REDUCED FOLATE TRANSPORT AND ANTIFOLATE RESISTANCE

使用经典的抗叶酸剂如甲氨蝶呤（MTX）和较新的药物如洛美沙星（DDATHF）和托莫得（ZD-1694）需要通过介导叶酸维生素摄取和流出的相同机制进行转运。 这些药物的疗效至少部分取决于它们的吸收和保留，并且为了实现最佳效果，不仅需要了解负责其运输的机制，而且还需要确定负责基因的调节方式。 在含有约2 μ M叶酸的标准组织培养基中培养的中国仓鼠肺成纤维细胞（DC-3F）不表达叶酸受体，并且在叶酸应激条件下不能表达它们，即使它们确实含有叶酸受体α（FR α）的mRNA。 然而，当选择在15 pM [6S]-亚叶酸（leucovorin）中生长时，这些细胞（DC-3F/FA 3）通过基因扩增和转录介导的事件的组合将FR α 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培养基叶酸浓度和细胞内叶酸池大小成正比和反比，并且在转录后被调节。 本申请中提出的研究将使用多种重组DNA技术和体外转录和翻译测定来确定负责DC-3F/FA 3细胞中响应于叶酸应激的FR α表达的转录和转录后调节的机制。 他们还将确定为什么DC-3F细胞对这种压力没有反应。

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.