Effect of 5-fluorouracil on the synthesis and translation of polyadenylic acid-containing RNA from regenerating rat liver.

Abstract

The effect of 5-fluorouracil (5-FUra) on the synthesis of polysomal RNA, the incorporation of the drug into polysomal RNA, and the translation of 5-FUra-modified polyadenylic acidcontaining RNA [poly(A)RNA] were measured in regenerating liver of partially hepatectomized rats treated with 5-FUra at 1 and 16 hr after surgery. Polysomal RNA was separated into poly(A)RNA and rRNA. The synthesis of poly(A)RNA, as measured by [3H]orotic acid incorporation, was unaffected by 5-FUra (20 mg/kg), while rRNA synthesis was decreased approximately 60%. The intracellular specific radioactivity of UTP was unaltered by drug treatment. Poly(A)RNA contained 2.68 pmol of 5-FUra per µg of RNA, and rRNA contained 0.54 pmol of 5-FUra per µg of RNA after administration of a 20 mg/kg dose of 5-FUra. Agarose-urea gel electrophoresis showed that poly(A)RNA labeled with [3H]-5-FUra had a modal distribution in size of 21S and was similar to that of control poly(A)RNA labeled with [3H]orotic acid. rRNA labeled with [3H]-5-FUra showed 28S and 18S RNA with an additional species at 38S in comparison to control rRNA. Control and 5-FUra-modified poly(A)RNA were translated in vitro in a cell-free wheat germ system. The products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and fluorography. Poly(A)RNA from rats treated with 20 mg of 5-FUra per kg stimulated translation by 240% in comparison to poly(A)RNA from control animals. The stimulatory effect was associated with an overall increase in the quantity of products translated and a preferential increase in five high-molecular-weight peptides. There was no change in the size or isoelectric composition of the proteins, and no new translation products were observed. Although the initial rate of translation of 5-FUra-modified poly(A)RNA was 4 times faster than that of control poly(A)RNA, the initiation step, as measured by the formation of methionylpuromycin, was the same for both species of RNA. Competition experiments with 7-methylguanosine monophosphate and 7-methylguanosine diphosphate, to measure the interaction of the 5′-cap of mRNA with initiation sites in the ribosome, also showed no differences between control and 5-FUra-modified poly(A)RNA.