Activation of multiple interleukin-1β converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis

Luis M. Martins, Timothy Kottke, Peter W. Mesner, Guriqbal S. Basi, Sukanto Sinha, Normand Frigon, Eric Tatar, Jay S. Tung, Karin Bryant, Atsushi Takahashi, Phyllis A. Svingen, Benjamin J. Madden, Daniel J Mc Cormick, William C. Earnshaw, Scott H Kaufmann

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Abstract

Recent genetic and biochemical studies have implicated cysteine- dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD- aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD- aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N- (N(α)-benzyloxycarbonylglutamyl-N(ε)-biotinyllysyl)-aspartic acid [(2,6- dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.

Original languageEnglish (US)
Pages (from-to)7421-7430
Number of pages10
JournalJournal of Biological Chemistry
Volume272
Issue number11
DOIs
StatePublished - Mar 14 1997

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Martins, L. M., Kottke, T., Mesner, P. W., Basi, G. S., Sinha, S., Frigon, N., Tatar, E., Tung, J. S., Bryant, K., Takahashi, A., Svingen, P. A., Madden, B. J., Mc Cormick, D. J., Earnshaw, W. C., & Kaufmann, S. H. (1997). Activation of multiple interleukin-1β converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis. Journal of Biological Chemistry, 272(11), 7421-7430. https://doi.org/10.1074/jbc.272.11.7421