Globin synthesis during erythroid cell maturation in alpha thalassemia.

Globin chain synthesis was examined in erythroid cells of increasing maturity, fractionated from bone marrow of two patients with hemoglobin H disease and in one alpha thalassemia 1 heterozygote. In contrast to beta thalassemia where a gradient of alpha/beta chain ratios increasing with erythroid cell maturation is observed, in alpha thalassemia the alpha/beta chain ratio remains constant throughout maturation. This finding suggests that in alpha thalassemia there is no modification of the imbalance in globin chain synthesis either by increased alpha chain production or decreased beta chain synthesis in erythroid precursors. Furthermore, the constant alpha/beta ratio reflects a limited degree of beta chain destruction, indicating that the ability of the excess beta chains to associate into tetramers protects them from proteolytic digestion.     

Relationship between cell-cycle state of erythroid progenitors and fetal hemoglobin synthesis.

DNA-synthesis state of circulating burst forming unit-erythroid (BFU-E) was evaluated in patients with sickle cell anemia and correlated with percent of fetal hemoglobin synthesized in the BFU-E-derived cells. Percentage of S-phase BFU-E inversely correlated with percent fetal hemoglobin synthesized in the BFU-E-derived cells (simple linear correlation coefficient, r = -0.8, P = 0.0302; polynomial regression, R = -0.99, P = 0.0002). This observation is of relevance to our understanding of the relationship between the developmental stage of the erythroid progenitors and expression of globin genes.     

Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3' to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had "adult" and not "fetal" progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.     

Relationship of mitochondrial membrane potential to hemoglobin synthesis during Friend cell maturation

Previous studies have shown that exposure to imidazole dissociates hemoglobin synthesis from other aspects of cell maturation in dimethylsulfoxide (DMSO)-treated mouse erythroleukemia (MEL) cells. In the present study, we have found that imidazole causes hyperpolarization of the mitochondrial membrane in MEL cells exposed to DMSO, in contrast to the depolarization observed with DMSO alone. Like the defect in hemoglobin synthesis, membrane hyperpolarization is reversible upon removal of imidazole and incubation of cells with DMSO alone. These correlations suggest that alterations in the electrostatic properties of the mitochondrial membrane, due directly or indirectly to the effects of imidazole, interfere with heme synthesis but not with other aspects of the maturation process in these developing erythroid cells.     

Stochastic expression of fetal hemoglobin in adult erythroid cells.

The expression of fetal hemoglobin, Hb F, in the adult cells is cellularly restricted both in vivo and in culture. Because, in cultures of erythroid progenitors, subclones that express or fail to express Hb F are derived from the same erythroid stem cell, a mechanism must exist whereby Hb F expression segregates in the progeny of erythroid progenitors during their differentiation. We present mathematical analyses of experimental data which suggest that expression of Hb F in human adult erythroid cells occurs on a stochastic basis. We quantified Hb F expression among the subclones of erythroid bursts (clones) in vitro by labeling subclones with fluorescent anti-Hb F antibodies. The observed data were compared with predictions from a stochastic model with the assumption that the expression of Hb F in a subclone occurs with a probability P equal to the frequency of Hb F-expressing subclones in an experiment. There was good fit between the observed and predicted data with respect to: (i) the relative frequencies of monomorphic F+, monomorphic F-, and bimorphic F+/F- bursts, respectively; (ii) the size distributions among F+, F- and F+/F- bursts; and (iii) the proportions of subclones expressing Hb F within bimorphic F+/F- bursts. Given the hypothesis that erythroid progenitors which have an active Hb F program are less differentiated than cells which do not proceed to express Hb F, the stochastic event indicated by our analyses may be the probability that adult erythroid progenitor cells undergo terminal differentiation at an earlier stage than usual.     

Effects of dexamethasone on fetal hemoglobin synthesis in peripheral blood erythroid burst-forming units

Colonies derived from erythroid burst-forming units (BFU-E) synthesize fetal hemoglobin (HbF) in amounts that far exceed in vivo levels. There is some evidence that HbF synthesis is controlled at the level of a primitive erythroid precursor cell. Dexamethasone may potentiate the development of BFU-E. Since a means of augmenting HbF production in sickle cell anemia or severe β-thalassemia would be of great therapeutic value, we studied the effects of dexamethasone on HbF and γ-globin chain synthesis in BFU-E from patients with sickle cell anemia and controls. HbF was measured by radioimmunoassay of BFU-E lysate and γ-chain synthesis by the incorporation of ³H-leucine into globin, which was then purified by gel filtration and column chromatography. Dexamethasone (10^?9 M) produced an increase in the number of BFU-E in 16 of 19 subjects when compared with numbers of BFU-E cultured with only erythropoietin. The individual BFU-E were larger and contained more subcolonies. Dexamethasone did not increase HbF or γ-chain synthesis, and there was no relationship between increased proliferation of BFU-E and augmented HbF production. Thus, although dexamethasone augmented the development of erythroid bursts, there was no increment in HbF.     

Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures.

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady-state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units-erythroid (BFU-E) colonies. There was a high correlation of gamma-globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma-globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma-globin expression in adult erythroid progenitor cell cultures.     

Fetal hemoglobin (HbF) synthesis in baboons, Papio cynocephalus. Analysis of fetal and adult hemoglobin synthesis during fetal development.

Fetal hemoglobin (HbF) and adult hemoglobin (HbA) synthesis was studied in fetal baboons, Papio cynocephalus, to determine the normal pattern of hemoglobin production during fetal development. Fetuses ranging from 53 to 180 days gestation (term gestation 184 days) were used. Erythroid cells were incubated with 3H-L-leucine, and the rates of globin chain synthesis and the distribution of radioactivity into hemoglobin intermediates and completed hemoglobin molecules were determined. Gamma chain synthesis accounted for approximately 97% of the total nonalpha chain synthesis up to 140 days gestation; beta chain synthesis accounted for the remainder. After 140 days gestation, approximately equal quantities of gamma and beta chain were synthesized in the bone marrow. Prior to 140 days gestation, total alpha chain synthesis was 30% greater than total non-alpha chain synthesis, while there was balanced chain synthesis after 140 days gestation. During the period of excess alpha chain synthesis, fetal erythrocytes contained a large pool of alpha-hemoglobin (alpha chain with heme attached) molecules uncombined with beta or gamma chains. In view of the possibility that alpha chains may have a lower affinity for gamma chains than beta chains, excess alpha chain synthesis may be required to maintain low levels of free gamma chains.     

Quantitation of hemoglobins within individual red cells: asynchronous biosynthesis of fetal and adult hemoglobin during erythroid maturation in normal subjects

We outline a method for estimating either HbF or HbA content in single erythrocytes and their precursors. Our method depends on microphotometric assay of darkfield reflectance arising from individual pericellular immunoprecipitates developed with anti-HbF or anti-HbA. When uniform-diameter latex microspheres were used to normalize comparisons between preparations, mean coefficient of variation for HbF reflectance among separate preparations of the same sample was < 3%. Reflectance is a faithful (r = 0.99) linear function of the logarithm of picograms per cell in samples with known HbF or HbA content. The following features emerged from such analyses. First, despite the use of antigenically-specific antihemoglobins from different sources, the least detectable quantity of HbF (3.2 pg) and HbA (14.8 pg) remained invariant. Second, these detection thresholds depends on antihemoglobin affinity constants but are little influenced by antibody concentration. Third, our procedure is equally valid for persons with normal HbF constant (mean +/- SD = 4.4 +/- 0.3 pg per cell, 15 subjects) and for those with much higher levels. Fourth, like the percentage of HbF- bearing cells, HbF content is usually unchanging in serial samples. Fifth, the utility of the method is evidenced in bone marrow analyses of five hematologically normal persons in whom HbF content, unlike HbA content, remained constant throughout maturation from erythroblasts to erythrocytes. In vivo HbF biosynthesis is thus normally completed long before HbA production.     

Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.     

Pyruvate kinase synthesis and degradation by normal and pathologic cells during erythroid maturation

Mature erythrocytes contain a specific isozyme of pyruvate kinase (R-PK) while immature erythroblasts coexpress R-PK and another isozyme, M2-PK. To determine what roles degradation and decreasing of synthesis played in the disappearance of M2-PK during erythroid maturation, M2-PK and R-PK synthesis and degradation were studied in erythroblasts from fetal liver and blood BFU-E-derived erythroblasts from healthy subjects, an erythroleukemic patient, and a patient with an erythrocyte PK hyperactivity associated with M2-PK persistence in mature erythrocytes. In normal erythroblasts, R-PK synthesis was constant throughout erythroid maturation, whereas M2-PK synthesis decreased to the point of becoming undetectable. R-PK degradation was very low, while M2-PK degradation was more pronounced and steady during erythroid maturation. In leukemic erythroblasts, total protein turnover was higher than in normal cells, but the M2-PK degradation rate was lower. In erythroblasts from the patient with M2-PK persistence in mature erythrocytes, M2-PK synthesis did not decline with cell maturation. In conclusion, our results emphasize the importance of the decrease of M2-PK synthesis in the disappearance of M2-PK during erythroid maturation. Further studies of patients with pathologic persistence of M2-PK synthesis will help in the understanding of this event involved in erythroid maturation.     

Hemoglobin switching in sheep and goats: induction of hemoglobin C synthesis in cultures of sheep fetal erythroid cells.

Synthesis of HbF (alpha2gamma2) is replaced by synthesis of Hb A (alpha2betaA2) shortly before birth in sheep homozygous for the betaA globin chain whereas Hb C (alpha2betaC2) is produced transiently during the neonatal period. We have obtained a Hb F-to-Hb C switch by generating erythroid colonies at high erythropoietin concentration in plasma clot cultures of mid-gestation fetal bone marrow or liver. Furthermore, high erythropoietin concentration appeared specifically to activate the gene for betaC and not those for betaA or betaB globin in colonies grown from cells of an animal heterozygous for the betaA and betaB genes. Erythropoietic stress in the form of periodic bleeding or erythropoietin injection in utero did not stimulate production of Hb C (alpha2betaC2) in fetal sheep until shortly before birth, and then only in two of six animals. Thus, factors other than erythropoietin may influence the potential for betaC globin synthesis in vivo in fetal sheep.     

The relationship of hemoglobin synthesis to erythroid colony and burst formation.

We have demonstrated that the cyclohexanone method for the extraction of hematin can be used to measure hemoglobin synthesis induced by erythropoietin (epo) in mouse bone marrow cells cultured in medium containing methyl cellulose. The time course of hemoglobin synthesis by mouse marrow cells showed two effects due to epo: an increase in hemoglobin synthesis at day 2, which corresponded to the formation of small erythroid colonies resulting from the CFU-E (colony-forming unit, erythroid), and a very large increase in hemoglobin synthesis, which was maximal at days 7-8 and corresponded to the formation of large erythroid colonies (bursts) resulting from the BFU-E (burst-forming unit, erythroid). The epo dose-response curves for CFU-E colony counts and day-2 hemoglobin synthesis were similar, and the cell-number-response curves for these two paramaters were parallel. The epo dose-response curve for BFU-E colony counts reached a plateau at an epo concentration between 3 and 5 units/ml, whereas the dose-response curve for 6-8-day hemoglobin synthesis did not reach a plateau even at an epo dose of 10 units/ml.     

c-kit ligand reactivates fetal hemoglobin synthesis in serum-free culture of stringently purified normal adult burst-forming unit- erythroid

We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)- free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (< 10% and < 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL- 3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL- induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL- 3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.     

Flow cytometric analysis of fetal hemoglobin in erythroid precursors of beta-thalassemia

Fetal hemoglobin (HbF), the major hemoglobin species in fetal life, drops to <1% in normal adults, where it is restricted to a few 'F-cells', which may increase in various acquired and genetic conditions, including thalassemia. Using flow cytometry, we studied the percentage of HbF-containing cells and their HbF content in RBC, reticulocytes (retics) and normoblasts (NRBC) present in the peripheral blood of patients with beta-thalassemia. Thiazol orange, a nucleic acid-specific dye, and anti-CD45 antibodies identified the various blood cells and antihuman HbF antibodies quantitated HbF. The results indicated that F-RBC were more numerous in beta-thalassemic (both transfused and nontransfused) patients than in normal donors, but, in most cases, their HbF content was comparable, suggesting that increased HbF in thalassemia is mainly due to higher %F-cells rather than an increased HbF per cell. Among the retics, the %F-cells and their HbF content were highest in immature retics and decreased with maturation to levels of RBC. This may reflect preferential maturation of F-retics into RBC in the circulation. The NRBC population contained the lowest %F-cells. This could be due to preferential maturation of F-NRBC, having more normal phenotype than non-F-NRBC, in the bone marrow into F-retics, while non-F-NRBC enter the circulation.     

In vitro induction of fetal hemoglobin in human erythroid progenitor cells

OBJECTIVE: Clinical heterogeneity among patients with sickle cell anemia (SCA) is influenced by the amount of fetal hemoglobin (HbF) within circulating erythrocytes. Current pharmacotherapy focuses on increasing HbF in order to reduce hemolysis and help prevent acute vaso-occlusive events. Hydroxyurea, a known S-phase-specific cytotoxic ribonucleotide reductase (RR) inhibitor, is an effective agent for HbF induction in patients with SCA, but the mechanisms by which hydroxyurea induces HbF in vivo have not been elucidated. MATERIALS AND METHODS: We adapted an in vitro assay for HbF induction, growing burst-forming unit erythroid (BFU-E) colonies in methylcellulose from peripheral blood of children with SCA and extracting the hemoglobin for high-performance liquid chromatography analysis of HbF. Hydroxyurea and other known RR inhibitors, along with cytotoxic agents that are not RR inhibitors, were tested for the ability to induce HbF using this in vitro assay. RESULTS: Hydroxyurea decreased the number of BFU-E colonies that grew in culture and significantly increased HbF from 13.6%+/-6.2% to 25.4%+/-8.0% at 50 microM HU (p=0.012). Three other known RR inhibitors also significantly induced HbF: 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (p=0.025), guanazole (p=0.008), and gemcitabine (p=0.028). Cytarabine and alkylating agents BCNU and 4-hydroperoxycyclophosphamide, which are cytotoxic agents but not RR inhibitors, reduced BFU-E colony number but did not significantly induce HbF. CONCLUSION: Hydroxyurea and other RR inhibitors significantly induce HbF in vitro in human erythroid progenitor cells. Inhibition of RR may be a critical mechanism by which hydroxyurea increases HbF in vivo in patients with SCA.     

Expression of cytoskeletal protein 4.1 during avian erythroid cellular maturation.

We have isolated a cDNA clone encoding part of protein 4.1, an integral component of the erythrocyte cytoskeleton. The recombinant was isolated by immunological screening of a chicken erythroid lambda gt11 cDNA library using a monoclonal antibody directed against protein 4.1. DNA blot analysis shows that the gene is present as a single copy per haploid chicken genome, while RNA blot analysis reveals the presence of a single mRNA of 7 kilobases in reticulocytes. Message of the same size (in reduced amounts) is also present in an erythroleukemic cell line transformed by avian erythroblastosis virus and is also present in vastly reduced quantities in nonerythroid hemopoietic cells. Immunoblotting and immunofluorescence experiments show that a subset of the chicken 4.1 variant proteins is preferentially expressed during in vitro differentiation of chicken erythroleukemic cells. These data indicate that the gene is both actively transcribed and translated during early erythroid cellular maturation.     

5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons.

In an attempt to stimulate Hb F synthesis in baboons by means other than erythropoietic stress, we considered the possibility that an agent that inhibits methylation of CpG sequences in DNA may be effective. 5-Azacytidine, a cytosine analogue that cannot be methylated, is such an agent. Animals whose packed red cell volume was maintained at approximately 20% by bleeding were given 10 daily intravenous injections of the drug (6 mg/kg) in 12 days. Hb F levels in these animals started to increase on day 5 of this regimen and peak levels, which were 6-30 times higher than those produced by bleeding alone, occurred 5-7 days after the last dose of the drug. In animals previously identified as genetically "high" or "low" Hb F responders, the maximal Hb F levels were 70-85% and 35-40% respectively. In dose-response studies 5-azacytidine given daily at 3-4 mg/kg produced maximal Hb F increases. The drug did not correlate the percentage (number) of Hb F-containing cells (F cells) beyond the maximal number achieved by bleeding alone and thus its main effect was to increase Hb F per F cell. The finding that Hb F synthesis can be modulated to such a high degree by a drug may have therapeutic implications--e.g., in sickle cell anemia, in which stimulation of Hb F synthesis may prevent sickling.