Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation.
Activation of caspases is the key event of apoptosis and new methods are needed to assay this event, particularly in situ, in individual cells. To measure in situ caspases activation in the present study we employed fam-VAD-fmk and fam-VEID-fmk, the fluorochrome (fam)-labeled inhibitors of caspases (FLICA), which through the fluoromethylketone (fmk) moiety bind to active center of the activated enzymes. The peptide moiety of these inhibitors defines their specificity; VAD is generic to most caspases and VEID is caspase-6 specific. The frequencies of cells showing caspases activation were compared with those showing DNA fragmentation (detected by the TUNEL assay) in the same cultures. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) or tumor necrosis factor-alpha combined with cycloheximide (TNF-alpha + CHX). The cells that bound FLICA had morphological changes typical of apoptosis. The intensity of their fluorescence was measured by laser scanning cytometry. Maximal rate of activation of the caspases, measured by the increase in frequency of the cells that bound fam-VAD-fmk, occurred between 30 and 90 min after the administration of TNF-alpha + CHX and between 2 and 4 h after the administration of CPT. In the CPT-treated cultures about 30% fewer cells bound fam-VEID-fmk than fam-VAD-fmk which suggests that the activation of caspase-6 was delayed or was not induced in some cells. A strong overall correlation between the cytometric assays of the apoptotic index based on the detection of caspases activation by the FLICA and the TUNEL assay was observed. The data indicate that FLICA offers a rapid and convenient method of assessing caspase's activation in individual cells and can also be used to estimate the frequency of apoptosis.
Bedner
E
E
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, 10595, USA.
Smolewski
P
P
Amstad
P
P
Darzynkiewicz
Z
Z
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002166:Camptothecin / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D018922:HL-60 Cells / Q000166:cytology / Q000201:enzymology; D006801:Humans; D020287:In Situ Nick-End Labeling; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003513:Cycloheximide; D020169:Caspases; D002166:Camptothecin