Methotrexate and other antifolate drugs used in cancer chemotherapy must be transported across membranes to be effective in killing tumor cells. The goals of the proposed work are to establish the mechanisms by which MTX and other folate compounds are transported across the plasma membrane of L1210 mouse leukemia cells and to characterize the binding proteins which mediate these processes. Specific projects are designed to utilize intact cells and artificial systems to evaluate an anion exchange mechanism proposed for the MTX influx carrier, to identify possible intracellular anions which might exchange for extracellular MTX, to further develop and characterize affinity-labeling agents for MTX transport proteins, to utilize these labeling agents both in identifying specific transport routes and in labeling transport proteins, to develop model anion substrates for the study of specific influx and efflux carriers, to separate and identify MTX efflux systems by their differential responsiveness to various drugs and ions, to characterize and purify to homogeneity the carrier proteins involved in MTX transport, and to initiate studies on the effects of irreversible inhibitors of folate transport on cell growth. Information derived from this project should contribute to the understanding of the inherent limitations of antifolate drugs due to transport restrictions, assist in the development of more effective drugs and drug regimens, provide information on the nature of drug resistance that occurs via alterations in transport and how this type of resistance might be overcome, and to ascertain whether drugs directed against transport systems might provide an effective means for killing cells via folate starvation. Information derived from this project should also be useful in assessing the role of folate transport systems in folate deficiencies, determine whether these systems can be induced to function more efficiently, and to determine how the consequences of reduced dietary folate can be minimized.
