Erythroid Progenitor Cell Cultures Research Articles

Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures

Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

UNC0638 induces high levels of fetal hemoglobin expression in β-thalassemia/HbE erythroid progenitor cells.

Increased expression of fetal hemoglobin (HbF) improves the clinical severity of β-thalassemia patients. EHMT1/2 histone methyltransferases are epigenetic modifying enzymes that are responsible for catalyzing addition of the repressive histone mark H3K9me2 at silenced genes, including the γ-globin genes. UNC0638, a chemical inhibitor of EHMT1/2, has been shown to induce HbF expression in human erythroid progenitor cell cultures. Here, we report the HbF-inducing activity of UNC0638 in erythroid progenitor cells from β-thalassemia/HbE patients. UNC0638 treatment led to significant increases in γ-globin mRNA, HbF expression, and HbF-containing cells in the absence of significant cytotoxicity. Moreover, UNC0638 showed additive effects on HbF induction in combination with the immunomodulatory drug pomalidomide and the DNMT1 inhibitor decitabine. These studies provide a scientific proof of concept that a small molecule targeting EHMT1/2 epigenetic enzymes, used alone or in combination with pomalidomide or decitabine, is a potential therapeutic approach for HbF induction. Further development of structural analogs of UNC0638 with similar biological effects but improved pharmacokinetic properties may lead to promising therapies and possible clinical application for the treatment of β-thalassemia.

Red Blood Cells Store and Release Interleukin-33

Interleukin-33 (IL-33) is a member of the IL-1 cytokine superfamily that potently drives production of a variety of cytokines and contributes to the pathogenesis of inflammatory diseases. The IL-33 is...

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

Quantification of HBG mRNA in primary erythroid cultures: prediction of the response to hydroxyurea in sickle cell and beta‐thalassemia

Increased expression of fetal hemoglobin (HbF) may ameliorate the clinical course of hemoglobinopathies like sickle cell disease (SCD) and β-thalassemia. Hydroxyurea (HU) can stimulate HbF production in these diseases but the response is highly variable indicating the utility of developing an in vitro test to predict the patient's response to HU. We assessed whether the HbF response of patients with SCD and thalassemia intermedia (TI) to HU correlates with HBG (both γ-globin genes) expression in their cultured erythroid progenitors following exposure to HU. We exposed primary erythroid cultures from peripheral blood mononuclear cells from 30 patients with SCD and 15 with TI to HU and measured HBG mRNA by real-time quantitative PCR. The same patients were then treated with HU and their HbF response after treatment with a stable dose of HU was compared with the mRNA results in cultured cells. The fold increase in HBG mRNA in erythroid progenitors was similar to the fold increase in HbF in vivo. Quantification of HBG mRNA in erythroid progenitor cell cultures from patients with SCD and TI is predictive of their clinical response to HU.

Evaluation of a Xeno-Free Protocol for Long-Term Cryopreservation of Cord Blood Cells

Cord blood is regarded as a powerful source for adult stem cells. Cord blood transplants have been used successfully to treat children and adults in autologous and allogeneic settings. Nevertheless, in many cases, the clinically relevant cell number (CD34+ cells and total leukocytes) is a limiting factor. To enable standardized cell banking and future in vitro expansion of adult stem/progenitor cells, elimination of serum, which inevitably differs from lot to lot and donor to donor, is highly desirable. Here, we demonstrate the feasibility of a xeno-free, chemically defined cryopreservation procedure for cord blood-derived cells over a period of 1 year. Cell recoveries with respect to retrieval of clinically relevant CD34+ cells, colony-forming units, and in vitro cultures of erythroid progenitor cells under standardized conditions were analyzed after 1 week or 1 year of cryopreservation and found to be very high and similar to the samples before freezing. The established xeno-free procedure is an important step toward using the full potential of adult stem cells from cord blood, enabling the elimination of serum-derived factors negatively influencing proliferation, differentiation, and survival of hematopoietic stem cells.

Combinatorial Effect of Pharmacological Inhibitors of Lsd-1 and DNMT on γ-Globin Synthesis in Cultured Baboon Bone Marrow Erythroid Progenitors

Abstract 249Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New therapies to increase HbF levels in patients are urgently needed. Previous studies from our laboratory have shown that decitabine, an inhibitor of DNA methyltransferase (DNMT), increases HbF to therapeutic levels in non-human experimental primates and SCD patients, thus demonstrating that drugs targeting repressive epigenetic modifications can be effective HbF-inducing agents. Both DNMT1 and Lysine Specific Demethylase-1, (LSD-1), a mono- and dimethyl- histone H3 K4 demethylating enzyme, are components of the DRED multiprotein complex, a repressor of γ-globin expression (Cui et al Mol Cell Biol 31:3298–3311, 2011). Experiments were performed to test the effect of the pharmacological LSD-1 inhibitor, tranylcypromine (TC), alone and in combination with the DNA methyltransferase inhibitor decitabine (DAC), on γ-globin expression. Baboon bone marrow (BM) CD34+ erythroid progenitors were isolated from BM aspirates of normal baboons and grown in a co-culture system with the AFT024 murine fetal liver cell line in Iscove’s media containing 30% fetal bovine serum, 200ng/ml SCF, 2U/ml epo, and 1μM dexamethasone. TC (0.5 to 5.0μM) was added to cultures on d7 and d10, while decitabine (0.1μM) was added on d7. On d14 erythroid cells were purified by immunomagnetic column chromatograpy using a baboon-specific anti-red blood cell antibody (BD Bioscience). Effects on globin expression were measured by biosynthetic radiolabelling of nascent globin chain synthesis in the presence of [3H] leucine followed by HPLC separation of individual globin chains. DNA was isolated and effect of the drugs on DNA methylation of the γ-globin promoter determined by bisulfite sequence analysis. Effects on erythroid differentiation were investigated by examination of Wright’s stained cytospin preparations. In three independent cultures TC induced dose-dependent increases in γ-globin chain synthesis (γ/γ+β). In addition TC and DAC in combination increased γ-globin chain synthesis in an additive manner (see Table).CD34+ cell sourceTC (μM)DAC (μM)γ/γ+βPA 7680000.0600.10.325.000.305.00.10.62PA 7482000.0900.10.352.500.165.000.302.50.10.545.00.10.68Bisulfite sequence analysis showed that increased γ-globin chain synthesis in TC-treated cultures was not associated with increased DNA hypomethylaton of the γ-globin promoter. Examination of Wright’s stained cytospin preparations of d14 cultures showed increased numbers of basophilic and polychromatic erythroblasts in cultures exposed to TC compared to untreated controls suggesting that TC reduced the rate of erythroid differentiation. In contrast, numbers of basophilic and polychromatic cells were reduced and orthochromatic cells were increased in cultures treated with TC and DAC compared to TC alone. We conclude that TC increased γ-globin chain synthesis in a dose-dependent manner in baboon erythroid progenitor cell cultures while TC and DAC, in combination, produced additive effects on γ-globin chain synthesis with minimal effects on erythroid differentiation. These results suggest that combinatorial use of epigenetic modifiers may be superior to single agents and support further studies to determine whether TC and other LSD-1 pharmacological inhibitors can effectively be used in combination with DNMT inhibition to increase HbF levels in vivo. Disclosures:No relevant conflicts of interest to declare.

Glucose Metabolism and Erythropoietin Stimulation of Erythroid Progenitor Cells

AbstractAbstract 4232Erythropoietin (EPO) is known for its activity in stimulating erythroid progenitor cell survival, proliferation and differentiation. EPO is produced in the interstitial cells of the kidney in a hypoxic responsive manner and is used in treatment of anemia in patients with chronic kidney disease. Suggestions that EPO may affect metabolic parameters led to a recent report that high dose erythropoietin treatment in mice resulted in reduced blood glucose level in addition to increased hematocrit (Hojman et al., Plos One 2009). To examine the link between EPO and metabolic processes further, we treated wild type C57Bl/6 mice fed normal diet (11 kcal% fat) and high fat diet (45 kcal% fat) with EPO (3,000 U/kg three times a week for 3 weeks). As expected from the stimulation of erythropoiesis by exogenous EPO, both groups showed the expected increase in hematocrit up to 80%. We also found that EPO decreased fasting blood glucose level in both normal diet fed mice and high fat diet fed mice, compared with mice treated with saline. An improvement in glucose tolerance was also observed. Since hematopoietic tissue is the major target of EPO, we speculate that EPO may stimulate glucose uptake in hematopoietic tissue, especially during erythropoiesis. To examine EPO stimulation and glucose uptake in vitro, we used cultures of primary human adult erythroid progenitor cells isolated from peripheral blood. We found that with EPO stimulation, glucose uptake per cell increased with erythroid differentiation and peaked in late erythroid progenitor cells at day 8 to 10, then decreased during terminal differentiation. This pattern was observed at both 21% O2 and 5% O2. EPO increased glucose uptake in a dose dependent manner up to 10 U/ml. Cellular glucose uptake appeared to follow EpoR expression and was maximal when EpoR levels, and therefore EPO response, were highest. Glucose uptake decreased as EpoR levels decreased with the progression of erythroid maturation. These data suggest that EPO stimulation regulates glucose uptake in erythroid progenitor/precursor cells depending on the stage of erythroid differentiation and that glucose uptake is reduced in late erythroid precursor cells with the down regulation of EpoR and loss of EPO dependence as the cells differentiate into mature erythrocytes. We also examined the effect of high glucose concentration on EPO response in primary human adult erythroid progenitor cell cultures. We found that high glucose concentration in erythroid progenitor cell cultures inhibited EPO stimulated differentiating erythroid progenitor cell proliferation. The combination of EPO with high dose glucose increased hemoglobinization as determined by the increase in benzidine positive cells, suggesting premature hemoglobin accumulation and differentiation. This activity of elevated glucose level suggests that elevated blood glucose concentration contributes to the blunted EPO response, giving rise to EPO resistance. The findings from this study provide us with new insight on EPO stimulation of erythropoiesis and the potential interplay between erythropoiesis and glucose metabolism. Disclosures:No relevant conflicts of interest to declare.

SiDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)−dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures

These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells. DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.

The G9A Histone Methyltransferase BIX-01294 Reactivates ε-Globin Expression and Blocks Erythroid Differentiation in Primary Baboon Erythroid Progenitor Cell Cultures

The development of new therapies to increase fetal hemoglobin (HbF) levels in patients with sickle cell disease and β-thalassemia depends on an increased understanding of the mechanism responsible for the developmental regulation of globin gene expression. A role for epigenetic modifications in the mechanism of of globin gene regulation is suggested by the presence of high levels of DNA methylation near the 5' regions of developmentally silenced ε- and γ-globin genes and the ability of pharmacological inhibitors of DNA methyltransferase (DNMTase) to reactivate ε- and γ-globin expression in adults. Whether additional epigenetic modifications associated with gene silencing and DNA methylation, such as histone H3 (lys9) dimethylation, are also involved is unknown. To investigate the hypothesis that histone H3 (lys9) dimethylation may function in the mechanism of developmental globin gene silencing, chromatin immunopreciptation assays were performed to determine the distribution of histone H3 (lys9) dimethyl and histone H3 (lys9) acetyl throughout the β-globin gene complex in purified primary baboon bone marrow (BM) erythroid cells from phlebotomized baboons expressing low levels (5–10%) of HbF and purified erythroid cells from erythroid progenitor cell cultures expressing high levels of HbF (30–50%). In BM erythroid cells, the level of histone H3 (lys9) acetyl associated with the β-globin gene was 10–20 fold higher than with the ε- and γ-globin genes, while the level of histone H3 (lys9) dimethyl associated with the ε- and γ-globin genes was 2–4 fold higher than with the β-globin gene. In erythroid cells from day 12 erythroid progenitor cell cultures, the level of histone H3 (lys9) acetyl associated with the highly expressed γ- and β-globin genes was 10–20 fold higher than with the silent ε-globin gene, while the level of histone H3 (lys9) dimethyl associated with the ε-globin gene was 2–4 fold higher than with the γ- and β-globin genes. Therefore a reciprocal relationship was observed between levels of histone H3 (lys9) acetylation and dimethylation associated with active and inactive globin genes. Experiments were performed to further investigate the role of histone H3 (lys9) dimethyl in ε-globin gene silencing by determining the effect of the G9A histone methyltransferase inhibitor BIX-01294 on ε-globin expression. Erythroid progenitor cell cultures derived from CD34+ BM cells of three individual baboons were treated with the varying doses of the DNMTase inhibitor decitabine (0.125–1.0μM), and BIX-01294 (1.25–5μM), alone and in combination. Changes in ε- globin were assessed by real time PCR using the ΔΔCT method with α-globin as the standard. Decitabine (0.5μM) increased ε-globin 25.8±7.7 fold while BIX-01294 (2.5μM) increased ε-globin 3.09±1.16 fold. Decitabine (1μM) and BIX-01294 (2.5μM) in combination increased ε-globin 55.7±24.9 fold. BIX-01294 enhanced ε-globin expression approximately twofold at all decitabine doses ranging from 0.125–1.0μM (mean increase=103± 44.7%). BIX-01294 also blocked terminal erythroid differentiation and allowed expansion of more primitive cells as evidenced by the presence of a large population of basophilic erythroblasts at late stages of culture (day 14). These results demonstrate that BIX-01294 reactivates expression of the silenced ε-globin gene and that synergistic reactivation can be achieved using combinations of BIX-01294 and decitabine. While these results are consistent with the hypothesis that epigenetic modifications are important in the mechanism of developmental globin gene silencing, the observation that BIX-01294 blocks erythroid differentiation suggests the possible involvement of a reprogramming mechanism.

Erythropoietin Receptor Expression: A Role for the Tal1/SCL and GATA-1 Complex

Tal1/SCL is required for hematopoietic stem cell development and is essential for normal erythropoiesis. Although Tal1-DNA binding is not required for hematopoietic development, Tal1 participates in a complex with other binding partners GATA-1, LMO2, Ldb1 and possibly Sp1. During erythropoiesis, Tal1 can activate expression via direct association with E-box-GATA DNA sequence motifs for lineage specific genes such as Protein 4.2. In mice, loss of Tal1 in adult erythropoiesis also affects TER119 expression and BFU-E growth resulting in anemia. We found that knock down of Tal1 expression in primary erythropoietin (EPO) stimulated hematopoietic progenitor cells in culture inhibited EPO receptor (EPOR) expression and erythroid differentiation, consistent with the anemia observed in mice with targeted deletion of adult Tal1 expression. In contrast, overexpression of Tal1 in erythroid cells increased both EPOR expression and erythroid differentiation. In fact, overexpression of EPOR was sufficient to increase differentiation in erythroid progenitor cell cultures. Increased EPOR expression by Tal1 was mediated by 3 conserved E-boxes in the 5′ UTR. In reporter gene assays in K562 erythroid cells, EPOR promoter activity was lost with the mutation of these E-boxes, which are located 75 bp downstream of the required GATA-1 binding motif in the human EPOR proximal promoter. Tal1/E2A dimer binding to the E-box region was demonstrated by gel mobility shift assay. Tal1 transactivated EPOR mRNA and chromatin immunoprecipitation (ChIP) analysis confirmed that Tal1 bound directly to the E-box region in intact erythroid cells. We previously showed that GATA-1 also transactivates EPOR gene expression and is required for high level of EPOR transcription activity. In ChIP assays, an antibody to Tal1 also pulled down chromatin containing the GATA-1 binding site as well as the E-box region and conversely, antibodies to GATA-1 pulled down chromatin corresponding to the GATA-1 binding site and the E-box region. These data show that the complex containing Tal1 and GATA-1 bound to the E-box region and to the GATA-1 site in the EPOR proximal promoter and that the complex has greater occupancy at the GATA-1 site in the proximal promoter. High-level Tal1 expression increased chromatin containing Tal1 or GATA-1 associated with the GATA-1 binding site as well as the down stream E-box region, with a greater proportionate increase in the E-box region. Hence, in addition to the GATA-1 binding site, the downstream E-box region is necessary for high-level EPOR expression. These data suggest that transactivation of the EPOR promoter via these sites is mediated by a Tal1 and GATA-1 containing complex and that during erythropoiesis EPO induction of both Tal1 and GATA-1 regulates the high expression of its own receptor. We hypothesize that the high binding of GATA-1 and Tal1 at the GATA site opens the upstream chromatin structure. The over-expression of Tal1 promotes the open status and facilitates the spreading of open chromatin along the chromatin to the downstream E-box region. Subsequent E-box binding by Tal1 and its interaction with GATA-1 maintains the open chromatin and activates transcription.

DNA Methylation and Globin Gene Expression.

Understanding how the human γ-globin gene is regulated has important clinical implications because increased levels of fetal hemoglobin (HbF) are beneficial to patients with sickle cell disease and β-thalassemia. DNA methylation is strongly implicated in developmental silencing of the γ-globin gene based on: an inverse correlation between DNA methylation of the γ-globin gene and its expression, the acquisition of CpG residues within the γ-globin 5’ region during evolution as the γ-globin gene was recruited to fetal stage expression, and the ability of pharmacological inhibitors of DNA methyltransferase (5-azacytidine; decitabine) to reactivate high-level expression of the γ-globin gene in experimental primates that led to clinical trials demonstrating that decitabine increased HbF to therapeutic levels in patients with sickle cell disease. Decitabine treatment in vivo decreases DNA methylation of the γ-globin gene and increases association of RNA polymerase II, acetyl Histone H3 and H4, and Histone H3 (lys4) trimethyl with the γ-globin gene, strongly suggesting that decitabine increases γ-globin gene transcription. These results are consistent with the hypothesis that γ-globin expression in adults is repressed by the binding of methyl DNA binding proteins to the methylated γ-globin promoter with subsequent recruitment of co-repressor complexes that actively repress γ-globin transcription. The reduction of γ-globin gene DNA methylation induced pharmacologically in adults by decitabine is linked to high level γ-globin expression, as is the complete loss of γ-globin gene methylation attained physiologically during erythroid differentiation of fetal liver hematopoietic progenitor cells. The mechanism of action of the drug has not been definitively established, however, and the role of DNA methylation in regulation of γ-globin gene expression remains an active area of investigation. High level γ-globin expression in baboon erythroid progenitor cell cultures without a reduction of γ-globin gene DNA methylation suggests the existence of alternative mechanisms of activation. In addition to reducing DNA methylation, decitabine activates the p38 MAP kinase pathway, increases p21WAF, and accelerates terminal erythroid differentiation. The role of these effects remains to be investigated. Increased understanding of the role of DNA methylation in γ-globin gene regulation is likely to impact the design of future therapies to increase HbF levels.

Expression of γ-Globin Is Reactivated by a Novel Mechanism in Baboon CD34+ Erythroid Progenitor Cell Cultures.

Elevated levels of fetal hemoglobin (HbF) reduce the symptoms of sickle cell disease and increase the life span of patients. Future pharmacologic therapies to increase HbF will depend on increased knowledge of the mechanism(s) regulating γ-globin gene expression. To investigate the relationship between DNA methylation, chromatin structure, and γ-globin gene regulation, DNA methylation of 5 CpG sites within γ-globin gene promoter, levels of acetyl-histone H3 and H4 and histone H3 lys4 trimethyl throughout the β-globin gene complex, and the pattern of γ-globin polypeptide chain synthesis were analyzed in1.primary baboon fetal liver (FL) erythroid cells,2.primary baboon bone marrow (ABM) erythroid cells from phlebotomized adults expressing low levels of HbF,3.ABM erythroid cells from adults expressing high levels of HbF following treatment in vivo with the DNA demethylating drug decitabine, and4.erythroid cells expressing high levels of HbF generated from CD34+ baboon BM erythroid progenitors in a liquid culture system.High levels of histone acetylation were associated with the ε- and γ-globin genes and low levels with the β-globin gene in FL erythroid cells while in ABM erythroid cells expressing low levels of HbF (6.69±1.93%) high levels were associated with the β-globin gene and low levels with the γ-globin gene. Histone H3 lys 4 trimethyl was enriched near the γ-globin gene in FL and the β-globin gene in ABM. The γ-globin gene was not methylated in FL. The level of methylation was similar in bled (75.1±8.26%) and normal (82.1±7.51%) ABM erythroid cells. The ratio of expression of 5′ Iγ- and 3′ Vγ-globin genes in the fetus (1.85) differed from bled adults (0.65). Expression of γ-globin was reactivated to similar levels in decitabine-treated baboons (51.5±4.50%) and in erythroid progenitor cell cultures (45.3±12.1%; d14). The Iγ- and Vγ-globin chains were expressed at the characteristic fetal ratio in erythroid progenitor cell cultures (1.76±0.21), but not in decitabine-treated baboons (0.76±0.32). Distribution of histone acetylation and histone H3 lys4 trimethyl was nearly identical in erythroid cells expressing high levels of HbF from decitabine-treated animals and from erythroid progenitor cultures and was characterized by high levels of histone H3 lys4 trimethyl associated with both the active γ- and β-globin genes and enrichment of histone H3 and H4 acetylation near the ε-, γ-, and β-globin genes. HbF reactivation in decitabine-treated baboons was associated with a reduction in the level of γ-globin gene methylation (33.5±9.43% dmC). Surprisingly, the level of γ-globin gene methylation in erythroid cells purified from erythroid progenitor cell cultures (79.5±9.80% dmC; d11) synthesizing high levels of γ-globin was not significantly different than in ABM erythroid cells expressing <2% HbF. These results demonstrate that reactivation of γ-globin expression in erythroid progenitor cell cultures is uniquely characterized by the Iγ/Vγ-globin chain ratio of fetal development. In contrast to the absence of γ-globin gene methylation in FL and reduced levels associated with reactivation of HbF following decitabine treatment, the level of γ-globin gene methylation in erythroid progenitor cell cultures was the same as in ABM. Our data strongly suggests that reactivation of γ-globin gene expression in erythroid progenitor cell cultures is achieved through a novel mechanism not dependent on loss of DNA methylation.

Fetal Hemoglobin Induction in Baboons (P. Anubis) Following Admistration of a Novel Decitabine Dinucleotide (S110) Compound.

Increased fetal hemoglobin (HbF) levels can ameliorate the symptoms of patients with sickle cell disease and increase their life span. The DNA methyltransferase (DNMT) inhibitor decitabine increases HbF in non-human primates (baboons) and sickle cell patients and offers great promise as an effective therapeutic agent for the treatment of this disease. The goal of this investigation was to develop and test new compounds that inhibit DNMT, increase HbF, and offer significant advantages compared to decitabine such as increased stablity, reduced cytotoxicity, and a lower potential for mutagenicity. S110 is decitabine-guanine dinucleotide developed by Supergen, Inc., that is cytosine deaminase resistant and more stable in plasma than decitabine. Experiments were performed to compare the effect of S110 and decitabine on γ-globin expression in human erythroid progenitor cell cultures derived from CD34+ peripheral blood cells. Cultures were treated with decitabine (1 X 10−6 M) or S110 (1 and 5 X 10−6 M) on day 8. Forty eight hours later, RNA was purified for real time PCR analysis of ε- and γ-globin gene expression by the ΔΔCT method using α-globin as a control. Effects on globin polypeptide chain expression was determined by HPLC analysis of freeze-thaw lysates prepared 72 hours following drug addition. Both S110 and decitabine increased expression of ε-globin and γ-globin mRNA and the γ/γ + β polypeptide chain ratio (see Table). Effect of S110 on ε-and γ-globin mRNA and the γ / γ + β chain ratio in Human Erythroid Progenitor Cultures (n=4)DRUGCONCENTRATIONε-globin mRNA (Fold increase)γ-globin mRNA (Fold increase)Globin chain ratio (γ/γ + β)Untreated3 +/− 3.3Decitabine1 X 10-6 M3 +/− 24.82 +/− 2.88 +/− 3.2S1101 x 10-6 M5 +/− 4.24 +/− 1.18 +/− 1.9S1105 X 10−69 +/− 34.535 +/− 1.52 +/− 2.9The effect of S110 on HbF levels in vivo was then tested in non-human primates. Two baboons (PA 7256; PA 7470, P. anubis) were phlebotomized for eight days to achieve a HCT of 20 followed by treatment with S110 (1mg/kg/d; sc) for ten days. Pretreatment HbF levels were 3.1% (PA 7256) and 3.5% (PA 7470). Following S110 treatment, peak HbF levels of 46.1% (PA 7256) and 75.5% (PA 7470) were attained that were similar to levels observed in animals treated with equivalent molar doses of decitabine. Bisulfite sequence analysis showed that the level of DNA methylation of 5 CpG sites within the γ-globin promoter in purified BM erythroblasts following S110 administration was significantly lower (41.5%, PA 7256; 16.3%, PA 7470) compared to erythroblasts from bled, untreated baboons (70.8%; n=4). Pharmacokinetics was also evaluated in parallel to assess systemic exposure. These data demonstrate that S110, a newly developed decitabine-guanine dinucleotide, effectively increased HbF and reduced DNA methylation in cultured human erythroid progenitor cells and in experimental non-human primates.

Hypoxia alters progression of the erythroid program

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Induction of Human γ Globin Gene Expression by Histone Deacetylase Inhibitors.

In previous studies we have showed that HDAC inhibitors including hydroxamic acid derivatives of short chain fatty acids butyryl hydroxamate, propionyl hydroxamate, subericbis hydroxamic acid (SBHA), and suberoylanilide hydroxamic acid (SAHA), are potent inducers of γ globin gene expression in in vitro luciferase assays and in cultures of human adult erythroid progenitor cells. In this present study, we used μLCR Aγ transgenic mice to test whether these compounds can also induce γ gene expression in vivo. We found that in addition to γ gene induction these compounds have considerable erythropoiesis activity. Thus, Propionyl and butyryl hydroxamate increased reticulocytes of mice by 71% and 139%, the in vivo BFUe counts by 75% and 51% and the in vivo γ gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. We conclude that Hydroxamic acid derivatives can stimulate both the in vivo erythropoiesis and fetal globin production in a thalassemic murine model. Cyclic depsipeptide FK228 is a highly potent histone deacetylase inhibitor, currently in clinical trials in cancer patients. We investigated whether FK228 also functions as inducer of human γ globin gene expression and compared Hb F induction by FK228 to that of four other HDAC inhibitors, including hydroxamic acids (TSA), synthetic benzamides (MS-275), and two cyclic tetrapeptides, Apicidin and HC-Toxin. Our results showed that FK228 is the most potent fetal hemoglobin inducer among all the HDAC inhibitors tested in our laboratory. In a concentration of 0.84 nanomolar, FK228 induces γ gene promoter activity in the dual luciferase assay by 7.81 fold. In the human erythroid progenitor cell cultures it increases the levels of γ mRNA by 8.48 fold in a concentration of 0.143 nM. In contrast, fetal hemoglobin induction by other HDAC inhibitors is achieved in concentrations that are 100 to 1000 fold higher. We conclude that FK228 is a promising compound for induction of Hb F in patients with sickle cell disease and thalassemia.

Cell culture and animal models to screen for promising fetal hemoglobin-stimulating compounds

Sickle cell anemia (SCA) and the thalassemias are globally the most common class of inherited single-gene disorders. Current treatment options are limited, especially in developing countries. More practical and cheaper therapies are urgently needed. Since high fetal hemoglobin (HbF) levels ameliorate the clinical symptoms of these diseases, one current approach is to use pharmcaological agents to reactivate the γ-globin genes and stimulate the production of HbF. Several in vitro and in vivo experimental models developed for this purpose are the subject of this review. The models include in vitro established erythroid-like cell lines and primary cultures (both in semisolid and liquid media) of erythroid progenitor cells obtained from normal donors and patients with SCA and β-thalassemia, as well as in vivo models in genetically modified (transgenic) and unmodified animals. These experimental systems are useful for large-scale screening of compounds for HbF-stimulating potential, for determining the mechanism of action of potent compounds at the cellular and molecular levels, and for studying the pharmacology, pharmacokinetics, and toxicology of the drugs. These models are essential to find, test, and develop new drugs that will be effective and safe for clinical use in adults and children.

Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells.

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.

Aluminium accumulation in chronic renal failure affects erythropoiesis

Summary: Aluminium (Al) toxicity has been associated with anaemia in exposed patients with chronic renal failure (CRF). the present study was undertaken to determine whether the ingestion of Al citrate was able to affect erythropoiesis in rats with normal or impaired renal function. the renal insufficiency was induced by surgical procedures and control rats were sham operated. Twenty‐four rats were allocated to four groups of six rats each: (A) Sham; (B) Sham+Al; (C) CRF; and (D) CRF+Al. the groups B and D received daily doses of Al citrate (0.5 μmol/g bodyweight) and the groups A and C, deionized water, via the intragastric route. At the end of the experimental period (15 weeks) cultures of late erythroid progenitor cells (CFU‐E) stimulated with erythropoietin were performed and haematological parameters determined. the liver, kidney, brain, bone and serum Al amounts were quantified. the results are expressed as median and interquartile range. the CFU‐E growth was found inhibited in B and D groups (A: 100; B: 74/54‐83; C: 86/54‐98; D: 46/39‐53 %). the haematocrit values were significantly diminished in rats with renal insufficiency when compared to controls (A: 42/40‐43; B: 45/42‐46; C: 37/32‐40 and D: 37/24‐39 %). Serum Al accumulation was observed in B and D groups receiving Al (A: 8/5‐12; B: 36/36‐44; C: 5/5‐6; D: 45/26‐132 μg Al/l). No differences among groups were found in the liver and kidney Al contents, but uraemic state favoured Al accumulation in brain (A: 6/5.0‐9.0; B: 4/3.8‐4.3; C: 2/1.0‐3.0; D: 15/12.0‐21.0 μg Al/g tissue) and bone (A: 29/27‐31; B: 30/29‐39; C: 42/33‐48; D: 68/56‐79 μg Al/g tissue). We suggest that the heavy accumulation of Al in the bone compartment may result in a protracted endogenous exposure of bone marrow cells, affecting the erythropoiesis in vivo.

Comparison of Hemin Enhancement of Burst-Forming Units-Erythroid Clonal Efficiency by Progenitor Cells from Normal and HIV-Infected Patients

The ability of peripheral-blood hematopoietic progenitor cells from AIDS patients and normal controls to respond to erythropoietin (Epo) was assessed for burst-forming units-erythroid (BFU-E). BFU-E colony formation from AIDS patients' peripheral blood responded to a wide range of Epo concentrations (0.5-4 U) in a similar manner as erythroid progenitors obtained from normal peripheral blood. The optimum dose response of BFU-E to Epo was 2 U which resulted in generation of 71 +/- 4 BFU-E in AIDS patients (n = 10), as compared to 77 +/- 5 BFU-E in normal donors (n = 3). The optimum concentration range of hemin enhancement of erythroid progenitor BFU-E was 10-50 microM. In all instances, Epo was essential for BFU-E growth. Inclusion of hemin at a concentration of 10 microM in AIDS patients' peripheral-blood erythroid progenitor cells resulted in enhancement of BFU-E by 136-215%. Similarly, inclusion of hemin (10-100 microM) in normal bone marrow erythroid progenitor cell cultures resulted in enhancement of BFU-E. Inclusion of an equivalent amount of iron or tin protoporphyrin to progenitors cells from AIDS patients' peripheral blood had no effect on the number of colonies observed. On the other hand, inclusion of another heme analogue, zinc protoporphyrin, in AIDS or normal cultures resulted in a 50% suppression of BFU-E colony formation. These results demonstrate that peripheral-blood mononuclear cells from AIDS patients retain the capacity to generate erythroid precursors such as BFU-E in the presence of Epo, and that hemin has a specific enhancement effect on growth of BFU-E colony formation obtained from peripheral blood or bone marrow cells.