Characterization of cell clones stably transfected with short form caspase-9: apoptotic resistance and Bcl-XL expression.
Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.
Yi
Xiaolan
X
Department of Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, 30912, USA.
Wang
Jinzhao
J
Seol
Dai-Wu
DW
Dong
Zheng
Z
D000255:Adenosine Triphosphate / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D017209:Apoptosis; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002999:Clone Cells / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology; D008928:Mitochondria / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D014162:Transfection; D015854:Up-Regulation; D051020:bcl-X Protein / Q000096:biosynthesis*
C494813:Bcl2l1 protein, rat; D051020:bcl-X Protein; D000255:Adenosine Triphosphate; D045304:Cytochromes c; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases