Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells.
J Biol Chem
; 277(1): 804-15, 2002 Jan 04.
Article
en En
| MEDLINE
| ID: mdl-11677238
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Neoplasias de la Mama
/
Paclitaxel
/
Caspasas
/
Antineoplásicos Fitogénicos
Límite:
Female
/
Humans
Idioma:
En
Revista:
J Biol Chem
Año:
2002
Tipo del documento:
Article
País de afiliación:
Estados Unidos