CARRIER MEDIATED ANTIFOLATE TRANSPORT AND RESISTANCE

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This proposal is a continuation of ongoing work in this laboratory to define the molecular basis for the transport of folates and antifolates mediated by the reduced folate carrier, RFC1. The objectives are to delineate carrier elements that are determinants of substrate binding and translocation and to characterize the functional properties of mutant RFC1 that underlie antifolate resistance to new generation antifolates due to impaired drug transport. A number of experimental approaches will be undertaken for which there are supporting publications, preliminary data and demonstrated expertise: (1) This laboratory has developed a panel of 26 clonal L1210 leukemia cell lines with defined mutations in RFC1 that result in impaired MTX transport and drug resistance. In several of these lines transport alterations are highly substrate specific. We will assess transport properties of selected thymidylate synthase (TS) and GARFT inhibitors to further explore the functional consequences of these mutations, substrate specificity and cross-resistance patterns. (2) RFC1 mutations will be identified that result in primary transport-related resistance to TS and GARFT inhibitors using single-step antifolate selection augmented by chemical mutagenesis emphasizing 5-formyltetrahydrofolate as the sole folate source. (3) RFC1 mutations that occur in the DNA from lymphoblasts of patients with ALL will be screened for mutations by single-strand conformation polymorphism followed by sequencing of suspect regions. (4) Site-directed mutagenesis will be employed to further characterize the functional role of RFC1 residues the substitution of which results in altered transport but with a high degree of substrate specificity, as has been demonstrated for a number of murine and human RFC1 amino acid residues. This approach will also be applied to explore the functional role of RFC1 regions and to identify potential ion- pairing interactions between highly conserved oppositely charged amino acids in different predicted transmembrane domains. A comprehensive analysis of folate/antifolate transport properties mediated by mutant murine and human RFC1s from all the preceeding studies will be undertaken after transfection into transport- deficient murine and human leukemia cell lines. This will include the effects of inorganic and organic anions on RFC1- mediated fluxes and transmembrane gradients to identify carrier elements that are anion-sensitive and/or participate in anion exchange. Finally, native and mutant, mouse and human, RFC1 will be functionally reconstituted into proteoliposomes to assess carrier transport properties under conditions in which the intravesicular composition is defined and in the absence of complicating parallel transport pathways.