Real‐time detection of cellular death receptor‐4 activation by fluorescence resonance energy transfer

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Abstract

Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL-DR4 in live cells in real-time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4-CFP and DR4-YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4-CFP and DR4-YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4-CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4-CFP/YFP reporter cells exhibited a colocalized expression of DR4-CFP and DR4-YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi-fluorescence microscopy for FRET analysis. The real-time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time-lapse fluorescence microscopy. Therefore, DR4-CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti-cancer drug screening to identify compounds that are capable of activating TRAIL pathways.

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