Biochemical mechanisms in the Killmann experiment: critique of the deoxyuridine suppression test

The degree of inhibition of [3H]thymidine incorporation into DNA by exogenous deoxyuridine is assayed in a procedure known as the deoxyuridine suppression test. We report studies of the biochemical basis of this phenomenon in phytohemagglutinin-stimulated lymphocytes, which suggest that its mechanism has not been fully understood. Results show that inhibition by deoxyuridine is caused only in part by expansion of the intracellular pools of nonradioactive dTMP and dTTP, which dilutes the specific radioactivity of the [3H]dTMP and [3H]dTTP derived from [3H]thymidine. Increased dTTP levels also inhibit thymidine kinase. In addition, thymidine kinase is competitively inhibited by intracellular deoxyuridine. Inhibition of thymidine kinase activity by both mebolites further decreases the specific radioactivity of [3H]dTMP and [3H]dTTP. Deoxyuridine also inhibits the incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into DNA in these cells. Exogenous deoxyuridine still inhibits [3H]thymidine incorporation in cells whose de novo thymidylate synthesis has been strongly inhibited by 5-fluorodeoxyuridine or methotrexate. In such drug-treated cells, exposure to high concentrations of exogenous deoxyuridine can partially overcome the inhibition of thymidylate synthetase with resulting increase in the severely depleted dTTP pools. This increase is associated with enhanced DNA synthesis, as measured by incorporation into DNA of labeled deoxyribonucleosides other than [3H]thymidine. We conclude that exogenous deoxyuridine has multiple effects on [3H]thymidine incorporation, which must be considered in interpretations of deoxyurindine suppression test results.