Cycloheximide-induced T-cell death is mediated by a Fas-associated death domain-dependent mechanism

Cycloheximide (CHX) can contribute to apoptotic processes, either in conjun ction with another agent (e.g. tumor necrosis factor-alpha) or on its own. However, the basis of this CHX-induced apoptosis has not been clearly estab lished. In this study, the molecular mechanisms of CHX-induced cell death w ere examined in two different human T-cell lines. In T-cells undergoing CHX -induced apoptosis (Jurkat), but not in T-cells resistant to the effects of CHX (CEM C7), caspase-8 and caspase-3 were activated. However, the Fas lig and was not expressed in Jurkat cells either before or after treatment with CHX suggesting that the activation of these caspases does not involve the Fas receptor. To determine whether CHX-induced apoptosis was mediated by a Fas-associated death domain (FADD)-dependent mechanism, a FADD-DN protein w as expressed in cells prior to CHX treatment. Its expression effectively in hibited CHX-induced cell death, suggesting that CHX-mediated apoptosis prim arily involves a FADD-dependent mechanism. Since CHX treatment did not resu lt in the induction of Fas or Fast, and neutralizing anti-Fas and anti-tumo r necrosis factor receptor-1 antibodies did not block CHX-mediated apoptosi s, these results may also indicate that FADD functions in a receptor-indepe ndent manner. Surprisingly, death effector filaments containing FADD and ca spase-8 were observed during CHX treatment of Jurkat, Jurkat-FADD-DN, and C EM C7 cells, suggesting that their formation may be necessary, but not suff icient, for cell death.