Abstract
The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The effect of doxorubicin in a cell-based assay and the addition of cytochrome c and ATP in a cell-free system have been used to test the functionality of the reporter for the detection of apoptosome formation. Here, our data established a drug- and cytochrome c/ATP-independent way of apoptosis induction relying on the expression of the biosensor itself to induce formation of apoptosome. Overexpression of Apaf-1 constructs led to increased split luciferase activity and caspase-3 activity in the absence of any drug treatment. Caspase-3 activity was significantly inhibited when caspase-9DN was co-overexpressed, while the activity of the Apaf1 biosensor was significantly increased. Our results show that the Apaf-1 biosensor does not detect etoposide-induced apoptosis.
Highlights
Apoptosis is the process of programmed cell death in multicellular organisms and is vital for the development and regulation of homeostasis
Complementary luciferase activity has been detected in HEK293T cells transfected with split luciferase constructs of apoptotic protease-activating factor 1 (Apaf-1) (Nluc/Apaf-1 and Cluc/Apaf-1) and treated with doxorubicin, a known inducer of apoptosis [10]
We investigated the cell death induced in cells transfected with green fluorescent protein (GFP), either untreated or treated with etoposide, to see the effect of transfection on cell death
Summary
Apoptosis is the process of programmed cell death in multicellular organisms and is vital for the development and regulation of homeostasis. Two main/well-studied pathways through which apoptosis occurs are the extrinsic and intrinsic pathways. The extrinsic pathway is associated with the engagement of cell surface death receptors by their specific ligands, and the intrinsic pathway is associated with the release of cytochrome c from mitochondria in response to cell stress or damage [3,4]. Following a non-receptor-mediated stimulus produced inside the cell, cytochrome c is released through the formation of some pores in the outer mitochondrial membrane into the cytoplasm. There, it activates the adaptor protein Apaf-1, which homo-oligomerizes to form a heptameric complex that recruits and activates procaspase-9
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