Effect of AGM and fetal liver-derived stromal cell lines on globin expression in adult baboon (P. anubis) bone marrow-derived erythroid progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction.     

Acetylation of human fetal hemoglobin occurs throughout erythroid cell maturation

The biosynthesis of human acetylated fetal hemoglobin (Hb F1) has been examined by incubating the following cell types with [3H]leucine: (a) burst-forming unit erythroid cells cultured from umbilical cord mononuclear cells, (b) infant bone marrow, (c) umbilical cord blood, and (d) peripheral blood cells from adults with elevated fetal hemoglobin. Newly synthesized Hb F1 was 18-20% that of Hb F0 in burst-forming unit erythroid cells which were immature, mature, or in an intermediate state of development. In infant marrow and in infant and adult peripheral blood the extant Hb F1 comprised 10.8 +/- 1.8% of the total Hb F. In marrow cells the specific radioactivity (cpm/mg) of Hb F1 was 1.4-2.0-times greater than that of Hb F0. In peripheral blood cells these ratios were slightly greater. [3H]Leucine-labeled infant bone marrow, umbilical cord blood, and adult peripheral blood cells were subjected to density gradient ultracentrifugation. The ratios of specific radioactivity for Hb F1/Hb F0 increased from 1.0-1.8 in the lightest cell zone to 5.2-9.0 in the more dense cells. Thus the biosynthesis of Hb F1 is enhanced in cells which are more mature than those responsible for the bulk of hemoglobin synthesis, and the acetylation of Hb F provides a marker of erythroid cell maturation.     

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E2 (PGE2) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of 35S-methionine labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE2 treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE2 was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE2 to culture dishes presumably constitutes an environmental change to promote the functional changes seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

MB-02 cells undergo fetal to adult globin gene switching in response to erythropoietin

The recently described MB-02 human cell line requires Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) for continuous growth and terminally differentiates into enucleate, hemoglobinized cells in response to erythropoietin. Here, analysis of globin production now demonstrates that uninduced MB-02 cells produce alpha globin and the fetal globin chains G gamma and A gamma in a ratio of 1:3. Addition of erythropoietin results in de novo synthesis of beta globin chains and a marked increase in total Hb/cell. Thus, the MB-02 cell line partially recapitulates the fetal to adult globin switch that occurs during erythroid and human fetal development and provides a new clonal human erythroid progenitor system for investigating the biochemical and molecular events involved in globin gene switching.     

Characterization of "fetal-type" acetylcholinesterase in hemin-treated K562 cell culture

The alteration of acetylcholinesterase (ACHE) activity, a marker enzyme of erythroid differentiation, was studied during the hemin-induced erythroid differentiation of K562 human leukemia cells in suspension culture. The kinetics of postinduction differentiation was followed by determining the hemoglobin (Hb) content and the ACHE activity of cells. Embryonic hemoglobins as well as small quantities of fetal Hb (HbF) were synthetized by stimulated cells. The peaks of ACHE activity preceded the highest level of Hb content and, following induction, reached their pinnacles at 72 and 120 hours, respectively. These data indicate that ACHE activity is an earlier and more sensitive marker for hemin-induced erythroid differentiation of K562 cells than is elevated Hb content. Electrophoretic mobility of ACHE from hemin-treated cells proved to be the fetal type, but after incubation with neuraminidase, the rate of migration decreased to the level of the adult type enzyme.     

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E₂ (PGE₂) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of ³⁵S-methione labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE₂ treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE₂ was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE₂ to culture dishes presumably constitutes an environmental change to promote the functional seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

Formation of transient polykaryons by fusion of erythrocytes of different developmental programs

We report here two methods of fusing erythroid cells from bullfrogs (Rana catesbeiana), using polyethylene glycol or calcium phosphate, which yield masses of polykaryons in which the cytoplasms and nuclei of tadpole and adult frog erythroid cells are intermixed. The masses of fused cells carry out protein synthesis in culture, including the assembly of normal hemoglobin (Hb) tetramers. In these polykaryons there is reactivation of the expression of specific Hbs that have previously been "turned off" in vivo as the result of either a developmental Hb switch or normal cellular differentiation and RBC maturation. For example, the products of fusion of tadpole erythroblasts with adult frog mature RBCs synthesize adult Hb, whereas neither cell population alone does so. Recent experiments have taken advantage of a Hb-expression polymorphism that we discovered in this species, such that some tadpoles have greatly reduced expression of one of the larval Hbs (Hb Td-4). Fusion of erythroblasts from such tadpoles with RBC from frogs that had expressed Hb Td-4 when they were tadpoles produces polykaryons that synthesize Hb Td-4, indicating there is a trans factor that stimulates Td-4 expression. Heterospecific erythroid cell polykaryons can be constructed in an analogous manner, facilitating the study of trans-acting factors that regulate specific globin gene expression during development.     

Human globin knock-in mice complete fetal-to-adult hemoglobin switching in postnatal development

Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.     

Initial function analysis of a novel erythroid differentiation related gene EDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened λ-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern ofEDRF1 by RT-PCR technique. And a clue to the function ofEDRF1 has been found from confirmation of high levels ofEDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function ofEDRF1 in K562 cells, sense and antisenseEDRF1 constructs were prepared and transfected into K562 cells. α-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [³H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence thatEDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.     

Initial function analysis of a novel erythroid differentiation related geneEDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened λ-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern ofEDRF1 by RT-PCR technique. And a clue to the function ofEDRF1 has been found from confirmation of high levels ofEDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function ofEDRF1 in K562 cells, sense and antisenseEDRF1 constructs were prepared and transfected into K562 cells. α-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [³H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence thatEDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.     

Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.