Summary
Caspase activity assays in multi-well plate formats represent powerful tools for understanding experimental modulation of the apoptotic response. These assays are configured to exploit functional, biochemical, and temporal differences in substrate specificity and selectivity, which are useful in defining the magnitude and mechanism of a compound or treatment effect. New advances in fluorescent and luminescent chemistries now enable single addition “add-mix-measure” determinations of caspase activity directly in the sample plate with unprecedented sensitivity. Unlike other more cumbersome or laborious techniques, caspase activity induction or inhibition measures are quantifiable and definitive. The highlighted techniques in this chapter are cost efficient and allow for the rapid exploration of thousands of combinations and conditions.
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
References
Tewari, M., Quan, L.T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D.R., Poirier, G.G., Salveson, G.S., and Dixit, V.M. (1995) Yama/CPP32, a mammalian homolog of Ced-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81, 801–809.
Casciola-Rosen, L.A., Miller, D.K., Anhalt, G.J., and Rosen, A. (1994) Specific cleavage of the 70-kD protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J. Biol. Chem. 269, 30757–30760.
Casciola-Rosen, L., Nicholson, D.W., Chong, T., Rowan, K.R. Thornberry, N.A., Miller, D.K., and Rosen, A. (1996) Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J. Exp. Med. 183, 1957–1964.
Martin, S.J., and Green, D.R. (1995) Protease activation during apoptosis: death by a thousand cuts? Cell 82, 349–352.
Chinnaiyan, A.M., and Dixit, V.M. (1996) The cell death machine. Curr. Biol. 6, 555–562.
Nicholson, D.W., and Thornberry, N.A. (1997) Caspases: killer proteases. Trends Biochem. Sci. 22, 299–306.
Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salveson, G., Thornberry, N.A., Wong, W.W., and Yaun, J.Y. (1996) Human ICE/CED-3 protease nomenclature. Cell 87, 171–173.
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. J. Biol. Chem 272, 17907–17911.
Kuida, K., Lippke, J.A., Ku, F., Harding, M.W., Livingston, D.J., Su, M.S., and Flavell, R.A. (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267, 2000–2003.
Li, P., Allen, H., Banerjee, S., Franklin, S., Herzog, L., Johnston, C., McDowell, J., Paskind, M., Rodman, L., Salfeld, J., Towne, E., Tracey, D., Wardell, S., Wei, F.-Y., Wong, W., Kamen, R., and Seshadri, T. (1995) Mice deficient in IL-1-converting enzyme are defective in production of mature IL-1 and resistant to endotoxic shock. Cell 80, 401–411.
Stennicke, H.R., and Salveson, G.S. (1997) Biochemical characteristics of caspases-3, -6, -7, and -8. J. Biol. Chem. 272, 25719–25723.
Garcia-Calvo, M., Peterson, E., Rasper, D., Vaillancourt, J.P., Zamboni, R., Nicholson, D.W., and Thornberry, N.A. (1999) Purification and catalytic properties of caspase family members. Cell Death. Differ. 6, 362–369.
Musio, M., Stockwell, B.R., Stennicke, H.R., Salvesen, G.S., and Dixit, V.M. (1998) An induced proximity model for caspase-8 activation. J. Biol. Chem. 273, 2926–2930.
Stennicke, H.R., Deveraux, Q.L., Humke, E.W., Reed, J.C., Dixit, V.M., and Salvesen, G.S. (1999) Caspase-9 can be activated without proteolytic processing. J. Biol. Chem. 274, 8359–8362.
Siegel, R.M., Frederiksen, J.K., Zacharias, D.A., Chan, F.K., Johnson, M., Lynch, D., Schweizer, A., Briand, C., and Grütterm, M.G. (2003) Crystal structure of caspase-2, apical initiator of the intrinsic apoptotic pathway. J. Biol. Chem. 278, 42441–42447.
Chan, F.K., Chun, H.J., Zheng, L., Siegel, R.M., Bui, K.L., and Lenardo, M.J. (2000) A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 288, 2351–2354.
Nicholson, D.W., and Thornberry, N.A. (2003) Life and death decisions. Science 299, 214–215.
Tsien, R.Y., and Lenardo, M.J. (2000) Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 288, 2354–2357.
Lin, C.-F., Chen, C.-L., Chang, W.-T., Jan, M.-S., Hsu, L.-J., Wu, R.-H., Tang, M.-J., Chang, W.-C., and Lin, Y.-S. (2004) Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramide and etoposide-induced apoptosis. J. Biol. Chem. 279, 40755–40761.
Wagner, K.W., Engels, I.H., and Deveraux, Q.L. (2004) Caspase-2 can function upstream of bid cleavage in the TRAIL apoptosis pathway. J. Biol. Chem. 279, 35047–35052.
Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A., Newmeyer, D.D., Wang, H.-G., Reed, J.C., Nicholson, D.W., Alnemri, E.S., Green, D.R., and Martin, S.J. (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchial activation of caspases-2, -3, -6, -7, -8, and 10 in a caspase-9-dependent manner. J. Cell Biol. 137, 469–479.
Liu, J., Bhalgat, M., Zhang, C., Diwu, Z., Hoyland, B., and Klaubert, D.H. (1999) Fluorescent molecular probes V: a sensitive caspase-3 substrate for fluorometric assays. Bioorg. Med. Chem. Lett. 9, 3231–3236.
O’Brien, M.A. Daily, W.J., Hesselberth, E.P., Moravec, R.A., Scurria, M., Klaubert, D.H., Bulleit, R.F., and Wood, K.V. (2005) Homogeneous, bioluminescent protease assays: caspase-3 as a model. J. Biomol. Screen. 10, 137–148.
Riss, T.L., and Moravec, R.A. (2004) Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev Technol 2, 51–62.
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
Niles, A.L., Moravec, R.A., Riss, T.L. (2008). Caspase Activity Assays. 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-339-4_11
Publisher Name: Humana Press
Print ISBN: 978-1-58829-457-9
Online ISBN: 978-1-59745-339-4
eBook Packages: Springer Protocols