The purpose of this application is to extend previous studies of the effects of 5-fluorouracil (FUra) on RNA metabolism in an effort to better understand the RNA mediated effects of FUra cytotoxicity. This study will focus on two FUra induced aberrations of mRNA metabolism, pre-mRNA splicing and mRNA half life. It is specifically proposed that the following be accomplished: 1. Apply the polymerase chain reaction to quantitate steady state levels of dihydrofolate reductase (DHFR) RNA splicing substrates as well as splicing intermediates in the pre-mRNA processing pathway, in FUra treated and control cells. Extend this assay to non-gene amplified cells so that it may eventually be applied in vivo. 2. Measure the half life of 3.5 kb DHFR mRNA in cells grown in the presence or absence of FUra. Assess whether an increased half-life after cell growth in medium containing FUra is an explanation for the previously observed increase in this specific DHFR mRNA. Identify the 3' noncoding element(s) present in 3.5 kb DHFR mRNA which appear to mediate this effect of FUra on half-life deletion mapping and DNA transfection. 3. Study the effect of FUra pretreatment on methotrexate cytotoxicity in cells expressing mRNAs with altered 3' noncoding regions to correlate alterations in mRNA half life with DHFR drug responsiveness.
