5FLUOROURACIL EFFECTS ON URACIL-RICH MRNA STABILITY

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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.