Abstract
Apoptosis is a distinct form of cell demise. Originally defined by cellular morphology, apoptosis can now be characterized at the molecular, biochemical, and cellular levels. The detection of apoptosis has become more important, not only in the interests of scientific investigation, but also because of its significance in clinical practice. For example, since apoptosis has been implicated in a variety of devastating diseases such as cancer, therapies targeting apoptosis are being developed. To evaluate the effectiveness of the treatment, one would have to assess the apoptotic response before, during, and after the therapy. For the typical apoptosis, a set of characteristics in cell structure and biochemistry has been well defined. In combination, these provide the basis for apoptosis detection in a given setting. The methodology for analyzing these characteristics is as diverse as the research subjects. Several books devoted to the methodology of apoptosis analysis have recently been published (1–3). Readers may find detailed experimental protocols in these books. This chapter provides an overview of the basic approaches used in analyzing apoptosis, the principle and the basic methodology, in order to provide a quick guide for readers to use when deciding which methods are available and appropriate for their own study. We start with the determination of cell viability and the morphology of dying cells. We then discuss approaches for examining apoptotic changes on the cell membrane, in the cytosol, and in the nucleus. We also summarize some of the common sources of reagents for apoptosis research.
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References
Reed JC, Abelson JN, Simon MI. Apoptosis. In: Abelson JN, Simon MI, eds. Methods in Enzymology. Vol. 322. San Diego: Academic Press; 2000.
Schwartz L, Ashwell J, Wilson L, et al. Apoptosis. In: Schwartz L, Ashwell J, eds. Methods in Cell Biology. Vol. 66. San Diego: Academic Press; 2001.
LeBlanc AC. Neuromethods. In: LeBlanc AC, ed. Apoptosis: Techniques and Protocols. 2nd ed. Vol. 37. Totowa, NJ: Humana Press; 2002.
Dong Z, Venkatachalam MA, Weinberg JM, et al. Protection of ATP-depleted cells by impermeant strychnine derivatives: Implications for glycine cytoprotection. Am J Pathol 2001;158:1021–8.
Dong Z, Patel Y, Saikumar P, et al. Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine. Lab Invest 1998;78:657–68.
Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239–57.
Krysko DV, Berghe TV, D'Herde K, et al. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 2008;44:205–21.
Savill J, Fadok V. Corpse clearance defines the meaning of cell death. Nature 2000;407:784–8.
Williamson P, van den Eijnde S, Schlegel RA. Phosphatidylserine exposure and phagocytosis of apoptotic cells. Methods Cell Biol 2001;66:339–64.
Pabla N, Huang S, Mi QS, et al. ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis. J Biol Chem 2008; 283:6572–83.
Jiang M, Pabla N, Murphy RF, et al. Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation. J Biol Chem 2007;282:2636–45.
Roy S, Nicholson DW. Criteria for identifying authentic caspase substrates during apoptosis. Methods Enzymol 2000;322:110–25.
Stennicke HR, Salvesen GS. Caspase assays. Methods Enzymol 2000;322:91–100.
Brooks C, Wang J, Yang T, et al. Characterization of cell clones isolated from hypoxia-selected renal proximal tubular cells. Am J Physiol Renal Physiol 2007;292:F243–52.
Wang J, Biju MP, Wang MH, et al. Cytoprotective effects of hypoxia against cisplatin-induced tubular cell apoptosis: Involvement of mitochondrial inhibition and p53 suppression. J Am Soc Nephrol 2006;17:1875–85.
Wei Q, Yin XM, Wang MH, et al. Bid deficiency ameliorates ischemic renal failure and delays animal death in C57BL/6 mice. Am J Physiol Renal Physiol 2006;290:F35–42.
Wei Q, Wang J, Wang MH, et al. Inhibition of apoptosis by Zn2+ in renal tubular cells following ATP depletion. Am J Physiol Renal Physiol 2004;287:F492–500.
Cao G, Pei W, Lan J, et al. Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures. J Neurosci 2001;21:4678–90.
Komoriya A, Packard BZ, Brown MJ, et al. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J Exp Med 2000;191:1819–28.
Brooks C, Ketsawatsomkron P, Sui Y, et al. Acidic pH inhibits ATP depletion-induced tubular cell apoptosis by blocking caspase-9 activation in apoptosome. Am J Physiol Renal Physiol 2005;289:F410–9.
Jiang M, Wei Q, Wang J, et al. Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis. Oncogene 2006;25:4056–66.
Gross A, Jockel J, Wei MC, et al. Enforced dimerization of Bax results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 1998;17:3878–85.
Eskes R, Desagher S, Antonsson B, et al. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 2000;20:929–35.
Wei MC, Lindsten T, Mootha VK, et al. tBid, a membrane-targeted death ligand, oligomerizes Bak to release cytochrome c. Genes Dev 2000;14:2060–71.
Yi X, Yin XM, Dong Z. Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed. J Biol Chem 2003;278:16992–9.
Desagher S, Osen-Sand A, Nichols A, et al. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 1999;144:891–901.
Zhao Y, Li S, Childs EE, et al. Activation of pro-death Bcl-2 family proteins and mitochondria apoptosis pathway in tumor necrosis factor-alpha-induced liver injury. J Biol Chem 2001;276:27432–40.
Lemasters JJ, Qian T, Elmore SP, et al. Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy. Biofactors 1998;8:283–5.
Reynolds IJ. Mitochondrial membrane potential and the permeability transition in excitotoxicity. Ann NY Acad Sci 1999;893:33–41.
Matsuyama S, Reed JC. Mitochondria-dependent apoptosis and cellular pH regulation. Cell Death Differ 2000;7:1155–65.
Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-x(l) during apoptosis. Proc Natl Acad Sci USA 1997;94:3668–72.
Ott M, Robertson JD, Gogvadze V, et al. Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci USA 2002;99:1259–63.
Wei Q, Dong G, Franklin J, et al. The pathological role of Bax in cisplatin nephrotoxicity. Kidney Int 2007;72:53–62.
Gross A, Yin XM, Wang K, et al. Caspase cleaved Bid targets mitochondria and is required for cytochrome c release, while Bcl-xl prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 1999;274:1156–63.
Brooks C, Wei Q, Feng L, et al. Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins. Proc Natl Acad Sci USA 2007;104:11649–54.
Kamo N, Muratsugu M, Hongoh R, et al. Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J Membr Biol 1979;49:105–21.
Zamzami N, Métivier D, Kroemer G. Quantitation of mitochondrial transmembrane potential in cells and in isolated mitochondria. Methods Enzymol 2000;322:208–13.
Cossarizza A, Salvioli S. Analysis of mitochondria during cell death. Methods Cell Biol 2001;63:467–86.
Cossarizza A, Baccarani-Contri M, Kalashnikova G, et al. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the j-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993;197:40–5.
Bernardi P, Scorrano L, Colonna R, et al. Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem1999;264:687–701.
Salvioli S, Ardizzoni A, Franceschi C, et al. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 1997;411:77–82.
Scorrano L, Petronilli V, Colonna R, et al. Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition and inhibits respiratory complex I. Implications for the mechanism of cytochrome c release. J Biol Chem 1999;274:24657–63.
Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980;284:555–6.
Loo DT, Rillema JR. Measurement of cell death. Methods Cell Biol 1998; 57:251–64.
Wyllie A. Apoptosis. An endonuclease at last. Nature 1998;391:20–1.
Dong Z, Saikumar P, Weinberg JM, et al. Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases. Am J Pathol 1997;151:1205–13.
Collins RJ, Harmon BV, Gobé GC, et al. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int J Radiat Biol 1992;61:451–3.
Oberhammer F, Wilson JW, Dive C, et al. Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 1993;12:3679–84.
Darzynkiewicz Z, Li X, Bedner E. Use of flow and laser-scanning cytometry in analysis of cell death. Methods Cell Biol 2001;66:69–109.
Li Y, Sharov VG, Jiang N, et al. Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat. Am J Pathol 1995;146:1045–51.
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Jiang, M. et al. (2009). Analysis of Apoptosis: Basic Principles and Protocols. In: Dong, Z., Yin, XM. (eds) Essentials of Apoptosis. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-381-7_31
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DOI: https://doi.org/10.1007/978-1-60327-381-7_31
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