Analysis of the Inhibitory Effects of VP-16-213 (Etoposide) and Podophyllotoxin on Thymidine Transport and Metabolism in Ehrlich Ascites Tumor Cells in Vitro1

These studies explore the effects of an epipodophyllotoxin on the membrane transport and metabolism of thymidine in Ehrlich ascites tumor cells. Uptake of 3H after exposure of cells to [3H]thymidine is characterized by a rapid initial velocity that approx imates membrane transport followed by a slower rate of uptake that parallels the accumulation of phosphorylated derivatives of thymidine, primarily thymidine triphosphate, within the cell. The high rate of thymidine transport relative to thymidine metabolism to the triphosphate within the cell decreases as the extracellular nucleoside concentration is reduced due to a much greater decrease in membrane transport than the subsequent metabolic step. Hence, as extracellular thymidine is decreased, transport becomes increasingly rate limiting to metabolism within the cell. VP-16-213 (etoposide) or podophyllotoxin inhibits the initial up take rate for thymidine and, as a consequence, inhibits the intracellular formation of thymidine triphosphate. When extracel lular thymidine is high, inhibitory effects on transport are tran sient, and the net rate of thymidine triphosphate accumulation within drug-treated cells rapidly approaches a velocity compa rable to that of control cells, indicating no direct VP-16-213 or podophyllotoxin effect on nucleoside and nucleotide phosphorylation. When extracellular thymidine is reduced so that trans port is rate limiting to metabolism, the duration of the inhibitory effects of VP-16-213 on thymidine triphosphate formation is prolonged. A secondary effect of VP-16-213 becomes manifest beyond 10 min of incubation with [3H]thymidine with the virtual complete cessation of thymidine incorporation into the acid pre cipitate without any change in the thymidine triphosphate level. This late effect is not observed with podophyllotoxin and indi cates a direct effect of VP-16-213 on DMA synthesis that is distinct from the earlier inhibitory effect on thymidine phosphorylation, which is secondary to membrane transport.