Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry

Background: The fluorochrome-labeled inhibitors of caspases (FLICA) were re cently 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 a ere to (a) explore if FLICA can be used to study intracellular loc alization of caspases, (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and la ser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that w ould 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-337, or HL-60 cells was induced by cam ptothecin (CPT) or tumor necrosis factor-alpha (TNF-alpha) combined with cy cloheximide (CI-L). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counters taining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluo rochromes 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 labe ling withstood cell fixation and permeabilization, which made it possible t o stain DNA and measure its content for identification of the cell cycle po sition of labeled cells. When fixed cells were treated with FLICA, both apo ptotic and nonapoptotic cells became strongly labeled and the labeling patt ern was consistent with the localization of caspases as reported in the lit erature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was lar gely (> 90%) prevented by the substrates of the caspases or by the unlabele d caspase inhibitors having the same peptide moiety as the respective FLICA . Conclusions: The detection of caspase activation combined with cell permeab ilization requires exposure of live cells to FLICA followed by their fixati on. Cell reactivity with the respective FLICA, under these conditions, iden tifies the activated caspases and makes it possible to correlate their acti vation with the cell cycle position and other cell attributes that can be m easured only after cell fixation/permeabilization. FLICA can also be used t o study intracellular localization of caspases, including their translocati on. Cytometry 44.73-82, 2001. (C) 2001 Wiley-Liss, Inc.