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Characterization of cell clones stably transfected with short form caspase-9: apoptotic resistance and Bcl-XL
Xiaolan Yi1, Jinzhao Wang, Dai-Wu Seol
1Department of Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, 30912, USA.
Abstract:
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.
Insights
This study shows that inhibiting caspase-9 and up-regulating Bcl-XL makes cells resistant to apoptosis during ATP depletion. This dual resistance occurs at both cytosolic caspase activation and mitochondrial levels.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Caspases are crucial for apoptosis initiation and progression.
- ATP depletion triggers caspase activation (caspase-9, -8, -3) and apoptotic morphology.
Purpose of the Study:
- To investigate the specific role of caspase-9 in ATP depletion-induced apoptosis.
- To determine the mechanisms of cellular resistance to apoptosis.
Main Methods:
- Transfection of renal epithelial cells with dominant-negative caspase-9S.
- Assessing apoptosis, caspase activation, and cytochrome c release in wild-type vs. caspase-9S clones.
- Analyzing Bcl-2 family protein expression (Bax, Bak, Bcl-2, Bcl-XL).
Main Results:
- Caspase-9S clones exhibited significant resistance to ATP depletion-induced apoptosis.
- Inhibition of caspase activation and reduced cytochrome c release were observed.
- Resistance extended to the mitochondrial level, with inhibited Bax translocation.
- Up-regulation of the anti-apoptotic protein Bcl-XL was noted in resistant clones.
Conclusions:
- Cells resistant to apoptosis can be achieved by inhibiting caspase-9 and up-regulating Bcl-XL.
- This resistance operates at both cytosolic (caspase inhibition) and mitochondrial (Bcl-XL mediated) levels.
- The findings provide insights into apoptotic pathway regulation and potential therapeutic strategies.
