Methotrexate and other antifolate drugs used in chemotherapy must be transported across the plasma membrane to be effective in killing tumor cells. The goals of this research project are to establish and characterize the kinetic properties, transport mechanisms energy requirements, regulatory mechanisms, binding components, and the physiological importance of transport systems which mediate the influx and efflux of methotrexate and other folate compounds by tumor cells. Our model systems include mouse L1210 cells and CCRF-CEM human lymphoblasts. These cells share a common transport system for methotrexate which mediates influx and efflux via an anion exchange mechanism and a probenecid-sensitive unidirectional efflux route. L1210 cells also contain a unidirectional bromosulfophthalein- sensitive efflux system for methotrexate which is absent in CCRF-CEM cells. Specific projects will involve: (A) L1210 cell lines which have adapted to low concentrations of folate by the overproduction of a high-affinity folate binding protein; an investigation of possible mechanisms for transport via this protein; a determination of whether cells that become resistant to methotrexate due to defects in the anion-exchange system acquire sufficient folate for growth via an overproduction of this high-affinity binding protein; and whether the latter form of resistance can be overcome with methotrexate/homofolate combination therapy; (B) the bromosulfophthalein-sensitive efflux route for methotrexate in L1210 cells; the mechanism by which this system is inhibited by vincristine; the possibility that cyclic AMP and cholate are also substrates for this system; and the selection of methotrexate resistant mutants which contain elevated amounts of this efflux activity; (C) the binding component of the exchange system for methotrexate in L1210 and CCRF-CEM cells; the isolation of cell lines adapted to low concentrations of folate that over-produce this protein; the refinement of methods for purification of this protein after affinity labeling with active esters of methotrexate; and the development of probes that will be useful in isolating the cDNA which codes for this protein; and (D) the binding component of the methotrexate transport system of L. casei; the construction and screening of plasmids containing the binding protein gene; the determination of the DNA sequence; and the derivation of the amino acid sequence and other biochemical properties. Information derived from this project should contribute to the understanding of 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 lead to new approaches in drug design from structural information on the transport proteins which mediate drug transport.
