Role of S-adenosylhomocysteine in adenosine-mediated toxicity in cultured mouse T lymphoma cells

Abstract

S-adenosylhomocysteine (SAH) is known to be a potent inhibitor of S-adenosylmethionine (SAM)-mediated reactions, of which SAH itself is a product. The immediate metabolic fate of SAH involves its hydrolysis to adenosine and L-homocysteine by the enzyme SAH hydrolase, but the reversibility of this reaction and its extremely low K eq in the hydrolytic direction suggest that under certain conditions of adenosine excess, SAH might accumulate with significant cytotoxic effects. We have used a model system consisting of cultured S49 mouse lymphoma cells together with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), to determine whether SAH is a mediator of adenosine cytotoxicity. Cells rendered resistant to adenosine-induced pyrimidine starvation by the addition of exogenous uridine or by the mutational loss of adenosine kinase are still sensitive to adenosine at concentrations >15 μM. We find that this effect is appreciably enhanced by the addition of L-homocysteine thiolactone to the culture medium. Cytotoxic concentrations of adenosine also cause significant elevations in intracellular levels of SAH, which are increased an additional several fold by 100μM exogenous L-homocysteine thiolactone. A fair correlation exists between a single time point determination of intracellular SAH and the degree of growth inhibition after 72 hr, but complicated time-dependent variations in SAH make it difficult to compare results obtained in the absence and presence of exogenous L-homocysteine thiolactone. In vivo DNA methylation in S49 cells is markedly inhibited by exposure of cells to concentrations of adenosine known to cause uridine-resistant cytotoxicity. This inhibition of methylation has been measured with short-term pulses of radiolabel, and correlates well with intracellular concentrations of SAH at all tested combinations of adenosine and L-homocysteine thiolactone. The results suggest that the uridine-resistant cytotoxic effects of adenosine on ADA-inhibited S49 cells are secondary to the inhibition of SAM-mediated methylation reactions by the adenosine metabolite SAH.