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Hirokazu Hirata^*, Atsushi Takahashi^*, Susumu Kobayashi^*, Shin Yonehara, Hirofumi Sawai^*, Toshiro Okazaki^*, Kokichi Yamamoto^*, and Masataka Sasada^*,

From the ^* Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan; the Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606, Japan; and the College of Medical Technology, Kyoto University, Kyoto 606, Japan

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.

¹Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole; DFF, DNA fragmentation factor; DISC, death-inducing signaling complex; DMQD-CHO, acetyl-Asp-Met-Gln-Asp-aldehyde; FSC, forward light scatter; ICE, IL-1β converting enzyme; NuMA, nuclear mitotic apparatus protein; PAK, p21-activated kinase; PARP, poly (ADP-ribose) polymerase; PKC, protein kinase C ; PS, phosphatidylserine; PT, permeability transition; VEID-CHO, acetyl-Val-Ileu-Asp-aldehyde; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone.

We thank Emad S. Alnemri for the generous gift of cDNAs for caspases; Nancy A. Thornberry for providing YV(bio)KD-aomk; Gerald M. Cohen for anti–caspase-7 Ab; Charles H. Yang for anti-NuMA Ab; Alastair Mackay for critical review of the manuscript and creative suggestions; Yuri A. Lazebnik, Scott H. Kaufmann, William C. Earnshaw, Eisuke Nishida, Fumiko Toyoshima, and Katsumi Takada for helpful discussion and thoughtful suggestions; and Akira Komoriya, Kuniko Takano, Akinori Maeda, and Kouhei Yamashita for help in flow cytometric analysis.

A. Takahashi is a Research Resident of the Japanese Foundation of Aging and Health.

Address correspondence to Atsushi Takahashi, The First Division, Department of Internal Medicine, Kyoto University Hospital, 54 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606, Japan. Phone: 81-75-751-4290; Fax: 81-75-751-4221; E-mail: atakahas{at}kuhp.kyoto-u.ac.jp

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