Biochemical correlates of responsiveness and collateral sensitivity of some methotrexate-resistant murine tumors to the lipophilic antifolate, metoprine.

The M5076 murine "ovarian" tumor which is naturally refractive to methotrexate was found to be highly responsive to the lipophilic antifolate, metoprine. M5076 cells were markedly deficient in mediated entry of methotrexate. This was in contrast to the L1210 leukemia, a tumor highly responsive to methotrexate but poorly responsive to metoprine. Two L1210 leukemia sublines, with acquired resistance to methotrexate by virtue of a deficiency in mediated entry of drug similar to that seen for M5076 cells, were found to be collaterally sensitive to metoprine. The insensitivity to methotrexate of the M5076 tumor and the two L1210 sublines is associated with low saturability (high Km) and reduced capacity (low Vmax) for mediated influx of drug. 5-Methyltetrahydrofolate, the major circulating folate in blood but not metoprine, shares this mediated route for entry. Therefore, a relatively low level of accumulation of this natural folate in these methotrexate-resistant tumors, in the face of a metoprine-induced blockade at the level of dihydrofolate reductase, probably accounts for the high sensitivity of these tumors to this lipophilic agent. Evidence for this notion was derived during transport and growth experiments in vitro using 5-formyltetrahydrofolate as a model folate coenzyme. The value for influx Vmax of this folate compound in a transport-deficient methotrexate-resistant subline compared to the parental L1210 was reduced to the same extent as that shown for methotrexate. Growth of this resistant L1210 subline showed a greater requirement for this model compound than did the parental line. Also, the concentration necessary for 50% inhibition by metoprine in the presence of this reduced folate was lower in the resistant subline. Inhibition of each cell line by metoprine, on the other hand, was the same when folic acid was used as the folate source. The implications of these findings for the use of lipophilic antifolates as alternative therapy for some methotrexate-resistant tumors are discussed.