Bcl-2 blocks apoptosis caused by pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly.
Pierisin-1, a 98-kDa protein that induces apoptosis in mammalian cell lines, is capable of being incorporated into cells where it ADP-ribosylates guanine residues in DNA. To investigate the apoptotic pathway induced by this unique protein, the bcl-2 gene was transfected into HeLa cells. Cy2-fluorescent pierisin-1 was incorporated into the resultant cells expressing Bcl-2 protein and ADP-ribosylated dG was detected to almost the same extent as in parent cells. However, bcl-2-transfected HeLa cells did not display apoptotic morphological changes, PARP cleavage, and DNA fragmentation, indicating acquisition of resistance. In parent HeLa cells, activation of caspase-9 and release of cytochrome c were observed after 8h treatment with 0.5ng/ml pierisin-1. Caspase substrate assays revealed further cleavage of Ac-DEVD-pNA, Ac-VDVAD-pNA, and Ac-VEID-pNA, suggesting activation of caspase-2, -3, and -6 in pierisin-1-treated HeLa cells. The caspase-3 inhibitor, Ac-DEVD-CHO, was also found to inhibit apoptosis. In contrast, this caspase activation was not observed in bcl-2-transfected HeLa cells. Our results thus indicate that pierisin-1-induced apoptosis is mediated primarily via a mitochondrial pathway involving Bcl-2 and caspases.
Kanazawa
Takashi
T
Cancer Prevention Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, 104-0045, Tokyo, Japan. tkanazaw@gan2.ncc.go.jp
Kono
Takuo
T
Watanabe
Masahiko
M
Matsushima-Hibiya
Yuko
Y
Nakano
Tsuyoshi
T
Koyama
Kotaro
K
Tanaka
Noriaki
N
Sugimura
Takashi
T
Wakabayashi
Keiji
K
D036002:ADP Ribose Transferases; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002080:Butterflies; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D019254:Genes, bcl-2; D006367:HeLa Cells; D006801:Humans; D019476:Insect Proteins / Q000494:pharmacology*
D003574:Cytochrome c Group; D019476:Insect Proteins; C113557:pierisin protein, insect; D000244:Adenosine Diphosphate; D036002:ADP Ribose Transferases; D020169:Caspases