Bax-induction alone is sufficient to activate apoptosis cascade in wild-type Bax-bearing K562 cells, and the initiation of apoptosis requires simultaneous caspase activation.
It has been reported that expression of Bax by Tet-On system induces apoptosis in Jurkat cells. The parental Jurkat cells have mutation of Bax gene and do not express Bax protein. Wild-type Bax-bearing cells express endogenous Bax protein and it is still unclear whether overexpression of Bax alone can sufficiently induce apoptosis in these cells in the absence of any cytotoxic stimulus. To investigate this, wild-type Bax-bearing K562 cells were transfected with Tet-On Bax-inducible system (pTet-On and pTRE-Bax plasmids), and Bax-inducible stable cell lines were established. Overexpression of Bax in wild-type Bax-bearing K562 cells without any cyctotoxic signal resulted in increase of apoptotic cells, caspase-3 activation, mitochondrial release of cytochrome c, and mitochondrial membrane potential change. Western blotting and confocal microscopy revealed that overexpression of Bax was detected in mitochondria. A pancaspase inhibitor, zVAD-fmk, which has no effect on mitochondrial cytochrome c release and mitochondrial membrane potential change inhibited the apoptotic events in the presence of overexpressed Bax in mitochondria. These findings suggest that Bax protein, when present above a threshold level, is sufficient to trigger an apoptosis cascade, and its initiation requires simultaneous caspase activation probably not mediated by mitochondrial cytochrome c release and mitochondrial membrane potential change in K562 cells.
Kobayashi
Tohru
T
The Second Department of Internal Medicine, Mie University School of Medicine, Tsu 514-8507, Japan. tohruk@clin.medic.mie-u.ac.jp
Sawa
Hidehiko
H
Morikawa
Jun
J
Ueno
Satoshi
S
Katayama
Naoyuki
N
Zhang
Wei
W
Shiku
Hiroshi
H
D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D020014:K562 Cells / Q000378:metabolism* / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D020169:Caspases; D013752:Tetracycline