FIGURE 1.

KD548-Fc induces the apoptotic cell death of tumor cells by specifically binding to DR4 and/or DR5. A, schematic diagram of KD548-Fc, the homodimeric Fc-fused form of KD548, generated by its C-terminal fusion to Fc of human IgG1. B, ELISA to analyze the binding specificity of KD548-Fc for the extracellular domains of the indicated TNF family receptors, in comparison with TRAIL and TNFα. C, competition ELISA. Shown is binding activity of KD548-Fc for the plate-coated extracellular domain of DR4 or DR5 in the absence or presence of 1 μm TRAIL. D, colocalization of KD548-Fc with cell surface-expressed DR4 and DR5. TRITC-labeled KD548-Fc was incubated for 1 h at 4 °C with HeLa cells transiently transfected with DR4ΔCD-YFP or DR5ΔCD-YFP fusion protein, respectively, and then visualized by confocal fluorescence microscopy. Nuclei were costained with DAPI. Bar, 20 μm. E and F, knockdown of DR4, DR5, or TNFR1 by siRNA transfection (E) and the effects on TRAIL- or KD548-Fc-mediated cell death (F) in HeLa cells. Cells untransfected (control) or transfected with the indicated siRNA for 36 h (E) were incubated with the indicated concentrations of TRAIL or KD548-Fc for 40 h prior to the MTT assay (F). DR4/DR5 siRNA indicates the cotransfection of DR4 and DR5 siRNAs. G, representative transmission electron microscopy images of HeLa cells, which were left untreated (control) or treated with either TRAIL (30 nm) or KD548-Fc (0.8 μm) for 20 h. Bar, 2 μm. H, oligonucleosomal DNA fragmentation assay for HeLa cells, pretreated for 1 h with Z-VAD-fmk, SP600125, or NAC and further left untreated (control) or treated with TRAIL (30 nm) for 15 h or KD548-Fc (0.8 μm) for 40 h. B, C, and F, data represent mean ± S.E. (error bars) of three independent experiments carried out in triplicate.