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Characterization of the Individual Transport Routes That Mediate the Influx and Efflux of Methotrexate in CCRF-CEM Human Lymphoblastic Cells¹

Division of Biochemistry, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037

The transport routes utilized by CCRF-CEM human lymphoblastic cells for the influx and efflux of methotrexate have been analyzed. Evidence was obtained for a single influx route for methotrexate: (a) Influx at 2 µM [³H]methotrexate was inhibited completely by high concentrations of unlabeled methotrexate, o-phthalate, and bromosulfophthalein, and the inhibition profile with each anion was monophasic; and (b) Pretreatment of the cells with an N-hydroxysuccinimide ester of methotrexate also blocked influx, and this inhibition was complete over a range of substrate concentrations from 2 to 50 µM. Influx was also saturable and proceeded with a maximum rate (V_max) of 4.3 pmol/min/mg protein (at 37°C) and with a K_t of 0.8 µM in an anion-deficient buffer and 4.6 µM in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonate-buffered saline. The ratio of V_max to the amount of carrier protein (0.3 pmol/mg protein) gave a turnover number for the transport system of 14.3/min. In contrast to influx, methotrexate efflux proceeded via three routes which could be separated by their sensitivity to specific inhibitors. The major portion of efflux occurred via the methotrexate influx carrier, the identity of which was established from its sensitivity to the N-hydroxysuccinimide ester of methotrexate and by its requirement for anions in the external medium. Methotrexate, adenosine monophosphate, and phosphate each stimulated efflux via this route and this stimulation was half-maximal at anion concentrations that approximated their K_i values for inhibition of methotrexate influx. A second efflux route was identified by its sensitivity to bromosulfophthalein. This route was relatively inactive and did not fluctuate significantly upon addition of various anions, glucose, or metabolic inhibitors. The third route was quantitated by its sensitivity to probenecid and its activity was increased in saline buffers and upon addition of glucose and was inhibited by oligomycin. Similar transport routes for methotrexate are present in L1210 mouse cells, although these two cell lines can be distinguished by the amount of transport protein and by the activity of the bromosulfophthalein-sensitive efflux route for methotrexate.

¹ This work was supported by Grants CH-229 from the American Cancer Society and CA 23970 from the National Cancer Institute. Research Institute of Scripps Clinic Manuscript 4019-BCR.

² To whom requests for reprints should be addressed, at Division of Biochemistry, BCR-7, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037.

Received 9/16/85. Revised 12/ 6/85. Accepted 12/27/85.