Support is requested to define the protein map of malignant human B lymphocytes at different stages of maturation and to identify differentiation-related proteins. Total cellular proteins from twenty leukemia and lymphoma cell lines will be analyzed, using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), representing seven different B cell maturation stages plus normal peripheral blood lymphocytes. Cells will be cultured in the presence or absence of two Protein Kinase C (PKC) activators with potential B cell differentiating activity; 12-0 tetradecanoylphorbol 13 acetate (TPA) and the Bryostatins for 120 hrs. 2D- PAGE will be conducted at two time points; 72 and 129 hrs. Elsie 4 Computer System will be used to analyze, measure and identify individual protein spots in different gels according to their isoelectric point, molecular masses, and integrated densities. This will enable us to detect qualitative and quantitative changes in cellular proteins induced by either agent in comparison with untreated (control cells) in each cell line. In order to related specific protein changes to differentiation, cells will also be analyzed by immunophenotypic markers at corresponding time points using stage-restricted B cell monoclonal antibodies and flow cytometry (FCM). DNA will also be stained with propidium iodide for cell cycle analysis by FCM to determine the growth-inhibitory capacity of each agent as a requirement for differentiation. The identification of differentiation-related proteins is an important step toward the uncovering of genetic mechanisms through which differentiation agents exert their effects.
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