Detection of Caspase Activation In Situ by Fluorochrome-Labeled Caspase Inhibitors

Abstract

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.

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

“…While there is evidence that FLICA binds to proteins of molecular weight [18][19][20][21][22][23], which is in concordance with the molecular weight of large subunits of caspases (4 -6), there are certain puzzling observations that cannot be easily explained by the initially proposed mechanism of the binding, i.e., through interactions with the caspase active center only. Thus, if caspase active centers were the only binding sites for these reagents, one would expect a significant level of protection of these sites by the unlabeled caspase inhibitors (z-VAD-FMK, z-DEVD-FMK) via competitive binding.…”

“…These reagents were designed as affinity ligands to react covalently with the reactive enzymatic center of activated caspases. During the past two years several articles have been published (19,(21)(22)(23), including the papers authored by us (18,24,25), in which these reagents have been used to probe for caspase activation. Based on a similar principle, other probes were developed to detect activation of intracellular serine proteases (26).…”

Impact of caspase activation in human spermatozoa

“…Any unbound inhibitor leaves the sperm (Vaux and Korsmeyer, 1999). In somatic cells various studies proved the association of fluorescence labeled caspase inhibitors with other parameters of apoptosis signaling, e.g., the disruption of Microscopy Research and Technique the transmembrane mitochondrial potential, the externalization of phosphatidylserine (EPS) and the DNA fragmentation rate (Amstad et al, 2001;Bedner et al, 2000;Pozarowski et al, 2003;Smolewski et al, 2001).…”

All that glitters is not gold: All that FLICA binds is not caspase. A caution in data interpretation—and new opportunities

“…One such methodology is based on use of the fluorochrome-labeled inhibitors of caspases (FLICA; also known under names CaspaTag, CaspACE, CaspGLOW, or FLIVO). FLICA were designed as affinity ligands to enzyme active centers to detect activation of caspases (2)(3)(4). Through the reactive fluoromethylketone (fmk) moiety they covalently bind to cysteine of the active center of caspase to form a thiomethyl ketone and thereby irreversibly inactivate the target enzyme (4,5).…”

“…Carboxyfluorescein (FAM) (1-4), fluorescein (FITC) (6), or sulforhodamine serves as a florescent tag to report its binding and localization in the cell. FLICA ligands have proven to be convenient and reliable reporters of caspase activation and induction of apoptosis (1)(2)(3)(4)6).…”