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Relationship of Reduced Folate Changes to Inhibition of DNA Synthesis Induced by Methotrexate in L1210 Cells in Vivo¹

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425 [D. G. P., M. B.], and Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [F. M. S.]

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/10⁶ 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/10⁶ 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.

¹ This research was supported in part by USPHS Grants CA 08748, CA 16153, CA 18856, and CA 22754 awarded by the National Cancer Institute and a grant from the Elsa V. Pardee Foundation.

² To whom requests for reprints should be addressed.

Received 8/30/88. Revised 2/16/89. Revised 4/18/89. Accepted 4/24/89.

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