Deoxyadenosine (AdR) appears to be central to the molecular events mediating immunodeficiency in children born with adenosine deaminase (ADA) deficiency but it is still uncertain whether lymphotoxicity is due to AdR directly inhibiting transmethylation reactions in which S-adenosylmethionine is the methyl group donor, or is due to phosphorylation of AdR to deoxyadenosine triphosphate (dATP) which then inhibits ribonucleotide reductase or is due to other mechanisms. Using AdR and the ADA inhibitor deoxycoformycin (dCF) and assessing cell viability, nucleoside incorporation into RNA and DNA, as well as measuring deoxyribonucleoside triphosphate (dNTP) concentrations and S-adenosylhomocysteine (SAH) hydrolase activity, we have studied various types of human lymphoid cells and demonstrated in them the relative importance of the above two mechanisms of AdR toxicity. Treatment of normal resting peripheral blood lymphocytes in culture with AdR and dCF resulted in impaired viability. Although elevated dATP levels as well as decreased SAH hydrolase activities were both observed, the failure of a known inhibitor of ribonucleotide reductase (hydroxyurea) to produce toxicity, and the inability of deoxycytidine (CdR) to achieve a rescue effect, point to another mechanism, possibly inhibition of trans-methylation or ATP depletion being the more likely causes of toxicity in resting lymphocytes. The same mechanism may well account for the rapid and severe lymphopenia in patients treated with dCF. On the other hand, in cultured lymphoblasts in the exponential phase of growth. AdR and dCF produced marked inhibition of growth and cell death both in a Thy-ALL line and in a c-ALL line, in the absence of significant inhibition of SAH hydrolase, but with a substantial elevation in dATP concentrations and depressed levels of the other dNTP. Minor toxicity occurred in a proliferating B lymphoblast line despite almost complete inactivation of SAH hydrolase. These observations indicate inhibition of ribonucleotide reductase as the more likely mechanism of toxicity in rapidly proliferating lymphocytes. Other T-cells actively synthesizing DNA, such as PHA-stimulated or MLC activated lymphocytes and T-lymphoid colony forming cells, are also likely to be affected by the same mechanism. Indeed in PHA-stimulated lymphocytes, deoxycytidine caused significant although incomplete rescue from toxicity due to dCF and AdR. In patients with ADA deficiency or treated with ADA inhibitors, both mechanisms could be operative. These observations are also relevant to the possible use of dCF and AdR as immunosuppressive agents and for the removal of T-cells or residual Thy-ALL blasts from bone marr
In four patients with Thy-acute lymphoblastic leukaemia changes in blast cell deoxynucleoside triphosphate concentrations and, in three, changes in blast cell S-adenosyl homocysteine hydrolase activity were measured during treatment with 2' deoxycoformycin, a potent inhibitor of adenosine deaminase. These studies were aimed at identifying the molecular basis of cell killing by this drug. In three patients an increase in blast deoxyadenosine triphosphate (dATP) concentration occurred which was found to be temporally related to cell killing and was accompanied by decreased concentrations of the other three deoxyribonucleoside triphosphates. In the one patient with Thy-ALL who responded poorly to treatment, the increase in dATP concentration was delayed and was not accompanied by a fall in the concentrations of the other deoxyribonucleoside triphosphates. Progressive inactivation of blast cell S-adenosyl homocysteine hydrolase was found to occur in the three patients tested but was maximal only after a substantial reduction of peripheral blast cell count. These results show that 2' deoxycoformycin has a potent cytoreductive effect in Thy-ALL and suggest that the molecular basis of this toxicity is related both to the intracellular accumulation of dATP with inhibition of ribonucleotide reductase. Inactivation of S-adenosyl homocysteine hydrolase may be of importance as an additional mechanism.
Summary. Deoxyribonucleoside triphosphate (dNTP) concentrations were measured in bone marrow and peripheral blood leucocytes from seven patients with acute Thy‐lymphoblastic leukaemia (Thy‐ALL), 12 patients with acute myeloblastic leukaemia and 15 patients with acute non‐T, non‐B lymphoblastic leukaemia (c‐ALL), and in thymocytes from patients with myasthenia gravis. Labelled thymidine and deoxycytidine incorporation into DNA was also studied. In Thy‐ALL, dNTP concentrations were markedly increased compared with those in the other acute leukaemias. The dNTP concentrations in thymocytes were, however, similar to those in Thy‐ALL. 3H‐nucleoside incorporation studies showed a marked difference in labelled deoxycytidine incorporation and particularly in the deoxycytidine/thymidine DNA labelling ratio between Thy‐ALL and the other cell types. We conclude that the pathways of DNA synthesis in Thy‐ALL blasts are different from those in the cells from other acute leukaemias and some but not all these differences may correspond to differences between normal cortical thymocytes and bone marrow cells.
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