Incorporation of Tritiated Uridine Into DNA of Ehrlich Ascites Tumor Cells

Abstract

Ehrlich ascites tumor cells were labeled with [5,6-³H] uridine in vivo during the exponential growth phase of the tumor in the mouse. Hydroxyapatite column chromatography of the total cell nucleic acid revealed a level of activity in the DNA approaching 50% of the incorporated activity of the RNA after 24 hours. After perchloric acid hydrolysis, the constituent bases of the DNA, separated by paper chromatography, contained more than 90% of the tritium radioactivity in the cytosine and thymine, at a ratio of approximately 2:1. Prior to digestion of the polymer, the level of label in the DNA was not sensitive to RNase, alkaline, or heat denaturation. Equilibrium density gradient centrifugation produced a single peak, coincidental for radioactivity and optical density at 260 nm. Our results indicate that tumor cells under replicative stress incorporated more than one-third of the tritium radioactivity of uridine into the DNA, whereas those at a growth plateau had less than 10% of the label in the DNA. This exogenous uridine radioactivity observed in the DNA represented neither a DNA-RNA hybrid, RNA primer pieces in DNA synthesis, nor any other RNase-sensitive species, but was apparently the consequence of amination and methylation of the tritium-labeled uracil moiety to satisfy the metabolic needs of the replicating cells for cytosine and thymine bases.