METABOLIC DETERMINANTS OF HEMATOPOIETIC CELL DEVELOPMENT

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While much is now known about specific cytokines that promote the survival and growth of hematopoietic stem cells and progenitors, relatively little is known about regulatory mechanisms that control the loss of self-renewal capacity, differentiation, and terminal maturation of these cells during normal blood cell development. In lower eukaryotes, e.g. S. cerevisiae and Dictyostelium, developmental switches are triggered by metabolic changes caused by nutrient deprivation, and studies of these organisms have shown that depletion of guanine ribonucleotides (e.g. GTP) is a critical metabolic event int he onset of development. Parallel studies of both human leukemia cell lines and purified, normal CD34+ marrow cells have also established a link between guanine ribonucleotide metabolism and the control of hematopoietic cell development. To understand how the regulation of intracellular guanylate pools serve as a metabolic signal for the development of hematopoietic cells, we propose studies with two central objectives. Our first objective is to understand how IMP-dehydrogenase (IMPDH), a highly conserved enzyme of intermediary purine metabolism which controls de novo guanine ribonucleotide synthesis, is regulated during the growth and development of hematopoietic cells. Using both the Il-3 dependent,murine hematopoietic cell line. 32D(cl3) and immuno-affinity purified CD34+ human marrow cells, we will characterize GTP synthesis, guanylate pools, and levels of IMPDH gene expression, protein, and activity under conditions of Il-3 stimulated growth, G-CSF induced myeloid maturation, and apoptosis following growth factor withdrawal. We will also investigate cytokine dependent post-translational modifications of IMPDH isoforms, examine the effects of intracellular pH changes and altered Il-3 signalling on IMPDH expression and activity, and determine how IMPDH over- and under-expression induced by gene transfection affect cellular maturation. Our second objective is to define mechanisms by which the regulation of guanylate pools influences hematopoietic cell development. We will examine the effects of IMPDH inhibition on GTP-dependent, ras mediated signalling pathways by measuring GTP-bound ras and IL-3 stimulated phosphorylation of raf. Because protein synthesis is highly GTP-regulated, we will also determine whether guanylate depletion and repletion affect the turnover and supply of short-lived proto oncogene proteins (e.g., c-myb) that influence hematopoietic cell growth and development. Finally, we will define the effects of guanylate depletion and repletion on the expression of transcription control genes, PU.1 and C/EBF, implicated in myeloid cell development, as well as myeloid specific genes (e.g., CD11b and lactoferrin). These basic studies of the control of hematopoietic cell development may lead to new insights relevant to the in vitro propagation and genetic manipulation of these cells for clinical purposes.