alpha-Amanitin and 5-fluorouridine inhibition of serum-stimulated DNA synthesis in quiescent AKR-2B mouse embryo cells.

Abstract

AKR-2B mouse embryo cells undergoing the serum-stimulated transition from a quiescent to a proliferating state exhibit an increase in the rate of hnRNA synthesis which appears to be mediated through an increase in the actual number of RNA polymerase II molecules. alpha-Amanitin, administered early in the prereplication interval following stimulation, effectively inhibits hnRNA synthesis, polysomal mRNA accumulation, polyribosome formation, and subsequent DNA synthesis, and cell division. Unexpectedly, alpha-amanitin treatment also produces almost complete inhibition of the synthesis of 45S rRNA precursor and the increase in accumulation of cytoplasmic rRNA following serum stimulation. In order to determine whether the inhibition of new ribosomal synthesis might in itself be sufficient to prevent serum-stimulated DNA synthesis, the effects of 5-fluorouridine (5-FU), a specific inhibitor of 45S rRNA processing, were investigated. If added within eight hours following serum stimulation, 5-FU was found to completely inhibit subsequent DNA synthesis. These results suggest that quiescent AKR-2B cells do not contain a sufficient excess of ribosomes to support the synthesis of proteins which are required for DNA synthesis in response to serum growth factors. Furthermore, an early polymerase II mediated synthesis of mRNA(s) coding for some factor(s) necessary for ribosomal gene transcription may be an essential step in the serum-stimulated synthesis of new ribosomes.