DEVELOPMENT OF OPTIMUM CONDITIONS FOR NIMC PRODUCTION

MITOCHONDRIAL RESPIRATION INHIBITION IN TUMOR CELLS MAY OCCUR IN TWO MANNERS. ONE MECHANISM INVOLVES THE SYNTHESIS OF NITRIC OXIDE (NO) BY MACROPHAGES OR IFN-GAMMA MEDIATED INDUCTION OF NO SYNTHESIS IN TUMOR CELLS. WE HAVE RECENTLY ESTABLISHED A MURINE MACROPHAGE CELL LINE, EA13.5, THAT RELEASES A SOLUBLE MEDIATOR, NIMC, THAT CAUSES TUMOR CELL RESPIRATION INHIBITION INDEPENDENT OF NO PRODUCTION AND INDEPENDENT OF IFN-GAMMA. PHASE I WILL INVOLVE DEVELOPMENT OF OPTIMAL CONDITIONS FOR EA13.5 CELL GROWTH AND PRODUCTION OF NIMC AND ESTABLISHMENT OF A PURIFICATION SCHEME. THE GROWTH AND TRIGGERING OF THE EA13.5 CELLS IN GAS PERMEABLE BAGS AND IN HOLLOW FIBER BIOREACTORS WILL BE EXAMINED; ONE OF THESE BIOREACTOR SYSTEMS WILL BE SELECTED FOR LARGE-SCALE CULTURE BASED ON EASE OF HANDLING, CELL PROLIFERATION AND VIABILITY, AND YIELD OF NIMC. THE CONDITIONS FOR INDUCTION OF NIMC WILL BE OPTIMIZED WITH RESPECT TO MEDIA, SIGNALS FOR INDUCTION, AND KINETICS OF PRODUCTION. PROTEIN PURIFICATION STEPS WILL BE EXAMINED AND OPTIMIZED FOR DESIGN OF A PURIFICATION SCHEME FOR NIMC. THE SUCCESSFL EXECUTION OF PHASE I WILL ENABLE ECONOMICAL PRODUCTION OF SUFFICIENT NIMC FOR LARGE-SCALE PROTEIN PURIFICATION STUDIES, AMINO ACID SEQUENCING, AND DNA PROBE CONSTRUCTION IN PHASE II.