The Use of Glucosamine and Actinomycin D in Radioactive Labeling of RNA in Cells in Culture

Abstract

Abstract Pretreatment of confluent cultures of mouse L cells or of well-differentiated nervous system cells in primary cultures with 20–120 mM glucosamine resulted in a stimulation of the uptake of tritiated uridine, but not of adenosine. A marked stimulation of the incorporation of radioactive uridine into acid-precipitable macromolecules was also obtained, while adenosine incorporation was unchanged. Cultures of L cells in log phase of growth were similarly affected by glucosamine pretreatment. Uridine and cytidine uptakes were stimulated by 50%. Tritiated uridine incorporation was stimulated in a biphasic manner, with maximal stimulation (115%) after 15–60 min of labeling and at later times an inhibition of incorporation. The stimulation of cytidine incorporation paralleled the stimulation of its uptake. The data indicate that there is: a) a glucosamine-induced stimulation of pyrimidine nucleoside uptake, b) a marked stimulation of tritiated uridine incorporation into RNA due to depletion of the cellular pools of unlabeled uridine nucleotides during glucosamine pre-treatment, and c) a decrease in the rate of RNA synthesis after several hours of glucosamine treatment, probably related to diminished intracellular supplies of uridine nucleotides. In the presence of glucosamine, high concentrations of actinomycin D could be used to increase nuclear retention of pulse-labeled nascent RNA. Cordycepin treatment did not result in similar retention of RNA. These techniques will be useful in autoradiographic and biochemical studies of nuclear RNA synthesis.