Background The fluorochrome‐labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308–313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. Methods Apoptosis of human MCF‐7, U‐937, or HL‐60 cells was induced by camptothecin (CPT) or tumor necrosis factor‐α (TNF‐α) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA‐specific fluorochromes was measured by LSC. Results Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF‐7 cells treated with CPT. FLICA binding was largely (>90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. Conclusions The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation. Cytometry 44:73–82, 2001. © 2001 Wiley‐Liss, Inc.

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the detection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a potent 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 by 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 staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK 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 versatile and specific tool for detecting activated caspases in living cells.

Resonance Raman (RR) spectra, obtained by ultraviolet laser excitation, are reported for 10 acylchymoptrypsins at pH 3.0, in which the acyl groups are derivatives of furylacrylic and thienylacrylic acids. Spectra are also shown of the sodium dodecyl sulfate (NaDodSO4) denatured acyl enzymes and the acid and ester analogues of the acyl groups. For most of the native acyl enzymes, the RR spectral profiles in the carbonyl stretching region suggest that the acyl groups bound to Ser-195 adopt two conformations, which are characterized by having either strong hydrogen bonds to the carbonyl oxygen or a nonbonding hydrophobic environment about the C==O group. It is also likely that in solution the ester and acid analogues of the acyl group adopt more than one conformation about the acryloyl linkages. Thus, the measured spectral parameters, such as the ethylenic double bond stretching frequency v C==C). For a series of compounds based on a given acyl group a correlation exists between (v C==C) and the measured absorption maximum (lambda max). Possible explanations are given for the observed changes in (v C==C) and (lambda max) when the acyl groups bind to the active site. A band appears near 1260 cm-1 in the RR spectra of some of the native acyl enzymes; it is not observed in the spectra of the NaDodSO4-treated intermediates or in the spectra of any model compounds.

The resonance Raman (RR) spectra, excited by

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