Advertisement

Measurement of Caspase Activation in Mammalian Cell Cultures

  • Magnus Olsson
  • Boris Zhivotovsky
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1133)

Abstract

The majority of caspases, cysteine-dependent aspartate-directed proteases, being in their activated state are involved in regulation of apoptosis by cleaving protein substrates harboring specific target motifs. Basically all biochemical and morphological changes in an apoptotic cell, including cell shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing, are consequence of caspase-mediated proteolysis. Thus, uncovering activities of unique caspases are key determinants of the apoptotic process. This chapter describes a set of experimental protocols available for characterization, quantification and inhibition of caspase activities in mammalian cell cultures, including immunoblotting, usage of synthetic substrates, flow cytometry, and microscopic techniques.

Key words

Apoptosis Caspase Caspase substrate SDS-PAGE Immunocytochemistry FACS 

Abbreviations

BSA

Bovine serum albumin

FACS

Fluorescence activated cell sorting

pAb

Polyclonal antibody

SDS

Sodium dodecyl sulfate

Tris

Tris(hydroxymethyl)aminomethane, mAb, monoclonal antibody

Notes

Acknowledgements

Work in the authors’ laboratory was supported by grants from the Swedish and Stockholm Cancer Societies, the Swedish Research Council, and the Swedish Childhood Cancer Foundation.

References

  1. 1.
    Degterev A, Boyce M, Yuan J (2003) A decade of caspases. Oncogene 22:8543–8567PubMedCrossRefGoogle Scholar
  2. 2.
    Fuentes-Prior P, Salvesen GS (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384:201–232PubMedCrossRefGoogle Scholar
  3. 3.
    Shi Y (2004) Caspase activation: revisiting the induced proximity model. Cell 117:855–858PubMedCrossRefGoogle Scholar
  4. 4.
    Luthi AU, Martin SJ (2007) The CASBAH: a searchable database of caspase substrates. Cell Death Differ 14:641–650PubMedCrossRefGoogle Scholar
  5. 5.
    Fridman A, Pak I, Butts BD, Hoek M, Nicholson DW, Mehmet H (2013) MerCASBA: an updated and refined database of caspase substrates. Apoptosis 18:369–371PubMedCrossRefGoogle Scholar
  6. 6.
    Liu X, Zou H, Slaughter C, Wang X (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89:175–184PubMedCrossRefGoogle Scholar
  7. 7.
    Li H, Zhu H, Xu CJ, Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501PubMedCrossRefGoogle Scholar
  8. 8.
    Fischer U, Janicke RU, Schulze-Osthoff K (2003) Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 10:76–100PubMedCrossRefGoogle Scholar
  9. 9.
    Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G, Murcia JM (1998) Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 273:33533–33539PubMedCrossRefGoogle Scholar
  10. 10.
    Kim MY, Zhang T, Kraus WL (2005) Poly(ADP-ribosyl)ation by PARP-1: ‘PAR-laying’ NAD + into a nuclear signal. Genes Dev 19:1951–1967PubMedCrossRefGoogle Scholar
  11. 11.
    Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B et al (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187:567–572PubMedCrossRefGoogle Scholar
  12. 12.
    Pozarowski P, Grabarek J, and Darzynkiewicz Z (2003) Flow cytometry of apoptosis. Curr Protoc Cytom Chapter 7, Unit 7 19Google Scholar
  13. 13.
    McStay GP, Salvesen GS, Green DR (2008) Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ 15:322–331PubMedCrossRefGoogle Scholar
  14. 14.
    Callus BA, Vaux DL (2007) Caspase inhibitors: viral, cellular and chemical. Cell Death Differ 14:73–78PubMedCrossRefGoogle Scholar
  15. 15.
    Smolewski P, Bedner E, Du L, Hsieh TC, Wu JM, Phelps DJ et al (2001) Detection of caspases activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44:73–82PubMedCrossRefGoogle Scholar
  16. 16.
    Tinel A, Tschopp J (2004) The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 304:843–846PubMedCrossRefGoogle Scholar
  17. 17.
    Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV et al (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19:107–120PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Magnus Olsson
    • 1
  • Boris Zhivotovsky
    • 1
  1. 1.Division of ToxicologyInstitute of Environmental Medicine, Karolinska InstitutetStockholmSweden

Personalised recommendations