Research Article: Hemin Augments Growth and Hemoglobinization of Erythroid Precursors from Patients with Diamond-Blackfan Anemia

Date Published: May 13, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Eitan Fibach, Memet Aker.


Diamond-Blackfan anemia (DBA) is congenital pure red-cell anemia due to a differentiation block in erythroid precursors. The disease is commonly caused by mutations in genes for ribosomal proteins. Despite the identification of disease causal genes, the disease pathogenesis is not completely elucidated. The ribosomal abnormalities are assumed to inhibit globin translation which may lead to excess free heme, stimulating a generation of free radicals and thereby damaging the precursors. We studied the effect of hemin (heme chloride) on cultured human erythroid precursors and found that contrary to aforementioned hypothesis, although hemin moderately stimulated free radicals, it did not cause apoptosis or necrosis. In erythroid precursors derived from DBA patients, hemin significantly stimulated growth and hemoglobinization. Thus, heme toxicity is unlikely to play a role in the pathophysiology of most DBA cases. Moreover, its beneficial effect in culture suggests a therapeutic potential.

Partial Text

DBA is a rare congenital form of pure red-cell anemia, characterized by macrocytic anemia, reticulocytopenia, and a block in erythroid differentiation at the proerythroblast stage, often in association with physical anomalies and growth retardation [1]. A large proportion of the patients carry mutations in genes encoding for ribosomal proteins, for example, RPS19, RPS24, and RPS17 [1]. Based on this genetic information, defects in ribosomal biogenesis are postulated to underlie the pathogenesis of the disease [1]. Although gross defects in ribosomal function are not compatible with viability, subtle defects may exhibit tissue specificity and impair only certain cellular functions where the requirement for high-level translation for a particular critical gene product is not fulfilled. In DBA, defects in ribosomal biogenesis are suspected to impair the initiation of globin translation, causing a mismatch between intracellular levels of globin chains and heme, a balance tightly coordinated under normal conditions. It has been suggested that a transient excess of heme is toxic to erythroid precursors via iron-mediated generation of free radicals [2].

The research was approved by the Hadassah—Hebrew University Medical Centre Human Experimentation Review Board. Peripheral blood samples were obtained from normal donors and patients that met the criteria of DBA [9]. Erythroid cells were cultured according to the two-phase liquid culture procedure as previously described [10]. In short, peripheral blood-derived mononuclear cells were first cultured in alpha medium supplemented with 10% fetal calf serum and 10% conditioned medium obtained from cultures of human bladder carcinoma cell line 5637 and 1 μg/mL cyclosporin A (phase I). After 6 days, nonadherent cells were harvested, washed, and suspended in phase II medium, containing alpha medium, 30% fetal calf serum, 1% bovine serum albumin, 10 μM β-mercaptoethanol, 1.5 mM glutamine, 10 μM dexamethasone, 5 ng/mL stem cell factor, and 1 U/mL human recombinant erythropoietin. Hemin (bovine, Sigma, St. Louis, MO) was prepared as previously described [7]. Intracellular Hb was quantified by HPLC as previously described [7]. Hemogbloin-containing cells were scored microscopically following staining with benzidine dihydrochloride [10].

To study the potential toxic effect of hemin on developing erythroid precursors, normal erythroid precursors were cultured according to the two-phase liquid culture procedure [10]. After 6 days in phase II culture, hemin (10–50 μM) was added for 16 hrs. The cells were then harvested and stained with DCF—a marker of free oxygen species (ROS), phycoerythrin-conjugated annexin-V—a marker of apoptosis which binds to phosphatidylserine exposed on the outer surface of the cells or propidium iodide—a marker of necrosis. The flow cytometry results (Figure 1) indicate that although ROS generation was modestly stimulated, it was not associated with apoptosis or necrosis.