Antifolate polyglutamylation and competitive drug displacement at dihydrofolate reductase as important elements in leucovorin rescue in L1210 cells.
Previous studies from this laboratory have shown that the addition of leucovorin to tumor cells dissociates methotrexate, but not methotrexate polyglutamates, from dihydrofolate reductase (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 inhibition 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 reductase activity. Growth inhibition (greater than 95%) by methotrexate (5-10 microM) following its intracellular polyglutamylation during a 3-h preexposure, or by continuous treatment with high levels of the lipophilic antifolate, trimetrexate (1 microM), was only slightly diminished by 10 microM leucovorin (15-25%). High-pressure liquid chromatographic analyses of the derivatives formed from radiolabeled (6S)-5-formyl tetrahydrofolate under these conditions showed an incomplete conversion to dihydrofolate and metabolism to predominantly 10-formyl tetrahydrofolate. Neither of the antifolates interfered appreciably with the metabolism of the folate derivatives 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 reductase activity 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 reductase activity. These findings support a role for the activation of dihydrofolate reductase 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 methotrexate polyglutamates, or in the presence of high levels of trimetrexate, it appears that the concentration of unbound drug within cells is a significant determinant of the extent of this competitive binding interaction. From these considerations, the high levels of methotrexate polyglutamates that accumulate in sensitive tumors relative to bone marrow and gastrointestinal cells would appear to represent an important factor for the selectivity of leucovorin rescue in vivo.
L H
LH
Matherly
C K
CK
Barlowe
I D
ID
Goldman
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D005493:Folic Acid Antagonists; D002955:Leucovorin / Q000494:pharmacology*; D007939:Leukemia L1210 / Q000201:enzymology; D008727:Methotrexate / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051379:Mice; D011099:Polyglutamic Acid / Q000378:metabolism; D011799:Quinazolines / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism; D016597:Trimetrexate
D005493:Folic Acid Antagonists; D011799:Quinazolines; D013763:Tetrahydrofolates; D011099:Polyglutamic Acid; D013762:Tetrahydrofolate Dehydrogenase; D002955:Leucovorin; D016597:Trimetrexate; D008727:Methotrexate