In this report, we have shown that a derivative of human Jurkat cells that does not express caspase 8 was resistant to the apoptosis induced by the activation of endogenous Fas or oligomerization of the Fas death domain. These results indicate that caspase 8 is indispensable for the Fas-mediated apoptotic cell death process, which agrees with the recent results that embryonal fibroblasts from caspase 8 null mice are resistant to the Fas-induced apoptosis (Varfolomeev et al., 1998
). On the other hand, oligomerization of FADD, an adaptor molecule that normally mediates the Fas signal to caspase 8, could kill cells that were deficient in caspase 8. This pathway does not use caspases to kill the cells, and seems to operate even in the cells (the parental Jurkat cells) that express caspase 8, because a broad caspase inhibitor could not prevent the cell death induced by oligomerization of FADD.
The Fas-induced cell death is so far thought to transduce only apoptotic signal. On the other hand, TNF was shown to transduce not only an apoptotic signal, but also a necrotic signal. In particular, treatment of mouse L929 cells with TNF produces oxidants and kills the cells by inducing necrotic morphological changes of the cells (Vercammen et al., 1998a
). This process was shown to be enhanced by caspase inhibitors. More recently, Vercammen et al. (1998b)
established mouse L929 cells overexpressing human Fas. When these cells were treated with the agonistic anti–Fas antibody, they underwent apoptosis. But, caspase inhibitors could not prevent the cell death process, and the cells died by necrosis. This result is apparently in contrast to our observation that the Fas engagement alone cannot kill the caspase 8–deficient human Jurkat cells. However, the sensitivity of the cells to necrosis may depend on the strength of the death signal evoked by Fas or FADD, the expression level of the downstream signal transducer for necrosis, and/or the balance between apoptotic and necrotic signals. In L929 cells, a downstream molecule(s) leading to necrosis is more abundant than in Jurkat cells, and the necrotic signal can be easily seen with the weak signal from the Fas receptor in the presence of caspase inhibitors, while its strong activation by direct oligomerization of FADD may be necessary to activate the necrotic pathway in Jurkat cells. FADD can also be activated by the TNF–TNF type I receptor system through an adaptor molecule called TRADD (TNFR1-associated death domain protein; Chinnaiyan et al., 1996
; Hsu et al., 1996
). It will be interesting to determine whether the TNF-induced necrosis in L929 cells is mediated by FADD or not. In addition, the TNF-induced necrosis in L929 cells can be inhibited by butylated hydroxyanisole, an anti–oxidant, suggesting an involvement of oxidants in this cell death process (Vercammen et al., 1998a
). Whether or not a similar oxidant(s) is activated during necrosis by oligomerization of FADD in Jurkat cells remains to be determined.
The FADD-mediated apoptosis occurs through recruitment of pro-caspase 8 to the FADD death effector domain, which leads to processing of caspase 8 and the activation of the downstream caspases, such as caspase 3, and a DNase (CAD; Nagata, 1997
; Enari et al., 1998
; Sakahira et al., 1998
). It would be interesting to examine whether or not the same region of FADD is responsible for the necrotic signal transduction and what kinds of molecules are activated by FADD. The JB-6 cells abundantly express Bcl-2, yet oligomerization of FADD killed the JB-6 cells, suggesting that the FADD-induced necrotic cell death cannot be inhibited by Bcl-2. This agrees with no inhibitory effect of Bcl-2 on the TNF-induced cytotoxicity in mouse L929 cells (Vanhaesebroeck et al., 1993
). In any case, the establishment of a system for studying necrosis in the absence of caspase activation will contribute to our understanding of the molecular mechanisms underlying necrosis.