Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: A caution in data interpretation
Article first published online: 18 AUG 2003
Copyright © 2003 Wiley-Liss, Inc.
Cytometry Part A
Volume 55A, Issue 1, pages 50–60, September 2003
How to Cite
Pozarowski, P., Huang, X., Halicka, D. H., Lee, B., Johnson, G. and Darzynkiewicz, Z. (2003), Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: A caution in data interpretation. Cytometry, 55A: 50–60. doi: 10.1002/cyto.a.10074
- Issue published online: 18 AUG 2003
- Article first published online: 18 AUG 2003
- Manuscript Revised: 30 JUN 2003
- Manuscript Accepted: 30 JUN 2003
- Manuscript Received: 4 JUN 2003
- Robert A. Welke Cancer Research Foundation, Inc.
- NCI. Grant Number: CA 28704
- cell necrobiology;
- active enzyme center;
- affinity ligand;
- annexin V;
- mitochondrial potential
Fluorochrome-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.
Apoptosis 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.
FLICA 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.
FLICA 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.