MOLECULAR BIOLOGY OF CYCLIC HEMATOPOIESIS

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The long-term goals are to define the molecular pathophysiology of cyclic hematopoiesis (CH) in gray collie dogs and evaluate the in vivo use of recombinant hematipoietic growth factors in treating cyclic hematipoiesis. To investigate the pathophysiology of cyclic hematipoiesis we will characterize a recently identified biochemical defect in phosphoinositide 2nd messenger production and investigate possible defects in production or actions of hematopoietic growth factors in CH dogs. The specific aims and methods are: 1) Investigate the biochemical basis for phospholipase C/protein kinase C-mediated protein phosphorylation defect(s) by measuring diacylglycerol and inositol phosphate production, protein kinase C activation and membrane translocation and compare G-protein regulation of phospholipase C in normal and CH dogs. 2) Characterize mechanisms regulating cyclic G-CSF production and determine if there is cyclic production of the hematopoietic growth factors IL-3, GM-CSF, M-CSF and the monokines IL-1 and hepatocyte activating factor with cDNA or oligonucleotide probes to quantitate mRNA production. Bone marrow and factor dependent cell lines will be used to quantitate levels of growth factors and monokines in serum and conditioned media from bone marrow cultures. 3) Compare the response of normal and CH dog bone marrow cells to recombinant growth factors with an in vitro CFU assay. 4) Administer recombinant hematipoietic growth factors to CH dogs to investigate in vivo actions on hematopoiesis and determine their potential for modulating hematopoiesis and stimulating growth of resting marrow cells. The complex in vivo control mechanisms regulating hematopoiesis, hematopoietic growth factor production and activation of the neuroendocrine axis by hematopoietic cells will be characterized in these studies. The potential therapeutic use of hematopoietic growth factors and growth factors combinations in stimulating growth of quiescent marrow cells in vivo will be systematically investigated. These studies will provide new information on hematopoietic stem cell regulation and disorders of blood cell production.