REDUCED FOLATE TRANSPORT AND ANTIFOLATE RESISTANCE

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The use of classical antifolates like methotrexate (MTX) and newer drugs such as lometrexol (DDATHF) and tomudex (ZD-1694) require transport by the same mechanisms that mediate uptake and efflux of the folate vitamins. The efficacy of these drugs is at least partially dependent upon their uptake and retention, and to achieve optimal effectiveness, it becomes necessary to not only understand the mechanisms responsible for their transport but also to determine the manner in which the genes responsible are regulated. Chinese hamster lung fibroblasts (DC-3F) cultured in standard tissue culture media containing approximately 2 muM folic acid do not express folate receptors and are incapable of expressing them under conditions of folate stress eventhough they do contain the mRNA for folate receptor alpha (FRalpha). When selected for growth in 15 pM [6S]-folinic acid (leucovorin), however, these cells (DC-3F/FA3) upregulate expression of FRalpha mRNA by 17 fold through a combination of gene amplification and transcription mediated events. In such cells the level of expression of the receptor itself is directly and inversely proportional to media folate concentrations and to the intracellular folate pool size and is regulated post transcriptionally. The studies proposed in this application will use a variety of recombinant DNA techniques and in vitro transcription and translation assays to determine the mechanisms responsible for both the transcriptional and post transcriptional regulation of FRalpha expression in DC-3F/FA3 cells in response to folate stress. They will also determine why DC-3F cells are not responsive to such stress.