The cells were then incubated for the indicated time periods with 0.5 M AP1510, and the cell viability was determined as described above. Changes of Mitochondrial Membrane Potential and Release of Cytochrome c in Caspase-independent Cell Death The death of JB6 cells induced by the oligomerization of FADD is accompanied by massive swelling of the mitochondria (Kawahara et al. mouse Fas were treated with Fas ligand or antiCmouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VADCfmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in m, as well as necrotic morphological changes. The presence of z-VADCfmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor. for 5 min. The supernatant was spun at 10,000 for 10 min, and the pellet, which was resuspended in buffer A, was used as the heavy membrane fraction containing mitochondria. The supernatant was further spun at 100,000 for 30 min, and the resultant supernatant was used as the S100 fraction. For Western blotting analysis, samples were mixed with an equal volume of 2 Laemmli’s sample buffer. After heating at Dolastatin 10 95C for 10 min, proteins were separated by electrophoresis in a 15C25% gradient polyacrylamide gel (Dai-ichi Chemical), and then they were transferred to a polyvinylidene difluoride membrane (Hybond P; Amersham-Pharmacia Biotech). The membrane was blocked with PBST (PBS supplemented with 0.05% Tween 20) containing 5% nonfat dry milk, followed by successive incubations with primary and secondary antibodies. Proteins were visualized with the enhanced chemiluminescence system (Renaissance; NEN Life Science Products Inc.). Results Requirement of DED for Necrotic Death Induced by FADD Oligomerization Artificial oligomerization of FADD can kill JB6 cells; this death is accompanied by necrotic morphological Rabbit polyclonal to ERO1L changes and occurs in a caspase-independent manner (Kawahara et al. 1998). FADD contains two distinct domains, the DD and DED (Boldin et al. 1995; Chinnaiyan et al. 1995), of which DED mediates the apoptotic cell death by recruiting caspase 8. To examine which domain is responsible for the caspase-independent cell death process, FADD-DD or FADD-DED was joined to two copies of FKBP, and the resulting expression plasmids were introduced into Jurkat or JB6 cells (Fig. 1 A). When the transformants were treated for 6 h with AP1510 to oligomerize the chimeric proteins, Jurkat cell transformants expressing FKBP-DED, but not FKBP-DD, were killed in a dose-dependent manner (Fig. 1 B). Oligomerization of FKBP-DED, but not FKBP-DD, also efficiently killed the JB6 cells. When the DED was truncated, its death-inducing activity was destroyed. As found with FKBP-FADD (Kawahara et al. 1998), the FKBP-induced death Dolastatin 10 of Jurkat cells was accompanied by caspase activation and apoptotic morphological changes. In contrast, the oligomerization of FKBP-DED in JB6 cells did not activate caspases, though it did induced necrotic morphological changes (data not shown). These results indicate that the FADD-DED is responsible for transducing not only the caspase-mediated apoptotic signal, but also the caspase-independent death signal. Inhibitory Effect of PDTC on Caspase-independent Cell Death To study the molecular mechanism of the caspase-independent necrotic pathway, we first screened various compounds for the ability to inhibit the FKBP-FADDCinduced death of JB6 cells. We found that PDTC inhibits the process in a dose-dependent manner. As shown in Fig. 2 A, AP1510 treatment killed 90% of the cells within 4 h. However, when the cells were preincubated with 80 M PDTC, 90% of the cells survived for at least 4 h. A similar inhibitory effect of PDTC was observed with JB6 cells expressing FKBP-DED. That is, death was substantially delayed by pretreating the cells with PDTC (Fig. 2 B). PDTC is known to work as an antioxidant (Liu et al. 1996). However, other antioxidants, such as butylated hydroxyanisole (BHA, 250 M) and nordihydroguaiaretic acid (25 M), showed little inhibitory effect on the FADD-induced death of JB6 cells (data not shown). Open in a separate window Open in a separate window Figure 2 Inhibitory effect of Dolastatin 10 PDTC on the cell death induced by FADD oligomerization. (A) Dose-dependent effect of PDTC. JB6 cell transformants expressing Dolastatin 10 FKBP-FADD were pretreated at 37C for 1 h with the indicated concentrations Dolastatin 10 of PDTC. The cells were divided into two samples, and each was incubated at 37C for 4 h, with or without 0.5 M AP1510. The WST-1 assay was carried out as described in Materials and Methods, and the cell viability of the PDTC-treated cells is expressed as the percent of the value obtained without AP1510. The experiments were performed in triplicate, and the average values were plotted with SD (bars). (B) Time-dependent cell death by FADD oligomerization. Two JB6 transformant clones (circles and triangles) expressing FKBP-DED were preincubated at 37C for 1 h, with (open symbols) or without (closed symbols) 80 M PDTC. The cells were then incubated for the indicated time periods with 0.5 M AP1510,.