Abstract

The present study encompasses the in vitro and in vivo proteomic analyses of homozygous sickle (SS) RBC membranes with and without clinically relevant hydroxyurea (HU) concentrations and the characterization of the significant HU-induced proteomic changes. HU is an effective oral drug for the management of sickle cell anemia (SS), in part, because it increases fetal hemoglobin (HbF) levels within sickle red blood cells (RBC) and thus reduces sickling. However, results from the Multicenter Study of HU (Charache et al., 1995) suggested that clinical symptoms in some SS patients often improved before a significant increase in HbF levels. This indicates that HU may be acting through alterations in additional cellular mechanisms that are yet to be identified. To identify important proteins involved in these pathways, we focused on protein profiling studies of SS RBC membrane proteome using Two-Dimensional Fluorescent Differential Gel Electrophoresis and tandem mass spectrometry. We demonstrated a strong dose dependent response of RBC membrane proteins to HU. We identified a significant increase in specific forms of RBC anti-oxidant enzymes, protein repair and degradation components after in vitro exposure to 50 and 100μM HU. We further demonstrated that HU-dependent in vitro post-translational modifications of the anti-oxidant enzyme catalase leads to its activation. We further proposed that though the anti-oxidant response may ease production of free radicals induced by low concentrations of HU (50 and 100μM), it is unable to offer protection at higher concentrations of HU (above 200μM) as indicated by a marked decrease in catalase activity in vitro. A significant decrease in actin and tubulin folding chaperonin TCP1 (subunit 2) in five patients receiving 400μM of HU also supports the damage caused to the anti-oxidant defense system at high concentrations of HU. These results suggest oxidative stress as an important clinical effect of high HU concentrations that are usually necessary to achieve satisfactory increase in HbF production, the main clinical benefit of HU. A significant increase in RBC membrane components and glycolytic enzymes at higher HU concentrations administered in vivo, for a period of four months to four years, may explain some of the clinical benefits of HU.