Biochemical basis for differential deoxyadenosine toxicity to T and B lymphoblasts: role for 5'-nucleotidase.

Abstract

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...

Decreased methionine synthesis in purine nucleoside-treated T and B lymphoblasts and reversal by homocysteine.

“…The deoxynucleosides were more potent inhibitors of methionine synthesis than were the cor- Rates of methionine synthesis were measured as in the legend to responding nucleosides, and T lymphoblasts were more sensitive to this methionine synthesis inhibition than were B lymphoblasts. These data, therefore, correlate with the results of growth studies that show that deoxynucleosides are more toxic to cultured lymphoblasts than are nucleosides and that T lymphoblasts are more sensitive to this toxicity than are B lymphoblasts (16)(17)(18). Moreover, since the adenine nucleosides inhibited methionine synthesis in both the T and B lymphoblasts and the guanine nucleosides inhibited methionine synthesis only in the T lymphoblasts, the data may provide a biochemical basis to explain the clinical differences between adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency.…”

Ecto-adenosine triphosphatase deficiency in cultured human T and null leukemic lymphocytes. A biochemical basis for thymidine sensitivity.

“…did not inhibit ecto-triphosphatase activity using ATP as substrate but -20% when dTTP was substrate (5,7). They have shown cultured leukemic T cell lines to be deficient in ecto-5'-nucleotidase compared with high levels in EBV-transformed B lymphocyte lines, and have correlated these findings with deoxynucleoside sensitivity and resistance.…”

“…We and others have reported that cultured leukemic lymphocytes of T and null cell type are extremely sensitive to growth inhibi-544 tion by TdR at concentrations two orders of magnitude below millimolar levels. The mechanisms of this increased sensitivity appear to reflect the ability of these leukemic cells to accumulate excess dTTP at low concentrations of exogenous TdR (5)(6)(7).…”

“…It has been postulated that these excess dNTP act as feedback inhibitors of ribonucleotide reductase, causing inhibition of DNA replication in lymphocyte precursors, resulting in immune defects (5,7,8 Deoxynucleoside inhibition of cell growth. This was performed as previously described by us (6).…”

Acute Lymphoblastic Leukemia

“…The levels of total dAdo-phosphorylating activity and of the two specific enzymes capable of catalyzing this reaction, dCyd kinase and Ado kinase, differ by no more than 2-to 4-fold in cytoplasmic extracts of a number of T-and B-cell lines (9,29). More rapid dAXP catabolism by B-cell lines than by T-cell lines has been proposed (7,10,61). However, the enzyme(s) specifi cally involved in dAXP catabolism has not been identified and, as with dAdo phosphorylation, only 2-to 4-fold differences in total cytoplasmic nucleotidase activity have been found in ex tracts of T-and B-cell lines (10).…”