Identification of the Bromosulfophthalein-sensitive Efflux Route for Methotrexate as the Site of Action of Vincristine in the Vinci ist ine-dependent Enhancement of Methotrexate Uptake in LI 210 Cells1

The mechanism by which vincristine enhances the uptake of methotrexate in leukemic LI 210 mouse cells has been investigated. Methotrex ate uptake after 30 min at 37°Cincreased 44% relative to untreated controls in cells suspended in a 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid/bicarbonate-buffered saline medium containing 20 jtM vin cristine. This stimulation was half-maximal at a vincristine concentration of 4 pM. An enhancement of methotrexate uptake by vincristine was also observed in the presence of glucose but a reversal could be achieved by prior treatment of the cells with taxol, a drug which prevents microtubule disassembly by vincristine. When the effect of vincristine was determined on the individual influx and efflux components for methotrexate, the increased uptake of methotrexate correlated with the inhibition of the unidirectional efflux route for methotrexate that is sensitive to bromosulfophtImlein. Inhibition of this route was half-maximal at 3 MMvincris tine and it exceeded 90% at high concentrations of the inhibitor. Trans port via the bidirectional exchange carrier for methotrexate was not affected by vincristine, while the unidirectional efflux route sensitive to probenecid was inhibited by vincristine but only at concentrations 10-fold higher than required to inhibit the bromosulfophthalein-sensitive route. Vincristine did not increase methotrexate uptake in CCRF-CEM lymphoblasts, a human cell line which contains much lower levels of bro mosulfophthalein-sensitive efflux route for methotrexate. Concentrations of vincristine which inhibited the bromosulfophthalein-sensitive efflux route of LI 210 cells by 80-90% had little or no effect on the intracellular pH or on intracellular levels of ATP, GTP, cyclic AMP, K*, or oxidized glutathione. A significant increase was observed in the cellular uptake of tetraphenylphosphonium ions, suggesting that vincristine causes a liyperpolarization of the plasma membrane.