Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be defected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing p ro-fluorescent substrates that become fluorescent upon cleavage by the casp ase. Most of these methods require the preparation of call extracts and, th erefore, are not suitable for the detection of active caspases within the l iving cell. Using FAM-VAD-FMK, we have developed a simple and sensitive ass ay for the defection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-asp artic acid-fluoromethyl ketone (zVAD-FMK), which is a patent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed b y flow cytometry, fluorescence microscopy,. or a fluorescence plate reader Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or stau rosporine. Non-stimulated negative control cells were not stained. Pretreat ment with the general caspase inhibitor zVAD-FMK blocked caspase-specific s taining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-ind uced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3 , -6, and -7, blocked caspase-specific cell staining, and led to the inhibi tion of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FM K had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versa tile and specific tool for detecting activated caspase, in living cells.