The long term aims of this project are to understand the control of the production and function of granulocytes and macrophages at both the cellular and molecular level and to define the molecular defects in those cells that lead to the formation of myeloid leukemia. The emphasis of the project has been on external regulation of these cells by molecular regulators and their cellular receptors since these provide intervention strategies for increasing the rate of hemopoietic recovery in cancer therapies, correcting defects in some hemopoietic diseases and controlling the behavior of myeloid leukemic cells. Previous progress on this grant has led in part to the current clinical use of at least two hemopoietic molecular regulators (GM-CSF and G-CSF) and the scene is now set for the use of the next generation of molecular regulators designed on the basis of a full molecular understanding of the interactions between these regulators and their receptor subunits. The present proposal is to use site-directed mutational strategies of cloned regulators and their receptor subunits to perform a detailed biochemical and biological analysis of these mutants. These results will allow a determination of the structural elements involved in specific binding of the regulator aggregation of receptor subunits and activation of the receptor for delivering proliferative and differentiative cellular signals. Emphasis will be placed on two particular regulatory systems (granulocyte-macrophage colony-stimulating factor, GM-CSF and leukemia inhibitory factor, LIF) that offer different perspectives on the types of cellular responses that can be expected. Monoclonal antibodies to receptor subunits will also be generated both to define important structural elements in the receptors and to determine by immunoprecipitation what signal transducing elements in cells become associated with activated receptor complexes. Naturally occurring and artificial soluble receptors will also be explored as potential regulators of the systemic effects of hemopoietic growth factors with particular emphasis on a serum LIF-binding protein that appears to prevent the systemic spread of this highly pleiotropic cytokine. Finally. in vivo models will be developed to test the effects of multiple hemopoietic regulators and multiple receptor stimulation on the behavior of hemopoietic cells as a prelude to future clinical trials. The outcome of this work is expected to be a more rational design of therapeutic strategies that can target specific aspects of the actions of extrinsic regulators on hemopoietic cells.
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