Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

J. Exp. Med. 191 (11): 1819-1828

Copyright © 2000 by the Rockefeller University Press.


Original Article

Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates

Akira Komoriyaa, Beverly Z. Packarda, Martin J. Brownb, Ming-Lei Wub, , and Pierre A. Henkartb

a OncoImmunin, Incorporated, Gaithersburg, Maryland 20877
b Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Experimental Immunology Branch, National Cancer Institute, Bldg. 10, Rm. 4B36, National Institutes of Health, Bethesda, MD 20892-1360.301-496-0887301-435-6404

ph8j{at}nih.gov

Abstract: To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

Key Words: apoptosis • caspase • PhiPhiLuxTM • thymocyte • lymphocyte


Abbreviations used in this paper: apaf-1, apoptotic protein activating factor 1; DTT, dithiothreitol; FADD, Fas-associated death domain protein; ICE, IL-1β–converting enzyme; PI, propidium iodide.

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry.
C. J. F. Van Noorden (2010)
Journal of Histochemistry & Cytochemistry 58, 481-497
   Abstract »    Full Text »    PDF »
Time-lapse observation of cellular function with fluorescent probe reveals novel CTL-target cell interactions.
M. Tomura, Y. S. Mori, R. Watanabe, M. Tanaka, A. Miyawaki, and O. Kanagawa (2009)
Int. Immunol. 21, 1145-1150
   Abstract »    Full Text »    PDF »
Granzyme B Activity in Target Cells Detects Attack by Cytotoxic Lymphocytes.
B. Z. Packard, W. G. Telford, A. Komoriya, and P. A. Henkart (2007)
J. Immunol. 179, 3812-3820
   Abstract »    Full Text »    PDF »
Differences and Similarities in Viral Life Cycle Progression and Host Cell Physiology after Infection of Human Dendritic Cells with Modified Vaccinia Virus Ankara and Vaccinia Virus.
A. Chahroudi, D. A. Garber, P. Reeves, L. Liu, D. Kalman, and M. B. Feinberg (2006)
J. Virol. 80, 8469-8481
   Abstract »    Full Text »    PDF »
Multiparameter Measurement of Caspase 3 Activation and Apoptotic Cell Death in NT2 Neuronal Precursor Cells Using High-Content Analysis.
M. Fennell, H. Chan, and A. Wood (2006)
J Biomol Screen 11, 296-302
   Abstract »    PDF »
{gamma}{delta} T Cells Regulate the Extent and Duration of Inflammation in the Central Nervous System by a Fas Ligand-Dependent Mechanism.
E. D. Ponomarev and B. N. Dittel (2005)
J. Immunol. 174, 4678-4687
   Abstract »    Full Text »    PDF »
Upstream Regulatory Role for XIAP in Receptor-Mediated Apoptosis.
J. C. Wilkinson, E. Cepero, L. H. Boise, and C. S. Duckett (2004)
Mol. Cell. Biol. 24, 7003-7014
   Abstract »    Full Text »    PDF »
Examining Intracellular Organelle Function Using Fluorescent Probes: From Animalcules to Quantum Dots.
D. B. Zorov, E. Kobrinsky, M. Juhaszova, and S. J. Sollott (2004)
Circ. Res. 95, 239-252
   Abstract »    Full Text »    PDF »
Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases: Review and Protocols.
W. M. Frederiks and O. R. F. Mook (2004)
Journal of Histochemistry & Cytochemistry 52, 711-722
   Abstract »    Full Text »    PDF »
Caspase-8 Activation Precedes Alterations of Mitochondrial Membrane Potential during Monocyte Apoptosis Induced by Phagocytosis and Killing of Staphylococcus aureus.
K. Weglarczyk, J. Baran, M. Zembala, and J. Pryjma (2004)
Infect. Immun. 72, 2590-2597
   Abstract »    Full Text »    PDF »
Nitrosative Stress in Rotated Three-Dimensional Colorectal Carcinoma Cell Cultures Induces Microtubule Depolymerization and Apoptosis.
L. M. Laguinge, S. Lin, R. N. Samara, A. N. Salesiotis, and J. M. Jessup (2004)
Cancer Res. 64, 2643-2648
   Abstract »    Full Text »    PDF »
Transcriptional activation of known and novel apoptotic pathways by Nur77 orphan steroid receptor.
A. Rajpal, Y. A. Cho, B. Yelent, P. H. Koza-Taylor, D. Li, E. Chen, M. Whang, C. Kang, T. G. Turi, and A. Winoto (2003)
EMBO J. 22, 6526-6536
   Abstract »    Full Text »    PDF »
On-Line Monitoring of Apoptosis in Insulin-Secreting Cells.
M. Kohler, S. V. Zaitsev, I. I. Zaitseva, B. Leibiger, I. B. Leibiger, M. Turunen, I. L. Kapelioukh, L. Bakkman, I. B. Appelskog, J. Boutet de Monvel, et al. (2003)
Diabetes 52, 2943-2950
   Abstract »    Full Text »    PDF »
Cystatin 10, a Novel Chondrocyte-specific Protein, May Promote the Last Steps of the Chondrocyte Differentiation Pathway.
Y. Koshizuka, T. Yamada, K. Hoshi, T. Ogasawara, U.-i. Chung, H. Kawano, Y. Nakamura, K. Nakamura, S. Ikegawa, and H. Kawaguchi (2003)
J. Biol. Chem. 278, 48259-48266
   Abstract »    Full Text »    PDF »
Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis.
S. S. Metkar, B. Wang, M. L. Ebbs, J. H. Kim, Y. J. Lee, S. M. Raja, and C. J. Froelich (2003)
J. Cell Biol. 160, 875-885
   Abstract »    Full Text »    PDF »
Dexamethasone-induced apoptosis of thymocytes: role of glucocorticoid receptor-associated Src kinase and caspase-8 activation.
M. C. Marchetti, B. Di Marco, G. Cifone, G. Migliorati, and C. Riccardi (2003)
Blood 101, 585-593
   Abstract »    Full Text »    PDF »
Role of Apical Caspases and Glucocorticoid-regulated Genes in Glucocorticoid-induced Apoptosis of Pre-B Leukemic Cells.
S. L. Planey, M. T. Abrams, N. M. Robertson, and G. Litwack (2003)
Cancer Res. 63, 172-178
   Abstract »    Full Text »    PDF »
Memory Functions and Death Proneness in Three CD4+CD45RO+ Human T Cell Subsets.
T. Ohara, K. Koyama, Y. Kusunoki, T. Hayashi, N. Tsuyama, Y. Kubo, and S. Kyoizumi (2002)
J. Immunol. 169, 39-48
   Abstract »    Full Text »    PDF »
Intracellular Calcium Oscillations Signal Apoptosis Rather than Activation in In Vitro Aged Mouse Eggs.
A. C. Gordo, P. Rodrigues, M. Kurokawa, T. Jellerette, G. E. Exley, C. Warner, and R. Fissore (2002)
Biol Reprod 66, 1828-1837
   Abstract »    Full Text »    PDF »
The Apoptotic Protease-Activating Factor 1-Mediated Pathway of Apoptosis Is Dispensable for Negative Selection of Thymocytes.
H. Hara, A. Takeda, M. Takeuchi, A. C. Wakeham, A. Itie, M. Sasaki, T. W. Mak, A. Yoshimura, K. Nomoto, and H. Yoshida (2002)
J. Immunol. 168, 2288-2295
   Abstract »    Full Text »    PDF »
Enzyme Cytochemical Techniques for Metabolic Mapping in Living Cells, with Special Reference to Proteolysis.
E. Boonacker and C. J. F. Van Noorden (2001)
Journal of Histochemistry & Cytochemistry 49, 1473-1486
   Abstract »    Full Text »    PDF »
Free Cholesterol Loading of Macrophages Is Associated with Widespread Mitochondrial Dysfunction and Activation of the Mitochondrial Apoptosis Pathway.
P. M. Yao and I. Tabas (2001)
J. Biol. Chem. 276, 42468-42476
   Abstract »    Full Text »    PDF »
Caspase 8 Activity in Membrane Blebs After Anti-Fas Ligation.
B. Z. Packard, A. Komoriya, T. M. Brotz, and P. A. Henkart (2001)
J. Immunol. 167, 5061-5066
   Abstract »    Full Text »    PDF »
Quantifying the Relationship Between Multiple Immunological Parameters and Host Resistance: Probing the Limits of Reductionism.
D. Keil, R. W. Luebke, and S. B. Pruett (2001)
J. Immunol. 167, 4543-4552
   Abstract »    Full Text »    PDF »
Inhibition of p38 Kinase Reveals a TNF-{{alpha}}-Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line.
J. Varghese, S. Chattopadhaya, and A. Sarin (2001)
J. Immunol. 166, 6570-6577
   Abstract »    Full Text »    PDF »
More than one way to go.
A. H. Wyllie and P. Golstein (2001)
PNAS 98, 11-13
   Full Text »    PDF »
Matrix Regulation of Skeletal Cell Apoptosis. ROLE OF CALCIUM AND PHOSPHATE IONS.
C. S. Adams, K. Mansfield, R. L. Perlot, and I. M. Shapiro (2001)
J. Biol. Chem. 276, 20316-20322
   Abstract »    Full Text »    PDF »
The In Vitro Fate of Rabbit Fetal Brain Cells after Acute In Vivo Hypoxia.
M. Derrick, J. He, E. Brady, and S. Tan (2001)
J. Neurosci. 21, RC138
   Abstract »    Full Text »    PDF »

To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882