REDUCED FOLATE TRANSPORT AND ANTIFOLATE RESISTANCE

传统的抗叶酸药物如甲氨蝶呤(MTX)和新药如洛美曲索(DDATHF)和托美地尔(ZD-1694)的使用需要通过相同的机制来调节叶酸维生素的吸收和外流。这些药物的疗效至少部分取决于它们的吸收和滞留，为了实现最佳效果，不仅需要了解它们的转运机制，而且还需要确定相关基因的调控方式。中国仓鼠肺成纤维细胞(DC-3F)在含有约2um叶酸的标准组织培养液中不表达叶酸受体，即使含有叶酸受体α(FRpha)的mRNA，也不能在叶酸应激条件下表达。然而，当选择在15 PM[6S]-叶酸(亚叶酸)中生长时，这些细胞(DC-3F/FA3)通过基因放大和转录调节事件的组合将FRpha mRNA的表达上调17倍。在这样的细胞中，受体本身的表达水平与叶酸浓度和细胞内叶酸池的大小成正比，并在转录后受到调节。本申请中提出的研究将使用各种重组DNA技术和体外转录和翻译分析来确定DC-3F/FA3细胞在叶酸胁迫下转录和转录后调控FRAlpha表达的机制。他们还将确定DC-3F细胞对这种压力没有反应的原因。

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.