The overall goal of this project is to establish the metabolic basis for the clinically observed sensitivity and resistance of tumor cells to treatment with L-asparaginase. This will require a thorough understanding of the importance and interaction of those cellular enzymes responsible for the metabolism of asparagine. In order to accomplish this objective, we propose to produce monoclonal antibodies to the enzymes involved in asparagine and glycine metabolism. These specific antibodies will enhance the purification, assay and comparison of the individual enzymes in these pathways from tumor and normal tissues. However, monoclonal antibody screening procedures will be developed and employed to obtain highly specific antibodies that inhibit the enzymatic activity of the key asparagine metabolizing enzymes. These specific inhibitory antibodies will then be introduced into isolated hepatocytes and tumor cells by fusion of liposomes or resealed erythrocyte ghosts containing the antibody. A nonfluorescent, fluoregenic aqueous space marker will be coincorporated into the antibody-containing liposomes or ghosts. The products of fusion of the liposomes or ghosts with hepatocytes or tumor cells will display intense fluorescence due to cytoplasmic esterase activity, and this will be measured using flow cytometry. The specifically inhibited cells will then be used to study the flow of labelled substrates through the pathways of asparagine and glycine metabolism. This will enable the identification of key in vivo control points in these pathways, and provide insight into the metabolic basis of the effectiveness of L-asparaginase therapy. With this knowledge it is hoped that we will be able to design therapeutic regimens of enhanced effectiveness.
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| Pfeiffer, N E; Wylie, D E; Schuster, S M (1987) Immunoaffinity chromatography utilizing monoclonal antibodies. Factors which influence antigen-binding capacity. J Immunol Methods 97:1-9 |
| Pfeiffer, N E; Mehlhaff, P M; Wylie, D E et al. (1986) Monoclonal antibodies specific for bovine pancreatic asparagine synthetase. Production and use in structural studies. J Biol Chem 261:1914-9 |
