The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein.

Methotrexate accumulation, subcellular distribution, metabolism, and cytotoxicity were studied in human epidermoid carcinoma (KB) cells that were exposed to a low extracellular concentration of methotrexate (25 nM) following culture in widely differing concentrations of folic acid. KB cells cultured in standard medium with a high folic acid concentration (2.3 microM) had high levels of cellular folate (21.4 pmol/10(6) cells). Five passages through low folate (2.7 nM) medium reduced the level of cellular folate to near physiologic levels (0.4-1.0 pmol/10(6) cells). In contrast to KB cells cultured in standard medium, in KB cells cultured in low folate medium, 1) methotrexate inhibited growth; 2) methotrexate uptake was markedly increased; 3) methotrexate polyglutamation was almost complete; 4) methotrexate binding to dihydrofolate reductase was markedly enhanced; and 5) significant methotrexate binding to a previously undescribed membrane-associated protein occurred. The amount of methotrexate bound to the membrane-associated protein from KB cells cultured in low folate medium equaled the quantities bound by dihydrofolate reductase. Further characterization of this membrane-associated protein indicated that it was soluble in solutions containing Triton X-100, was capable of binding folic acid as well as methotrexate, had an apparent Mr of 160,000 by gel filtration in the presence of Triton X-100, and was precipitated by antiserum to human placental folate receptor. This membrane-associated protein may play an important role in the uptake and metabolism of methotrexate under physiologic conditions.