Abstract
Apoptosis is an energy-dependent enzymatic cell suicide process. It almost always involves the activation of caspases. In this chapter, we systemically introduce methodologies to assay caspases dependent biochemical and morphological changes in vitro breast cancer cell lines and in vivo breast cancer tissues. In addition, mitochondrial involvement is crucial to distinguish two different apoptotic pathways. Methodology to assay dissipation of mitochondrial transmembrane potential, an early event of mitochondrial involvement, is also included. Of note, since apoptotic features may not appear to the same extent depending on the context of cell types and the death-inducing insults, a common practice is to use more than one method to assess apoptosis, qualitatively and quantitatively.
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References
Johnstone RW, Ruefli AA, Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108:153–164
Chinnaiyan AM et al (2000) Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc Natl Acad Sci U S A 97:1754–1759
Gasco M, Shami S, Crook T (2002) The p53 pathway in breast cancer. Breast Cancer Res 4:70–76
Lowe SW, Ruley HE, Jacks T, Housman DE (1993) p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957–967
Janicke RU, Sprengart ML, Wati MR, Porter AG (1998) Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273:9357–9360
Liu X, Zou H, Slaughter C, Wang X (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89:175–184
Mandal D, Moitra PK, Saha S, Basu J (2002) Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes. FEBS Lett 513:184–188
Crawford ED, Wells JA (2011) Caspase substrates and cellular remodeling. Annu Rev Biochem 80:1055–1087
Martin DA, Siegel RM, Zheng L, Lenardo MJ (1998) Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signal. J Biol Chem 273:4345–4349
Boatright KM et al (2003) A unified model for apical caspase activation. Mol Cell 11:529–541
Tait SW, Green DR (2010) Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11:621–632
Chai J et al (2001) Crystal structure of a procaspase-7 zymogen: mechanisms of activation and substrate binding. Cell 107:399–407
Riedl SJ et al (2001) Structural basis for the activation of human procaspase-7. Proc Natl Acad Sci U S A 98:14790–14795
Chang HY, Yang X (2000) Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev 64:821–846
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501
Grasl-Kraupp B et al (1995) In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21:1465–1468
Reers M, Smith TW, Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30:4480–4486
Smiley ST et al (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci U S A 88:3671–3675
Sakahira H, Enari M, Ohsawa Y, Uchiyama Y, Nagata S (1999) Apoptotic nuclear morphological change without DNA fragmentation. Curr Biol 9:543–546
Didenko VV, Ngo H, Baskin DS (2003) Early necrotic DNA degradation: presence of blunt-ended DNA breaks, 3′ and 5′ overhangs in apoptosis, but only 5′ overhangs in early necrosis. Am J Pathol 162:1571–1578
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Sun, Y., Zong, WX. (2016). Cellular Apoptosis Assay of Breast Cancer. In: Cao, J. (eds) Breast Cancer. Methods in Molecular Biology, vol 1406. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3444-7_11
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DOI: https://doi.org/10.1007/978-1-4939-3444-7_11
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