Relationship of reduced folate changes to inhibition of DNA synthesis induced by methotrexate in L1210 cells in vivo.

Reduced folate levels and DNA synthesis were examined in L1210 cells in mice after exposure to a wide range of methotrexate doses. A radioenzymatic assay based upon entrapment of tissue 5,10-methylenetetrahydrofolate (CH2FH4), and other reduced folates after cycling to this form, into a stable ternary complex with thymidylate synthase and [3H]-5-fluoro-2'-deoxyuridine 5'-monophosphate was used to estimate reduced folates. DNA synthesis was estimated from incorporation of [3H]-2'-deoxyuridine into DNA. The predominant reduced folate in cells from untreated animals was 10-formyltetrahydrofolate (10-CHOFH4; 3.59 pmol/10(6) cells). The four other reduced folates measured, tetrahydrofolate (FH4), CH2FH4, dihydrofolate (FH2), and 5-methyltetrahydrofolate (5-CH3FH4), were present in nearly equal amounts yielding a total reduced folate level of 6.24 pmol/10(6) cells. When methotrexate was administered s.c. at doses of 1.5, 12, and 400 mg/kg, the level of FH2 increased dramatically and total tetrahydrofolates measured decreased extensively within 1 h. DNA synthesis was completely inhibited during the first 1-2 h after administration of each dose of methotrexate with onset of recovery after 4 and 20 h at 1.5 and 12 mg/kg and not at all after the highest dose. Both FH4 and CH2FH4 were extensively depleted at 12 and 400 mg/kg methotrexate but considerably less depletion of CH2FH4, and none of FH4, was observed at 1.5 mg/kg during a period when DNA synthesis was essentially abolished. The metabolically linked 5-CH3FH4 and 10-CHOFH4 pools were also extensively depleted following treatment with methotrexate. While FH2 levels expanded extensively after drug treatment, the total increase did not account for the extent of depletion of the combined tetrahydrofolate pools. The change in concentration with time of any one folate pool was apparently not sufficient to explain completely the duration of inhibition of DNA synthesis; however, sustained inhibition of DNA synthesis was generally associated with maintenance of an expanded FH2 pool and delay in repletion of the combined tetrahydrofolate pools. Discussion is presented with respect to the impact of these results on changing notions of the mode of action of classical antifolates.