Inhibition of Methylation by Adenosine in Adenosine Deaminase-Inhibited, Phytohemagglutinin-Stimulated Human Lymphocytes

Abstract

Abstract Adenosine toxicity was studied in phytohemagglutinin-stimulated, normal human, peripheral lymphocytes in order to determine whether inhibition of S-adenosylmethionine (AdoMet)-mediated transmethylation reactions might contribute to the immune defect associated with heritable deficiency of the enzyme adenosine deaminase (ADA). Cells were treated with 20 µM erythro-9-(2-hydroxy-3-nonyl)adenine to inhibit ADA and with 20 µM uridine to prevent the pyrimidine starvation known to be caused by adenosine in this system. The further addition of 25 to 100 µM adenosine inhibited the uptake of radiolabeled thymidine and leucine into acid precipitable material, and also increased intracellular levels of S-adenosylhomocysteine (AdoHcy) from 3- to 8-fold. The formation in these experiments of AdoHcy, a potent inhibitor of AdoMet-mediated transmethylation reactions, presumably occurs through the action of S-adenosylhomocysteine hydrolase. Although the physiologic function of this enzyme is to hydrolyze AdoHcy, the reversibility of the reaction and an equilibrium constant favoring synthesis allow net synthesis of AdoHcy under conditions of adenosine excess. Inclusion of 100 to 200 µM L-homocysteine thiolactone in our experiments enhanced AdoHcy accumulation and also potentiated the inhibitory effects of adenosine on radiolabeled thymidine and leucine uptake. Cytotoxic concentrations of adenosine also caused significant inhibition of in vivo DNA methylation, a process known to be AdoMet-mediated, and a good correlation between this effect and AdoHcy levels was noted. These findings indicate that adenosine toxicity in cultured human lymphocytes is at least in part due to AdoHcy accumulation and resultant inhibition of essential AdoMet-mediated methylation reactions. The relevance of these findings to the pathogenesis of the immune defect in ADA-deficient patients is not established but deserves consideration.