Hemin Augments Growth and Hemoglobinization of Erythroid Precursors Derived from Patients with Diamond-Blackfan Anemia (DBA).

DBA is a congenital form of pure red cell anemia characterized by a macrocytic anemia, reticulocytopenia, and a block in erythroid differentiation at the proerythroblast stage, often in association with physical anomalies and growth retardation. About 25% of the patients carry mutations in genes that encode for proteins (RPS19, RPS24 and RPS17) that bind to the 40S subunit of the ribosome. The resultant defect in ribosomal biogenesis has been proposed to impair the initiation of globin translation, leading to mismatch between intracellular levels of heme and globin chains. It has been hypothesized that the transient excess of intracellular free heme resulting from the delay in globin synthesis exerts direct toxicity to erythroid precursors and plays a major role in pathogenesis of DBA through apoptosis of proerythroblasts (Keel et al., Science319;825,2008).

Free hemin, however, is not necessarily toxic to developing erythroid precursors. Exogenously supplied hemin is readily taken up by erythroid cells in culture and its iron is incorporated into hemoglobin or stored in ferritin (Fibach et al., J Cell Physiol130;460,1987). Following addition of succinylacetone, a potent inhibitor of heme synthesis, exogenously supplied hemin can replace intracellularly synthesized heme and be incorporated into de novo formed hemoglobin (Fibach et al., Blood85;2967,1995). Hemin supplementation to semi-solid cultures promotes the growth of normal erythroid precursors (e.g., Lu and Broxmeyer, Exp Hematol11;721,1983). We showed in a two-phase liquid culture that exogenous hemin promotes normal erythropoiesis by accelerating the proliferation and hemoglobinization of erythroid precursors in the presence or absence of transferrin (Fibach et al., Blood85;2967,1995). This effect was particularly prominent during the early stages of hemoglobinization, when iron-uptake and heme synthesis are rate-limiting. In the present study we show that surplus hemin (10 - 50 mM) supplemented to cultures at early stage of erythroid development is well tolerated. Although the generation of reactive oxygen species (measured by staining with dichlorofluorescein diacetate) was modestly (50 ± 15%, N=4) increased, it was not associated with increased apoptosis, as measured by binding of annexin V, nor necrosis as measured by propidium iodide staining.

Having demonstrated the growth and differentiation promoting potential of exogenous hemin on normal erythroid precursors and lack of overt toxicity, we studied the effect of exogenous heme in cultures of erythroid cells derived from six patients with DBA. We show that hemin, added as heme chloride or heme arginate, circumvented the primary defect and significantly stimulated (4 - 20-fold, p<0.001) ) the growth of DBA erythroid cells and their hemoglobinization.

In conclusion, our results show that

• exogenous hemin is taken up by developing erythroid cells and can supplement or substitute endogenously synthesized heme;

• excess heme stimulates free radical generation moderately but does not cause apoptosis or necrosis;

• addition of hemin to cultured erythroid precursors derived from normal donors stimulates their growth and hemoglobinization, and in DBA, in contrast to the recently proposed scheme, heme can actually restore the growth and differentiation potential of the DBA-erythroid precursors.

The beneficial effect of hemin on DBA erythroid precursors may be related to its effect on translation initiation factors, such as eIF-2 , and suggests a therapeutic potential.