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Reversal of Enzymic Phenotype of Thymidine Metabolism in Induced Differentiation of U-937 Cells¹

First Department of Internal Medicine, Kagawa Medical School, Ikenobe, Miki, Kagawa 761-07, Japan

Exposure of U-937 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in specific alterations in thymidine metabolism. Within 24 h after treatment with 1.62 x 10^-9 M TPA, the reciprocal alteration in the activities of opposing enzymes of thymidine metabolism observed during normal cell culture growth was reversed. In TPA-treated cells, the activities of anabolic enzymes thymidine kinase (EC 2.7.1.75) and thymidylate synthase (EC 2.1.1.45) declined with time linearly to 20 and 16% of those of untreated cells by 72 h. Incorporation of [³H]thymidine and [³H]deoxyuridine into acid-insoluble fractions also decreased in parallel with the decline in enzyme activities. In contrast, the activities of catabolic enzymes thymidine phosphorylase (EC 2.4.2.4) and dihydrothymine dehydrogenase (EC 1.3.1.2) increased. The rise in thymidine phosphorylase activity peaked at 48 h with 406% elevation over the control. The activity of dihydrothymine dehydrogenase was not altered for the first 24 h, but it increased up to 338% by 96 h. Immunotitration of dihydrothymine dehydrogenase with monoclonal antibody against this enzyme showed that the rise in activity in the differentiated cells was due to the increase in the amount of enzyme protein. No significant difference was observed in the K_m values for the substrate of each enzyme between untreated and TPA-treated cells. These metabolic alterations during induced differentiation were in line with the changes in cell morphology and accompanied by an accumulation of the cells in G₁ at the expense of S phase. These observations indicate that induced differentiation of U-937 cells results in a reversal of the enzymic phenotype of thymidine metabolism and suggest that emergence of thymidine metabolic imbalance may serve as an early marker of differentiation of these cells.

¹ This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (59480265).

² To whom requests for reprints should be addressed.

Received 6/16/89. Accepted 9/ 6/89.