This is a proposal for a competing renewal of an OIG Awarded September 1985 following fifteen years of continuous NCI RO-1 funding. This period of stable support was utilized to pursue one of the major objectives of the OIG strategy, the opportunity to reorient to a new investigative approach - the study of membrane transport of folates and antifolates at the molecular level, now a major thrust of our current and planned research. This required several years of developmental activities to establish new collaborations, the acquisition of new expertise and technologies and the recruitment and training of new personnel. This substantial investment in time and resources has now blossomed into exciting new areas of increasingly productive research. Concurrently, other complex and innovative studies at the biochemical and quantitative biological levels made possible by this grant have further elucidated basic elements of antifolate actions. Major contributions include: cloning and initial characterization of two distinct CDNA'S for folate binding proteins (FBP) in L1210 cells; increased expression of one FBP by genomic amplification or rearrangement; the identification of a FBP genomic rearrangement in novel cell lines selected for resistance to MTX with immobilization of the THF-cofactor carrier and membrane protein alterations distinct from the carrier. Other studies clarified elements of the mechanism and selectivity of leucovorin rescue, the role of endogenous folates as feed-back inhibitors of key folate requiring enzymes, the identification of THF cofactor pools that are not oxidized to DHF after antifolates and the role of the relative catalytic activities of thymidylate synthase (TS) and DHFR as determinants of THF cofactor depletion after antifolates. Proposed studies focus on molecular aspects of the transport of folates. Emphasis is on sequencing rearranged segments of the FBP gene that appear to regulate expression, characterization of folate binding sites and transfection of FBPs into L1210 cells to clarify their function and localization. Other studies will focus on cloning the THF-cofactor carrier in upregulated lines circumventing the difficulties in purifying this protein (i.e. use of subtractive cloning, oligonucleotide probes to enzymes with similar structural specificity, etc). Possible interactions between FBP and other cellular transport elements will be assessed by crosslinking and co-immunoprecipitation. Expression of transporters will be assessed in normal and malignant human tissues as a function of cell growth and differentiation and to establish possible new leads in selectivity. Finally, biochemical and network thermodynamic modeling approaches will continue to explore effects of folate cofactors on folate-dependent reactions, interactions among new antifolates that act at the levels of DHFR, TS or the transformylases and folate interconversions as a function of cycle phase. Biochemical and molecular mechanisms by which intracellular folates regulate transport of folates will also be explored.
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