5FLUOROURACIL EFFECTS ON URACIL-RICH MRNA STABILITY

描述：虽然5-氟尿嘧啶（FU）已用于治疗 癌症近四十年来，药物的抗肿瘤活性机制 还没有完全被理解。 FU很容易被掺入所有种类的 然而，RNA是一种精确的机制，描述了随之而来的毒性， 仍有待确定。 一些已知的最不稳定的mRNA含有 选择性靶向mRNA的腺苷酸、尿苷酸富集元件（ARE） 快速降解。 由于大多数含有ARE的mRNA编码蛋白质， 参与细胞生长、分化等关键过程 和发展，干扰稳定性调节可能是有害的 到细胞。 本申请将调查是否将FU纳入 含有ARE的mRNA增强其稳定性。 FU对 将研究含有三种不同战神的mRNA的稳定性。 的 将c-myc、GM-CSF或c-jun战神与基因的一部分在读码框内融合 编码长寿的β珠蛋白mRNA。 将嵌合基因 下游的血清诱导，c-fos启动子，并被转染 NIH 3 T3成纤维细胞。 静止细胞将用血清+ Fu. 新转录的mRNA将整合FU和mRNA 将从转录物衰变计算半衰期。 多重 聚合酶链反应（PCR）试验将用于评价稳定性 从肿瘤细胞中选择数量的含ARE和缺乏ARE的mRNA 用FU处理的线。 差异显示PCR将确定哪些mRNA 随着FU被掺入mRNA中而差异稳定。 克隆 对差异稳定的mRNA进行测序， 差异稳定的mRNA的比例包含ARE。 的 尿嘧啶和FU取代的战神对反式作用蛋白因子的亲和力 将通过凝胶电泳检测与ARE结合并调节mRNA稳定性的蛋白质 迁移率变动分析。 将测定暴露于FU的细胞中的 游离蛋白因子与结合蛋白因子，以确定 暴露于FU。 对独特的抗肿瘤活性的完整理解 FU的使用可以提高其临床有效性，并有助于设计新的 抗癌药

DESCRIPTION: Although 5-fluorouracil (FU) has been used in the treatment of cancer for nearly four decades, the drug's mechanism of anti-tumor activity is not completely understood. FU is readily incorporated into al species of RNA, however, a precise mechanism that describes the ensuing toxicity remains to be established. A number of the most labile mRNas known contain an adenylate, uridylate-rich element (ARE) that selectively targets the mRNA for rapid degradation. Since most ARE-containing mRNas encode for proteins that are involved in critical processes such as cell growth, differentiation and development, interference with stability regulation may be deleterious to the cell. This application will investigate if incorporation of FU into ARE-containing mRNAs enhance their stability. The effect that FU has on the stability of mRNAs containing three different AREs will be studied. The c-myc, GM-CSF or c-jun AREs were fused in frame to portions of a gene encoding the long-lived beta globin mRNA. The chimeric genes were placed downstream from the serum-inducible, c-fos promotor and were transfected into NIH 3T3 fibroblasts. Quiescent cells will be induced with serum plus FU. The newly transcribed mRNAs will incorporate the FU and the MRNA half-life will be calculated from the transcript decay. A multiplex polymerase chain reaction (PCR) assay will be used to evaluate the stability of a select number of ARE-containing and ARE-lacking mRNAs from tumor cell lines treated with FU. Differential display PCR will determine what mRNAs are differentially stabilized as FU becomes incorporated into mRNA. Cloning and sequencing the differentially stabilized mRNAs will determine what proportion of the differentially stabilized mRNAs contain the ARE. The affinity of uracil- and FU-substituted AREs to trans-acting protein factors that bind to the ARE and regulate mRNA stability will be examined by a gel mobility shift assay. Cells exposed to FU will be assayed for the amount of free versus bound protein factors to determine if the affinity following exposure to FU. A complete understanding of the unique anti-tumor activity of FU may enhance its clinical effectiveness and aid in the design of new anti-cancer drugs.