Home LiteratureArticle Details
PMID: 12126059 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Review

Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases.

Human cell ·Vol. 15 ·No. 1 ·2002-03-00 ·Pages 1-12

Grabarek J, Amstad P, Darzynkiewicz Z

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 FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 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.

MeSH Terms
Affinity Labels Apoptosis Caspase Inhibitors Caspases/analysis Cells, Cultured Enzyme Inhibitors Flow Cytometry Fluoresceins Fluorescent Dyes Humans Microscopy, Fluorescence
Chemicals
Affinity Labels Caspase Inhibitors Enzyme Inhibitors Fluoresceins Fluorescent Dyes Caspases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Grabarek Jerzy
New York Medical College, Hawthorne, NY 10532, USA.
Amstad Paul
Darzynkiewicz Zbigniew
References (64)
64 references, click to expand
  1. Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors.
    Biotechniques. 2001 Sep;31(3):608-10, 612, 614, passim PMID: 11570504
  2. Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells.
    J Cell Sci. 2001 Feb;114(Pt 4):663-70 PMID: 11171371
  3. The S-phase cytotoxicity of camptothecin.
    Exp Cell Res. 1991 Mar;193(1):27-35 PMID: 1995300
  4. Fluorescent molecular probes V: a sensitive caspase-3 substrate for fluorometric assays.
    Bioorg Med Chem Lett. 1999 Nov 15;9(22):3231-6 PMID: 10576694
  5. Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor.
    Int J Oncol. 2002 Feb;20(2):225-33 PMID: 11788882
  6. Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis.
    Exp Cell Res. 2000 Nov 1;260(2):248-56 PMID: 11035919
  7. Apoptosis, oncosis, and necrosis. An overview of cell death.
    Am J Pathol. 1995 Jan;146(1):3-15 PMID: 7856735
  8. 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. 2000 Feb 25;255(1):125-32 PMID: 10666341
  9. Flow cytometry of apoptotic cell death.
    J Immunol Methods. 2000 Sep 21;243(1-2):167-90 PMID: 10986414
  10. Apoptosis. Its significance in cancer and cancer therapy.
    Cancer. 1994 Apr 15;73(8):2013-26 PMID: 8156506
  11. 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. 2000 Jun 30;121(2):223-8 PMID: 10876036
  12. Slide-based laser scanning cytometry.
    Acta Cytol. 1997 Jan-Feb;41(1):123-43 PMID: 9022736
  13. Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry.
    Cytometry. 2001 May 1;44(1):73-82 PMID: 11309811
  14. Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin.
    Int J Oncol. 1999 Nov;15(5):861-71 PMID: 10536167
  15. Spontaneous in contrast to CD95-induced neutrophil apoptosis is independent of caspase activity.
    J Trauma. 2001 Jun;50(6):982-8 PMID: 11426111
  16. Mitochondrial release of caspase-2 and -9 during the apoptotic process.
    J Exp Med. 1999 Jan 18;189(2):381-94 PMID: 9892620
  17. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias.
    Leukemia. 1993 May;7(5):659-70 PMID: 8483318
  18. Noncaspase proteases in apoptosis.
    Leukemia. 2000 Sep;14(9):1695-703 PMID: 10995018
  19. Sequential activation of caspases and serine proteases (serpases) during apoptosis.
    Cell Cycle. 2002 Mar-Apr;1(2):124-31 PMID: 12429921
  20. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation.
    Exp Cell Res. 2000 Aug 25;259(1):308-13 PMID: 10942603
  21. Autoradiography with tritiated methotrexate and the cellular distribution of folate reductase.
    Science. 1966 Mar 25;151(3717):1528-30 PMID: 5909582
  22. Nuclear localization of procaspase-9 and processing by a caspase-3-like activity in mammary epithelial cells.
    Eur J Cell Biol. 2000 May;79(5):358-64 PMID: 10887967
  23. 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. 1999 Feb;32(1):25-37 PMID: 10371301
  24. Biochemical pathways of caspase activation during apoptosis.
    Annu Rev Cell Dev Biol. 1999;15:269-90 PMID: 10611963
  25. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
    Br J Cancer. 1972 Aug;26(4):239-57 PMID: 4561027
  26. ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-alpha.
    Immunol Lett. 1997 Jan;55(1):5-10 PMID: 9093874
  27. Caspase inhibitors.
    Cell Death Differ. 1999 Nov;6(11):1081-6 PMID: 10578177
  28. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.
    EMBO J. 1997 May 1;16(9):2271-81 PMID: 9171342
  29. Development and application of a GFP-FRET intracellular caspase assay for drug screening.
    J Biomol Screen. 2000 Oct;5(5):307-18 PMID: 11080689
  30. Effect of protease inhibitors on early events of apoptosis.
    Exp Cell Res. 1996 Mar 15;223(2):372-84 PMID: 8601414
  31. Activation of caspase-1 in the nucleus requires nuclear translocation of pro-caspase-1 mediated by its prodomain.
    J Biol Chem. 1998 Sep 11;273(37):23621-4 PMID: 9726961
  32. Caspase knockouts: matters of life and death.
    Cell Death Differ. 1999 Nov;6(11):1043-53 PMID: 10578172
  33. Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells.
    Cytometry. 2000 Jun 1;40(2):151-60 PMID: 10805935
  34. During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential.
    Exp Cell Res. 2000 Jun 15;257(2):290-7 PMID: 10837143
  35. Human ICE/CED-3 protease nomenclature.
    Cell. 1996 Oct 18;87(2):171 PMID: 8861900
  36. Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.
    Oncogene. 1997 Jun 12;14(23):2741-52 PMID: 9190889
  37. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.
    J Biol Chem. 1997 Jul 18;272(29):17907-11 PMID: 9218414
  38. Prodomain-dependent nuclear localization of the caspase-2 (Nedd2) precursor. A novel function for a caspase prodomain.
    J Biol Chem. 1998 Sep 18;273(38):24535-42 PMID: 9733748
  39. Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo.
    J Immunol Methods. 1999 Jun 24;226(1-2):43-8 PMID: 10410970
  40. Laser-scanning cytometry: A new instrumentation with many applications.
    Exp Cell Res. 1999 May 25;249(1):1-12 PMID: 10328948
  41. Mechanism of action of camptothecin.
    Ann N Y Acad Sci. 1996 Dec 13;803:44-9 PMID: 8993499
  42. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis).
    Cytometry. 1997 Jan 1;27(1):1-20 PMID: 9000580
  43. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis.
    Cancer Res. 1993 Sep 1;53(17):3976-85 PMID: 8358726
  44. Mammalian caspases: structure, activation, substrates, and functions during apoptosis.
    Annu Rev Biochem. 1999;68:383-424 PMID: 10872455
  45. The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling.
    J Cell Biol. 1998 Mar 23;140(6):1485-95 PMID: 9508780
  46. Nucleolar segregation as an early marker for DNA damage; an experimental study in rats treated with 4-hydroxyaminoquinoline 1-oxide.
    Virchows Arch. 1995;426(3):295-300 PMID: 7773509
  47. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates.
    J Exp Med. 2000 Jun 5;191(11):1819-28 PMID: 10839799
  48. Features of apoptotic cells measured by flow cytometry.
    Cytometry. 1992;13(8):795-808 PMID: 1333943
  49. DNA strand breaks occurring during apoptosis - their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors.
    Int J Oncol. 1992 Nov;1(6):639-48 PMID: 21584593
  50. Catalytic properties of the caspases.
    Cell Death Differ. 1999 Nov;6(11):1054-9 PMID: 10578173
  51. Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones.
    Biochemistry. 1994 Apr 5;33(13):3934-40 PMID: 8142397
  52. Caspase structure, proteolytic substrates, and function during apoptotic cell death.
    Cell Death Differ. 1999 Nov;6(11):1028-42 PMID: 10578171
  53. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.
    Nature. 1994 Sep 22;371(6495):346-7 PMID: 8090205
  54. Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat T cells.
    Exp Cell Res. 1999 Feb 1;246(2):491-500 PMID: 9925765
  55. 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. 2000 Nov;48(11):1521-30 PMID: 11036095
  56. Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis.
    J Immunol Methods. 2002 Jul 1;265(1-2):111-21 PMID: 12072182
  57. Nucleolar segregation during apoptosis of haemopoietic stem cell line FDCP-Mix.
    Cell Death Differ. 1999 Apr;6(4):334-41 PMID: 10381627
  58. CpG-specific common commitment in caspase-dependent and -independent cell deaths.
    Mol Cell Biol Res Commun. 2000 Jan;3(1):33-41 PMID: 10683315
  59. Stathmo-apoptosis: arresting apoptosis by fluorochrome-labeled inhibitor of caspases.
    Int J Oncol. 2001 Oct;19(4):657-63 PMID: 11562738
  60. Measurement of caspase activity in individual cells reveals differences in the kinetics of caspase activation between cells.
    Cell Death Differ. 2001 Jan;8(1):38-43 PMID: 11313701
  61. Caspases: their intracellular localization and translocation during apoptosis.
    Cell Death Differ. 1999 Jul;6(7):644-51 PMID: 10453075
  62. Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells.
    Biochemistry. 1999 Oct 19;38(42):13906-11 PMID: 10529236
  63. Inhibition of human caspases by peptide-based and macromolecular inhibitors.
    J Biol Chem. 1998 Dec 4;273(49):32608-13 PMID: 9829999
  64. Insights into UV-induced apoptosis: ultrastructure, trichrome stain and spectral imaging.
    Micron. 2002;33(2):157-66 PMID: 11567885
Article Info
Journal
Human cell
Abbr.
Hum Cell
ISSN
0914-7470
DOI
10.1111/j.1749-0774.2002.tb00094.x
Published
2002-03-00
Pages
1-12
Language
English
Region
Japan
NLM ID
8912329
Subset
IM
Grants
NCI NIH HHS · R01 CA028704 · United States
NCI NIH HHS · R01 CA028704-25 · United States
NCI NIH HHS · CA 28 704 · United States
External Links
PubMed source DOI: 10.1111/j.1749-0774.2002.tb00094.x View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2