoglobin accumulation, and the expression of genes characteristic for immature cells. Sustained proliferation of erythroid progenitors could be induced equally well from purified erythroid burst-forming units (BFU-E), from CD34+ blast cells, and from bone marrow depleted from CD34+ cells.

The Glucocorticoid Receptor Cooperates With the Erythropoietin Receptor and c-Kit to Enhance and Sustain Proliferation of Erythroid Progenitors In Vitro

Although erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid progenitors. We examined the role of corticosteroids in the in vitro expansion of primary human erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone, cooperated with Epo and stem cell factor to induce erythroid progenitors to undergo 15 to 22 cell divisions, corresponding to a 105- to 106-fold amplification of erythroid cells. Dex acted directly on erythroid progenitors and maintained the colony-forming capacity of the progenitor cells expanded in liquid cultures. The hormone delayed terminal differentiation into erythrocytes, which was assayed by morphology, hemoglobin accumulation, and the expression of genes characteristic for immature cells. Sustained proliferation of erythroid progenitors could be induced equally well from purified erythroid burst-forming units (BFU-E), from CD34+ blast cells, and from bone marrow depleted from CD34+ cells.