Several lines of clinical and experimental evidence suggest that elevated levels of fetal hemoglobin (HbF)may improve the clinical course of individuals with sickle cell disease and beta thalassemia. A number of cytotoxic drugs have been shown to enhance gamma globin synthesis (and HbF levels) in experimental animals and patients with hemoglobinopathies, although the mechanism of action of these agents have yet to be clearly delineated. The K562 human erythroleukemia cell line has been shown to express constitutively low levels of embryonic and fetal but not adult hemoglobin, and can be reversibly induced to preferentially increase gamma globin gene expression in response to hydroxyurea. We are therefore using the K562 cell as a model system to understand the mechanism by which hydroxyurea (and similar agents) induces fetal hemoglobin synthesis. K562 cells have been grown in the presence of 25mM hemin and 0 25, 50, 100 and, 150 mM hydroxyurea. Nuclear extracts have been prepared from these and other non-erythroid (control) cells. A 420 bp fragment 5'of the normal A- gamma gene has been subcloned into an expression vector, and has been used to examine the differential binding characteristics of the nuclear protein extracts. We will employ gel-retardation electrophoresis, DNA-footprinting (DNase I and methylation protection), and ion exchange and affinity chromatography to investigate the interaction of both specific and non-specific protein/gamma gene promoter DNA. Our previous results suggests that extracts obtained from hydroxyurea-induced K562 cells contains an activity not found in uninduced or hemin-induced (K562) extracts that specifically binds to the region extending from -139 to -260 of the A-gamma CAP site. Furthermore, using synthetic oligonucleotides, we have sublocalized the binding site to the region between - 190 and - 160 of the A-gamma globin gene promoter. It is hoped that the further identification, characterization and purification of this putative binding protein(s) would not only extend the current knowledge of the molecular basis of the fetal to adult """"""""switch"""""""", but also suggest a novel pharmacological approach to the reversal of this switch in several clinically significant hemoglobinopathies.
