Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells

Timothy J. Kottke, April L. Blajeski, X. Wei Meng, Phyllis A. Svingen, Sandrine Ruchaud, Peter W. Mesner, Scott A. Boerner, Kumiko Samejima, Nicholas V. Henriquez, Tamie J. Chilcote, Janet Lord, Michael Salmon, William C. Earnshaw, Scott H. Kaufmann

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62 Scopus citations

Abstract

MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nM paclitaxel, 37 ± 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, β-catenin, gelsolin, protein kinase Cδ, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.

Original languageEnglish (US)
Pages (from-to)804-815
Number of pages12
JournalJournal of Biological Chemistry
Volume277
Issue number1
DOIs
StatePublished - Jan 4 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Kottke, T. J., Blajeski, A. L., Wei Meng, X., Svingen, P. A., Ruchaud, S., Mesner, P. W., Boerner, S. A., Samejima, K., Henriquez, N. V., Chilcote, T. J., Lord, J., Salmon, M., Earnshaw, W. C., & Kaufmann, S. H. (2002). Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells. Journal of Biological Chemistry, 277(1), 804-815. https://doi.org/10.1074/jbc.M108419200