Inhibition of Sugar Uptake by Methotrexate in Cultured Ehrlich Ascites Carcinoma Cells¹

Methotrexate (MTX) decreases adenosine 5′-triphosphate pools and inhibits glycolysis in cultured Ehrlich ascites carcinoma cells. The purpose of this study was to examine the effects of MTX on sugar transport and phosphorylation. Cells that were grown in continuous spinner cultures were incubated in media with and without 2 × 10^-6 m MTX for 24 hr and assayed at intervals for rates of uptake of 3-O-methyl-d-glucose (to measure the rates of sugar transport), of 2-deoxy-d-glucose (to measure the rates of sugar transport and phosphorylation), and of glucose into total cellular material (to compare with the data obtained with the non-metabolizable sugars). MTX progressively and profoundly inhibited 2-deoxy-d-glucose uptake; the effect was on the V_max and the apparent K_m for 2-deoxy-d-glucose was normal. Adenosine 5′-triphosphate depletion by glucose deprivation inhibited 2-deoxy-d-glucose uptake to a similar degree. MTX progressively inhibited the rates of protein synthesis. Hexokinase activity in cell extracts was normal during first 6 hr and decreased after 24 hr. Cycloheximide progressively decreased 2-deoxy-d-glucose uptake rates, which corresponded to the decreases of hexokinase activity in cell extracts. Rates of 3-O-methyl-d-glucose transport were decreased by up to 50% in MTX-treated and in adenosine 5′-triphosphate-depleted cells. This decrease was not due to a change in the saturability characteristics of the transport system, to inhibition of protein synthesis, or to effects on a ouabain-sensitive adenosine-5′-triphosphatase. Hypoxanthine prevented the inhibitions of 3-O-methyl-d-glucose and of 2-deoxy-d-glucose uptakes by MTX. Glucose uptakes into total cellular material were reduced by 50% in cells incubated with MTX for 24 hr, by 30% in cells incubated with cycloheximide for 24 hr, and by 30% in glucose-deprived cells.