Inhibitionof Sugar Uptakeby Methotrexatein CulturedEhrlich AscitesCarcinomaCells1

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 x i0@ M MTX for 24 hr and assayed at intervals for rates of uptake of 3-0- methyl-D-glucose (to measure the rates of sugar transport), of 2-deoxy-D-glucose (to measure the rates of sugar trans port and phosphorylation), and of glucose into total cellular material (to compare with the data obtained with the non metabolizabie sugars). MTX progressively and profoundly inhibited 2-deoxy-D-glucose uptake; the effect was on the V,nax and the apparent K,0, for 2-deoxy-D-glucose was nor mal. Adenosine 5'-triphosphate depletion by glucose dep rivation inhibited 2-deoxy-D-glucose uptake to a similar degree. MTX progressivelyinhibited the rates of protein synthesis. Hexokinase activity in cell extracts was normal during first 6 hr and decreased after 24 hr. Cycioheximide progressively decreased 2-deoxy-D-glucose uptake rates, which correspondedto the decreasesof hexokinaseactivity in cell extracts. Rates of 3-O-methyi-D-glucose transport were decreased by up to 50% in MTX-treated and in adeno sine 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-giucose 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 incu bated with cycioheximide for 24 hr, and by 30% in glucose deprived cells.