In vitro DNA synthesis by bone marrow cells and PHA-stimulated lymphocytes. Suppression by nonradioactive thymidine of the incorporation of 3H-deoxyuridine into DNA: enhancement of incorporation when inadequate vitamin B12 or folate is corrected.

Previous studies demonstrated that excess deoxyuridine (dU) added to short-term bone marrow and PHA-stimulated lymphocyte cultures, blocks the incorporation of radioactive thymidine into DNA via the salvage pathway. In the current study, we investigated the effects of added thymidine (TdR) in varying concentrations (10(-6) to 1 mumol) on the incorporation of 3H-dU into thymine-DNA, i.e. we executed 'thymidine suppression tests.' Increasing concentrations of exogenous TdR caused progressive inhibition of 3H-dU incorporation into DNA, and decreasing 3H-dU incorporation was parallelled by increasing incorporation of added 14C-TdR. These findings demonstrate reciprocity of the salvage and the de novo pathways of thymine-DNA synthesis, presumably mediated by thymidine-triphosphate (dTTP), the common end product of both pathways, via feedback inhibition. In patients with folate and/or vitamin B12 deficiency, the addition of appropriate vitamins to marrow and lymphocyte cultures enhanced the incorporation of 3H-dU into DNA. As predicted, this was not observed in normal subjects. The enhancing effect of these vitamins on in vitro incorporation of 3H-dU into DNA by deficient cell systems was similar to their correcting effect on abnormal dU suppression. These findings support the theoretical concept that the dU suppression test defines biochemical megaloblastosis due to deficiency of folate and vitamin B12.