scholarly journals The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion.

Haematologica ◽  
2020 ◽  
pp. 0-0
Author(s):  
Audrey Astori ◽  
Gabriel Matherat ◽  
Isabelle Munoz ◽  
Emilie-Fleur Gautier ◽  
Didier Surdez ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Close Correlation ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Functional Studies ◽  
Healthy Donors ◽  
Epigenetic Regulator ◽  
Smad7 Expression

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFs-dependent and mediated by SMAD7, a TGFa-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFsssuperfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

MicroRNAs 155 and 451 as Possible Key Molecules for Human Erythroid Differentiation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4071-4071
Author(s):  
Tsukuru Umemura ◽  
Shizuka Masaki ◽  
Rie Ohtsuka ◽  
Yasunobu Abe ◽  
Koichiro Muta
Keyword(s):  
Red Blood Cells ◽  
Internal Control ◽  
Blood Cells ◽  
Human Peripheral Blood ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Expression Levels ◽  
Final Maturation ◽  
Normal Human ◽  
U6 Rna

Abstract MicroRNAs (miRNAs) are 18–25-nucleotide noncoding RNAs which play important roles for cell death, proliferation, development and differentiation. MiRNA is an important molecule to regulate genes by suppressing the translation or inducing instability of miRNAs, and is consist of the network system to regulate gene functions in combination with transcription factors. Many recent works demonstrated that some of miRNAs are playing key roles for hematopoiesis and leukemogenesis. In this study, we analyzed the expression of miRNAs(miRNA-155, miRNA-221, miRNA-223, miRNA-451) during differentiation of purified normal human eryhroid progenitors in the liquid culture system. Cells increased almost 500-folds in a number, and differentiated to benzidine-positive mature erythroblasts after days 7 to 9 which were partly red blood cells on days 12 to 14. Since mature erythroid cells loose cellular nucleic acids at the final maturation stages, we measured changes in U6 RNA contents as the internal control for assays of miRNA. Each expression levels of miRNAs were normalized using U6 RNA contents. Analyses of miRNA expressions using quantitative real-time reversetranscriptase polymerase chain reaction have shown that the expression level of miRNA-155 decreased about 200-folds from day 3 to day 12 with almost 87.5% reduction between days 3 and 5. On the other hand, the expression levels of miRNA-451 increased about 270-folds by day 12 in parallel to an increase in benzidine-positive cell numbers. To extend our observation on the up-regulation of miRNA-451 in mature blood cells, we analyzed the miRNA-451 levels in each mature blood cells (red blood cells, granulocytes, lymphocytes and monocytes, platelets) purified from normal human peripheral blood by using a density centrifugation method. miRNA-451 was expressed in red blood cells about 104 folds more than in granulocytes, about 102 folds more than in platelets. Moderate down-regulations of miRNAs 221 and 223 were observed. In conclusion, our observations suggest that the down-regulation of miRNA-155 and the up-regulation of miRNA-451 are key events for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be useful tools for specifying the differentiation stage of each erythroid cells.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 344-344
Author(s):  
Daniel Garcia Santos ◽  
Matthias Schranzhofer ◽  
José Artur Bogo Chies ◽  
Prem Ponka
Keyword(s):  
Red Blood Cells ◽  
Heme Oxygenase ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Heme Biosynthesis ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Wild Type ◽  
Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

Investigation of the Role of Heme Oxygenase 1 During Erythroid Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1997-1997
Author(s):  
Daniel Garcia dos Santos ◽  
Jesse Eisenberg ◽  
Matthias Schranzhofer ◽  
Jose Artur Bogo Chies ◽  
Prem Ponka
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Physiological Mechanism ◽  
Erythroid Differentiation ◽  
The Body ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells

Abstract Abstract 1997 Poster Board I-1019 Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is by heme oxygenases (HO1 and HO2) that catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of sodium arsenite (NaAsO2), HO1 inducer, on heme and iron metabolism in MEL cells induced to erythroid differentiation. MEL cells treated with NaAsO2 displayed a significant reduction in globin expression and increased ferritin levels. Moreover, NaAsO2treatment decreased levels of transferrin receptor in cell membranes. These effects triggered by NaAsO2 could be prevented by the addiction of tin-protophorphyrin (SnPP), HO1 activity inhibitor. Using a siRNA specifically targeting HO1, we observed an increase in globin expression together with a small decrease in the expressin of ferritin in DMSO-induced MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during erythroid differentiation. In summary, our results showing that NaAsO2-induced HO1 in erythroid cells cause a defect in erythroid differentiation suggest that HO1 could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Early events in erythroid differentiation: accumulation of the acidic peroxidoxin (PRP/TSA/NKEF-B)

1995 ◽  
Vol 312 (3) ◽  
pp. 699-705 ◽  
Author(s):  
T Rabilloud ◽  
R Berthier ◽  
M Vinçon ◽  
D Ferbus ◽  
G Goubin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Cell Systems ◽  
Transformed Cell ◽  
Early Stages ◽  
Antioxidant Protein

The acidic peroxidoxin [also named thiol-specific antioxidant protein (TSA) or protector protein (PRP)], which plays a role in the response against oxidative stress, is one of the major proteins of red blood cells. In this work, we show that this protein is induced at early stages of erythroid differentiation prior to haemoglobin accumulation, which suggests that it may play a role at the erythroblast stage, where haemoglobinized, nucleated and genetically active cells are submitted to a maximally dangerous oxidative stress. The early accumulation of this protein has been demonstrated both on transformed cell systems and on normal differentiating human erythroid cells. This suggests that this protein may play an important role in the differentiation of the erythroid cells.

The Role of Heme Oxygenase 1 in Erythroid Differentiation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3847-3847
Author(s):  
Daniel Garcia dos Santos ◽  
Matthias Schranzhofer ◽  
Jose Artur Bogo Chies ◽  
Prem Ponka
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Tissue Injury ◽  
Erythroid Differentiation ◽  
The Body ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Transferrin Receptors ◽  
Erythroid Cells ◽  
Wild Type

Abstract Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75–80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Production and fate of erythroid cells in anaemic Xenopus laevis

1979 ◽  
Vol 35 (1) ◽  
pp. 403-415
Author(s):  
N. Chegini ◽  
V. Aleporou ◽  
G. Bell ◽  
V.A. Hilder ◽  
N. Maclean
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Xenopus Laevis ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Complete Recovery ◽  
Cell Counting ◽  
Erythroid Cells ◽  
Spleen Cells ◽  
Sudden Release

Adult Xenopus laevis, rendered anaemic by phenylhydrazine injection, have been studied during the recovery from such anaemia. Electron microscopy of liver and spleen sections indicates that both of these organs are active in the phagocytosis and destruction of the old damaged red blood cells. May-Grunwald and Giemsa staining of liver and spleen cells following anaemia has been used to show that erythropoiesis also occurs in both liver and spleen, and this has been confirmed by electron-microscope studies of these organs. Cell counting and radiolabelling of the new population of circulating erythroid cells in the period following phenylhydrazine injection suggests that a sudden release of basophilic erythroblasts from liver and spleen is followed by mitosis of this new cell population in circulation, and that no further release of erythroid cells from these organs is likely until complete recovery has occurred.

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

2002 ◽  
Vol 20 (5) ◽  
pp. 467-472 ◽  
Author(s):  
Thi My Anh Neildez-Nguyen ◽  
Henri Wajcman ◽  
Michael C. Marden ◽  
Morad Bensidhoum ◽  
Vincent Moncollin ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Large Scale ◽  
Ex Vivo ◽  
Erythroid Cells

Differential Requirements for Survivin in Hematopoietic Cell Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1257-1257
Author(s):  
Yanfei Xu ◽  
Sandeep Gurbuxani ◽  
Ganesan Keerthivasan ◽  
Amittha Wickrema ◽  
John D. Crispino
Keyword(s):  
Cell Cycle ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Caspase 3 ◽  
Terminal Differentiation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Stage Of Development ◽  
Primary Mouse

Abstract The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1581-1581
Author(s):  
Rodwell Mabaera ◽  
Christine Richardson ◽  
Sarah Conine ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Maximal Response ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Potent Inducer ◽  
Cellular Dna ◽  
Globin Gene Expression

Abstract 5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980’s there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

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