The mechanism of action of methotrexate. II. Augmentation by vincristine of inhibition of deoxyribonucleic acid synthesis by methotrexate in Ehrlich ascites tumor cells

Incorporation of deoxyuridine into DNA persists in Ehrlich ascites tumor cells in vitro even after high affinity intracellular binding sites have been saturated with methotrexate (MTX). Further suppression of deoxyuridine metabolism requires exposure of cells to extracellular MTX to achieve intracellular MTX levels in excess of the tightly bound fraction. Vincristine (10 μM) augments the steady state level of intracellular MTX, and this is associated with an increased inhibition of deoxyuridine incorporation into DNA. Exposure of cells to vincristine in the absence of MTX, or under conditions in which high affinity binding sites are completely associated with MTX but intracellular MTX in excess of this component is not present, did not alter the incorporation of deoxyuridine into DNA over 35 min. These observatons relate to other studies which suggest that vincristine acts as a metabolic poison to inhibit energy dependent processes which limit the net accumulation of MTX, an effect which occurs in the absence of an alteration in the unidirectional influx or the extracellular MTX level. These data further indicate a crucial role for intracellular MTX in excess of the usual tightly bound fraction in the inhibition of DNA synthesis, and suggest a rational basis for the design of chemotherapeutic regimes with MTX and vincristine.