The objective of this research project is to elucidate the role of colony-stimulating factor (CSF) in the modulation of myelopoiesis.
The specific aims i nclude (1) the purification and characterization of human CSF from different sources, (2) the production of monoclonal antibody and the establishment of CSF in myelopoiesis. CSF will be purified from human sources including human pancreatic carcinoma cell line MIA PaCa-2, human lung, placenta and umbilical cord conditioned media by the newly established methods of high performance liquid chromatography. The purified CSF will be characterized which will include: amino acid composition analysis, HPLC profile of tryptic peptides, carbohydrate side chain structure-function and amino acid sequence. Chemical modification studies will also be carried out to determine the biological active site and binding site. Monoclonal antibody against purified human CSF will be prepared and the RIA for human CSF will be established. The RIA will be used to study the immunological heterogeneity among human CSFs and will also be used to purify CSF by the method of antibody affinity chromatography. The mechanism of action of CSF will be studied by using purified CSF and promyelocytic leukemia cell line HL-60 or enriched human marrow CFU-GM as responsive cells. The binding of CSF to the receptor and their interaction will be studied, which will include the binding kinetics, the identification of receptor by crosslinking experiments, and the purification and phosphorylation of the receptor. Using the same myeloid differentiation system, the role of glutamine and polyamine metabolism in myelopoiesis will be elucidated by determining enzyme activities involved in glutamine utilization and purine and pyrimidine biosynthesis as well as ornithine decarboxylase in polyamine metabolism after the induction by CSF. The project utilizes a multidisciplinary approach in experimental hematology involving biochemistry, immunology and cell biology.
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