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The cytotoxic actions of 5-fluorouridine (FUrd) have been evaluated in K-562 erythroleukemia cells, focussing on RNA-directed actions. FUrd was employed such that little DNA-directed cytotoxicity was seen. Substantial inhibition of cellular proliferation was observed at concentrations of FUrd which did not inhibit significantly the activity of thymidylate synthase and which were reversed by less than 10% by exogenous thymidine. In contrast, the syntheses of both poly A- and poly A+ RNAs were substantially reduced. The effects of FUrd on rRNA included reduction by greater than 90% of mature rRNA following a 2 h exposure to 1 microM FUrd, which persisted for at least 48 h, and the appearance of partially processed nuclear rRNA precursors incapable of being metabolized to mature rRNA. FUrd also decreased the levels of several mRNAs, including those for the proto-oncogenes c-myc and c-abl, and for gamma-globin, by 40 to 70%. In contrast to the effects of FUrd on rRNA, decreases in mRNA levels were reversible, and within 12 h following a 2 h exposure to 1 microM FUrd, mRNA levels for each of these three mRNAs were back to those present in untreated control cells. mRNAs did not respond in a connected fashion to FUrd. Thus, levels of beta-actin mRNA were unchanged and levels of ornithine decarboxylase mRNA were increased by exposure to FUrd. These findings demonstrate that FUrd acted in multifarious ways to alter mRNA synthesis and longevity. Inhibitors of individual RNA polymerases were used to analyze the degree to which the FUrd-induced inhibition of RNA metabolism was linked to cytotoxicity. Both actinomycin D, which specifically interfered with the incorporation of FUrd into rRNA transcripts, and alpha-amanitin, which specifically inhibited incorporation of FUrd into mRNA transcripts, decreased the cytotoxicity of FUrd, suggesting that incorporation of FUrd into both mRNA and rRNA precursors plays a role in the RNA-directed cytotoxic actions of FUrd. However, the antagonism provided by actinomycin D was greater than that produced by alpha-amanitin, demonstrating that inhibition of rRNA synthesis is the predominant mechanism of cytotoxicity in K-562 cells exposed to FUrd.