Abstract
In situ ligation (ISL) is a simple and specific technique for apoptosis labeling in tissue sections. In its most economical version ISL uses ordinary PCR-labeled DNA fragments as probes. In tissue sections these makeshift probes are ligated to apoptotic DNA breaks by T4 DNA ligase. The approach can selectively label 5′PO4 DNA breaks with blunt ends, and is the histological equivalent of electrophoretic apoptotic ladder detection. The main drawback of this technique is its low speed, as it requires 18 h-incubation for efficient labeling. Here, we describe an easy modification of ISL which reduces the incubation time to 1 h and converts ISL into a rapid detection method taking ~3 h overall. Signal enhancement is achieved by a new type of isothermal amplification reaction which generates “zebra tails”— long and labeled extensions of the probes attached to DNA breaks.
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References
Nagata S, Nagase H, Kawane K et al (2003) Degradation of chromosomal DNA during apoptosis. Cell Death Differ 10:108–116
Staley K, Blaschke A, Chun J (1997) Apoptotic DNA fragmentation is detected by a semiquantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ 4:66–75
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
Hornsby PJ, Didenko VV (2011) In situ ligation: a decade and a half of experience. Methods Mol Biol 682:49–63. doi:10.1007/978-1-60,327-409-8_5
Didenko VV (2002) Detection of specific double-strand DNA breaks and apoptosis in situ using T4 DNA ligase. Methods Mol Biol 203:143–151
Didenko VV, Hornsby PJ (1996) Presence of double-strand breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis. J Cell Biol 135:1369–1376
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
Loo DT (2011) In situ detection of apoptosis by the TUNEL assay: an overview of techniques. Methods Mol Biol 682:3–13. doi:10.1007/978-1-60,327-409-8_1
Charriaut-Marlangue C, Ben-Ari Y (1995) A cautionary note on the use of the TUNEL stain to determine apoptosis. Neuroreport 7:61–64
Wolvekamp MC, Darby IA, Fuller PJ (1998) Cautionary note on the use of end-labeling DNA fragments for detection of apoptosis. Pathology 30:267–271
Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H 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
Sloop GD, Roa JC, Delgado AG et al (1999) Histologic sectioning produces TUNEL reactivity. A potential cause of false-positive staining. Arch Pathol Lab Med 123:529–532
Pulkkanen KJ, Laukkanen MO, Naarala J, Yla-Herttuala S (2000) False-positive apoptosis signal in mouse kidney and liver detected with TUNEL assay. Apoptosis 5:329–333
Bassotti G, Villanacci V, Fisogni S et al (2007) Comparison of three methods to assess enteric neuronal apoptosis in patients with slow transit constipation. Apoptosis 12:329–332
Lawrence MD, Blyth BJ, Ormsby RJ et al (2013) False-positive TUNEL staining observed in SV40 based transgenic murine prostate cancer models. Transgenic Res 22:1037–1047. doi:10.1007/s11248-013-9694-7
Haunstetter A, Izumo S (2012) Strategies to prevent apoptosis. In: Hasenfuss G, Marban E (eds) Molecular approaches to heart failure therapy, 3rd edn. Springer Science & Business Media, Berlin
Galluzzi L, Aaronson SA, Abrams J et al (2009) Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 16:1093–1107
Apoptosis Detection Using Terminal Transferase and Biotin-16-dUTP (TUNEL Enzyme Method). (2016) http://www.ihcworld.com/_protocols/apoptosis/tunel_enzyme.htm.
Didenko VV (2011) In situ ligation simplified: using PCR fragments for detection of double-strand DNA breaks in tissue sections. Methods Mol Biol 682:65–75. doi:10.1007/978-1-60,327-409-8_6
Maunders MJ (1993) DNA and RNA ligases (EC 6.5.1.1, EC 6.5.1.2, EC 6.5.1.3). Methods Mol Biol 16:213–230. doi:10.1385/0-89,603-234-5:213
Hauser P, Wang S, Didenko VV (2017) Apoptotic bodies: selective detection in extracellular vesicles. Methods Mol Biol 1554:200–193. doi:10.1007/978-1-4939-6759-9_12
Schlissel M, Constantinescu A, Morrow T et al (1993) Double-strand signal sequence breaks in V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev 7:2520–2532
van Gent DC, Hoeijmakers JHJ, Kanaar R (2001) Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2:196–206
Longhese MP, Guerini I, Baldo V, Clerici M (2008) Surveillance mechanisms monitoring chromosome breaks during mitosis and meiosis. DNA Repair 7:545–557
Sweeney PJ, Walker JM (1993) Proteinase K (EC 3.4.21.14). Methods Mol Biol 16:305–311. doi:10.1385/0-89,603-234-5:305
Yang L, Didenko VV, Noda A et al (1995) Increased expression of p21Sdi1 in adrenocortical cells when they are placed in culture. Exp Cell Res 221:126–131
Koda M, Takemura G, Kanoh M et al (2003) Myocytes positive for in situ markers for DNA breaks in human hearts which are hypertrophic, but neither failed nor dilated: a manifestation of cardiac hypertrophy rather than failure. J Pathol 199:229–236
Okada H, Takemura G, Koda M et al (2005) Myocardial apoptotic index based on in situ DNA nick end-labeling of endomyocardial biopsies does not predict prognosis of dilated cardiomyopathy. Chest 128:1060–1062
Schoppet M, Al-Fakhri N, Franke FE et al (2004) Localization of osteoprotegerin, tumor necrosis factor-related apoptosis-inducing ligand, and receptor activator of nuclear factor-kappa B ligand in Mönckeberg’s sclerosis and atherosclerosis. J Clin Endocrinol Metab 89:4104–4112
Audo I, Darjatmoko SR, Schlamp CL et al (2003) Vitamin D analogues increase p53, p21, and apoptosis in a xenograft model of human retinoblastoma. Invest Ophthalmol Vis Sci 44:4192–4199
Al-Fakhri N, Chavakis T, Schmidt-Woll T et al (2003) Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties. J Biol Chem 384:423–435
Matsuoka R, Ogawa K, Yaoita H et al (2002) Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia-reoxygenation: the association of apoptosis and cell membrane disintegrity. Heart Vessels 16:241–248
Guerra S, Leri A, Wang X et al (1999) Myocyte death in the failing human heart is gender dependent. Circ Res 85:856–866
Leri A, Claudio PP, Li Q et al (1998) Strech-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2 to Bax protein ratio in the cell. J Clin Invest 101:1326–1342
Murata I, Takemura G, Asano K et al (2002) Apoptotic cell loss following cell proliferation in renal glomeruli of Otsuka Long-Evans Tokushima Fatty rats, a model of human type 2 diabetes. Am J Nephrol 22:587–595
Acknowledgment
I am grateful to Candace Minchew for her outstanding technical assistance.
This research was supported by grant R01 NS082553 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health and by grants R21 CA178965 from the National Cancer Institute, National Institutes of Health and R21 AR066931 National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health.
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Didenko, V.V. (2017). Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation. In: Didenko, V. (eds) Fast Detection of DNA Damage. Methods in Molecular Biology, vol 1644. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7187-9_15
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DOI: https://doi.org/10.1007/978-1-4939-7187-9_15
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