J. Biochem, 2001, Vol. 129, No. 1 35-41
© 2001 Japanese Biochemical Society
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Activation of Caspase-3 by Lysosomal Cysteine Proteases and Its Role in 2,2'-Azobis-(2-Amidinopropane)Dihydrochloride (AAPH)—Induced Apoptosis in HL-60 Cells1
*Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University 1677-1 Yoshida, Yamaguchi 753-8515
Institute of Medical Science, Kurashiki Medical Center 250 Bakuro-cho, Kurashiki 710-8522;
Doonan Institute of Medical Science Hakodate 041-8502
2To whom correspondence should be addressed. Tel: +81-83-933-5856, Fax: +81-83-933-5820, E-mail: utsumi{at}agr.yamaguchi-u.ac.jp
We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca2+ in the presence of inorganic phosphate (Pi), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.
1This work was supported in part by a Grant-in-Aid for Scientific Research (No. 4743) from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from the Japanese Gerontologi-cal Society and the Japan Keirin Association.
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