scholarly journals Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or beta-thalassemia

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1630-1635
Author(s):  
E Fibach ◽  
LP Burke ◽  
AN Schechter ◽  
CT Noguchi ◽  
GP Rodgers
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Cell Number ◽  
Model Systems ◽  
Beta Thalassemia ◽  
Mean Corpuscular Hemoglobin ◽  
Normal Individuals ◽  
Culture Procedure

Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood- derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5- fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta- thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.

scholarly journals Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or beta-thalassemia

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1630-1635 ◽  
Author(s):  
E Fibach ◽  
LP Burke ◽  
AN Schechter ◽  
CT Noguchi ◽  
GP Rodgers
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Cell Number ◽  
Model Systems ◽  
Beta Thalassemia ◽  
Mean Corpuscular Hemoglobin ◽  
Normal Individuals ◽  
Culture Procedure

Abstract Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood- derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5- fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta- thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.

Maintenance of elevated fetal hemoglobin levels by decitabine during dose interval treatment of sickle cell anemia

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3905-3908 ◽  
Author(s):  
Joseph DeSimone ◽  
Mabel Koshy ◽  
Louise Dorn ◽  
Donald Lavelle ◽  
Linda Bressler ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Absolute Neutrophil Count ◽  
Neutrophil Count ◽  
Fetal Hemoglobin ◽  
Dose Interval ◽  
Cell Number ◽  
Drug Dose ◽  
Hematologic Toxicity ◽  
Cumulative Toxicity

We have previously demonstrated that 5-aza-2′-deoxycytidine (decitabine) augments fetal hemoglobin (HbF) levels in patients with sickle cell anemia (SS) who did not respond to hydroxyurea (HU). The present study was designed to determine the effect of repeated decitabine dosing on HbF levels and hematologic toxicity over a 9-month treatment period. Seven patients (5 HU nonresponders) were entered. One patient had α-thalassemia sickle cell anemia. Decitabine was administered by intravenous infusion at a starting dose of 0.3 mg/kg per day, 5 days a week for 2 weeks, followed by a 4-week observation period. If the absolute neutrophil count dropped below 1000, the dose was reduced by 0.05 mg/kg per day in the next cycle. A drug dose was obtained for each patient, and it resulted in an elevated HbF without neutropenia (absolute neutrophil count nadir greater than 1500) or evidence of cumulative toxicity. Average HbF and average maximal HbF levels attained during the last 20 weeks of treatment for the 6 SS patients increased to 13.93% ± 2.75% and 18.35% ± 4.46%, respectively, from a pretreatment mean of 3.12% ± 2.75%. Mean and mean maximal hemoglobin (Hb) levels increased from 7.23 ± 2.35 g/dL to 8.81 ± 0.42 g/dL and 9.73 ± 0.53 g/dL, respectively. Individual maximal F-cell number observed during the trial was 69% ± 10.12%. The absence of cumulative toxicity may allow shorter intervals between drug treatments, which may lead to higher hemoglobin and HbF levels after several treatment cycles and, therefore, to greater clinical improvement.

scholarly journals Augmentation of fetal hemoglobin (HbF) synthesis in culture by human erythropoietic precursors in the marrow and peripheral blood: studies in sickle cell anemia and nonhemoglobinopathic adults

Blood ◽  
1978 ◽  
Vol 52 (6) ◽  
pp. 1115-1124
Author(s):  
K Kidoguchi ◽  
M Ogawa ◽  
JD Karam ◽  
AG Martin
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Fetal Hemoglobin ◽  
Culture Cell ◽  
Control Mechanisms ◽  
Normal Individuals ◽  
Individual Variations ◽  
Gel Isoelectric Focusing ◽  
Clonal Assay

We cultured marrow and peripheral blood erythropoietic precrusors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, F, and S in patients with and without sickle cell anemia. Hb was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing techniques, and quantitated by autoradiographic methods. Comparison of marrow late (CFU-E) and early (BFU-E) precursors from patients without hemoglobinopathies showed that preferential synthesis of HbF is limited to early precursors. Simultaneous examinations of Hb synthesis by blood and marrow early erythropoietic precursors confirmed the similarity of the biosynthetic capabilities of the precursors from the two sources. Increasing concentrations of erythropoietin (Ep) in culture corresponded with increases in the percentages of HbF synthesized by blood BFU-E of normal individuals. HbF biosynthesis by blood BFU-E from sickle cell anemia patients was significantly higher than that synthesized by nonanemic individuals and showed significant individual variations. HbF synthesis in patients with sickle cell anemia was partially dependent on Ep concentrations in culture. Cell culture of circulating erythropoietic precursors in man appears to provide a unique tool for studying the control mechanisms of Hb synthesis in man.

scholarly journals Effects of hydroxyurea on hemoglobin F and water content in the red blood cells of dogs and of patients with sickle cell anemia

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 212-216 ◽  
Author(s):  
EP Orringer ◽  
DS Blythe ◽  
AE Johnson ◽  
G Jr Phillips ◽  
GJ Dover ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Water Content ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Hemoglobin Concentration ◽  
Mean Corpuscular Hemoglobin ◽  
Cell Disease ◽  
Hemoglobin F ◽  
The Mean

A rationale for clinical trials of hydroxyurea (HU) treatment in sickle cell disease is that the agent increases red blood cell (RBC) fetal hemoglobin content. However, an additional effect of HU is to raise the mean corpuscular volume (MCV). To investigate the action of HU in a species that makes no electrophoretically distinguishable fetal hemoglobin, we treated dogs with the drug and compared their response to that of five patients with sickle cell anemia. Both dogs and patients had an increase in MCV, but the effect of HU treatment on the mean corpuscular hemoglobin concentration (MCHC), density, and water content of the RBCs differed in the two species. The dog RBCs became low in MCHC, high in ion and water content, and low in mean density. Thus, HU can raise MCV and lower MCHC without influencing fetal hemoglobin synthesis. A different pattern was seen in the sickle cell patients during HU treatment. Although the MCV of their RBCs increased, there was no change in MCHC, ion content, or mean density. A notable change in the sickle cell patients' blood was that two subpopulations of cells were nearly eliminated during HU treatment; the hypodense reticulocyte fraction and the hyperdense fraction that contains irreversibly sickled cells. These findings lead us to suggest that trials of HU in sickle cell disease must recognize the possibility that any beneficial effect of this agent might be due not only to an increase in hemoglobin F alone, but perhaps also to the associated increase in MCV or the altered RBC density profile.

scholarly journals Augmentation of fetal hemoglobin (HbF) synthesis in culture by human erythropoietic precursors in the marrow and peripheral blood: studies in sickle cell anemia and nonhemoglobinopathic adults

Blood ◽  
1978 ◽  
Vol 52 (6) ◽  
pp. 1115-1124 ◽  
Author(s):  
K Kidoguchi ◽  
M Ogawa ◽  
JD Karam ◽  
AG Martin
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Fetal Hemoglobin ◽  
Culture Cell ◽  
Control Mechanisms ◽  
Normal Individuals ◽  
Individual Variations ◽  
Gel Isoelectric Focusing ◽  
Clonal Assay

Abstract We cultured marrow and peripheral blood erythropoietic precrusors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, F, and S in patients with and without sickle cell anemia. Hb was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing techniques, and quantitated by autoradiographic methods. Comparison of marrow late (CFU-E) and early (BFU-E) precursors from patients without hemoglobinopathies showed that preferential synthesis of HbF is limited to early precursors. Simultaneous examinations of Hb synthesis by blood and marrow early erythropoietic precursors confirmed the similarity of the biosynthetic capabilities of the precursors from the two sources. Increasing concentrations of erythropoietin (Ep) in culture corresponded with increases in the percentages of HbF synthesized by blood BFU-E of normal individuals. HbF biosynthesis by blood BFU-E from sickle cell anemia patients was significantly higher than that synthesized by nonanemic individuals and showed significant individual variations. HbF synthesis in patients with sickle cell anemia was partially dependent on Ep concentrations in culture. Cell culture of circulating erythropoietic precursors in man appears to provide a unique tool for studying the control mechanisms of Hb synthesis in man.

Hydroxyurea-Mediated Reactivation of Fetal Genes in Primary Erythroid Cell Cultures from Beta-Thalassemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3809-3809
Author(s):  
Roberta Calzolari ◽  
Aurelio Maggio ◽  
Alice Pecoraro ◽  
Vito Borruso ◽  
Antonio Troia ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Sickle Cell ◽  
Cell Cultures ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Alternative Mechanism ◽  
Rna Fish ◽  
F Cells

Abstract Fetal hemoglobin (HbF) is frequently increased in the hemoglobinopathies such as sickle cell anemia and b-thalassemia. Epidemiological studies have indicated that an increase in HbF ameliorates the clinical symptoms of these diseases (Rodgers and Rachmilewitz - Bri J. Haemat. 91: 263 -1995). In sickle cell anemia, HbF containing red blood cells have a lower concentration of sickle hemoglobin (HbS), and the HbF itself inhibits HbS polymerization, decreasing cell sickling process (Eaton and Hofrichter - Science 268:1142 -1995). In b-thalassemia patients, HbF partially compensate HbA deficiency and could potentially improve RBC survival resulting in an increase of hemoglobin levels. Hydroxyurea (HU) is one of the pharmacological agents currently used to stimulate HbF synthesis in patients with sickle cell anemia and more recently has been tested in clinical trials for b-thalassemia patients too (Olivieri et al. Ann. NY Acad Sci850:100-1998; Rigano et al. Hemogl.21(3): 219- 1997; Dixit et al. Ann. Haematol. 84: 441 -2005). The mechanism involved in the HU-mediated changes is still unclear. It may involve a selection of a minor pre-existing subpopulation of F-cells that has a growth and/or survival advantage (cellular mechanism). This mechanism may be particularly effective for cells derived from patients with hemoglobinopathies, where F-cells may be resistant to “ineffective erythropoiesis”. An alternative mechanism could involve stimulation of HbF in all or the majority of cell-population by direct induction of g genes (molecular mechanism). Here we report the analysis on thalassemia patients homozygoutes for Lepore genotype that present high levels of fetal hemoglobin. We combined the use of primary erythroid cell culture from peripheral blood stem cells of these patients, with primary transcript in situ hybridization (RNA-FISH) of the g and b globin genes to investigate the mechanism of action of hydroxyurea in adult erythroid cells. RNA-FISH on erythroid cell cultures from these patients reveals that the majority of cells express one g allele only (g: 75.2 %, g:g 19.6%). The analysis in hydroxyurea-treated cultures shows the increase of cells transcribing both g-alleles, indicating the reactivation of fetal genes (g: 58.1%, g:g 40%). This evidence suggests that the molecular mechanism is involved directly on fetal genes reactivation to increase fetal hemoglobin production in HU-treated patients.

scholarly journals Effects of hydroxyurea on hemoglobin F and water content in the red blood cells of dogs and of patients with sickle cell anemia

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 212-216 ◽  
Author(s):  
EP Orringer ◽  
DS Blythe ◽  
AE Johnson ◽  
G Jr Phillips ◽  
GJ Dover ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Water Content ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Hemoglobin Concentration ◽  
Mean Corpuscular Hemoglobin ◽  
Cell Disease ◽  
Hemoglobin F ◽  
The Mean

Abstract A rationale for clinical trials of hydroxyurea (HU) treatment in sickle cell disease is that the agent increases red blood cell (RBC) fetal hemoglobin content. However, an additional effect of HU is to raise the mean corpuscular volume (MCV). To investigate the action of HU in a species that makes no electrophoretically distinguishable fetal hemoglobin, we treated dogs with the drug and compared their response to that of five patients with sickle cell anemia. Both dogs and patients had an increase in MCV, but the effect of HU treatment on the mean corpuscular hemoglobin concentration (MCHC), density, and water content of the RBCs differed in the two species. The dog RBCs became low in MCHC, high in ion and water content, and low in mean density. Thus, HU can raise MCV and lower MCHC without influencing fetal hemoglobin synthesis. A different pattern was seen in the sickle cell patients during HU treatment. Although the MCV of their RBCs increased, there was no change in MCHC, ion content, or mean density. A notable change in the sickle cell patients' blood was that two subpopulations of cells were nearly eliminated during HU treatment; the hypodense reticulocyte fraction and the hyperdense fraction that contains irreversibly sickled cells. These findings lead us to suggest that trials of HU in sickle cell disease must recognize the possibility that any beneficial effect of this agent might be due not only to an increase in hemoglobin F alone, but perhaps also to the associated increase in MCV or the altered RBC density profile.

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