Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of gamma-globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically-mediated regulation of the expression of human gamma-globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including beta-thalassemia and sickle cell anemia.     

The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.     

Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation

The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching.     

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.     

alpha-Amino-N-butyric acid stimulates fetal hemoglobin in the adult

The effect of alpha-amino-N-butyric acid (alpha ABA) on fetal hemoglobin production in the adult was examined in vivo after being administered to normal and anemic baboons and in erythroid progenitor cell cultures. Infusion of alpha ABA for five days resulted in four- to fivefold increases in the level of F reticulocytes of normal or chronically anemic baboons. The induction of HbF by alpha ABA was strikingly enhanced by the administration of 5-azacytidine. The addition of alpha ABA in culture produced a concentration-related increase of HbF in baboon CFUe and e-cluster colonies. In addition to the induction of HbF, alpha ABA stimulated the growth of all classes of erythroid progenitors in vivo or in culture. The activation of gamma-globin gene expression by alpha ABA is attributed to an interaction between regulatory sites of globin chromatin modified by alpha ABA and the immature intracellular environment of the expanding erythropoiesis. The combination of chromatin modification, DNA methylation, and the immature intracellular environment of rapid erythroid regeneration may explain the synergistic induction of HbF by alpha ABA and 5-azacytidine.     

Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells

The present study aimed to determine whether rapamycin could increase the expression of gamma-globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti-tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors and patients with beta-thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, gamma-globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four beta-thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human gamma-globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including beta-thalassaemia and sickle cell anaemia.     

Methyl binding domain protein 2 mediates gamma-globin gene silencing in adult human betaYAC transgenic mice

The genes of the vertebrate beta-globin locus undergo a switch in expression during erythroid development whereby embryonic/fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal gamma-globin gene. The gamma-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human beta-globin locus as a yeast artificial chromosome (betaYAC) transgene. To elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2-/- mice were generated by breeding betaYAC mice with MBD2-/- mice. Adult betaYAC/MBD2-/- mice continue to express the gamma-globin gene at a level commensurate with 5-azacytidine treatment, 10- to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of gamma-globin expression is consistently higher in MBD2-/- mice in 14.5- and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic/fetal erythroid development. DNA methylation levels are modestly decreased in MBD2-/- mice. MBD2 does not bind to the gamma-globin promoter region to maintain gamma-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of gamma-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells.     

On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.     

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 silencing in humans

Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a "switching" model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.     

p38 MAP kinase activation mediates gamma-globin gene induction in erythroid progenitors

OBJECTIVE: Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on gamma-globin gene activity was studied. METHOD: Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on gamma-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on gamma-globin mRNA synthesis was analyzed. RESULTS: NB and TSA activated p38 MAPK and increased gamma-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gamma-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased gamma-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. CONCLUSION: This study supports a novel role for p38 MAPK in gamma-globin regulation in human erythroid progenitors.     

Effective erythropoiesis and HbF reactivation induced by kit ligand in beta-thalassemia

In human beta-thalassemia, the imbalance between alpha- and non-alpha-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in beta-thalassemia produced inconsistent results. Here, we investigated the in vitro response of beta-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major beta-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of gamma-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in beta-thalassemia major). These studies indicate that in beta-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of gamma-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.     

Locus control region-A gamma transgenic mice: a new model for studying the induction of fetal hemoglobin in the adult.

All pharmacologic agents that induce fetal hemoglobin (Hb) have been discovered with in vivo studies of humans, macaques, and baboons. We tested whether transgenic mice carrying human fetal (gamma) globin genes provide a model for studying the pharmacologic induction of HbF in the adult. In initial studies, phenylhydrazine-induced hemolytic anemia, 5-azacytidine, butyrate, or combinations of these treatments failed to activate the human gamma-globin gene in a transgenic mouse line carrying a 4.4-kb G gamma globin gene construct that is expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human A gamma gene linked to the locus control region (LCR) regulatory sequences and expressing heterocellularly HbF (about 25%, gamma-positive cells) were used. Treatments with erythropoietin, 5-azacytidine, hydroxyurea, or butyrate resulted in induction of gamma gene expression as documented by measurement of F-reticulocytes, the gamma/gamma + beta biosynthetic ratio and the level of steady state gamma mRNA. Administration of erythropoietin or butyrate to transgenic mice carrying a muLCR-beta (human) globin construct, failed to increase human beta-globin expression. These results suggest that the muLCR-A gamma transgenic mice provide a new model for studying the induction of fetal Hb in the adult.     

Role of cyclic nucleotides in fetal hemoglobin induction in cultured CD34+ cells

OBJECTIVE: In vivo, several drugs have been shown to increase fetal hemoglobin (HbF), including 5-azacytidine (AZA), sodium butyrate (SB), and hydroxyurea (HU). Studies in K562 cells suggest that cyclic guanosine monophosphate (cGMP) is required for HbF induction; however, the role of cyclic nucleotides in HbF induction in primary erythroid cultures has not been established. METHODS: CD34-selected peripheral blood monocytes cultured in a semi-solid serum-free system that mimics in vivo F-cell production are utilized to explore the role of cyclic adenosine monophosphate (cAMP) and cGMP in HbF induction in response to HU, AZA, and SB. RESULTS: In serum-free CD34 cultures, HU, SB, and AZA all markedly stimulate FNRBC production up to 30-fold, associated with induction of gamma-globin mRNA and total HbF protein. Guanylate cyclase inhibition results in only minimal blunting of HbF induction by each agent. In contrast, adenylate cyclase inhibition markedly reduces HU, SB, and AZA-mediated FNRBC induction and gamma-globin mRNA induction. The adenylate cyclase activator forskolin modestly induces FNRBC production and augments the action of standard induction agents. HU, AZA, and SB, however, fail to significantly stimulate adenylate cyclase themselves. CONCLUSIONS: In human CD34(+) cultures, cAMP production is required for full induction of HbF by HU, SB, and AZA, while perturbation of cGMP production has only minimal effects. These findings are in marked contrast to data in K562 cells where cGMP production is critical for HbF induction while cAMP stimulation blunts HbF response, and suggest that these agents may share a common induction pathway.