Tumor necrosis factor-alpha and Fas activate complementary Fas-associated death domain-dependent pathways that enhance apoptosis induced by gamma-irradiation

Activation of either tumor necrosis factor receptor I or Fas induces a low level of programmed cell death in LNCaP human prostate cancer cells. We hav e shown that LNCaP cells are entirely resistant to gamma-radiation-induced apoptosis, but can be sensitized to irradiation by TNF-alpha. Fas activatio n also sensitized LNCaP cells to irradiation, causing nearly 40% cell death 72 h after irradiation. Caspase-8 was cleaved and activated after exposure to tumor necrosis factor (TNF)-alpha. However, after exposure to anti-Fas antibody caspase-8 cleavage occurred only between the 26-kDa N-terminal pro domain and the 28-kDa C-terminal region that contains the protease componen ts. Although anti-Fas antibody plus irradiation induced apoptosis that coul d be blocked by the pancaspase inhibitor zVAD, there was no measurable casp ase-8 activity after exposure to anti-Fas antibody. The effector caspases-6 and -7, and to a lesser extent caspase-3, were activated by TNF-alpha, but not by anti-Fas antibody. Anti-Fas antibody, like TNF-or also activated se rine proteases that contributed to cell death. Exposure of LNCaP cells simu ltaneously to TNF-alpha and anti-Fas antibody CH-11 resulted in marked enha ncement of apoptosis that occurred very rapidly and was still further augme nted by irradiation. Rapid apoptosis that ensued from combined treatment wi th TNF-alpha, anti-Fas antibody, and irradiation was completely blocked eit her by zVAD or expression of dominant negative Fas-associated death domain. Our data shows that there are qualitative differences in caspase activatio n resulting from either TNF receptor 1 or Fas, Simultaneous activation of t hese receptors was synergistic and caused rapid epithelial cell apoptosis m ediated by the caspase cascade.