MOLECULAR REGULATION OF FOLATE AND ANTIFOLATE TRANSPORT

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The goal of this project is to characterize the structure and regulation of the "classical" methotrexate/tetrahydrofolate cofactor membrane transporter in human tumor cells. This system is critical to the antitumor effectiveness of methotrexate and related antifolates. For methotrexate, an agent widely used in the chemotherapy of human cancers, defects in the regulation or biosynthesis of the transport system result in decreased antifolate accumulation within tumor cells and appear to be key factors in the development of drug resistance. In earlier experiments, K562 human erythroleukemia cells with increased uptake capacities for methotrexate and reduced folates were selected and a highly glycosylated transporter protein (GP-MTX) was identified and purified. Most recently, antiserum to GP-MTX was prepared, and several putative GP-MTX cDNAs from a human lymphoblastic leukemia cDNA library were isolated. Based on these initial results, we propose to (i) verify and characterize the putative full length cDNAs for GP-MTX by in vitro transcription/translation, dideoxy sequencing, and eukaryotic expression. In addition, we propose to (ii) identify and characterize the biochemical and molecular bases for impaired methotrexate transport in drug resistant K562 erythroleukemia and CCRF-CEM lymphoblastic leukemia cell variants at the levels of GP-MTX structure, biosynthesis, and/or regulation. These studies provide a framework for the study of regulation of the transport system for methotrexate and tetrahydrofolate cofactors in both drug sensitive and resistant human tumor cells. They should clarify the mechanistic bases for transport-mediated resistance to methotrexate. In this fashion, the identification of membrane-linked biochemical or molecular "markers" for methotrexate sensitivity or resistance in vitro should promote the more effective clinical use of this and related agents, including the detection and treatment of drug resistant tumors.