Real-time detection of cellular apoptosis using a rat C6 glioma cell-based assay system

Abstract

Caspase-3 is a key mediator of apoptosis in mammalian cells. Cells expressing caspase-3 substrate peptides have become powerful and increasingly common components of cell-based assay systems. We developed a cell-based assay to measure staurosporine (STP)-induced caspase-3 activity in live rat glioma C6 cells. The caspase-3 sensing system was constructed to include a nuclear export signal (NES), followed by the amino acid sequence Asp-Glu-Val-Asp (DEVD) and a green fluorescent protein (GFP) fused to the N-terminal site of a nuclear localization signal (NLS). Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of GFP from the cytosol to the nucleus. After 8 h of 0.5 mu M STP treatment, caspase-3 activity was assessed by monitoring the translocation of GFP to the nucleus due to cleavage of the NES from the GFP by caspase-3. Finally, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. Analysis of caspase-3 activity using real-time monitoring can potentially be used to screen for apoptotic molecules in living cells.