An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells.

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.