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

Abstract

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.

Cytotoxic Activity of Cobalamin in Cultured Malignant and Nonmalignant Cells

“…Adenosylco balamin is required in many rearrangement reactions and in the ribonucleotide reduction reaction which provides the building blocks for the synthesis of DNA in microor ganisms [20], Vitamin B|2 was able to correct defective DNA-thymine synthesis in vitamin B|2-defective marrow [28]. In patients with folate and vitamin Bi2 deficiency, the addition of these vitamins to marrow and lymphocyte cultures enhanced the incorporation of 3H-deoxyuridine into DNA [29]. However, it is not known whether the cytotoxic effect of adenosylcobalamin was associated with the positive and negative control of DNA synthesis.…”

Influence of Cobalamin on the Survival of Mice Bearing Ascites Tumor

“…It has been shown that the addition of vitamin B,i to food in amounts far in excess of need or absorbability appears to be without hazard. Cyanocobalamin has caused no toxic ity in animals at levels several thousand times their nutri tional requirements [29], Toxicity tests in our laboratory indicated that these three cobalimin derivatives were Values are means ± SD of survival times (days) for n = I0. Significance of the difference between control and experimental values: a p < 0.05; b p < 0.01; cp < 0.001.…”

Derangement of DNA Synthesis in Erythroleukaemia. Normal Deoxyuridine Suppression and Impaired Thymidine Incorporation in Bone Marrow Culture

“…dTMP, which, after a sequence of phosphorylation forms dTTP and is uti lized for DNA synthesis, is in turn produced either by de novo synthesis from carbamylasparate or by phosphorylation of exogen ous thymidine by the salvage pathway. The two alternative pathways of thymidylate synthesis, the de novo and the salvage path ways, are interrelated by a common end product, dTTP which exerts a regulatory in fluence on both pathways by a feedback in hibition [17], This also accounts for the re ciprocity of the two pathways [9], In defi ciency of vitamin B12 and folate or in the presence of folate antagonists such as MTX, the impairment of the de novo pathway leads to increased activity of the salvage en zyme, thymidine kinase [16,22], which in turn promotes increased incorporation of exogenous thymidine into DNA. In con trast, the megaloblastoid erythroblasts in ery throleukaemia marrows showed reduced incorporation of exogenous sH-TdR as compared to normal as well as vitamin B12/ folate-deficient megaloblastic bone mar rows.…”

“…The effect of methotrexate (MTX) on 3H-TdR incorporation into DNA was studied by preincu bating replicate cultures with and without MTX (10~4 to 10"eM) followed by addition of 1 ,«Ci 3H-TdR (21 Ci/mmol) and incubating the cultures for a further 3 h. DNA extraction and measure ment of radioactivity were done as described above [9,14,21].…”