The goal of this project is to understand the metabolism of asparagine and glycine in tumor cells. This will be done in order to explain the sensitivity of some tumors to treatment with L-asparaginase. Our data indicates that the basis of the L-asparaginase sensitivity may be in the dependence of some tumor cells for asparagine-dependent glycine synthesis. Therefore, we have begun to study the enzymes of both asparagine and glycine metabolism in both tumor and normal cells. The experimental approach to be used relies on the production of inhibitory monoclonal antibodies to four of the key enzymes of asparagine and glycine metabolism. These antibodies will be microinjected into tumor cells, and the resultant changes in metabolism monitored. The enzymes to which inhibitory monoclonal antibodies are to be made are asparagine synthetase, cytosolic serine hydroxymethyltransferase, cytosolic asparagine-glyoxylate aminotransferase, and L-asparaginase. The antibodies will be selected for their potency as inhibitors of the various reactions, so that they can be used to produce measurable responses in amino and keto acid metabolism. We have already prepared inhibitory monoclonal antibodies to asparagine synthetase, and they have been characterized. We now have several antibodies that potently and selectively inhibit the various reactions catalyzed by asparagine synthetase. Some of these antibodies cause total inhibition of the enzymatic reaction of stoichemetric concentrations. In addition, we have demonstrated that the microinjection technique necessary for the proposed experiments can be accomplished with the cells to be used, with minimal effects on the cells.
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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 |
