The purpose of this application is to delineate the mechanisms which control the cellular production of adult and fetal hemoglobin (HbA and HbF). Three principles underlie this proposal: 1) HbF is confined in adults to a subpopulation of red cells (F cells) which contain both HbF and HbA; 2) increases postnatally in HbF levels are primarily associated with increases in F cell production not increased HbF in all red cells; and 3) increases in F cell production ameliorates the clinical severity of sickle cell (SS) disease and certain forms of thalassemia.
Our Specific Aims focus on three categories: First, how are F cells different from non-F cells? Using single cell immunoassays with monoclonal antibodies to human HbF the mean corpuscular hemoglobin, mean cell volume and concentration of HbS in F cells and non F cells in SS patients will be measured and the variables which control preferential F cell survival in SS disease will be determined. By autoradiographic and fluorescent flow cytometry techniques it will be determined whether F cells skip terminal cell division during maturataion and whether precursors of F cells have unique antigens (surface receptors) or different erythrocyte enzyme activities than non-F cells. Second, using comparisons between sibs with SS disease, the number of genetic loci which control F cell production and the genetic control of HbF levels in F cells will be analyzed. Having separated F erythroblasts from non-F erythroblasts using immunoadsorption techniques, differences in chromatin structure (DNAse I hypersensitive sites) and the methylation patterns of CpG dinucleotides around the Gamma-Delta-Beta-globin gene complex will be assessed. Third, the effect of cell-cycle specific agents (Hydroxyurea, 5-azacytidine) on increasing HbF production in severely affected SS patients will be studied. Five to 15 SS patients will be treated with varying doses of hydoxyurea to determine the rate of onset and duration of increased F cell production. Dose-response analyses of hydroxyurea versus % F cell production will be measured and the toxicity of drug regiments which maintain elevated F cell production levels will be monitored. The in vitro erythroid culture system will be used to analyze the effect of cell-cycle specific drugs on erythroid maturation and HbF production. The study of F cells can serve as a model for understanding how differentiation is controlled at the cellular level and how cell divisions affect differential gene expression.
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