Vanadate elevates fetal hemoglobin in human erythroid precursors by inhibiting cell maturation.

Increased fetal hemoglobin (HbF) in erythroid precursors of patients with beta-hemoglobinopathies (sickle cell anemia and beta-thalassemia), in which adult hemoglobin synthesis is defective, ameliorates the clinical symptoms of the underlying diseases. The production of erythroid precursors depends on the action of erythropoietin (EPO), which prevents their apoptosis and stimulates their proliferation. EPO binds to its surface receptor, induces its homodimerization, and initiates a cascade of phosphorylation and dephosphorylation of a series of proteins by kinases and phosphatases, respectively. Vanadate inhibits various phosphatases, including those that are involved in the EPO pathway, thereby intensifying the signal. In this study, we investigated the effect of vanadate on the proliferation and maturation of human erythroid precursors in culture. When vanadate was added to cells derived from normal donors, cell maturation was delayed, as indicated by cell morphology, cell growth kinetics, the rate of appearance of hemoglobin-containing cells, and the expression of surface antigens (CD117, CD71, and glycophorin A). Analysis by high-performance liquid chromatography and flow cytometry of the hemoglobin profile of vanadate-treated normal cells revealed a higher proportion of HbF than was found in untreated cells. When vanadate was added to cells derived from patients with beta-thalassemia, a significant increase in HbF was observed. The results suggest that intensification of the EPO signal by vanadate results in maturation arrest and increased HbF production. Thus, inhibitors that are more specific and less toxic than vanadate may present a novel option for elevating HbF in patients with beta-hemoglobinopathies, as well as for intensifying the EPO response in other forms of anemia.

Hemoglobin switch in the newborn: a flow cytometry analysis.

BACKGROUND: Hemoglobin (Hb) production switches upon birth from fetal Hb (HbF) to adult Hbs. HbF production is reactivated in sickle cell anemia and beta-thalassemia, where increased HbF ameliorates clinical symptoms and HbF-stimulating drugs are used for treatment. Understanding of the switch is therefore of basic interest and important for rational drug design. We studied the switch by determining changes after birth in the frequency and HbF-content of HbF-containing red blood cells (F-RBC) and reticulocytes (F-retics). METHODS: Blood samples of neonatal cord, babies in their first year and adults were stained with both phycoerythrin-conjugated anti-human HbF antibodies and the nucleic acid dye thiazol-orange, and analyzed by flow-cytometry. RBC and retics were distinguished by their different thiazol orange-derived fluorescence and their HbF content was determined by the intensity of phycoerythrin-derived fluorescence. RESULTS: The frequency of both F-RBC and F-retics decreases with time after birth. In most cases, the percentage of F-RBC was higher, but their HbF-content was lower than that of retics. The HbF content of the F-RBC and the F-retics showed a gradual decrease with age. CONCLUSIONS: The results can be explained by two models: (A) A pool of homogenous stem cells that undergo gradual changes in their globin transcription pattern. (B) A pool of heterogeneous stem cells with varying HbF potentials that become active in an age-dependent manner: first, cells with a high HbF potential and then cells with a gradually lower HbF potential. In both models, these changes are induced by a biological clock and environmental factors.

Flow cytometric analysis of hydroxyurea effects on fetal hemoglobin production in cultures of beta-thalassemia erythroid precursors.

An increase in fetal hemoglobin (Hb F) ameliorates the clinical symptoms of the underlying disease in the beta hemoglobinopathies-sickle cell anemia and beta-thalassemia (thal). Hydroxyurea (HU) can elevate Hb F production in erythroid cells and is the agent currently in clinical use for patients with sickle cell anemia; it is presently being tested in clinical trials for thalassemia. We have developed a two-phase liquid culture system that mimics the in vivo hematological changes that are observed in patients treated with HU. Adding HU during the second phase of the culture increases the proportion of Hb F, increases the levels of total hemoglobin (Hb) content per cell and increases cell size, but it decreases the numbers of cells and the total amount of Hb produced. In the present study we developed and utilized a double labeling procedure for flow cytometric analysis of the cellular distribution of Hb F. Cells exposed to various concentrations of HU on day 6 of the second phase of the culture were harvested on day 12, and stained simultaneously with fluorochrome-conjugated monoclonal antibodies specific for human glycophorin A, an erythroid specific marker, and human Hb F. Both the percentage of the Hb F-containing cells and their intensity of fluorescence were recorded. The latter value gives a semi-quantitative estimation of the mean cellular Hb F content. The results indicated that cultures derived from different beta-thalassemic patients differ in their response to HU. In most patients, low doses of HU decreased the percentage of Hb F-cells as well as their Hb F content. At high doses, some patients showed an increase in both parameters, while others showed an increase in the percentage of Hb F-cells with minimal increase in their mean Hb F content, while still other patients showed little effect at all. In all patients, high doses of HU caused a decrease in cell numbers. These results suggest that HU has mixed effects on erythroid precursors. Both the two-phase liquid culture and the flow cytometric analysis procedures described herein provide the experimental tools for screening of Hb F-inducing drugs and for evaluating patients' cell response prior to treatment.