Activation-induced Aggregation and Processing of the Human Fas Antigen

Abstract

Fas (APO1/CD95) is a type 1 transmembrane protein critically involved in receptor-mediated apoptosis. Previous studies have shown that Fas exists in monomeric form in resting cells and aggregates upon cross-linking to form a complex that serves to recruit additional signaling molecules to the cell membrane. To study the molecular fate of the Fas antigen following receptor activation, a monoclonal antibody specific for the cell death domain of Fas has been generated. This monoclonal antibody (3D5) could be used in Western blot analysis using total cell lysates to identify different forms of Fas antigens without immunoprecipitation. High molecular mass (>200 kDa), SDS-and ␤-mercaptoethanol-resistant Fas aggregates were formed immediately following receptor cross-linking, and a 97-kDa band (p97) was detected about 2 h later. p97 could be detected by antibodies against either the death domain or the C terminus. However, p97 could not be precipitated by antiextracellular domain antibodies. Thus, p97 most likely represents a processed form of the high molecular weight Fas aggregates. Although p97 generation followed a similar time course as CPP32 activation and poly(ADP-ribose) polymerase cleavage, it could not be inhibited by cysteine protease, calpain, or proteasome inhibitors.

DNA-damaging reagents induce apoptosis through reactive oxygen species-dependent Fas aggregation

“…Fas expression increased the sensitivity of L1210 cells to cisplatin, suggesting that lower extents of DNA damage, which are unable to trigger the mitochondrial death pathway, activate Fas-FADD pathway. FADD-dependent, but FasL-independent, cell death has been reported in apoptosis induced by UV, cycloheximide, c-Jun N-terminal kinase, and cytotoxic drugs (Kamitani et al, 1997;Rehemtulla et al, 1997;Aragane et al, 1998;Bennett et al, 1998;Micheau et al, 1999;Tang et al, 1999;Chen and Lai, 2001;Han et al, 2001). Through the use of same cell line with selective mutation, we further confirmed the critical role of caspase-8 and Fas in DNA damage-induced apoptosis.…”

“…UV, cycloheximide, c-Jun N-terminal kinase, and cytotoxic drugs are also reported to trigger FADDdependent, but FasL-independent, apoptosis in cancer cells (Kamitani et al, 1997;Rehemtulla et al, 1997;Aragane et al, 1998;Bennett et al, 1998;Micheau et al, 1999;Tang et al, 1999;Chen and Lai, 2001;Han et al, 2001). The mechanism of how FADD-capase-8 cascade is activated under those stress stimuli is not completely clear.…”

The role of the ubiquitin-proteasome pathway in apoptosis

“…56 Jurkat cells exposed to combinations of proteasome inhibitors and an anti-Fas antibody had higher levels of apoptosis than either treatment alone. 115 In cells from patients with CLL which resisted apoptosis despite treatment with TNF, cellular sensitivity was restored after treatment with lactacystin. 61 Inhibition of NF-kB activation with a proteasome inhibitor enhanced apoptosis caused by TRAIL, anti-APO-1, TNFa, and doxorubicin, and cell lines as well as primary leukemia cells resistant to doxorubicin-induced apoptosis could be sensitized with this proteasome inhibitor.…”

Death of Smooth Muscle Cells and Expression of Mediators of Apoptosis by T Lymphocytes in Human Abdominal Aortic Aneurysms

“…Consistent with the data obtained by immunohistochemistry, AAAs but not normal aortas contained appreciable amounts of perforin, Fas, and FasL (Figure 7a through 7c). Recent work by Kamitani et al 13 has shown that certain anti-Fas antibodies recognize activated aggregates of human Fas. Indeed, immunoblots using these antibodies (3D5 and G254-274) disclosed higher-molecular-weight Fas aggregates, particularly a 97-kDa fragment in extracts of …”