A novel method for fluorescence microscopic demonstration of caspase activity

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We have developed a 4-methoxy-2-naphthylamide (MNA)-based substrate, Z-Asp(OME)-Glu(OME) -Val-Asp(OME) -MNA (DEVD-MNA), which is relatively specific for caspase-3, one of the major caspases that triggers multiple apoptotic cellular degeneration. MNA, released by caspase-3 showed fluorescence with an exitation wave length of 340nm and emission wave length of 425nm. Using this substrate, we first determined the time course of caspase-3-like enzyme activity in cell lysates of murine osteoclast-like cells treated with nitric oxide (NO)-releaser, NOC18. Caspase-3-like enzyme activity showed a transient peak at 8 h after NOC18 treatment and decreased thereafter. MNA forms a Schiff-base complex with 5-nitrosalycylaldehyde (NSA) under mild acidic condition (〜pH 6.0), and the complex grows as needle-like fluorescence crystals. When DEVD-MNA and NSA were applied to NOC18-treated murine osteoclast-like cells (8 h), small needle-like fluorescence crystals were formed in the cells. The crystal formation was completely inhibited with caspase inhibitor DEVD- fenylmethylketone (FMK). Thus, intracellular activation of caspase(s) was visualized with this fluorescence substrate. After 16 h of NOC18-treatment, the crystal formation in cells was markedly reduced, and typical apoptotic morphologies of nuclear condensation and cell shrinkage, were observed. The efficiency of the crystal formation correlated well with changes in caspase-3-like enzyme activity measured by DEVD-MNA in vitro. The visualized caspase activity preceded the apoptotic morphological changes. The advantages of the method we have developed are (1) stained cells with this fluorescence substrate were fixable and (2) among mixed cell populations, such as osteoclast culture, cells undergoing apoptotic degeneration could be specifically detected.