Molecular Pharmacology, Vol 18, 281-286, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Modulation of Methotrexate Toxicity by Thymidine: Sequence-Dependent Biochemical Effects

¹ Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223

Biochemical consequences of treatment with combinations of methotrexate (MTX) and thymidine (dThd) were examined in four mammalian cell lines in vitro. When 40 µM dThd was added to medium, 1 h after a lethal dose of MTX, fibroblasts and lymphoblasts were partially protected, but N₁S₁ tumor cells were not. Cytotoxicity of this combination correlated with cellular thymidylate synthetase activity, and in the presence of MTX, followed by dThd, N₁S₁ tumor cells accumulated dihydrofolate, whereas dihydrofolate accumulation was less extensive in the other cell lines. In fibroblasts, lymphoblasts, and N₁S₁ tumor cells, MTX caused a marked accumulation of cellular deoxyuridylate. This effect was decreased, but not eliminated, by administration of dThd 1 h after MTX. The effects of administration of dThd (40 µM) 2 h before MTX were markedly different from the effects of dThd after MTX; no accumulation of deoxyunidylate or dihydrofolate was observed, and the dThd pretreatment conferred a greater degree of protection from MTX toxicity than dThd "rescue" in fibroblasts, lymphoblasts, and N₁S₁ tumor cells. In N₁S₁ cells MTX had a pronounced antipunine effect, as measured by the decrease in dGTP pools; this effect was largely prevented by dThd pretreatment, but dThd administration after MTX restored the dTTP pool without abolishing the antipunine effect of the MTX. The abolition of the MTX antipurine effect by dThd pretreatment is attributed to decreased cellular thymidylate synthetase activity consequent upon the reduction in deoxyunidylate caused by dThd pretreatment. In cells of the well-differentiated Morris hepatoma 8999S, pretreatment with 40 µM dThd gave no protection from MTX, though partial protection was obtained at 100 µM dThd.

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ACKNOWLEDGMENT The author thanks Mr. L. Soliven for skilled technical assistance.