Hydroxyurea (HU) has been shown to increase the proportion of fetal hemoglobin (Hb F) in most patients with sickle cell anemia. This therapeutic effect generally occurs at doses >= 15 mg/kg/day. We have previously shown that a lower dosage regimen of HU (#10 mg/kg/day Y 4 d/wk) increased total hemoglobin (Hb) levels in some patients with thalassemia intermedia by preferentially increasing b-globin biosynthesis. To further characterize these apparent dose-dependent effect of HU, we used a two-phase liquid erythroid culture system to examine the effect of HU dosage, developmental stage at which HU was given, and duration of HU exposure on various hematological parameters. HU treatment elicited multiple effects on the erythroid cultured cells: (1) Depending on the phase of the culture period at which HU was added, there was a significant dose-dependent increase in Hb F levels; (2) Low doses of HU (from 0 to 25 mM) increased Hb levels by up to 2.7 fold when added at days 3-6 of the phase II cultured period. In contrast, Hb levels declined significantly in response to a high dose of HU (100mM) added at days 3-6, due to its cytotoxic effects; (3) There was no significant change in Hb levels in response to HU exposure during the late stage of phase II culture (>=9-12 days); (4) HU exposure during days 0 - 3 of phase II culture increased the number of erythroid colonies, to a maximum of 5-fold at 5mM HU; (5) up-regulation of GATA-2 and down-regulation of GATA-1; 6) Treatment with 100 mM HU upregulated various genes related to cell cycle control and apoptosis, as determined by cDNA microarray analysis. Our results suggest that HU exerts concentration-dependent effects on Hb F production and erythropoiesis and that these two effects can be dissociated.
