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D.M. Robertson, W.M. Petroll, J.V. Jester, H.D. Cavanagh; Caspase Activation in Human Corneal Epithelial Cells Using FRET . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2102.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Activation of caspase 3, a cysteine protease involved in the apoptotic cascade, precedes nuclear degradation and cell death. The purpose of this experiment was to develop a fluorescence–resonance energy transfer (FRET) probe to dynamically evaluate caspase 3 activation in telomerase–immortalized human corneal epithelial (hTCEpi) cells. Methods: Enhanced yellow fluorescent protein, EYFP, was amplified from pEYFP by PCR using a forward primer containing a 4 amino acid sequence specific for the caspase 3 proteolytic cleavage site DEVD. The fragment was directionally cloned into the N terminus of an enhanced cyan fluorescent protein expression plasmid, pECFP–N1, with the resulting sequence ECFP–DEVD–EYFP. ECFP–DEVG–EYFP, a mutant substituting a Glycine for Aspartic Acid at the fourth amino acid residue and ECFP–EYFP, cleavage site omitted, were used as controls. hTCEpi cells were plated and transiently transfected using Lipofectamine. 1µM staurosporine was used to induce apoptosis. Cells were imaged using a Nikon microscope equipped with individual CFP and YFP excitation and emission filters and digitally acquired using a Photometrics CoolSnap Camera. The average regional pixel intensity for CFP and YFP emission using the CFP excitation filter was measured for each cell using MetaMorph Software and the ratio of YFP/CFP emission was calculated. Results: Prior to induction of apoptosis, ratio imaging analysis of cells transfected with either the DEVD, DEVG, or CFP–YFP FRET probe, showed a 2.43 ± 0.68 ratio of YFP/CFP emission with CFP excitation. After addition of staurosporine to activate caspase 3, the DEVD transfected cells showed a marked decrease in the ratio of YFP/CFP emission to 1.01 ± 0.06 in single cells. Cells transfected with the control FRET probes containing either the DEVG or CFP–YFP linker showed no change. Conclusions: These findings indicate that FRET can be used to study dynamic activation of caspase 3 in living hTCEpi cells. Stable cell lines expressing FRET probes for caspase 3 and the upstream effector caspase 9 will enable caspase activation to be evaluated temporally and spatially in a 4D epithelial cell culture model.
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