Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells

Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3',5'-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 muM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.     

Nitric oxide and cyclic GMP levels in sickle cell patients receiving hydroxyurea

Recent studies suggest that nitric oxide (NO) may partly be responsible for the beneficial effect of hydroxyurea (HU) in sickle cell disease (SCD) patients. NO stimulates cyclic guanosine monophosphate (cGMP) production, which mediates vasodilatation. We investigated the association between NO, cGMP and fetal haemoglobin (HbF) levels after HU administration. Our data showed that chronic HU significantly increased NO, cGMP, and HbF levels in SCD. Recently it was shown that HbF production was stimulated by cGMP-dependent protein kinase. Our results suggest that NO stimulates cGMP production, which then activates a protein kinase and increases the production of HbF.     

Levels of endothelial, neutrophil and platelet-specific factors in sickle cell anemia patients during hydroxyurea therapy.

It has been shown that the clinical course of sickle cell (SS) patients can be ameliorated by administration of hydroxyurea (HU). Induction of hemoglobin F (HbF) is thought to be the mechanism responsible for clinical improvement in some patients. However, HU has a variable effect on HbF production and there exists no good correlation between the extent of HbF increase and clinical response. On the other hand, the degree of adherence of SS to vascular endothelium and neutrophil counts correlate well with clinical severity. Being a cytotoxic drug, used in myeloproliferative diseases, HU may alter proliferation among various cell lines. Moreover, HU has been reported to reduce red blood cell (RBC) adhesion receptor expression in young SS individuals and induces changes in endothelial cells in vitro. It should be conceived that in addition to its effects on HbF production, HU may change the clinical symptoms of SS patients by affecting the degree of adherence of different blood cells, by influencing the activity of endothelium as well as the activity of white blood cells (WBC) and platelets. To analyze whether several of the determinants of adhesion are modulated by HU treatment we studied the levels of endothelial activity (soluble vascular adhesion molecule-1, (sVCAM-1), interleukin-8 (IL-8), fibronectin, neutrophil activity (sL-selectin, sIL-6 receptor-alpha, myeloperoxidase) and platelet activity (von Willebrand factor) in relation to clinical symptoms, hematological data and HbF levels in 8 SS patients before and during 5 months of HU therapy. Steady state sVCAM-1 levels are increased compared to normal controls and a significant decrease is noted during HU treatment, suggesting a decrease in the interactions between RBC and vascular endothelium. The IL-8 levels are comparable to those in normal controls and remain unaffected by HU therapy. Intercurrent infection and crises reveal striking increases in IL-8 which are accompanied by leukocytosis, but otherwise the IL-8 levels do not correlate with hematological data. HU has no demonstrable effect on fibronectin or soluble neutrophil adhesion molecules, but the levels of myeloperoxidase decrease significantly while WBC counts do not, implying a reduction in neutrophil activity which may help attenuate the propagation phase of a vasoocclusive crisis.     

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

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

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.     

Enhanced fetal hemoglobin production by phenylacetate and 4- phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia

In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.     

Influence of hydroxyurea on fetal hemoglobin production in vitro

Cytotoxic drugs increase circulating fetal hemoglobin levels. We examined the mechanism by measuring the fetal hemoglobin produced per BFU-E-derived erythroblast following hydroxyurea treatment in vivo and in vitro. Treatment of four sickle cell patients increased the percentage of circulating F reticulocytes. The frequencies of bone marrow or peripheral blood BFU-E or CFU-E-derived colonies and their fetal hemoglobin content were unaffected. In all cases, the number of erythroid cells/progenitor-derived colony increased. To explore further the effect of hydroxyurea on fetal hemoglobin production, we added 50 mumol/L hydroxyurea to cultures of peripheral blood BFU-E-derived erythroblasts on 1 of 9 days (day 5 through 13) to nine samples. These BFU-E were derived from the peripheral blood of normal donors, sickle trait donors, and sickle cell anemia patients and from the bone marrows of monkeys. This concentration of hydroxyurea was selected so that the frequency of BFU-E and their size was moderately decreased. Addition of hydroxyurea to these progenitor-derived erythroid cells had no effect on fetal hemoglobin content per cell. Neither did transient exposure of progenitors to hydroxyurea prior to culture in nontoxic concentrations (0 to 500 mumol/L) result in a significant increase in fetal hemoglobin content in progenitor-derived erythroblasts. These data suggest that hydroxyurea does not directly alter the HbF program expressed by progenitor-derived erythroid cells. Instead, it enhances hemoglobin F content secondarily, possibly by inducing alterations in erythropoiesis.     

Zileuton induces hemoglobin F synthesis in erythroid progenitors: role of the L-arginine-nitric oxide signaling pathway

Induction of fetal hemoglobin (Hb F) is an important therapeutic tool in ameliorating complications of sickle cell disease. Nitric oxide has been implicated in the mechanism of Hb F synthesis induced by hydroxyurea (HU). This study examined whether zileuton (ZL), a structural analog of hydroxyurea, possessed Hb F-inducing properties and the potential role nitric oxide plays. ZL caused a dose-dependent increase in gamma-globin expression in K562 cells. This effect was confirmed by a dose-dependent increase in Hb F synthesis in erythroid progenitors from individuals with sickle cell anemia and normal hemoglobin genotypes. l-arginine had no effect on Hb F production; however, it dose-dependently inhibited ZL's ability to induce Hb F. The nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) inhibited l-arginine's effect and restored ZL-mediated increase in Hb F synthesis. In addition, 8-PCPT-cGMP (8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate) inhibited ZL-mediated induction of Hb F synthesis. When comparing l-NMMA effects alone on ZL and HU, a partial reversal of increased Hb F synthesis was seen only with HU. Neither l-arginine alone nor l-arginine in combination with l-NMMA effected hydroxyurea-mediated induction of Hb F synthesis. This study demonstrates that ZL induces Hb F through a mechanism that involves l-arginine/nitric oxide/cGMP in a manner distinctly different from HU.     

Hydroxyurea induces the eNOS-cGMP pathway in endothelial cells

Hydroxyurea is a cell-cycle-specific drug that has been used to treat myeloproliferative diseases and sickle cell anemia. We have recently shown that hydroxyurea, like nitric oxide (NO)-donor compounds, increased cGMP levels in human erythroid cells. We show now that hydroxyurea increases endothelial-cell production of NO; this induction of NO in human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC) is blocked by competitive inhibitors of NO synthase (NOS), such as NG-nitro-L-arginine-methyl ester (L-NAME) and NG-nitro-L-arginine. It is dependent on cAMP-dependent protein kinase (PKA) and protein kinase B (PKB/Akt) activity. We found that hydroxyurea dose- and time-dependently induced rapid and transient phosphorylation of eNOS at Ser1177 in a PKA-dependent manner; inhibitors of PKB/Akt could partially abrogate this effect. In addition, hydroxyurea induced cAMP and cGMP levels in a dose-dependent manner, as well as levels of intracellular calcium in HUVECs. These studies established an additional mechanism by which rapid and sustained effects of hydroxyurea may affect cellular NO levels and perhaps enhance the effect of NO in myeloproliferative diseases.     

Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease

Fetal hemoglobin (HbF) level and the HbF responses to hydroxyurea (HU) vary among patients with sickle cell disease and are, at least in part, genetically regulated. We hypothesized that siblings with sickle cell disease are likely to share the same parental beta-like globin gene clusters with their cis-acting regulatory sequences and therefore, if regulation of this response is linked to the beta-globin gene cluster, might have concordant HbF responses to HU. Accordingly, we studied 26 families (30 sib pairings), 20 with sickle cell anemia (three families had three siblings) and 6 families with HbS-beta-thalassemia (one family had three siblings, and one family consisted of monozygotic twins), to see if siblings with sickle cell disease had discordant or concordant changes in HbF during HU treatment. Intraclass correlation coefficients (r) showed a high, positive correlation between sibs for HbF levels before and during HU treatment and a concordant change in HbF response from baseline to treatment-associated levels. Changes in mean corpuscular volume (MCV) paralleled HbF levels, while the expected correlations between treatment-associated fall in leukocyte count and increase in MCV were also present. Our results provide additional evidence that some elements that regulate HbF expression are linked to the beta-globin gene cluster.     

Modulation of erythrocyte arginase activity in sickle cell disease patients during hydroxyurea therapy

An elevated erythrocyte arginase activity with a corresponding decrease in nitric oxide (NO) level has been implicated in the pathophysiology of sickle cell disease (SCD). Recent studies have shown that hydroxyurea (HU) increases the production of NO, which increases the soluble guanylate cyclase activity and fetal haemoglobin (HbF) synthesis. To study the effects of HU on the arginase and nitric oxide synthase (NOS) activities in SCD patients, we compared levels of arginase activity and NO metabolites in red blood cells and plasma, respectively, from 23 patients with SCD (HbSS) receiving HU therapy, with those of 12 SCD patients not receiving HU treatment. Patients on HU therapy showed significantly lower arginase activity than that of HbSS patients not on HU therapy (1.36+/-0.2 U/10(8) cells vs. 3.31+/-0.29 U/10(8) cells). NOS activity was higher in patients on HU therapy than in untreated patients (0.72+/-0.4 nmol/ml/min vs. 0.35+/-0.15 nmol/ml/min, P<0.05). Among the HU-treated patients, the decreased level of arginase activity correlated (r=0.71) with HbF level as well as the mean corpuscular haemoglobin content. These data suggest that one of the beneficial effects of HU in vivo may involve the regulation of arginase activity and a concomitant induction of NOS activity, which may lead to an increased production of NO. The outcome of this study may lead to the development of improved NO-based treatments for SCD.     

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

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 alpha-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.     

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.     

Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease

Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD. For HbS polymerization inhibition, the hypothesis of an “HbF per red blood cell (HbF/RBC) threshold” requires accurate measurement of HbF in individual RBC. To date, HbF detection methods are limited to a qualitative measurement of RBC populations containing HbF - the F cells, which are variable. We developed an accurate method for HbF quantification in individual RBC. A linear association between mean HbF content and mean RBC fluorescence by flow cytometry, using an anti-Human-HbF antibody, was obtained from non-SCD subjects presenting homogeneous HbF distribution. This correlation was then used to measure HbF/RBC. Hydroxyurea (HU) improves SCD clinical manifestations, mainly through its ability to induce HbF synthesis. The HbF distribution was analyzed in 14 SCD patients before and during HU treatment. A significant decrease in RBC population containing less than 2 pg of HbF/RBC was observed. Therefore, we tested associations for %RBC above different HbF/RBC thresholds and showed a decrease in the pathognomonic vaso-occlusive crisis incidence from the threshold of 4 pg. This quantity was also correlated with the level of sickle RBC after in vitro deoxygenation. This new method allows the comparison of HbF/RBC distributions and could be a useful tool to characterize baseline patients HbF distribution and therapeutic response to HbF inducers.     

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.     

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease

The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug-response in sickle cell disease.     

Erythropoietic activity in patients with sickle cell anaemia before and after treatment with hydroxyurea

In this project we have prospectively studied the erythropoietic activity in patients with sickle cell anaemia (SS) before and after treatment with hydroxyurea (HU). Some of the patients were enrolled in a double-blind placebo controlled trial of HU in patients with SS and others were enrolled in an open label study. Determinants of erythropoietic activity included the reticulocyte count, red blood cell (RBC) survival by the 51Cr method, plasma 59Fe clearance, plasma iron turnover (PIT), erythron transferrin uptake (ETU), RBC production/destruction rate, and RBC Fe utilization. Therapy with HU increased the mean corpuscular volume (MCV), haemoglobin (Hb)F, RBC survival and t1/2 59Fe clearance; it decreased the reticulocyte count, the white blood cell (WBC) count, ETU, and the PIT. Most of the changes in parameters of erythropoiesis could be explained by the increase in 51Cr RBC survival after therapy with HU. Together the data showed that in selected patients the net effect of HU on Hb level was a function of the difference between the suppressive effect of HU (decreased RBC production) and the increase in RBC survival. In the majority of patients who responded to HU, there was a preferential effect on RBC survival.     

High fetal hemoglobin production in sickle cell anemia in the eastern province of Saudi Arabia is genetically determined

Homozygous sickle cell disease in the eastern province of Saudi Arabia is clinically mild. Circulating fetal hemoglobin levels of 16.0 +/- 7.4% were found in these anemic patients, but only 1.09 +/- 0.97% in their sickle trait parents. To determine whether these sickle cell anemia patients inherit an increased capacity to synthesize fetal hemoglobin, a radioimmunoassay of fetal and adult hemoglobin was performed on erythroid progenitor (BFU-E)-derived erythroblasts from Saudi Arabian sickle cell patients and their parents. Mean fetal hemoglobin content per BFU-E-derived erythroblast from Saudi Arabian sickle cell patients was 6.2 +/- 2.4 pg/cell or 30.4 +/- 8.6% fetal hemoglobin (normal 1.1 +/- 0.7 pg/cell and 5.1 +/- 1.8%). Linear regression analysis of % HbF in peripheral blood versus % HbF per BFU-E- derived cell showed a positive correlation with an r of 0.65. The variance of the intrinsic capacity to produce HbF may account for almost 40% (r2) of the variance of circulating fetal hemoglobin but other factors, particularly selective survival of F cells, must also contribute significantly. Despite virtually normal HbF levels in sickle trait parents of these Saudi patients, mean fetal hemoglobin production per BFU-E-derived erythroblast in these individuals was elevated to 3.42 +/- 1.79 pg/cell or 16.1 +/- 6.4% fetal hemoglobin, and the magnitude of fetal hemoglobin production found in parents correlated with that of the patients. These data indicate that the high fetal hemoglobin in Saudi sickle cell disease is genetically determined but expressed only during accelerated erythropoiesis. Further evidence of such genetic determination was provided by analysis of DNA polymorphisms within the beta-globin gene cluster on chromosome 11. This revealed a distinctive 5' globin haplotype (+ + - + +) on at least one chromosome 11 in all high F SS and AS tested. The precise relationship of this haplotype to HbF production in this population remains to be defined.     

In vitro induction of fetal hemoglobin in human erythroid progenitor cells

OBJECTIVE: Clinical heterogeneity among patients with sickle cell anemia (SCA) is influenced by the amount of fetal hemoglobin (HbF) within circulating erythrocytes. Current pharmacotherapy focuses on increasing HbF in order to reduce hemolysis and help prevent acute vaso-occlusive events. Hydroxyurea, a known S-phase-specific cytotoxic ribonucleotide reductase (RR) inhibitor, is an effective agent for HbF induction in patients with SCA, but the mechanisms by which hydroxyurea induces HbF in vivo have not been elucidated. MATERIALS AND METHODS: We adapted an in vitro assay for HbF induction, growing burst-forming unit erythroid (BFU-E) colonies in methylcellulose from peripheral blood of children with SCA and extracting the hemoglobin for high-performance liquid chromatography analysis of HbF. Hydroxyurea and other known RR inhibitors, along with cytotoxic agents that are not RR inhibitors, were tested for the ability to induce HbF using this in vitro assay. RESULTS: Hydroxyurea decreased the number of BFU-E colonies that grew in culture and significantly increased HbF from 13.6%+/-6.2% to 25.4%+/-8.0% at 50 microM HU (p=0.012). Three other known RR inhibitors also significantly induced HbF: 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (p=0.025), guanazole (p=0.008), and gemcitabine (p=0.028). Cytarabine and alkylating agents BCNU and 4-hydroperoxycyclophosphamide, which are cytotoxic agents but not RR inhibitors, reduced BFU-E colony number but did not significantly induce HbF. CONCLUSION: Hydroxyurea and other RR inhibitors significantly induce HbF in vitro in human erythroid progenitor cells. Inhibition of RR may be a critical mechanism by which hydroxyurea increases HbF in vivo in patients with SCA.