Analysis of caspases that are activated during apoptosis in leukemia U937 cells in response to geranylgeraniol

Affinity labeling showed that active caspases with molecular masses of 20 kDa, 19 kDa, and 17 kDa were formed upon treatment of human leukemia U937 cells with GGO. These caspases are quite similar to those activated by treatments with other apoptosis-inducers, such as VP16 and camptothecin, suggesting that similar caspases, such as caspases 3 and 6, are activated during apoptosis in U937 cells that is induced by a variety of apoptosis-inducing stimuli. An inhibitor of caspases, Z-Asp-CH2DCB, inhibited DNA fragmentation in response to GGO in vivo by blocking the cleavage of 20-kDa to 17-kDa peptides. This cleavage is catalyzed by caspase 3 itself or by a caspase-3-like protease. In contrast, other inhibitors of caspases such as Z-DEVD-FMK and Z-VAD-FMK, inhibited the processing of the caspase 3 precursor p32 to 20-kDa and 17-kDa peptides, a result which suggests that these inhibitors inhibited other upstream caspases. Treatment of U937 cells with GGO resulted in the release of cytochrome c from mitochondria prior to DNA fragmentation and the release of cytochrome c was inhibited by Zn2+ ions and by a chelator of Ca2+ ions but not by inhibitors of caspases such as Z-Asp-CH2DCB or Z-VAD-FMK. These results suggest that intracellular free Ca2+ ions, and some caspases that are inhibited by Zn2+ ions, but not by Z-Asp-CH2DCB or Z-VAD-FMK are necessary for the release of cytochrome c that is caused by the treatment with GGO.