Figure 6.

PBOX-15/TRAIL-induced apoptosis occurs in a caspase-dependent manner and is associated with reduced expression of c-FLIP and IAPs in Jurkat ALL cells. Jurkat cells (500,000 cells/ml) were treated with vehicle [0.2% (v/v) ethanol] or PBOX-15 (1 μM) ± TRAIL (20 ng/ml) for 24 h. Western blot analysis was then used to examine (A) cleavage of initiator pro-caspases-8 and -9 and effector pro-caspase-3, (B) expression of c-FLIP or (C) expression of IAPs (cIAP-1, cIAP-2, XIAP and livin). Levels of GAPDH were used as a loading control. Blots are representative of three independent experiments. (D) Jurkat or Nalm-6 cells (500,000 cells/ml) were pre-treated for 1 h with a solvent control [0.1% (v/v) DMSO], a caspase-8 inhibitor (20 μM z-IETD-fmk), a caspase-9 inhibitor (20 μM z-LEHD-fmk) or a general caspase inhibitor (50 μM z-VAD-fmk). The cells were subsequently treated with vehicle (0.2% (v/v) ethanol) or PBOX-15 (1 μM) ± TRAIL (20 ng/ml) for 24 h. The DNA content of cells fixed in ethanol and stained with propidium iodide was assessed by flow cytometry. Cells in the subG₀/G₁ phase (<2N DNA) were deemed apoptotic, while cells with 4N quantities of DNA were considered to be in the G₂/M phase of the cell cycle. Values represent the mean ± SEM for three independent experiments.