Figure 4.

TK/GCV-induced apoptosis involves brefeldin A-inhibitable aggregation of CD95 leading to recruitment of FADD and caspase-8 to CD95. SH-EP pLXSN cells [TK−, containing wild type (WT) FADD] were compared with SH-EP pHyTK11 cells (TK+, containing FADD-WT) and with SH-EP pHyTK11/FADD32 cells (TK+, containing FADD-DN). (A) TK/GCV induces aggregation of CD95 as demonstrated by immunoprecipitation. Cells were treated with 10 μM of GCV for 36 h. CD95 was crosslinked and immunoprecipitated by using anti-APO-1 at the limiting concentration of 0.5 μg/ml and the excess concentration of 2 μg/ml, respectively, and protein A-Sepharose. The precipitate was subjected to immunoblot analysis for CD95. (B) TK/GCV induces DISC formation. Cells (2 × 10⁷) were treated with 10 μM of GCV for the indicated time. Immunoprecipitation was performed by using anti-APO-1 IgG3 mAb (2 μg/ml) and protein A-Sepharose. Immunoblot was performed as described for FADD and caspase-8 protein expression. Similar results were obtained in two separate experiments. (C) TK/GCV-induced DISC formation is inhibited by brefeldin A. Experimental conditions were as for B except that 1 μg/ml of brefeldin A was added to GCV during the incubation period of 24 h.