Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry.
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-alpha (TNF-alpha) 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.
Smolewski
P
P
Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10532, USA.
Bedner
E
E
Du
L
L
Hsieh
T C
TC
Wu
J M
JM
Phelps
D J
DJ
Darzynkiewicz
Z
Z
D000590:Amino Acid Chloromethyl Ketones; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018922:HL-60 Cells; D006801:Humans; D007834:Lasers; D009842:Oligopeptides; D013379:Substrate Specificity; D020298:U937 Cells
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C024098:6-carboxyfluorescein; D020169:Caspases