Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Background: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. Methods: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring din-finished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. Results: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase-3-deficient MCF7-Fas cells and a caspase-6-specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase-3 activation consistently, preceded caspase-6 by similar to 30 mill following induction of apoptosis. Conclusions: We have developed a novel FRET-based probe for simultaneous detection of two caspase activities in living cells Using flow cytometry. Simultaneous detection of two caspase activities using this probe has clearly provided information of the ordering of caspase-3 and -6 in the apoptotic pathway. (c) 2006 International Society for Analytical Cytology.