Effects of formycin B on human lymphocyte deoxyribonucleic acid synthesis: Optimization of cell culture conditions

Abstract

We evaluated the effect of formycin B, an inhibitor of purine nucleoside phosphorylase (PNP), on DNA synthesis in phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes using various culture conditions. We found that the dose-response curve for formycin B inhibition of lymphocyte PNP activity was similar to that for the inhibition of DNA synthesis when formycin B was added to the cultures at the time of maximum DNA synthesis. Under these conditions formycin B inhibited protein synthesis less than DNA synthesis. We also evaluated the effects of substrates of PNP added at the time of maximum DNA synthesis; deoxyguanosine was inhibitory, whereas guanosine enhanced thymidine incorporation into DNA. To assess the potential use of formycin B in exploring lymphocyte toxicity in PNP deficiency, we evaluated its effects on DNA synthesis in combination with other nucleosides. Deoxyguanosine and formycin B, when used together, were not synergistic in their combined effects on DNA synthesis. Further, deoxycytidine did not prevent the inhibition by formycin B of DNA synthesis. Our results suggest a relationship between formycin B inhibition of PNP and DNA synthesis in PHA-stimulated lymphocytes. The role of formycin B in a human cell model of PNP deficiency, however, is limited by its effects on other enzyme systems and its relatively high K i for PNP. Its potential, if any, for exploration of lymphocyte toxicity in PNP deficiency remains to be defined.