Paclitaxel-induced apoptosis depends on caspase-9. (A) Effects of paclitaxel on caspase-9–deficient Jurkat cells. Lysates of caspase-9–deficient and caspase-9–reconstituted cells treated for 48 hours with 1 μM paclitaxel or left untreated were subjected to immunoblot analysis using caspase-9 and β-actin antibodies. Caspase-9 was cleaved in proficient cells upon stimulation with paclitaxel as indicated by the decrease of its proform. (B) Caspase-9–deficient and – reconstituted Jurkat cells were treated with increasing concentrations of paclitaxel for 48 hours or were left untreated. Analysis of hypodiploid nuclei (left panel) demonstrates that caspase-9–proficient but not caspase-9–deficient cells undergo apoptosis in response to paclitaxel. Treatment of caspase-9–reconstituted Jurkat cells with the pancaspase inhibitor QVD-oPh (20 μM) revealed that paclitaxel-induced DNA degradation is caspase dependent (right panel). (C,D) Caspase-9–deficient and – reconstituted Jurkat cells were treated as described in panel B. Dead cells were measured by cellular uptake of the nonvital dye PI (left panels). Mitochondrial membrane depolarization (middle panels), PS exposure (right panels), and cell death were observed only in caspase-9–proficient cells (C). Consistently, these effects can be blocked by the caspase inhibitor QVD-oPh (D). (E) Upon long-term treatment with paclitaxel, caspase-9–deficient cells also succumb to a caspase-independent cell death. Cells were treated for 4 days with 1 μM paclitaxel in the presence or absence of QVD-oPh and analyzed as described. Results in panels B-E show the mean values (± SD).