Analysis of a mitochondrial apoptotic pathway using Bid-targeted ribozymes in human MCF7 cells in the absence of a caspase-3-dependent pathway.
In normal cells, tumor necrosis factor-alpha (TNF-alpha) activates caspase 8 in both mitochondrion-dependent and mitochondrion-independent apoptotic pathways. It is believed that these two pathways converge, with resultant activation of effector caspases, such as caspase 6 and caspase 7. However, the precise mechanism of the activation of caspases 6 and 7 remains unknown. In this study, in order to focus on the mitochondrion-dependent pathway, we employed MCF7 human breast carcinoma cells, which do not have a functional mitochondrion-independent (caspase 3-dependent) pathway. We specifically targeted the transcript of Bid, a proapoptotic facilitator that is a substrate of caspase 8 in the mitochondrial pathway. In the TNF-alpha-treated MCF7 cells that expressed Bid-targeted ribozymes, the release of cytochrome c and the activation of caspase 9, but not of caspase 8, was delayed. Furthermore, the proteolysis of procaspase 7 was also delayed in Bid ribozyme-expressing cells. Because MCF7 cells are caspase 3 deficient, the direct cross-talk between caspase 8 and caspase 3 does not take place. Therefore, it became clear for the first time that caspase 9 by itself can activate caspase 7 in the absence of the caspase 3-dependent pathway in TNF-alpha-induced apoptosis by the use of specific ribozymes.
Onuki
Reiko
R
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Higashi, Tsukuba Science City 305-8562, Japan.
Kawasaki
Hiroaki
H
Baba
Tadashi
T
Taira
Kazunari
K
D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D001483:Base Sequence; D001943:Breast Neoplasms / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D004792:Enzyme Precursors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D045304:Cytochromes c; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases