The objectives of this research proposal are to further elucidate the structural and functional properties of the folate receptor system recently described in our laboratory and to apply this information to the uptake and metabolism of folate and the chemotherapeutically active analogue of folate, methotrexate (MTX), presently used to treat a wide variety of human cancers. The folate receptor is made up of a membrane component and a soluble component. Studies are outlined that are designed to determine the functional regulation of these components by folate, the role of the soluble component in folate homeostasis, the mechanism of receptor mediated cellular uptake and the mechanism of the recently observed conversion of the membrane component to the soluble component. Studies are outlined that are also designed to determine the role of the folate receptor in the cellular uptake of MTX, the effect of incubation of cells with MTX on the folate receptor and cellular folate composition and to determine if MTX cytotoxicity is related to folate depletion, disturbance of cellular folate coenzyme distribution or occupancy of the folate receptor by methotrexate polyglutamates with resultant inhibition of transport of physiologic folates. The role of the folate receptor in the efflux of MTX from cells and the role of the soluble component of the folate receptor (that circulates in human serum) in the enterohepatic circulation of MTX (folate) will also be studied. MTX is most cytotoxic to cells in the proliferative phase of the cell cycle. The relationship of expression of the folate receptor during proliferation and differentiation of cells will be compared to the cellular uptake and cytotoxicity of MTX. In addition to inhibition of dihydrofolate reductase, MTX may also interfere with the activity of the cobalamin-dependent methionine synthetase - the other enzyme known to be responsible for intracellular production of tetrahydrofolate. The relationship of methionine synthetase activity to the level of the folate receptor and to MTX (folate) uptake will be studied under conditions where the activity of methionine synthetase can be reversibly altered. Deficient methionine synthetase activity results in homocystinemia and the level of homocysteine in tissue culture media and human serum will be used to monitor for the level of methionine synthetase activity after exposure of cells and humans to varying concentrations of MTX.
