title: Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation.
abstract: BACKGROUND
  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.
  
  
  METHODS
  Apoptosis of HL-60, Jurkat, MCF-7 and T-24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H(2)O(2)). 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
authors.forename: P
authors.initials: P
authors.lastname: Pozarowski
authors.forename: X
authors.initials: X
authors.lastname: Huang
authors.forename: D H
authors.initials: DH
authors.lastname: Halicka
authors.forename: B
authors.initials: B
authors.lastname: Lee
authors.forename: G
authors.initials: G
authors.lastname: Johnson
authors.forename: Z
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D004396:Coloring Agents / Q000494:pharmacology; D004249:DNA Damage; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019169:Jurkat Cells; D008024:Ligands; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D018384:Oxidative Stress; D011419:Propidium / Q000494:pharmacology; D011485:Protein Binding; D012107:Research Design; D013223:Statistics as Topic; D013997:Time Factors; D019772:Topotecan / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D008024:Ligands; D011419:Propidium; D019772:Topotecan; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases