This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 μM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 μM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrofolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (~30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 μM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effeect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyltetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5,10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate. These findings demonstratate that treatment of cells with methotrexate or trimetrexate suppresses the flow of one-carbon units through the de novo nucleotide and amino acid biosynthetic pathways, even when high levels of reduced folate cofactors are present. This appears to involve effects on specific folate-dependent biosynthetic reactions, including thymidylate synthase and the purine transformylases by methotrexate and/or dihydrofolate polyglutamates that accumulate in drug-treated cells. These inhibitory effects may account for the failure of 5-formyltetrahydrofolate to rescue tumor cells which have metabolized methotrexate to polyglutamates. Furthermore, the lack of appreciable build-up of methotrexate polyglutamates in normal cells of the bone marrow and gastrointestinal tract may account for the ability of reduced folates to reverse antifolate effects in these tissues and, hence, may account for the selectivity of rescue.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1987|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology