Inhibition by methotrexate (MTX) polyglutamates (PGS) of folate-dependent biosyntheses in L1210 Leukemia cells

The inhibition of folate-dependent pathways by MTX PGS was evaluated in folate-depleted L1210 cells incubated with (6S)5-formyl(CHO)tetrahydrofolate(FH4)(5μM). The accumulation of MTX PGS during exposure to MTX (10μM;3h) inhibited cell growth (>70%) under these conditions. In the presence of 5-CHO-FH4, carbon transfer from 14C-formate or 3-14C-serine into purines, dTMP, and amino acids was suppressed following MTX-pretreatment, suggesting the formation of only low levels of FH4 to drive these reactions. In cells treated with MTX (6S)5-CHO-[3H]-FH4 was metabolized predominantly to 10-CHO-[3H]-FH4. While intracellular dihydrofolate (FH2) increased 10-fold, indicating a block at FH2 reductase by MTX PGS, FH2 represented only 20% of the total metabolites of 5-CHO-FH4. The incorporation of 14C from 5-[14C]-CHO-FH4 into serine and methionine was not affected by the presence of intracellular MTX PGS, however, carbon transfer into dTMP and purine nucleotides was reduced (50-60%). These findings demonstrate that MTX pretreatment inhibits de novo nucleotide and amino acid biosynthetic pathways even when high levels of reduced folates are present. The data suggest a suppression of dTMP synthase and the purine transformylase(s) by MTX and/or FH2 PGS that accumulate in drug-treated cells. Inhibition of the purine biosynthetic steps appears to trap 10-CHO-FH4, limiting FH4 for the synthesis of dTMP, serine, and methionine