REDUCED FOLATE TRANSPORT AND ANTIFOLATE RESISTANCE

使用传统的抗叶酸药物，如甲氨蝶呤（MTX）和较新的药物，如乐美曲醇（DDATHF）和托莫替（ZD-1694），需要通过与叶酸维生素的吸收和排出相同的机制进行运输。这些药物的疗效至少部分依赖于它们的吸收和保留，为了达到最佳效果，不仅需要了解它们的转运机制，还需要确定相关基因的调节方式。在含有约2mum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，并且在叶酸胁迫条件下不能表达叶酸受体，尽管它们含有叶酸受体α (frα)的mRNA。然而，当选择在15pm [6S]-叶酸（亚叶酸素）中生长时，这些细胞（DC-3F/FA3）通过基因扩增和转录介导事件的结合，将frα mRNA的表达上调了17倍。在这些细胞中，受体本身的表达水平与培养基中叶酸浓度和细胞内叶酸池大小成反比，并在转录后受到调节。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试验，以确定DC-3F/FA3细胞在叶酸胁迫下frα表达的转录和转录后调控机制。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.