The long term objective of this research project is to identify and characterize the molecular mechanisms involved in the regulation of eythropoiesis. To this end in the present proposal, the following investigations have been planned: First, we wish to characterize the intracellular mitogenic and transforming factors present in the peripheral blood mononuclear cells in polycythemia vera with respect to their role in the autonomous proliferation which characterizes this disorder. Using both conventional liquid chromatography and reversed phase-HPLC, as well as SDS-PAGE, we plan to isolate the growth factors we have previously identified for amino-terminal sequencing, amino acid composition and the production of synthetic peptides and antibodies to them. Using well defined assays for these growth factors, we will identify the cells in which they are expressed and whether they are secreted in the urine. We will also examine their ability to influence the proliferation of normal hematopoietic progenitor cells and the proliferation and function of marrow fibroblasts and vascular endothelial cells. The conditions influencing the production of these growth factors will be examined including the expression of particular proto-oncogenes by their cells of origin. Second, the fate and function of pure recombinant human erythropoietin will be studied in two different models. The metabolism of native and desialated erythropoietin will be studied in vivo in the rat and in vitro using rat hepatic parenchymal and endothelial cells as well as human and bovine vascular endothelial cells. The receptor-ligand interaction of erythropoietin and its target cells will be studied using erythroblasts isolated from the spleens of the phenylhydrazinetreated mice by centrifugal elutriation. In particular, we wish to examine the role of calcium in the interaction of erythropoietin and its target cells and will do this both with respect to receptor-ligand binding and subsequent intracellular events. The latter will be evaluated using a fluorescent intracellular calcium probe and a sensitive digital imaging system capable of analyzing changes in intracellular free calcium in single, living cells.
