Molecular Toxicology Protocols pp 623-632

Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

Detection of Programmed Cell Death in Cells Exposed to Genotoxic Agents Using a Caspase Activation Assay



Many toxins that individuals are exposed to cause DNA damage. Cells that have sustained DNA damage may attempt to repair the damage prior to replication. However, if a cell has sustained serious damage it cannot repair, it will commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late enough in the PCD pathway that a cell can no longer avoid cell death, but still earlier than PCD-associated morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells. The analysis and documentation is performed using fluorescence microscopy.

Key words

Apoptosis Programmed cell death (PCD) Caspase Inhibitor Fluorescence DNA damage Genotoxic agent 


  1. 1.
    Schins RP (2002) Mechanisms of genotoxicity of particles and fibers. Inhal Toxicol 14:57–78PubMedCrossRefGoogle Scholar
  2. 2.
    Snyder RD, Green JW (2001) A review of the genotoxicity of marketed pharmaceuticals. Mutat Res 488:151–169PubMedCrossRefGoogle Scholar
  3. 3.
    Weisburger JH (2001) Antimutagenesis and anticarcinogenesis from the past to the future. Mutat Res 480–481:23–35PubMedCrossRefGoogle Scholar
  4. 4.
    Zito R (2001) Low doses and thresholds in genotoxicity: from theories to experiments. J Exp Clin Cancer Res 20:315–325PubMedGoogle Scholar
  5. 5.
    Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147:742–758PubMedCrossRefGoogle Scholar
  6. 6.
    Lincz LF (1998) Decipher the apoptotic pathway: all roads lead to death. Immunol Cell Biol 76:1–19PubMedCrossRefGoogle Scholar
  7. 7.
    Jenner P, Olanow CW (1996) Oxidative stress and the pathogenesis of Parkinson’s disease. Neurology 47:161–170CrossRefGoogle Scholar
  8. 8.
    Paradis E, Douillard H, Koutroumanis M, Gooryer C, LeBlanc A (1996) Amyloid beta peptide of Alzheimer’s disease downregulates bcl-2 and upregulates bax expression in human neurons. J Neurosci 16:7533–7539PubMedGoogle Scholar
  9. 9.
    Hetts SW (1998) To die or not to die: an overview of apoptosis and its role in disease. JAMA 279:300–307PubMedCrossRefGoogle Scholar
  10. 10.
    Zimmerman KC, Bonzon C, Green DR (2001) The machinery of programmed cell death. Pharmacol Ther 92:57–70CrossRefGoogle Scholar
  11. 11.
    Zou H, Henzel J, Lui X, Lutschg A, Wang X (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90: 405–413PubMedCrossRefGoogle Scholar
  12. 12.
    Li P, Nijhawan D, Budihardjo I et al (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479–489PubMedCrossRefGoogle Scholar
  13. 13.
    Cai J, Yang J, Jones DP (1998) Mitochondrial control of apoptosis: the role of cytochrome c. Biochim Biophys Acta 1366:139–149PubMedCrossRefGoogle Scholar
  14. 14.
    Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9: 459–470PubMedCrossRefGoogle Scholar
  15. 15.
    Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates and functions during apoptosis. Annu Rev Biochem 68:383–424PubMedCrossRefGoogle Scholar
  16. 16.
    Constantinou C, Papas KA, Constantinou AI (2009) Caspase-independent pathways of programmed cell death: the unravelling of new targets of cancer therapy? Curr Cancer Drug Targets 9:717–728PubMedCrossRefGoogle Scholar
  17. 17.
    Merle-Béral H, Barbier S, Roué G, Bras M, Sarfati M, Susin SA (2009) Caspase-independent type III PCD: a new means to modulate cell death in chronic lymphocytic leukemia. Leukemia 23:974–977PubMedCrossRefGoogle Scholar
  18. 18.
    Schrader K, Huai J, Jöckel L, Oberle C, Borner C (2010) Non-caspase proteases: triggers or amplifiers of apoptosis. Cell Mol Life Sci 67: 1607–1616PubMedCrossRefGoogle Scholar
  19. 19.
    Favreau DJ, Meessen-Pinard M, Desforges M, Talbot PJ (2012) Human coronavirus-induced neuronal programmed cell death is cyclophilin D-dependent and potentially caspase-dispensable. J Virol 86:81–93PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    McCarthy NJ, Evan GI (1998) Methods for detecting and quantifying apoptosis. Curr Top Dev Biol 36:259–278PubMedCrossRefGoogle Scholar
  21. 21.
    Köhler C, Orrenius S, Zhivotovsky B (2002) Evaluation of caspase activity in apoptotic cells. J Immunol Methods 265:97–110PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2014

Authors and Affiliations

  • Madhu Gupta
    • 2
  • Madhumita Santra
    • 2
  • Patrick P. Koty
    • 1
  1. 1.Department of PediatricsWake Forest University School of MedicineWinston-SalemUSA
  2. 2.Department of PediatricsWake Forest University School of MedicineWinston-SalemUSA

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