Abstract
Classical apoptotic cell death can be defined by certain morphological and biochemical characteristics that distinguish it from other forms of cell death. One such feature, which is a hallmark of apoptosis, is DNA fragmentation. In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180-bp oligomers and appear as a DNA ladder when run on an agarose gel. Here, we present commonly used methods such as conventional agarose gel electrophoresis to analyze fragmented nuclei in cells. The various methods used are dependent on the extent of fragmentation or the amount of fragmented nuclei in a sample. Determining whether a cell exhibits DNA fragmentation can provide information about the type of cell death occurring and the pathways activated in the dying cell.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Clarke, P. G. H. (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat. Embryol. 181, 195–213.
Steller, H. (1995) Mechanisms and genes of cellular suicide. Science 267, 1445–1449.
Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462.
Vaux, D. L. (1993) Toward an understanding of the molecular mechanisms of cell death. Proc. Natl. Acad. Sci. USA 90, 786–789.
Wyllie, A. H. (1980). Glucocorticoid induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556.
Zakeri, Z. F., Quaglino, D., Latham, T., and Lockshin, R. A. (1993) Delayed internucleosomal DNA fragmentation in programmed cell death. FASEB J. 7, 470–478.
Williamson, R. (1970) Properties of rapidly labeled deoxyribonucleic acid fragments isolated from the cytoplasm of primary cultures of embryonic mouse liver cells. J. Mol. Biol. 51, 157–168.
Williams, J. R., Little, J. B., and Shipley, W. U. (1974) Association of mammalian cell death with a specific endonucleolytic degradation of DNA. Nature 252, 754–756.
Sikorska, M., and Walker, P. R. (1998) Endonuclease activities and apoptosis, in When Cells Die (Lockshin, R. A., Zakeri, Z., Tilly, J. L., eds.), Wiley-Liss, Inc., New York, NY, pp. 211–242.
Walker, P. R., Leblanc, J., Smith, B., Pandey, S., and Sikorska, M. (1999) Detection of DNA fragmentation and endonucleases in apoptosis. Methods 17, 329–338.
Walker, P. R., Kokileva, L., LeBlanc, J., and Sikorska, M. (1993) Detection of the initial stages of DNA fragmentation apoptosis. BioTechniques 15, 1032–1040.
Walker, P. R., Weaver, V. M., Lach, B., LeBlanc, J., and Sikorska, M. (1994) Endonuclease activities associated with HMW and internucleosomal DNA fragmentation in apoptosis. Exp. Cell Res. 213, 100–106.
Walker, P. R., and Sikorska, M. (1994) Endonuclease activities, chromatin structure and DNA degradation in apoptosis. Biochem. Cell Biol. 72, 615–623.
Jochová, J., Quaglino, D., Zakeri, Z., Woo, K., Sikorska, M., Weaver, V., and Lockshin, R. A. (1997) Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands of Manduca sexta. Devel. Gen. 21, 249–257.
Brown, D. G., Sun, X. M., and Cohen, G. M. (1993) Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J. Biol. Chem. 268, 3037–3039.
Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184.
Arends, M. J., Morris, R. G., and Wyllie, A. H. (1990) Apoptosis: the role of the endonuclease. Am. J. Pathol. 136, 593–608.
Montague, J. W., Bortner, C. D., Hughes, F. M. Jr., and Cidlowski, J. A. (1999) A necessary role for reduced intracellular potassium during the DNA degradation phase of apoptosis. Steroids 64, 563–569.
Bursch, W., Kleine, L., and Tenniswood, M. (1990) The biochemistry of cell death by apoptosis. Biochem. Cell Biol. 68, 1071–1074.
Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501.
Umansky, S. R., Beletsky, I. P., Korol, B. A., Lichtenstein, A. V., and Neilpovich, P. A. (1988) Molecular mechanisms of DNA degradation in dying rodent thymocytes. Mol. Cell Biol. 7, 221–228.
Beletsky, I. P., Matyasova, J., Nikonova, L. V., Skalka, M., and Umansky, S. R. (1989) On the role of Ca, Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues. Gen. Physiol. Biophys. 8, 381–398.
Karasavvas, N., Erukulla, R. K., Bittman, R., Lockshin, R., and Zakeri, Z. (1996) Stereospecific induction of apoptosis in U937 cells by N-octanoylsphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis. Eur. J. Biochem. 236, 729–737.
Olive, P. L., Banath, J. P., and Durand, R. E. (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat Res. 122, 86–94.
Lee, R. F., and Steinert, S. (2003) Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat Res. 544, 43–64.
Lee, R. F., and Steinert, S. (2003) Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res. 544, 43–64.
Kagan, T., and Zakeri, Z. (1998) Detection of apoptotic cells in the nervous system, in Methods in Molecular Medicine, Vol. 22, Neurodegeneration Methods and Protocols (Harry, J., ed.), Humana Press, Totowa, NJ, pp. 105–124.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Matassov, D., Kagan, T., Leblanc, J., Sikorska, M., Zakeri, Z. (2004). Measurement of Apoptosis by DNA Fragmentation. In: Brady, H.J.M. (eds) Apoptosis Methods and Protocols. Methods in Molecular Biology, vol 282. Humana Press. https://doi.org/10.1385/1-59259-812-9:001
Download citation
DOI: https://doi.org/10.1385/1-59259-812-9:001
Publisher Name: Humana Press
Print ISBN: 978-0-89603-873-8
Online ISBN: 978-1-59259-812-0
eBook Packages: Springer Protocols