The effect of methotrexate on levels of dUTP in animal cells.

Abstract

The amounts of intracellular nucleotides of dThd and dUrd were measured in cultured human lymphoblasts with and without treatment with methotrexate (plus hypoxanthine) to inhibit thymidylate synthetase. dTTP fell from approximately 40 pmol/10(6) cells (untreated) to approximately 1 pmol/10(6) cells with drug treatment. dUMP, increased approximately 10(3)-fold with methotrexate. dUTP was not detected in the cell without drug treatment (less than or equal to 0.3 fmol/10(6) cells), but was easily measured at approximately 0.2 pmol/10(6) cells in drug-inhibited cells. Thus the ratio dUTP/dTTP incrased from less than or equal to 10(-5) in untreated cells to approximately 0.2 in cells treated with methotrexate, indicating that dUMP may be incorporated in substantial amounts into the DNA of the drug-treated cells. The magnitude of these effects on nucleotide pools suggests the possibility that the normal nucleolytic mechanism for removal of dUMP from DNA may play a part in the toxic effects on the cell that result from depression of thymidylate synthetase activity.