title: Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer.
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.
authors.affiliation: Department of Biomedical Engineering, City University of New York/City College, 160 Convent Avenue, Steinman Hall, T-434, New York, NY 10031, USA.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Dereli-Korkut
authors.forename: Harmeet
authors.initials: H
authors.lastname: Gandhok
authors.forename: Ling Ge
authors.initials: LG
authors.lastname: Zeng
authors.forename: Sidra
authors.initials: S
authors.lastname: Waqas
authors.forename: Xuejun
authors.initials: X
authors.lastname: Jiang
authors.forename: Sihong
authors.initials: S
authors.lastname: Wang
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016503:Drug Delivery Systems; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011993:Recombinant Fusion Proteins