HbF Synthesis Research Articles

Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin.

Reactivation of fetal hemoglobin (HbF) expression using pharmacological agents represents a potential strategy for the therapy of beta-thalassemia, sickle cell disease, HbE and other beta-hemoglobinopathies. However, the drugs currently available have low efficacy and specificity and are associated with high toxicity. We describe the development of stable cellular genomic reporter assays (GRAs) based on the green fluorescence protein (EGFP) gene under the Ggamma-globin promoter in the intact human beta-globin locus. We show that human erythroleukemic cell lines stably transfected with a Ggamma-EGFP beta-globin locus construct can maintain a uniform basal level of EGFP expression over long periods of continuous culture and that induction of EGFP expression parallels the induction of the endogenous globin genes. We compared the EGFP-induction potency of a number of chemotherapeutic agents, including histone deacetylase inhibitors and DNA-binding agents. We show that hydroxyurea and butyrate result in moderate levels of induction (70-80%) but with an additive inductive effect. Among the DNA-binding agents tested, cisplatin was the most potent inducer of HbF expression, (442+/-32%), a level which is comparable to hemin (764+/-145%). These results indicate that cellular GRAs containing Ggamma-EGFP-modified beta-globin locus constructs can be used to develop novel inducers of HbF synthesis for the therapy of beta-hemoglobinopathies.

Fetal Hemoglobin Synthesis Determined by γ-mRNA/γ-mRNA + β-mRNA Quantitation in Infants at Risk for Sudden Infant Death Syndrome Being Monitored at Home for Apnea

Fetal hemoglobin (HbF) levels in the hemolysates obtained from infants who died from sudden infant death syndrome (SIDS) are reported to be markedly increased compared with controls. This finding could have been explained by increased HbF synthesis caused by episodes of hypoxemia in the SIDS infants. A prospective study in a group of infants being monitored at home after an apparent life-threatening event (ALTE) and considered at increased risk for SIDS was conducted with an improved ribonuclease protection assay. The ribonuclease protection assay allowed for the quantitation of [gamma/(gamma+beta)]-globin mRNAs, which has a highly significant correlation with the levels of HbF synthesis. Thirty-five infants who were admitted for an ALTE were included in the study. All infants were at home under surveillance with a cardiorespiratory monitor and followed in an apnea clinic with monthly appointments. Seventy-three blood samples were obtained between 38 and 61 weeks of postconceptional age. For control purposes, a similar group of 37 normal infants (99 samples) whose HbF synthesis was previously determined were included. Mean [gamma/(gamma+beta)]-globin mRNAs were increased in the ALTE group at 42 to 45 and 46 to 49 weeks of postconceptional age (mean: 55.2 +/- 17.4% and 33.9 +/- 14%) in comparison with HbF synthesis in controls (mean: 42.6 +/- 13.7% and 23.6 +/- 9.8%). The data obtained in this report from infants who were considered at risk for SIDS show that HbF synthesis is increased between 42 and 49 weeks of postconceptional age. Determining HbF synthesis as described in this study may have value as a marker for episodes of hypoxemia for certain infants who are at risk for SIDS.

HbF reactivation in sibling BFU-E colonies: synergistic interaction of kit ligand with low-dose dexamethasone

Mechanisms underlying fetal hemoglobin (HbF) reactivation in stress erythropoiesis have not been fully elucidated. We suggested that a key role is played by kit ligand (KL). Because glucocorticoids (GCs) mediate stress erythropoiesis, we explored their capacity to potentiate the stimulatory effect of KL on HbF reactivation, as evaluated in unilineage erythropoietic culture of purified adult progenitors (erythroid burst-forming units [BFU-Es]). The GC derivative dexamethasone (Dex) was tested in minibulk cultures at graded dosages within the therapeutical range (10(-6) to 10(-9) M). Dex did not exert significant effects alone, but synergistically it potentiated the action of KL in a dose-dependent fashion. Specifically, Dex induced delayed erythroid maturation coupled with a 2-log increased number of generated erythroblasts and enhanced HbF synthesis up to 85% F cells and 55% gamma-globin content at terminal maturation (ie, in more than 80%-90% mature erythroblasts). Equivalent results were obtained in unicellular erythroid cultures of sibling BFU-Es treated with KL alone or combined with graded amounts of Dex. These results indicate that the stimulatory effect of KL + Dex is related to the modulation of gamma-globin expression rather than to recruitment of BFU-Es with elevated HbF synthetic potential. At the molecular level, Id2 expression is totally suppressed in control erythroid culture but is sustained in KL + Dex culture. Hypothetically, Id2 may mediate the expansion of early erythroid cells, which correlates with HbF reactivation. These studies indicate that GCs play an important role in HbF reactivation. Because Dex acts at dosages used in immunologic disease therapy, KL + Dex administration may be considered to develop preclinical models for beta-hemoglobinopathy treatment.

Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin

Pharmacologic stimulation of fetal hemoglobin (HbF) expression may be a promising approach for the treatment of beta-thalassemia. In this study, we have investigated the HbF-inducing activity and molecular mechanisms of specific histone deacetylase (HDAC) inhibitors in human K562 erythroleukemia cells. Apicidin was the most potent agent compared with other HDAC inhibitors (trichostatin A, MS-275, HC-toxin, suberoylanilide hydroxamic acid [SAHA]) and previously tested compounds (butyrate, phenylbutyrate, isobutyramide, hydroxyurea, 5-aza-cytidine), leading to a 10-fold stimulation of HbF expression at nanomolar to micromolar concentrations. Hyperacetylation of histones correlated with the ability of HDAC inhibitors to stimulate HbF synthesis. Furthermore, analysis of different mitogen-activated protein (MAP) kinase signaling pathways revealed that p38 signaling was activated following apicidin treatment of cells and that inhibition of this pathway abolished the HbF-inducing effect of apicidin. Additionally, activation of the Agamma-globin promoter by apicidin could be inhibited by p38 inhibitor SB203580. In summary, the novel HDAC inhibitor apicidin was found to be a potent inducer of HbF synthesis in K562 cells. The present data outline the role of histone hyperacetylation and p38 MAP kinase signaling as molecular targets for pharmacologic stimulation of HbF production in erythroid cells.

Induction of fetal hemoglobin synthesis by valproate: modulation of MAP kinase pathways.

Valproate has been found to stimulate fetal hemoglobin (HbF) synthesis in patients with sickle cell disease. In accordance with these clinical observations, we found a moderate induction of HbF synthesis in K562 erythroid cells in vitro. Investigation of the role of the mitogen-activated protein kinase (MAPK) pathways by Western blot analysis and use of specific kinase inhibitors suggests that inhibition of ERK pathway and activation of the p38 pathway may contribute to the HbF-inducing activity of valproate.

Superoxide Dismutase Activity in Hydroxyureatreated E^|^beta; Thalassaemia

The aim of the present study was to examine the activity of superoxide dismutse (SOD) and the level of fetal hemoglobin (HbF) in hydroxyrea (HU) -treated Eβ thalassaemia. We measured SOD level, HbF, mean corpuscular volume (MCV), packed cell volume (PCV) and hemoglobin (Hb) in Eβ thalassaemia patients treated with HU (dose 30mg/kg/day) for 90 consecutive days. There was an increase in HbF synthesis without any increase in Hb in HU-treated patients. The decreased SOD level in long-tem HU therapy in Eβ thalassaemic patients suggests that HU plays some role in inhibiting the superoxide radical generation in thalassamic erythrocytes. HU may act as an inhibitor of oxidative damage of red cells in E thalassaemia.

K + transport in red blood cells from human umbilical cord

The current study was designed to characterise K + transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na +/K + pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K + transport was dominated by a bumetanide (10 μM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 μM), calyculin A (100 nM) and Cl − removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 μM) – all consistent with mediation via the K +-Cl − cotransporter (KCC). KCC activity in HbF cells was also O 2-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO 2 of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K + accumulation via the Na +/K + pump. These findings are relevant to the mechanism by which O 2 regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.

Effects of hydroxurea, stem cell factor, and erythropoietin in combination on fetal hemoglobin in the baboon.

Augmentation of the level of fetal hemoglobin (HbF) is considered therapeutic for patients with sickle cell disease. The objective of this study was to determine the effect of treatment with a combination of erythropoietin (Epo), stem cell factor (SCF), and hydroxyurea (HU) on HbF levels. The effect of treatment with a combination of Epo, SCF, and HU on HbF, F-cell numbers, and globin chain synthesis was evaluated in a baboon model. Treatment with a combination of SCF+Epo resulted in a two-fold increase in HbF, F-cells, and F-reticulocytes compared to Epo alone. The combination of SCF+Epo+HU resulted in an additional two-fold increase in HbF, whereas F-cells and F-reticulocytes increased only 25% compared to the SCF+Epo regimen. Measurement of differential globin chain synthesis indicated that the SCF+Epo+HU treatment also increased the I gamma/V gamma (homologous to human G gamma and A gamma) synthetic ratio toward the fetal ratio. HU can effectively augment growth factor-induced HbF synthesis in vivo. Because I gamma/V gamma ratios are unaffected by erythropoietic stress and similar increases in this ratio have only been observed following administration of 5-azacytidine, we suggest that these two agents may share a common mechanism of action involving the recruitment of a similar target cell population to terminal erythroid differentiation.

Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate

Mechanisms underlying fetal hemoglobin (HbF) reactivation in adult life have not been elucidated; particularly, the role of growth factors (GFs) is controversial. Interestingly, histone deacetylase (HD) inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF. We developed a novel model system to investigate HbF reactivation: (1) single hematopoietic progenitor cells (HPCs) were seeded in serum-free unilineage erythroid culture; (2) the 4 daughter cells (erythroid burst-forming units, [BFU-Es]), endowed with equivalent proliferation/differentiation and HbF synthesis potential, were seeded in 4 unicellular erythroid cultures differentially treated with graded dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated in terminal erythroblasts by assay of F cells and gamma-globin content (control levels, 2.4% and 1.8%, respectively, were close to physiologic values). HbF was moderately enhanced by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor treatment (up to 5%-8% gamma-globin content), while sharply reactivated in a dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The stimulatory effects of KL on HbF production and erythroid cell proliferation were strictly correlated. A striking increase of HbF was induced by combined addition of KL and NaB or TSA (40%-43%). This positive interaction is seemingly mediated via different mechanisms: NaB and TSA may modify the chromatin structure of the beta-globin gene cluster; KL may activate the gamma-globin promoter via up-modulation of tal-1 and possibly FLKF transcription factors. These studies indicate that KL plays a key role in HbF reactivation in adult life. Furthermore, combined KL and NaB administration may be considered for sickle cell anemia and beta-thalassemia therapy.

Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate

AbstractMechanisms underlying fetal hemoglobin (HbF) reactivation in adult life have not been elucidated; particularly, the role of growth factors (GFs) is controversial. Interestingly, histone deacetylase (HD) inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF. We developed a novel model system to investigate HbF reactivation: (1) single hematopoietic progenitor cells (HPCs) were seeded in serum-free unilineage erythroid culture; (2) the 4 daughter cells (erythroid burst-forming units, [BFU-Es]), endowed with equivalent proliferation/differentiation and HbF synthesis potential, were seeded in 4 unicellular erythroid cultures differentially treated with graded dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated in terminal erythroblasts by assay of F cells and γ-globin content (control levels, 2.4% and 1.8%, respectively, were close to physiologic values). HbF was moderately enhanced by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor treatment (up to 5%-8% γ-globin content), while sharply reactivated in a dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The stimulatory effects of KL on HbF production and erythroid cell proliferation were strictly correlated. A striking increase of HbF was induced by combined addition of KL and NaB or TSA (40%-43%). This positive interaction is seemingly mediated via different mechanisms: NaB and TSA may modify the chromatin structure of the β-globin gene cluster; KL may activate the γ-globin promoter via up-modulation of tal-1 and possibly FLKF transcription factors. These studies indicate that KL plays a key role in HbF reactivation in adult life. Furthermore, combined KL and NaB administration may be considered for sickle cell anemia and β-thalassemia therapy.

Induction of Globin Synthesis in K562 Cells Is Associated with Differential Expression of Transcription Factor Genes

ABSTRACT: Globin gene switching may be mediated by proteins expressed during different stages of development. Their identification may clarify the mechanisms of the conversion from fetal to adult globin production and lead to new approaches to reversing or retarding the γ- to β-globin gene switch. To explore this hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25, 2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and 72 h. Erythroid differentiation was verified by benzidine staining and by measuring the activity of a transduced Aγ-globin gene promoter linked to a luciferase reporter gene. Using differential display polymerase chain reaction (PCR), total mRNA extracted from induced cells at each time point of induction was reverse transcribed in the presence of A, G, and C anchored primers and 16 arbitrary primers, calculated to amplify approximately 50% of expressed genes. Amplified mRNAs from induced and uninduced cells were separated in polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to represent either up- or downregulated mRNA species in induced K562 cells were identified. Sixty-four of these fragments had more than 95% homology to known GenBank sequences. Seventeen fragments with characteristics of transcription factors were cloned. These include differentiation-related gene-1 (drg-1), PAX 3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), α-NAC transcriptional coactivator, LIM domain protein, and trophoblast hypoxia regulating factor. Differential expression of all 17 fragments over 72 h was confirmed by reverse Northern dot blot analysis, semiquantitative PCR using nested primers, and Northern analysis. Erythroid maturation in induced K562 cells is associated with differential expression of numerous genes. Some encode transcription factors that could effect the initiation of HbF synthesis. Almost half of the differentially expressed clones contained cDNAs of unidentified open reading frames and these are the object of continued study.

HbF synthesis during stress erythropoiesis as determined by gamma-mRNA/non-alpha-mRNA quantification.

To determine whether a quantitative relationship exists between globin mRNAs and their translation product during stress erythropoiesis in infants with increased production of fetal hemoglobin (HbF), we measured and compared the relative amounts of the mRNAs of alpha-, beta-, and gamma-globins and their protein synthesis. The synthesis of globin in immature red cells was determined by the incorporation of [3H]leucine, followed by separation and quantification of the polypeptides by C4-reverse phase HPLC. The relative proportions of the mRNAs of the globins were determined by RNase protection assay. A comparison of blood samples from 17 infants expected to have increased production of HbF in relation to their developmental age (five infants of diabetic mothers, two infants with intrauterine growth retardation, one infant with bronchopulmonary dysplasia, and seven infants with cyanotic heart disease) revealed a very significant correlation (r2 = 0.994; p < 0.001) between the ratio of globin mRNAs encoding HbF ([gamma/(gamma + beta)] mRNAs) and the ratios of the de novo synthesis of HbF [gamma/(gamma + beta)]. When only the 10 infants who had increased HbF synthesis are included, the correlation remains unchanged (r2 = 0.997, p < 0.001). The data demonstrated that under conditions of erythropoietic stress, when HbF production is increased, there is a close relationship between the quantification of gamma-globin mRNA and gamma-globin synthesis. The usual methods of determining HbF synthesis can be replaced by globin mRNA determination, which can be performed rapidly with a minimal amount of blood.

Induction of fetal hemoglobin synthesis with recombinant human erythropoietin in anemic patients with heterozygous beta‐thalassemia during pregnancy

Objective: Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous β-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis. Methods: Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells. Results: RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron. Conclusions: RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous β-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated. J. Matern.-Fetal Med. 1999;8:1–7. © 1999 Wiley-Liss, Inc.

Induction of fetal hemoglobin synthesis with recombinant human erythropoietin in anemic patients with heterozygous beta-thalassemia during pregnancy.

Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous beta-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis. Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells. RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron. RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous beta-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated.

Pharmacological induction of foetal haemoglobin synthesis in sickle-cell disease.

In the present review the role and regulation of foetal haemoglobin (HbF) in sickle-cell disease as well as the clinical results of therapy aimed at augmenting HbF synthesis are briefly discussed. Enhanced levels of HbF have an inhibitory effect on sickling and ameliorate the clinical course of sickle-cell disease. This knowledge has incited a search for pharmacological agents capable of stimulating HbF production. Cytostatic drugs, in particular hydroxyurea, but also other agents such as erythropoietin, butyrate and its analogues have been shown to induce HbF production. The mechanisms of action and the clinical effects of these agents are summarized. Only hydroxyurea has shown significant clinical effects in terms of reduction of pain-crises, chest syndrome and transfusions in sickle-cell patients. However, not all patients experience clinical amelioration and it appears difficult to predict which patient will gain from hydroxyurea therapy. Further studies need to expand the clinical experience with hydroxyurea, other HbF-inducing agents and combinations of these drugs. Moreover, they will hopefully provide criteria to enable appropriate selection of those patients with sickle-cell anaemia who will benefit clinically from HbF stimulation, also taking into account the risks and costs of long-term pharmacological therapy.

QUANTITATIVE CONCORDANCE BETWEEN GLOBIN mRNAs AND SYNTHESIS OF FETAL HEMOGLOBIN (HbF) AND ADULT HEMOGLOBIN (HbA) DURING HYPOXEMIC STRESS ERYTHROPOIESIS. † 1293

Increased production of HbF can be considered a marker for hypoxemia, since increased levels of HbF synthesis have been found in newborn infants following maternal hypoxemia, placental insufficiency, and diabetes, as well as in infants with broncho-pulmonary dysplasia and children with congenital cyanotic heart disease. To examine if a quantitative relationship exists between the mRNAs encoding globins and their translation into proteins during hypoxemic erythropoietic stress, the relative amounts of the mRNAs and the relative amounts of the α,β,γ-globin synthesis in samples obtained from cord blood from infants of insulin-dependent diabetic mothers, intrauterine growth-retarded infants of preeclamptic mothers and older infants with cyanotic heart disease were measured and compared. The synthesis of globins in reticulocytes was measured by the incorporation of [3H]-leucine followed by the separation and quantitation of the polypeptides on a C4-reverse phase HPLC. The relative proportions of the mRNAs of globins were determined by RNase protection assay. Total RNA isolated from blood (50μl) was incubated with [32P]-labelled cRNA probes ofα,β,γ-globins, followed by RNase A/T1 digestion, separation of the protected fragments and densitometry of the radioactive bands by phosphorimaging. The study population showed an increase in HbF synthesis, compared to controls. There was a strong correlation (r2=0.993; n=16) between the ratio of mRNAs encoding HbF and HbA and the ratio of de novo synthesis of HbF and HbA. Therefore, under conditions of stress erythropoiesis there is a close correlation between quantitation of globin mRNAs and HbF& HbA synthesis. As well, globin mRNA quantitation could replace the usual method of HbA & HbF synthesis determination.

Fetal hemoglobin as a possible marker of susceptibility to working conditions for certain men exposed to industrial pollutants

In 43 from 222 men (19.4%) employed in a coking plant a higher than normal concentration of fetal hemoglobin (HbF) was found, whereas an elevated level of HbF was detected in only two from 87 persons (2.3%) working in the cold rolling mill of the steel mill ‘Huta Sendzimira’, Krakow, Poland. Subjects employed in the coking mill worked in a much more polluted atmosphere than those in the cold rolling mill. It is speculated that synthesis of HbF may be a marker of the effect of unfavorable working conditions on some susceptible persons.

QUANTITATIVE CONCORDANCE BETWEEN GLOBIN mRNA AND SYNTHESIS OF FETAL AND ADULT HEMOGLOBIN DURING HEMOGLOBIN SWITCHOVER IN PERINATAL PERIOD. • 1725

During the perinatal period the switchover from fetal (HbF) to adult hemoglobin (HbA) synthesis in humans, γ-globin levels decline as the proportion of β-globin increases under the influence of a developmental clock. In order to examine if a quantitative relationship existed between the mRNAs encoding globins and their translation into proteins during the period of switchover, the relative amounts of the mRNAs and the relative amounts ofα, β, γ-globin synthesis in cord blood samples were measured and compared. The synthesis of globins in reticulocytes was measured by the incorporation of [3H]-leucine followed by the separation and quantitation of the polypeptides on a C4-reverse phase HPLC equipped with an integrator. The relative proportions of the mRNAs of globins were determined by RNase protection assay. Total RNA isolated from cord blood was incubated with [32P]-labelled cRNA probes (of equivalent specific activity) of α, β, γ-globins overnight at 50°C, followed by RNAse A (10 μg/ml) /T1 (200 U/ml) digestion at 25°C, separation of the protected fragments on 6% urea-polyacrylamide gels and densitometry of the radioactive bands by phosphorimaging. A comparison cord blood samples from 18 newborn infants of different gestational ages (27-40 weeks; birth weight: 850-4035 g) with no congenital anomalies, revealed a strong and significant correlation (r2= 0.934) between the ratio of mRNAs encoding HbF and HbA and the ratio of de novo synthesis of HbF and HbA. There was a linear relationship between the proportions of mRNAs encoding HbF and HbA with the proportional synthesis of HbF and HbA throughout the gestational age studied. This information can be useful for studies furthering the understanding of the mechanisms regulating globin gene expression.

Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

Fetal Hemoglobin Induction by Acetate, a Product of Butyrate Catabolism

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma-chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF-containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two-carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.