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 k Da, 19 kDa, and 17 kDa were formed upon treatment of human leukemia U937 ce lls with GGO. These caspases are quite similar to those activated by treatm ents with other apoptosis-inducers, such as VP16 and camptothecin, suggesti ng that similar caspases, such ns caspases 3 and 6, are activated during ap optosis in U937 cells that is induced by a variety of apoptosis-inducing st imuli. 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 pep tides. This cleavage is catalyzed by caspase 3 itself or by a caspase-3-lik e pretense. In contrast, other inhibitors of caspases such as Z-DEVD-FMK an d 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 inhi bited other upstream caspases. Treatment of U937 cells with GGO resulted in the release of cytochrome c from mitochondria prior to DNA fragmentation a nd 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 ol Z-V AD-FMK. These results suggest that intracellular free Ca2+ ions, and some c aspases that are inhibited by Zn2+ ions bill not by Z-Asp-CH2DCB or Z-VAD-F MK are necessary for the release of cytochrome c that is caused by the trea tment with GGO.