Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.
References
1.
Green, D. (1998). Apoptotic pathways: The roads to ruin. Cell94:695.
2.
Tsien, R. (1998). The green fluorescent protein. Annu. Rev. Biochem.67:509.
3.
Pollok, B., Heim, R. (1999). Using GFP in FRET-based applications. Trends Cell Biol.9:57.
4.
Xu, X., Gerard, A., Huang, B., Anderson, D., Payan, D., Luo, Y. (1998). Detection of programmed cell death using fluorescence energy transfer. Nucl. Acid Res.26:2034.
5.
Mahajan, N., Linder, K., Berry, G., Gordon, G., Heim, R., Herman, B. (1998). Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis. Chem. Bio.6:401.
6.
Thomberry, N., Lazebnik, Y. (1998). Caspases: enemies within. Science281:1312.
7.
Zheng, T., Flavell, R. (1999). AllIs well that ends dead. Nature400:410.
8.
Yuan, J., Yanker, B. (1999). Caspase activity sows the seeds of neuronal death. Nat. Cell Biol.1:E44.
9.
Zapata, J., Takahashi, R., Salvensen, G..Reed, J. (1998). Granzyme release and caspase activation in activated human T-lymphocytes. J. Bio. Chem.273:6919.
10.
Grynkiewicz, G., Poenie, M., Tsien, R. ('1985). A new generation of Ca2 I indicators with greatly improved fluorescence properties. J. Bio. Chem.260:3440.
11.
Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. (1995). A novel assay for apoptosis: Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods184:39.
12.
Miyawaki, A., Lopis, J., Heim, R., McCaffery, J., Adams, J., Ikura, M., Tsien, R. (1997). Fluorescent indicators for Ca+ based on green fluorescent proteins and calmodulin. Nature388:882.
13.
Heim, R. (1999). Green fluorescent protein forms for energy transfer. Methods Enzymol.302:408.
14.
Talanian, R., Quinlan, C., Trautz, S., Hackett, M., Manovich, J., Banach, D., Ghayur, T., Brady, K., Wong, W. (1997). Substrate specificities of caspase family proteases. J. Biol. Chem.272:9677.
15.
Margolin, N., Raybuck, S., Wilson, K., Chen, W., Fox, T., Gu, Y., Livingston, D. (1997). Substrate and inhibitor specificity of interleukin-1β-converting enzyme and related caspases. J. Biol. Chem.272:7223.
16.
Enari, M., Hug, H., Nagata, S. (1995). Involvement of an ICE-like protease in apoptotic cells. EMBO J.14:6148.
17.
Ju, S-T., Panka, J., Cui, H., Ettinger, R., El-Khatib, M., Sherr, D., Stanger, B., Marshak-Rothstein, A. (1995). CD95 (CD95)/CD95L interactions required for programmed cell death after T cell activation. Nature373:444.
18.
Schlegel, J., Peters, I., Orrenius, S., Miller, D., Thornberry, N., Yamin, T-T., Nicholson, D. (1996). CPP-32/apopain is a key interleukin-1l3-converting enzyme-like protease involved in CD95-mediated apoptosis. J. Biol. Chem.271:1841.
19.
Dolle, R., Hoyer, D., Prasad, C., Schmidt, S., Helaszek, R., Miller, R., Ator, M. (1994). P1 aspartate-based a-((2,6-dichlorobenzoylfoxy) methyl ketones as potent time-dependent inhibitors of interleukin-l(-converting enzyme. J. Med. Chem.37:563.
20.
Jacobson, M., Weil, M., Raff, M. (1996). Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J. Cell Biol.133:1041.
21.
Zhang, J-H., Chung, T., Oldenburg, K. (1999). A simple parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen.4:67.
22.
Shifrin, V., Anderson, P. (1999). Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis. J. Biol. Chem.274:13985.
23.
Pintado, E., Baquero-Leonis, D., Conde, M., Sobrino, F. (1995). Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes. Biochem. Pharmacol.18:227.
24.
Courtney, M., Coffey, E. (1999). The mechanism of ara-C-induced apoptosis of differentiating cerebellar granule neurons. Eur. J. Neurosci.11:1073.
25.
Chandler, J., Cohen, G., MacFarlane, M. (1998). Different subcellular distribution of caspase-3 and caspase-7 following CD95-induced apoptosis in mouse liver. J. Biol. Chem.273:10815.
