Abstract
Mouse L1210 cell variants were selected for resistance to 5, 10-dideazatetrahydrofolate, a potent inhibitor of the first folate-dependent enzyme in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. The drug-resistant phenotype selected was conditional to the folate compound used to support growth: grown on folic acid cells were 400-fold resistant, whereas they were 2.5-fold more sensitive to 5,10-dideazatetrahydrofolate than wild-type L1210 cells when grown on folinic acid. In folic acid-containing media, polyglutamation of 5, 10-dideazatetrahydrofolate was markedly reduced, yet folylpolyglutamate synthetase activity was not different from that in parental L1210 cells. Resistance was due to two changes in membrane transport: a minor increase in the Km for 5, 10-dideazatetrahydrofolate influx, and a major increase in folic acid transport. Enhanced folic acid transport resulted in an expanded cellular content of folates which blocked polyglutamation of 5,10-dideazatetrahydrofolate. We propose that polyglutamation of 5,10-dideazatetrahydrofolate is limited by feedback inhibition by cellular folates on folylpolyglutamate synthetase, an effect which reflects a mechanism in place to control the level of cellular folates. Although the primary alteration causative of resistance is different from those reported previously, all 5, 10-dideazatetrahydrofolate resistance phenotypes result in decreased drug polyglutamation, reflecting the centrality of this reaction to the action of 5,10-dideazatetrahydrofolate.
Highlights
Mouse L1210 cell variants were selected for resistance to 5,10-dideazatetrahydrofolate, a potent inhibitor of the first folate-dependent enzyme in de novo purine synthesis, glycinamide ribonucleotide formyltransferase
We describe a unique mechanism by which murine leukemic L1210 cells develop resistance to (6R)DDATHF: an expansion of the intracellular folate pool with consequent blockade of the synthesis of (6R)-DDATHF polyglutamates
The inhibition of growth of L1210/D3 cells was reversed by either hypoxanthine or aminoimidazole carboxamide, indicating that the target of (6R,6S)-DDATHF in these cells was glycinamide ribonucleotide formyltransferase (GARFT), as was the case in parental L1210 cells [34]
Summary
Compared with methotrexate (MTX), the classical antifolate targeted toward dihydrofolate reductase, (6R)-DDATHF, ZD1694, and MTA are metabolized more rapidly and extensively to long chain polyglutamates by the enzyme folylpolyglutamate synthetase (FPGS) (2, 9, 14 –16) The metabolism of these drugs to their polyglutamate derivatives is essential for their cellular retention and these polyglutamates have been reported to be substantially more potent inhibitors of their respective target enzymes [4, 6, 7, 14, 15]. We describe a unique mechanism by which murine leukemic L1210 cells develop resistance to (6R)DDATHF (and cross-resistance to ZD-1694): an expansion of the intracellular folate pool with consequent blockade of the synthesis of (6R)-DDATHF polyglutamates This unexpected mutant phenotype appears due to a suspected but heretofore unproven feedback control mechanism on the synthesis of celbonucleotide formyltransferase; MTX, methotrexate; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; 2-ME, 2-mercaptoethanol; MTA, N-{4-[2-(2-amino-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid; FCS, fetal calf serum; CHO, Chinese hamster ovary; FdUMP, 5-fluoro-2Ј-deoxyuridine 5Ј-monophosphate; FUdR, 5-fluorodeoxyuridine. Lular (anti)folate polyglutamates, presumably by direct effects of cellular folates on FPGS
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