SOURCES AND ACTIONS OF HUMAN GMCSF AND MULTI-CSF

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During the past two years, the genes for five human hematopoietic growth factors have been cloned. Recombinant proteins were produced and characterized. Although much is known about these factors at a gene and protein level, there is less information on the identify of the cells that synthesize them, the physiological mechanism by which synthesis is regulated, and their mechanism of action. We plan to investigate the relative important of cells known to produce HGF's by comparing their capacity to synthesize the different factors under unstimulated conditions and after stimulation with a variety of inducing agents. This will be accomplished by analyzing RNA by Northern blots, protein in Western blots and immunoprecipitations and, in the case of GMCSF, by radioimmune assay, as well. We intend to identify producer cells in tissue sections and cytospin preparations of hematopoietic organs by using powerful in situ RNA hybridization techniques combined with immunoenzymatic staining. We will begin to investigate the mechanism of action of HGF's by using highly purified radiolabeled GMCSF to study receptor binding on normal and leukemic cells. Our longer term aims are to characterize the receptor by electrophoresis of solubilized membranes containing receptor crosslinked to radiolabeled GMCSF. Finally, we wish to clone the gene for the GMCSF receptor by using biotinylated GMCSF, fluorescent microsphere and fluorescence activated cell sorting of murine L cells that express the receptor after transformation with genomic DNA. It is our conviction that these studies will constitute important steps toward defining rational criteria for the therapeutic use of GMCSF and the other growth factors as they become available. Furthermore, we hope to gain insight into the mechanism by which proliferation and differentiation are controlled in normal and leukemic cells.