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

Abstract

BackgroundFluorochrome‐labeled inhibitors of caspases (FLICA, e.g., FAM‐VAD‐FMK, FITC‐VAD‐FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.MethodsApoptosis of HL‐60, Jurkat, MCF‐7 and T‐24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H2O2). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase‐3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z‐VAD‐FMK or z‐DEVD‐FMK.ResultsFLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase‐3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM‐VAD‐FMK, FITC‐VAD‐FMK or FAM‐DEVD‐FMK binding to apoptotic cells could not be inhibited by z‐VAD‐FMK or z‐DEVD‐FMK, respectively, when the unlabeled inhibitors were added post‐induction of apoptosis.ConclusionsFLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases. Cytometry Part A 55A:50–60, 2003. © 2003 Wiley‐Liss, Inc.

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

“…FLICA, thus, binds to the enzymatic active center of their respective activated caspases, but this contribution is minimal with respect to the overall cell staining. While the extent of FLICA binding by cell constituents other than caspases may vary between cell types and may also depend on inducer of apoptosis, the data of Kuzelova et al (8) and our findings (7) reveal that specificity of staining of individual caspases with FLICA is far from that of the originally anticipated or advertised by the vendors. These findings (7,8) call for re-examination of interpretation of the data obtained with these reagents.…”

“…While the extent of FLICA binding by cell constituents other than caspases may vary between cell types and may also depend on inducer of apoptosis, the data of Kuzelova et al (8) and our findings (7) reveal that specificity of staining of individual caspases with FLICA is far from that of the originally anticipated or advertised by the vendors. These findings (7,8) call for re-examination of interpretation of the data obtained with these reagents. While FLICA binding reports caspase activation and is a marker of apoptosis, the overall fluorescence intensity of apoptotic cell labeled with this ligand does not represent its binding to enzyme active center of caspases.…”

“…We postulated, therefore, that FLICA binding sites may involve other cell constituents in addition to the activated caspases (7). In fact, only a minor fraction of total FLICA binding to apoptotic could be attributed to its interaction with the respective caspases (7).…”

“…Namely, the data (7,8) point out that during apoptosis, in association with activation of caspases, some proteins undergo modifications that lead to their ability to react with FLICA, most likely in a covalent manner. Known reactivity of fmk moiety with thiol groups suggests that the modifications may involve intra-or inter-molecular opening of the disulfide cysteine bridges.…”

In vitro measurement of cell death with the annexin A5 affinity assay

“…Induction of apoptosis leads to the activation of caspases by proteolytic cleavage of the inactive zymogen into the active, cleaved caspase. Activated caspases can be detected by western blot with antibodies to cleaved caspase fragments and by flow cytometry using fluorescent probes 17,18 . Caspases are activated early in the apoptotic process before PS expression, so with this method it is possible to detect cells at the onset of apoptosis 8 .…”