adenosine-kinase and 2--deoxyadenosine-triphosphate

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Kinase; Antigens, CD34; Apoptosis; Cells, Cultured; Deoxyadenine Nucleotides; Deoxycytidine Kinase; Enzyme Inhibitors; Humans; Infant; Precursor Cells, T-Lymphoid; Thymus Gland

Thymocyte development past the CD4(-)CD8(-) stage is markedly inhibited in adenosine deaminase-deficient (ADA-deficient) murine fetal thymic organ cultures (FTOCs) due to the accumulation of ADA substrates derived from thymocytes failing developmental checkpoints. Such cultures can be rescued by overexpression of Bcl-2, suggesting that apoptosis is an important component of the mechanism by which ADA deficiency impairs thymocyte development. Consistent with this conclusion, ADA-deficient FTOCs were partially rescued by a rearranged T cell receptor beta transgene that permits virtually all thymocytes to pass the beta-selection checkpoint. ADA-deficient cultures were also rescued by the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine (5'A5'dAdo), indicating that the metabolite responsible for the inhibition of thymocyte development is not adenosine or deoxyadenosine, but a phosphorylated derivative of an ADA substrate. Correction of ADA-deficient FTOCs by 5'A5'dAdo correlated with reduced accumulation of dATP, implicating this compound as the toxic metabolite. In ADA-inhibited FTOCs rescued with a Bcl-2 transgene, however, dATP levels were superelevated, suggesting that cells failing positive and negative selection continued to contribute to the accumulation of ADA substrates. Our data are consistent with dATP-induced mitochondrial cytochrome c release followed by apoptosis as the mechanism by which ADA deficiency leads to reduced thymic T cell production.

Topics: Adenosine Deaminase; Adenosine Kinase; Adenosylhomocysteinase; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Survival; Deoxyadenine Nucleotides; Deoxyadenosines; Hydrolases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Proto-Oncogene Proteins c-bcl-2; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Receptor, Adenosine A3; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Purinergic P1; Thymus Gland

The biochemical and metabolic effects of deoxycoformycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 X 10(-3) M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxycoformycin achieved was 0.4 to 0.5 microM when the extracellular concentration was 1 microM. At 10 microM extracellular concentration, the intracellular concentration was 3-4 microM. Although deoxycoformycin at very low concentrations (1 or 10 microM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphosphate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabolism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or deoxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may account, at least in part, for the differences that are observed between the pharmacologic inhibition of adenosine deaminase, and the inherited deficiency of adenosine deaminase.

Topics: Adenosine Deaminase Inhibitors; Adenosine Kinase; Chromatography, High Pressure Liquid; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxycytidine Kinase; DNA; Humans; Nucleoside Deaminases; Pentostatin; Phosphorylation; Ribonucleosides; RNA; T-Lymphocytes; Vidarabine

Accumulation of intracellular deoxyadenosine triphosphate and inactivation of the enzyme S-adenosylhomocysteine hydrolase by deoxyadenosine have been suggested as molecular mechanisms for lymphoid toxicity of inherited or acquired deficiency of adenosine deaminase. The relative roles of these two deoxyadenosine-mediated effects for lymphotoxicity have been explored by employing mutant human T- and B-lymphoblasts deficient in either adenosine kinase, deoxycytidine kinase, or both. At low concentrations (less than 25 mumol/L) of deoxyadenosine or ara-adenine, deoxycytidine kinase deficiency decreases growth sensitivity of human T-lymphoblasts to deoxyadenosine approximately fourfold, and to ara-adenine approximately twofold. Loss of both activities completely eliminates deoxyadenosine phosphorylation and cellular dATP accumulation, and decreases deoxyadenosine growth sensitivity approximately 200-fold and ara-adenine sensitivity approximately 80-fold. The inactivation by deoxyadenosine of intracellular S-adenosylhomocysteine hydrolase activity of human adenosine deaminase-deficient B-lymphoblasts and wild-type or deoxycytidine kinase-deficient T-lymphoblasts is comparable, despite the differing toxicity of this compound for these cell lines. Adenosine kinase deficiency in T-lymphoblasts results in resistance to 2'-deoxyadenosine--but not ara-adenine--associated inactivation of S-adenosylhomocysteine hydrolase, and this compound produces comparable degrees of inactivation of S-adenosylhomocysteine hydrolase in both the wild-type and double mutant cells, despite markedly different growth sensitivity. For B-lymphoblasts, 2'-deoxyadenosine together with adenosine produces comparable growth inhibition of wild-type and adenosine kinase-deficient cells, and this inhibition is more marked than with adenosine alone, but is independent of S-adenosylhomocysteine hydrolase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Kinase; Adenosylhomocysteinase; B-Lymphocytes; Cell Line; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxycytidine Kinase; Humans; Hydrolases; Phosphotransferases; Purine Nucleosides; T-Lymphocytes; Vidarabine

Topics: Adenosine Kinase; Cell Line; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxycytidine; Deoxycytidine Kinase; Humans; Kinetics; Leukemia, Lymphoid; Mutation; Phosphotransferases; T-Lymphocytes