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KMID : 1034820110070020177
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Molecular & Cellular Toxicology
2011 Volume.7 No. 2 p.177 ~ p.184
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Real-time detection of cellular apoptosis using a rat C6 glioma cell-based assay system
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Jung Kyoung-Hwa
Song Young-Me
Das Nando Dulal
Park Kyoung-Sun
Choi Mi-Ran
Hwang Sang-Youn
Lee Eun-Kyu
Lee Moon-Kwon
Choo Jae-Bum
Kim Kyoung-Suk
Kim Moo-Soung
Lee Sang-Rin
Chai Young-Gyu
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Abstract
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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 Nterminal 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 μM 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.
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KEYWORD
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Caspase-3, Apoptosis, Staurosporine (STP), Time-lapse measurements, Rat glioma C6 cells
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