Summary
A number of methods have been developed to identify the cells that undergo apoptosis by analyzing the morphological, biochemical, and molecular changes that take place during this universal biological process. The best recognized biochemical hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the fluorescein-labeled CaspaTag pan-caspase in situ detection kit.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Danial, N. N., and Korsmeyer, S. J. (2004) Cell death: critical control points. Cell 116, 205–19.
Green, D. R., and Evan, G. I. (2002) A matter of life and death. Cancer Cell 1, 19–30.
Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–62.
Evan, G. I., and Vousden, K. H. (2001) Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342–8.
Chen, Q., Gong, B., and Almasan, A. (2000) Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ 7, 227–33.
Gong, B., and Almasan, A. (2000) Apo2 ligand/TNF-related apoptosis-inducing ligand and death receptor 5 mediate the apoptotic signaling induced by ionizing radiation in leukemic cells. Cancer Res 60, 5754–60.
Kumar, S. (1999) Mechanisms mediating caspase activation in cell death. Cell Death Differ 6, 1060–6.
Walker, N. P., Talanian, R. V., Brady, K. D., Dang, L. C., Bump, N. J., Ferenz, C. R., Franklin, S., Ghayur, T., Hackett, M. C., Hammill, L. D., and et al. (1994) Crystal structure of the cysteine protease interleukin-1 beta-converting enzyme: a (p20/p10)2 homodimer. Cell 78, 343–52.
Rotonda, J., Nicholson, D. W., Fazil, K. M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E. P., Rasper, D. M., Ruel, R., Vaillancourt, J. P., Thornberry, N. A., and Becker, J. W. (1996) The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat Struct Biol 3, 619–25.
Wilson, K. P., Black, J. A., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and et al. (1994) Structure and mechanism of interleukin-1 beta converting enzyme. Nature 370, 270–5.
Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., and Nicholson, D. W. (1997) 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 272, 17907–11.
Hengartner, M. O. (2000) The biochemistry of apoptosis. Nature 407, 770–6.
Fischer, U., Janicke, R. U., and Schulze-Osthoff, K. (2003) Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 10, 76–100.
Gong, B., Chen, Q., Endlich, B., Mazumder, S., and Almasan, A. (1999) Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth Differ 10, 491–502.
Mazumder, S., Chen, Q., Gong, B., Drazba, J. A., Buchsbaum, J. C., and Almasan, A. (2002) Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells. Mol Cell Biol 22, 2398–409.
Mazumder, S., Gong, B., and Almasan, A. (2000) Cyclin E induction by genotoxic stress leads to apoptosis of hematopoietic cells. Oncogene 19, 2828–35.
Ray, S., and Almasan, A. (2003) Apoptosis induction in prostate cancer cells and xenografts by combined treatment with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and CPT-11. Cancer Res 63, 4713–23.
Masri, S. C., Yamani, M. H., Russell, M. A., Ratliff, N. B., Yang, J., Almasan, A., Apperson-Hansen, C., Li, J., Starling, R. C., McCarthy, P., Young, J. B., and Bond, M. (2003) Sustained apoptosis in human cardiac allografts despite histologic resolution of rejection. Transplantation 76, 859–64.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press Inc.
About this protocol
Cite this protocol
Mazumder, S., Plesca, D., Almasan, A. (2008). Caspase-3 Activation is a Critical Determinant of Genotoxic Stress-Induced Apoptosis. In: Mor, G., Alvero, A.B. (eds) Apoptosis and Cancer. Methods in Molecular Biology™, vol 414. Humana Press. https://doi.org/10.1007/978-1-59745-339-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-339-4_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-457-9
Online ISBN: 978-1-59745-339-4
eBook Packages: Springer Protocols