Abstract
Purine and pyrimidine deoxyribonucleoside metabolism was studied in G1 and S phase human thymocytes and compared with that of the more mature T lymphocytes from peripheral blood. Both thymocyte populations have much higher intracellular deoxyribonucleoside triphosphate (dNTP) pools than peripheral blood T lymphocytes. The smallest dNTP pool in S phase thymocytes is dCTP (5.7 pmol/10(6) cells) and the largest is dTTP (48 pmol/10(6) cells), whereas in G1 thymocytes, dATP and dGTP comprise the smallest pools. While both G1 and S phase thymocytes have active deoxyribonucleoside salvage pathways, only S phase thymocytes have significant ribonucleotide reduction activity. We have studied ribonucleotide reduction and deoxyribonucleoside salvage in S phase thymocytes in the presence of extracellular deoxyribonucleosides. Based on these studies, we propose a model for the interaction of deoxyribonucleoside salvage and ribonucleotide reduction in S phase thymocytes. According to this model, extracellular deoxycytidine at micromolar concentrations is efficiently salvaged by deoxycytidine kinase. However, due to feedback inhibition of deoxycytidine kinase by dCTP, the maximal level of dCTP which can be achieved is limited. The salvage of both deoxyadenosine and deoxyguanosine (up to 10(-4) M) is completely inhibited in the presence of micromolar concentrations of deoxycytidine, whereas the salvage of thymidine is unregulated resulting in large increases in dTTP levels. Moreover, significant amounts of the salvaged deoxycytidine is used for dTTP synthesis resulting in further increase of dTTP pools. The accumulated dTTP inhibits the reduction of UDP and CDP while stimulating GDP reduction and subsequently also ADP reduction. The end result of the proposed model is that S phase thymocytes in the presence of a wide range of extracellular deoxyribonucleoside concentrations synthesize their pyrimidine dNTP by the salvage pathway, whereas purine dNTPs are synthesized primarily by ribonucleotide reduction. Using the proposed model, it is possible to predict the relative intracellular dNTP pools found in fresh S phase thymocytes.
Highlights
Purine andpyrimidine deoxyribonucleoside metab- osine deaminase and purine nucleoside phosphorylase defiolism was studied iGn I and S phase human thymocytes ciencies (1-7)
Numerous studies have shown that T leukemic and compared with thatof the more mature Tlympho- cells as well as immature T cells of the thymus areexquisitely cytes from peripheral blood
Activities of deoxyribonucleotide degradation enzymes are low in T leukemic cells and in thymocytes (12-17), whereas the activity of the deoxyribonucleoside salvage enzyme, deoxycytidine kinase, is highest in thymocytes and decreases during T lymphocyte differentiation (15, 17)
Summary
From the Division of Immunology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. Thymocytes is dCTP (5.7 pmol/106cells) and the largest is dTTP (48 pmol/106 cells)w, hereas inGI thymocytes, dATP and dGTP comprise the smallest pools While both GI and S phase thymocytes have active deoxyribonucleoside salvage pathways, only S phase thymocytes have significant ribonucleotide reduction activity. The end result of the proposed model is that S phase thymocytes in the presenceof a wide range of extracellular deoxyribonucleoside concentrations synthesize their pyrimidine dNTP by the salvage pathway, whereas purine dNTPs are synthein vitro studies of deoxyribonucleotide metabolism in cultured cell lines emphasize the contribution of ribonucleotide reduction and de-emphasize the contribution of deoxyribonucleoside salvage. We have studied the salvage of individual proposed model, it is possible to predict the relative purine and pyrimidine deoxyribonucleosides inrelation to intracellular dNTP pools found in fresh S phase thy- ribonucleotide reduction, deoxyribonucleoside triphosphate mocytes. Poly[d(A,T)],poly[d(G,C)], and Escherichia coli DNA polymerase I were purchased from Miles Chemical Co
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