The effect of methotrexate on 5-fluorouracil metabolism has been studied extensively. Most cells exposed to methotrexate will accumulate greater quantities of 5-fluorouracil which is primarily the consequence of the antipurine effect of methotrexate and subsequent increased intracellular quantities of phosphoribosyl-pyrophosphate. The mechanism by which synergistic cell killing occurs following this drug sequence, however, is not known. The areas that can be investigated to search for the cause of this increased cytotoxicity include inhibition of thymidylate synthetase, FdUTP incorporation into DNA, FUDP-sugar production and FUTP incorporation into RNA. 5-Fluorouracil does interfer with RNA processing, however the precise nature of the RNA effect of 5-fluorouracil is inknown. The recent availability of cell lines which have increased gene copies of dihydrofolate reductase and therefore synthesize enormous amounts of mRNA for this protein provides a means by which the effect of 5-fluorouracil on mRNA structure and function can be evaluated. The incorporation of [3H] 5-fluorouracil into RNA classes (including DHFR-mRNA) and the effect of 5-fluorouracil on DHFR-mRNA will be evaluated by gel electrophetic methods, C-DNA probes and S-1 mapping. The effect of alternating the availability of purine and pyrimidine bases by antimetabolite pretreatment will also be evaluated. The rate at which FUTP is incorporated into RNA, not the total amount of FUTP incorporated, has been found by us to be associated with the cessation of RNA synthesis. We plan to use detailed RNA studies as outlined in hopes of defining what RNA structures appear to be sensitive to this rapid incorporation of FUTP, for example are small uracil rich RNA species primarily affected by the density of FUTP substitution, and will thus result in changes in secondary and tertiary structure. In addition we will evaluate the effects of other base substitution on RNA structure and function by the methods described. Specifically, the purine analogues will be evaluated.
