Figure 3

Effect of IAP on the cytochrome c-dependent generation of effector caspase activity. Extracts were prepared from the cell lines KTCTL-30 or CAKI-1 (top) or primary clear cell RCC (two examples are shown at the bottom). (A and B) Extracts were incubated without cytochrome c (open circles) or with cytochrome c (closed circles). To some samples recombinant XIAP was added together with cytochrome c and was found to inhibit the generation of caspase activity (squares). To evaluate the efficacy of DIABLO peptide, one sample was incubated with cytochrome c, XIAP and DIABLO peptide (300 μ M); DIABLO at least partially relieved the inhibition by XIAP (triangles). In (B), a control peptide of the same composition but the reverse orientation was added instead of the DIABLO peptide (300 μ M; together with cytochrome c and XIAP, open triangles). Effector caspase activity was measured as free AMC every 5 min, individual measurements are shown. (C and D) Examples of the effect of DIABLO peptide in extracts from primary RCC. Samples from patients (#51 and #54, see Table 3) were incubated without cytochrome c (open circles), with cytochrome c (50 μg ml⁻¹, closed circles) or with cytochrome c plus DIABLO peptide (300 μ M, filled squares). Effector caspase activity was measured as free AMC as above.