Fig. 5.

Inhibitors of 5-LO induce apoptosis in MCL cell lines and CLL primary cells. The effect of AA861 (5-LO inhibitor) and MK886 (FLAP inhibitor) were investigated using SP53 and Z138 MCL cell lines and primary CLL cells. In A, SP53 cells were treated for 24 h with 10 μm MK886 plus or minus the polycaspase inhibitor Z-VAD-fmk (100 μm), and apoptosis was assessed by flow cytometry with annexin V binding/PI staining as described under “Experimental Procedures.” In B, Z138 cells were treated with AA861 (50 and 100 μm) for 24 h before quantifying the PS⁺ (high annexin V binding) cells. Numbers in the lower left, upper left, and right quadrants refer to the percentage of cells that were viable, apoptotic, and necrotic, respectively. In C, Z138 cells were treated for 24 h with AA861 (10 μm) plus and minus Z-VAD-fmk (100 μm). Nuclei were stained with Hoechst 33342 and visualized by confocal microscopy using a Zeiss LSM 510 microscope and a 405 nm laser (blue diode) at 40× magnification with a 1.3 zoom. The blown up images are taken from the center of each cell field, and representative apoptotic nuclei are indicated with arrows. In D, the time course for AA861 induction of cell death (PS⁺) in SP53 cells is shown with 50 and 100 μm AA861. In E, concentration-effect curves for AA861-inducible cell death in primary CLL cells (after 18 h; n = 3; ±S.E.) are shown plus (▴) or minus (□) 200 μm Z-VAD-fmk. In F, the immunoblots show AA861-induced concentration-dependent caspase-9 and caspase-3 processing in Z-138 cells (E). The pro- and cleaved forms of caspase (Casp)-9 and -3 are indicated with arrows, and the effects of Z-VAD-fmk (200 μm) on caspase-9 processing are also shown. W.B., Western blotting.