title: Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells.
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, beta-catenin, gelsolin, protein kinase Cdelta, 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.
authors.affiliation: Division of Oncology Research, Mayo Clinic, Mayo Graduate School, Rochester, Minnesota 55905, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kottke
authors.forename: April L
authors.initials: AL
authors.lastname: Blajeski
authors.forename: X Wei
authors.initials: XW
authors.lastname: Meng
authors.forename: Phyllis A
authors.initials: PA
authors.lastname: Svingen
authors.forename: Sandrine
authors.initials: S
authors.lastname: Ruchaud
authors.forename: Peter W
authors.initials: PW
authors.lastname: Mesner
authors.forename: Scott A
authors.initials: SA
authors.lastname: Boerner
authors.forename: Kumiko
authors.initials: K
authors.lastname: Samejima
authors.forename: Nicholas V
authors.initials: NV
authors.lastname: Henriquez
authors.forename: Tamie J
authors.initials: TJ
authors.lastname: Chilcote
authors.forename: Janet
authors.initials: J
authors.lastname: Lord
authors.forename: Michael
authors.initials: M
authors.lastname: Salmon
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
authors.forename: Scott H
authors.initials: SH
authors.lastname: Kaufmann
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D017239:Paclitaxel / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D017239:Paclitaxel