Glutathione depletion in human T lymphocytes: analysis of activation-associated gene expression and the stress response

Glutathione depletion achieved by continuous exposure of mitogen-activated human T lymphocytes to L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, leads to a marked inhibition of the proliferative response. Concanavalin A-activated T cells treated with buthionine sulfoximine failed to exhibit the increase in glutathione content normally observed in activated T cells and were depleted of cellular glutathione over 4 days of culture. On Day 3 of culture, DNA synthesis was inhibited by greater than 75%. In addition, total RNA synthesis was dramatically reduced in the glutathione-depleted cells being inhibited by 26, 61, and 82% on Days 2, 3, and 4, respectively. Despite this global reduction in RNA synthesis, no specific effects on mRNA expression of a number of critical T cell genes required for activation and/or proliferation were detected. In contrast to a recent report of GSH depletion leading to down-regulation of ras mRNA expression in a number of transformed cell lines, glutathione depletion did not influence N-ras mRNA expression in T lymphocytes. No influence of glutathione depletion on the induction of histone mRNA expression was observed. However, consistent with previous studies on regulation of histone mRNA expression, histone transcript levels were reduced when DNA synthesis was markedly inhibited. A cellular stress response, characterized by an increase in mRNA levels of the two stress response genes, HSP70 and gadd 153, was evident in glutathione-depleted unstimulated cells. Additionally, in these cells at 48 hr, we observed a 3.5-fold increase in the steady-state level of mRNA encoding the catalytic subunit of gamma-glutamylcysteine synthetase, the enzyme inhibited by buthionine sulfoximine.