Abstract
Activation of caspases is the key event of apoptosis and different approaches were developed to assay it To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLlCA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:l stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.
Similar content being viewed by others
References
Alnemri ES, Livingston DI, Nicholson DW et al: Human ICE/CED-4 protease nomenclature. Cell 87: 171–173, 1996.
Kaufmann SH, Desnoyers S, Ottaviano Y et al: Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 53: 37–3985, 1993.
Lazebnik YA, Kaufmann SH, Desnoyers S et al: Cleavage of poly(ADP-ribose) polymerase by proteinase with properties like ICE. Nature 371: 346–347, 1994.
Budihardjo I, Oliver H, Lutter M et al: Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15: 269–290, 1999
Earnshaw WC, Martins LM, Kaufmann SH: Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383–424, 1999.
Nicholson, DW. Caspase structure, proteolytic substrates and function during apoptotic cell death. Cell Death Differ 6: 1028–1042, 1999.
Zhang TS, Hunort S, Kuida K et al: Caspase knockouts: matters of life and death. Cell Death Differ 6: 1043–1053, 1999.
Stennicke HR Salvesen GS: Catalytic properties of caspases. Cell Death Differ 6: 1060–1066, 1999.
Zhivotovsky B, Samali A, Gahm A et al: Caspases: their intracellular localization and translocation during apoptosis. Cell Death Differ 6: 644–651, 1999.
Li X, Darzynkiewicz Z: Cleavage of poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis. Exp Cell Res 255: 125–132, 2000.
Li X, Du L, Darzynkiewicz Z: During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondria1 electrochemical potential. Exp Cell Res 257: 290–297, 2000.
Tanaka M, Momoi T, Marunouchi T: In situ detection of activated caspase-3 in apoptotic granule neurons in the developing cerebellum in slice cultures and in vivo. Brain Res Dev Brain Res; 121: 223–228, 2000.
Gorman AM, Hirt UA, Zhivotovsky B et al: Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo. J Immunol Methods; 226: 43–48, 1999.
Liu J, Bhalgat M, Zhang C et al: Fluorescent molecular probes V a sensitive caspase-3 substrate for fluorometric assays. Bioorg Med Chem Lett 9: 3231–3236, 1999.
Hug H, Los M, Hirt W et al: Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells. Biochemistry 38: 13906–13911, 1999.
Komoriya A, Packard BZ, Brown MJ et al: Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J Exp Med 191: 1819–1828, 2000.
Belloc F, Belaund-Rotureau MA, Lavignolle V et al Flow cytometry of caspase-3 activation in preapoptotic leukemic cells. Cytometry 40: 151–160, 2000.
Jones J, Heim JR, Hare E et al: Development and application of a GFP-FRET intracellular caspase assay for drug screening. J Biomol Screen 5: 307–318, 2000.
Morgan MJ, Thorburn A: Measurement of caspase activity in individual cells reveals differences in the kinetics of caspase activation between cells. Cell Growth Differ 8: 38–43, 2001.
Bedner E, Smolewski P, Amstad P et al: Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res 260: 308–313, 2000
Smolewski P, Bedner E, Du L et al: Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry. Cytometry 2001; 44: 73–82.
Amstad PA, Yu G, Johnson GL et al: Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques 31: 608–616, 2001.
Ostrowski K, Barnard E, Darzynkiewicz Z: Localization of acetylcholinesterase activity using a 34-labeled irreversible inhibitor. Exp Cell Res 31: 8999, 1963.
Darzynkiewicz Z, Barnard, EA: Specific proteases of mast cells. Nature 213: 1198–1203, 1967.
Darzynkiewicz Z, Rogers AW, Barnard EA et al: Autoradiography with tritiated methotrexate and the cellular distribution of folate reductase. Science 131: 1538–1530, 1966.
Kunstle G, Leist M, Uhlig S et al: ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-alpha. Immunol Let 55: 511, 1997.
Ekert PG, Silke J, Vaux DL Caspase inhibitors. Cell Death Differ 6: 1081–1086, 1999.
Thornberry NA, Peterson EP, Zhao JJ et al: Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry 33: 3934–3940, 1994.
Garcia-Calvo, M, Peterson EP, Leiting B et al: Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 273: 32608–32613, 1998.
Kamentsky LA, Burger DE, Gershman RJ et al: Slide-based laser scanning cytometry. Acta Cytol 41: 123–143, 1997.
Darzynkiewicz Z, Bedner E, Li X et al: Laser scanning cytometry. A new instrumentation with many applications. Exp Cell Res 249: 1–12, 1999.
Thornberry NA, Rano TA, Peterson EP et al: A combinatorial approach defines specificities of members of the caspase family and granzyme B. J Biol Chem 272: 17907–17911, 1997.
Mancini M, Nicholson DW, Roy S et al: The caspase-3 precursor has a cytosolic and mitochondria1 distribution: implications for apoptotic signaling. J Cell Biol 140: 1485–1495, 1998.
Susin SA, Lorenzo HK, Zamzani N et al: Mitochondria1 release of caspase-2 and -9 during apoptotic process. J Exp Med 189: 381–393, 1999.
Collusi PA, Harvey NL, Kumar S: Prodomain-dependent nuclear localization of the caspase-2 (Nedd-2) precursor. A novel function for a caspase prodomain. J Biol Chem 273: 24535–24542, 1998.
Mao PL, Jiang Y, Wee BY et al: Activation of caspase-1 in the nucleus requires nuclear translocation of pro-caspase-1 mediated by its prodomain. J Biol Chem 273: 23621–23624, 1998.
Ritter PM, Marti A, Blanc C et al: Nuclear 1ocaIization of procaspase-9 and processing by caspase-3-like activity to mammary epithelial cells. Eur J Cell Biol 79: 358–364, 2000.
Imazawa T, Nishikawa A, Tada M et al: Nucleolar segregation as an early marker for DNA damage; an experimental study in rats treated with 4-hydroxyaminoquinolone 1-oxide. Virchows Arch 426: 295–300, 1995.
Raipert S, Bennion G, Hickman JA et al: Nucleolar segregation during apoptosis of hematopoietic stem cell line FDCP-Mix. Cell Death Differ 6: 334–341, 1999.
Miller ML, Andriga A, Dixon K et al: Insights into W-induced apoptosis: ultrastructure, trichrome staining and spectral imaging. Micron 33: 157–166, 2002.
Horky M, Wurzer G, Kotala V et al: Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells. J Cell Sci 114: 663–670, 2001.
Halicka HD, Bedner E, Darzynkiewicz Z: Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis. Exp Cell Res 260: 248–256, 2000.
Torres-Montaner A, Bolivar J, Astole A et al: Immunohistochemical detection of ribosomal transcription factor UBF and AgNOR staining identify apoptotic events in neoplastic cells of Hodgkin’s disease and in other lymphoid cells. J Histochem Cytochem 48: 1521–1530, 2000.
Takahashi A, Hirata H, Yonehara S et al: Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine and CrmA-sensitive caspase-8. Oncogene 14: 2741–2752; 1997.
Johnson DE: Noncaspase proteases in apoptosis. Leukemia, 14: 1695–1703, 2000.
Faleiro L, Kobayashi R, Fearnhead H, Lazebnik Y Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO Journal 16: 2271–2281; 1997.
Chow S, Slle E, MacFarlane M et al: Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat cells. Exp Cell Res 246: 491–500; 1999.
Darzynkiewicz Z, Bruno S, Del Bino G et al: Features of apoptotic cells measured by flow cytometry. Cytometry 13: 795–808, 1992.
Smolewski P, Grabarek J, Halicka HD et al: Assay of caspases activation in situ combined with probing plasma membrane integrity detects three distinct stages of apoptosis. J Immunol Meth 2002
Darzynkiewicz Z, Juan G, Li X et al: Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27: 1–20, 1997.
Majno G, Joris I. Apoptosis, oncosis and necrosis. An overview of cell death. Am J Pathol 146: 316, 1995.
Vermes I, Haanen C, Reutelingsperger C: Flow cytometry of apoptotic cell death. J Immunol Meth 243: 167–190, 2000.
Harter L, Keel M, Hentze H et al: Spontaneous in contrast to CD95induced neutrophil apoptosis is independent of caspase activity. J Trauma 50: 982–88, 2001.
Qi L, Sit KH. CpG-specific common commitment in caspase-dependent and -independent cell death. Mol Cell Biol Res Commun 3: 33–41, 2000.
Gorczyca W, Bruno S, Darzynkiewicz RJ et al: DNA strand breaks occurring during apoptosis: their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol 1: 639–648, 1992.
Kerr JFR, Wyllie AH, Curie AFt Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257, 1972.
Kerr JFR, Winterford CM, Harmon V Apoptosis. Its significance in cancer and cancer therapy. Cancer 73: 2013–2026, 1994.
Del Bino G, Darzynkiewicz Z, Degraef C et al: Comparison of methods based on annexin V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells. Cell Prolif 32: 25–37, 1999.
Hara S, Halicka HD, Bruno S et al: Effect of protease inhibitors on early events of apoptosis. Exp Cell Res 232: 372–384, 1996.
Smolewski P, Grabarek J, Phelps DJ et al: Stathmoapoptosis: arresting apoptosis by fluorochrome-labeled inhibitor of caspases. Int J Oncol 19: 657–663, 2001.
Darzynkiewicz Z, Traganos F, Kimmel M: Assay of cell cycle kinetics by multivariate flow cytometry using the principle of stathmokinesis. In: Techniques in Cell Cycle Analysis. JW Gray and Z Darzynkiewicz Eds. Humana Press, Clifton, NJ, USA, 1997, pp. 291–336.
Del Bino G, Lassota P, Darzynkiewicz Z: The S-phase cytotoxicity of camptothecin. Exp Cell Res 193: 27–35, 1991.
Deptala A, Li X, Bedner E et al: Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin. Int J Oncol 15: 861–871, 1999.
Liu LF, Duann P, Lin C-P et al: Mechanism of action of camptothecin. Ann NY Acad Sci 803: 44–49, 1996.
Gorczyca W, Bigman K, Mittelman A et al: Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7: 659–670, 1993.
Grabarek J, Johnson GL, Lee BW et al: Sequential activation of caspases and serine proteases (serpases) during apoptosis. Cell Cycle 1: 2002 (in press)
Grabarek, J, Dragan M, Lee BW et al: Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int J Oncol 20: 225–233, 2002.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Grabarek, J., Amstad, P. & Darzynkiewicz, Z. Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases. Hum Cell 15, 1–12 (2002). https://doi.org/10.1111/j.1749-0774.2002.tb00094.x
Published:
Issue Date:
DOI: https://doi.org/10.1111/j.1749-0774.2002.tb00094.x