Antifolate Polyglutamylation and Competitive Drug Displacement at Dihydrofolate ReducÃ-aseas Important Elements in Leucovorin Rescue in L1210 Cells1

Previous studies from this laboratory have shown that the addition of leucovorin to tumor cells dissociates methotrexate, but not methotrexate polyglutamates, from dihydrofolate reduc Ã-ase(L. H. Matherly, D. W. Fry, and I. D. Goldman, Cancer Res., 43: 2694-2699, 1983). To further assess the importance of these interactions to leucovorin rescue, antifolate growth inhibi tion toward L1210 cells in the presence of leucovorin was correlated with the metabolism of (6S)-5-formyl tetrahydrofolate to dihydrofolate as a measure of dihydrofolate reducÃ-aseactivity. Growth inhibition (greater than 95%) by methotrexate (5-10 UM) following its intracellular polyglutamylation during a 3-h preexposure, or by continuous treatment with high levels of the lipophilic antifolate, trimetrexate (1 /UM),was only slightly dimin ished by 10 MMleucovorin (15-25%). High-pressure liquid Chro matographie analyses of the derivatives formed from radiolabeled (6S)-5-formyl tetrahydrofolate under these conditions showed an incomplete conversion to dihydrofolate and metabolism to pre dominantly 10-formyl tetrahydrofolate. Neither of the antifolates interfered appreciably with the metabolism of the folate deriva tives to polyglutamates. Growth inhibition in the presence of leucovorin correlated with the accumulation of dihydrofolate (1.5-2.2 nmol) from radiolabeled (6S)-5-formyl tetrahydrofolate, reflecting continued suppression of dihydrofolate reducÃ-aseac tivity at these drug concentrations. With lower equitoxic levels of the trimetrexate (7.5 nM), the provision of leucovorin allowed for a restoration of cell growth to a level greater than 90% of control. Under these conditions, control levels of dihydrofolate (0.2 nmol) were formed from radiolabeled cofactor, consistent with sustained dihydrofolate reducÃ-aseactivity. These findings support a role for the activation of dihydrofolate reducÃ-ase as an important component of the reversal of the effects of diaminoantifolates by leucovorin, presumably by a competitive displacement of drug from the enzyme. Since no displacement occurs in cells which have accumulated methotrex ate polyglutamates, or in the presence of high levels of Irimetrexate, il appears that the concenlralion of unbound drug wilhin cells is a significanl determinant of the extent of this competitive binding interaclion. From these considerations, Ihe high levels of methotrexate polyglutamates thai accumulate in sensitive tu mors relative lo bone marrow and gaslrointestinal cells would Received 6/27/85; revised 10/9/85; accepted 10/11/85. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by Research Grant CA-16906 and Training Grant CA-09340 from the National Cancer Institute, NIH. 'Recipient of a Junior Faculty Research Award from the American Cancer Society. appear lo represent an importanl factor for the selectivity of leucovorin rescue in vivo.