Structure/function analysis of p55 tumor necrosis factor receptor and Fas-associated death domain - Effect on necrosis in L929sA cells

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor rec eptor (TNF-R55), In contrast, triggering of the fibrosarcoma cell line L929 sA gives rise to characteristic cellular changes resulting in necrosis, The intracellular domain of TNF-R55 can be subdivided into two parts: a membra ne-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis, Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defin ed by swelling of the cells, rapid membrane permeabilization, absence of nu clear condensation, absence of DNA hypoploidy, and generation of mitochondr ial reactive oxygen intermediates, is also confined to the DD, The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp( OMe)-fluoromethylketone on TNF-induced necrosis was also observed with rece ptors solely containing the DD, TNF-R55-mediated necrosis is not affected b y the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to C rmA, while the cytotoxicity due to over-expression of the deletion mutant F ADD-(1-111) lacking the DD is prevented by CrmA These results demonstrate t hat the death domain of FADD can elicit an active necrotic cell death pathw ay.