Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20-to 45-fold greater capacity to synthesize deoxyadenosine nucleotides than B lymphoblasts at deoxyadenosine concentrations of 50-300 ,uM. During the synthesis of dATP, T lymphoblasts accumulate large quantities of dADP, whereas B lymphoblasts do not accumu ate dADP. Enzymes affecting deoxyadenosine nucleotide synthesis were assayed in these cells. No substantial differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7. Deoxyadenosine and adenosine concentrations are increased in individuals with adenosine deaminase deficiency and severe combined immunodeficiency disease (1, 2). The accumulation of deoxyadenosine leads to the increased concentrations of dATP and dADP in erythrocytes, peripheral blood lymphocytes, and bone marrow cells of these patients (2-4). The elevated levels of deoxyadenosine nucleotides are believed to provide the biochemical basis for the immune dysfunction observed in the enzyme deficiency state.Although the precise mechanism for the immune dysfunction is unclear, several studies in vitro have attempted to determine the underlying molecular pathology. The addition of deoxyadenosine reduces the response of peripheral blood lymphocytes to mitogen stimulation when adenosine deaminase is inhibited (1, 5). The combination of deoxyadenosine and adenosine deaminase inhibition is also cytotoxic to T lymphoblasts but not B lymphoblasts (6, 7). Deoxyadenosine-mediated cytotoxicity in T lymphoblasts is accompanied by increased concentrations of dATP.Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. These studies have revealed a major difference in the capacity of T lymphoblasts to accumulate deoxyadenosine nucleotides compared to B lymphoblasts, possibly because of a difference in activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). MATERIALS AND METHODSReagents. Deoxyadenosine, deoxyinosine, ATP, dATP, dADP, dAMP, dIMP, Tris base, dithiothreitol, and EDTA were purchased from Sigma. Adenine and hypoxanthine were obtained from Calbiochem. Erythro-9-[3-(2-hydroxynonyl)]-adenine (EHNA) was a gift from G. B. Deoxyadenosine Metabolism. T and B lymphoblasts were removed from flasks in which they were grown in continuous culture. Cells were washed twice in normal saline and 100 mM Tris-HCl, pH 7.4. Cells were suspended in Eagle's minimal essential medium containing 10% dialyzed horse serum, 1.2 mM potassium phosphate at pH 7.4, 25 mM Tris-HCl at pH 7.4, and 0 or 5.0 ,uM EHNA. Cell counts were performed on the cell suspensions.Aliquots (50 ,l) of cell suspension (2.0-6.0 X 106 cells per ml) were incubated at 37°C for 20-35 min, removed and pl...
A B S T R A C T In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with <1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function.Dr. Mitchell is the recipient of a scholar award from the Leukemia Society of America.
The toxicity of the deoxyribonucleosides, 2′-deoxyadenosine, 2′- deoxyguanosine, and thymidine, for human T lymphoblasts is mediated by the accumulation of the corresponding deoxyribonucleoside triphosphate (dATP, dGTP, or dTTP, respectively). We have examined whether leukemic cells of non-T-cell origin are capable of accumulating deoxyribonucleotides in culture and whether this capability correlates with the activities of purine metabolizing enzymes in these cells. We have found that non-T, non-B acute lymphoblastic leukemia cells with low ecto-5′-nucleotidase and high adenosine deaminase activities increase their dATP pools by greater than tenfold when exposed to deoxyadenosine and an inhibitor of adenosine deaminase in culture. Cells from 2 of 9 patients with chronic lymphocytic leukemia and 4 of 11 patients with acute nonlymphoblastic leukemia achieved similar elevations in dATP, but there was no relationship between dATP accumulation and adenosine deaminase, purine nucleoside phosphorylase, or ecto-5′-nucleotidase activities. Treatment of four individuals with acute lymphoblastic leukemia with the adenosine deaminase inhibitor, 2′- deoxycoformycin, resulted in elevations in plasma deoxyadenosine concentrations and in increments in lymphoblast dATP levels that were similar to those measured in lymphoblasts cultured with deoxyadenosine and deoxycoformycin prior to treatment. In vitro incubations of leukemic cells with deoxyribonucleosides may provide a rational basis for the use of these compounds as chemotherapeutic agents.
The toxicity of the deoxyribonucleosides, 2′-deoxyadenosine, 2′- deoxyguanosine, and thymidine, for human T lymphoblasts is mediated by the accumulation of the corresponding deoxyribonucleoside triphosphate (dATP, dGTP, or dTTP, respectively). We have examined whether leukemic cells of non-T-cell origin are capable of accumulating deoxyribonucleotides in culture and whether this capability correlates with the activities of purine metabolizing enzymes in these cells. We have found that non-T, non-B acute lymphoblastic leukemia cells with low ecto-5′-nucleotidase and high adenosine deaminase activities increase their dATP pools by greater than tenfold when exposed to deoxyadenosine and an inhibitor of adenosine deaminase in culture. Cells from 2 of 9 patients with chronic lymphocytic leukemia and 4 of 11 patients with acute nonlymphoblastic leukemia achieved similar elevations in dATP, but there was no relationship between dATP accumulation and adenosine deaminase, purine nucleoside phosphorylase, or ecto-5′-nucleotidase activities. Treatment of four individuals with acute lymphoblastic leukemia with the adenosine deaminase inhibitor, 2′- deoxycoformycin, resulted in elevations in plasma deoxyadenosine concentrations and in increments in lymphoblast dATP levels that were similar to those measured in lymphoblasts cultured with deoxyadenosine and deoxycoformycin prior to treatment. In vitro incubations of leukemic cells with deoxyribonucleosides may provide a rational basis for the use of these compounds as chemotherapeutic agents.
Adenosine deaminase (ADA) is an enzyme in the purine catabolic pathway that has been used as an enzymatic marker of T cell lymphoblastic malignancies due to its high specific activity in thymocytes and immature T cells. We have investigated whether the level of ADA activity in lymphoid leukemic cells correlates with the amount of ADA- specific RNA and/or immunoreactive protein in these cells as an initial step toward characterizing the nature of the genetic regulation of ADA expression during differentiation. We have found a good correlation between the steady state levels of ADA-specific RNA and ADA- immunoreactive protein in T lymphoblastic leukemic cell lines, mature T cell lines, a B lymphoblast cell line, and leukemic cells directly isolated from four patients with acute lymphoblastic leukemia and three patients with chronic lymphocytic leukemia. Southern blot analysis of DNA from these cells shows no evidence for differences in ADA gene copy number or gene rearrangement to account for the variability in ADA expression. We conclude that levels of ADA in lymphoid leukemic cells are directly related to the amount of ADA-specific mRNA present. These findings imply that ADA expression in leukemic cells reflects either the transcriptional activity of the ADA gene or the stability of ADA mRNA in these cells.
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