The growth and proliferation of normal murine peritoneal exudate macrophages (PEM) in vitro is strictly dependent on a macrophage-specific growth factor known as colony-stimulating factor (CSF-1). By contrast, transformed macrophage tumor cell lines do not require exogenous CSF-1 for growth in culture. Recent studies show that tyrosine phosphorylation of certain membrane-bound proteins may be involved in the control of cell proliferation induced by specific growth factors. In addition, several oncogene products have been shown to be structurally related to either specific growth factors or growth factor receptors. We propose to use both normal macrophages and established macrophage cell lines with different growth phenotypes as models to investigate the role of protein phosphorylation in the regulation of macrophage proliferation. First, we will develop two dimensional gel electrophoresis and autoradiography techniques to identify the CSF-1 induced and/or spontaneous phosphorylated components from both mormal macrophages and established macrophage-monocyte tumor cell lines. Thin layer plate electrophoresis techniques will be used to characterize the chemical nature of the phosphorylated amino acids. Second, a monoclonal antibody against a phosphotyrosine moiety will be developed as a probe, along with """"""""Western blot"""""""" techniques, to facilitate the identification and characterization of tyrosine-phosphorylated-proteins. We will study whether the production of autocrine CSF-1 or CSF-like activity and tyrosine phosphorylation are linked to oncogenic transformation of macrophages during the early phase of development. We will establish, by tumor virus transformation, immortalized mouse bone marrow-derived macrophages to study whether these cell lines produce and secrete autostimulatory activity. We will also examine and identify phosphorylated components from these cell lines. The hypothesis of our work is that autocrine growth factor, tyrosine phosphorylation and ligand-receptor interaction play important roles in the cellular transformation and oncogenesis of bone marrow-derived hematopoietic cells.
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