Related Experiment Videos
Use of Bid-ribozymes to characterize and Bid-affiliated apoptotic pathway
1Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Japan.
Abstract:
Multiple cell and stimulus specific apoptosis pathways have been identified (1) revealing a complexity of molecular events involved. Tumor necrosis factor (TNF alpha) is known to activate mitochondria dependent or independent pathways for cell death. These pathways converge to activate effector caspases, caspases 3, 6 and 7 (2). The intermediate events including the substrates and effectors however are not clearly understood. In this report, we have employed ribozyme technology to elucidate some of these intermediate events. We show here that Bid, a pro-apoptotic facilitator that plays an important role in the mitochondrial pathway, can be targeted by Bid-specific ribozymes. MCF7 breast carcinoma cells stably transfected with expression plasmids encoding active (but not inactive or mock) Bid ribozymes showed delayed response to TNF alpha. This was accompanied by decreased activation of caspases 7 and 9, but not of caspase 8. The data assign caspase 9 as an upstream activator of caspase 7 in TNF alpha-induced Bid-mediated apoptosis.
Insights
Ribozyme technology targeting Bid protein delayed tumor necrosis factor (TNF alpha)-induced cell death in breast cancer cells. This study clarifies the roles of caspase 9 and caspase 7 in TNF alpha-mediated apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis pathways are complex, involving multiple molecular events.
- Tumor necrosis factor alpha (TNF alpha) triggers cell death via mitochondrial dependent or independent pathways.
- Intermediate events in TNF alpha-induced apoptosis, including substrates and effectors, require further elucidation.
Purpose of the Study:
- To utilize ribozyme technology to investigate intermediate events in apoptosis.
- To target the pro-apoptotic protein Bid using Bid-specific ribozymes.
- To understand the role of Bid in TNF alpha-induced cell death.
Main Methods:
- Employed ribozyme technology to target Bid.
- Used MCF7 breast carcinoma cells stably transfected with Bid ribozyme expression plasmids.
- Assessed cellular response to TNF alpha and measured caspase activation (caspase 7, 9, and 8).
Main Results:
- Active Bid ribozymes, but not inactive or mock versions, delayed TNF alpha-induced cell death in MCF7 cells.
- Decreased activation of caspase 9 and caspase 7 was observed in cells expressing active Bid ribozymes.
- Caspase 8 activation was not significantly affected by Bid ribozyme expression.
Conclusions:
- Bid-specific ribozymes can modulate TNF alpha-induced apoptosis.
- Caspase 9 acts as an upstream activator of caspase 7 in TNF alpha-induced apoptosis involving Bid.
- This study provides insights into the molecular mechanisms of Bid-mediated apoptosis.