Advertisement

Electron Microscopic Detection of DNA Damage Labeled by TUNEL

  • Antonio Migheli
Part of the Methods in Molecular Biology book series (MIMB, volume 203)

Abstract

A number of exogenous and endogenous toxic agents may damage DNA, leading to genomic instability and transcriptional infidelity. Genetic or acquired defects in DNA repair mechanisms also contribute to exacerbate DNA damage. Progressive accumulation of DNA injury may alter the genetic control of cell proliferation and cause cancer. Alternatively, increased cell death may be a more likely consequence if DNA damage affects postmitotic cells, e.g., neurons (1). As a matter of fact, DNA damage is suspected to play a major role in the neuronal death that characterizes brain ischemia (2) and various neurodegenerative diseases (1,3).

Keywords

Terminal Deoxynucleotidyl Transferase Cobalt Chloride Saline Sodium Citrate Label Solution Formvar Coated Nickel Grid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Rolig R. L. and McKinnon P. J. (2000) Linking DNA damage and neurodegeneration. Trends Neurosci. 23, 417–424.PubMedCrossRefGoogle Scholar
  2. 2.
    Love S. (1999) Oxidative stress in brain ischemia. Brain Pathol. 9, 119–131.PubMedCrossRefGoogle Scholar
  3. 3.
    De la Monte S. M., Luong T., Neely T. R., Robinson D. and Wands J. R. (2000) Mitochondrial DNA damage as a mechanism of cell loss in Alzheimer’s disease. Lab. Invest. 80, 1323–1335.PubMedCrossRefGoogle Scholar
  4. 4.
    Kerr J. F. R., Wyllie A. H. and Currie A. R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257.PubMedCrossRefGoogle Scholar
  5. 5.
    Budihardjo I., Oliver H., Lutter M., Luo X. and Wang X. (1999) Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269–290.PubMedCrossRefGoogle Scholar
  6. 6.
    Enari M., Sakahira H., Yokoyama H., Okawa K. and Nagata S. (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50.PubMedCrossRefGoogle Scholar
  7. 7.
    Majno G. and Joris I. (1995) Apoptosis, oncosis, and necrosis: an overview of cell death. Am. J. Pathol. 146, 3–15.PubMedGoogle Scholar
  8. 8.
    Wang K. K. (2000) Calpain and caspase: can you tell the difference? Trends Neurosci. 23, 20–26.PubMedCrossRefGoogle Scholar
  9. 9.
    Hayashi R., Ito Y., Matsumoto K., Fujino Y. and Otsuki Y. (1998) Quantitative differentiation of both free 3′-OH and 5′-OH DNA ends between heat-induced apoptosis and necrosis. J. Histochem. Cytochem. 46, 1051–1059.PubMedGoogle Scholar
  10. 10.
    Didenko V. V. and Hornsby P. J. (1996) Presence of double-strand breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis. J. Cell Biol. 135, 1369–1376.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    Gold R., Schmied M., Rothe G., Zischler H., Breitschopf H., Wekerle H. and Lassmann H. (1993) Detection of DNA fragmentation in apoptosis: application of an in situ nick translation to cell culture systems and tissue sections. J. Histochem. Cytochem. 41, 1023–1030.PubMedGoogle Scholar
  13. 13.
    Wijsman J. H., Jonker R. R., Keijzer R., Van de Velde C. J. H., Cornelisse C. J. and Van Dierendonck J. H. (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J. Histochem. Cytochem. 41, 7–12.PubMedGoogle Scholar
  14. 14.
    Gorczyca W., Gong J. and Darzynkiewicz Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53, 1945–1951PubMedGoogle Scholar
  15. 15.
    Migheli A., Cavalla P., Marino S. and Schiffer D. (1994) A study of apoptosis in normal and pathologic nervous tissue after in situ end-labeling of fragmented DNA. J. Neuropathol. Exp. Neurol. 53, 606–616.PubMedCrossRefGoogle Scholar
  16. 16.
    Grasl-Kraupp B., Ruttkay-Nedecky B., Koudelka H., Bukowska K., Bursch W. and Schulte-Hermann R. (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.PubMedGoogle Scholar
  17. 17.
    van Lookeren Campagne M., Lucassen P. J., Vermeulen J. P. and Balasz R. (1995) NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal rat brain. Eur. J. Neurosci. 7, 1627–1640.PubMedCrossRefGoogle Scholar
  18. 18.
    Mundle S., Gao X. Z., Khan S., Gregory S. A., Preisler H. D. and Raza A. (1995) Two in situ labeling techniques reveal different patterns of DNA fragmen tation during spontaneous apoptosis in vivo and induced apoptosis in vitro. Anticancer Res. 15, 1895–1904.Google Scholar
  19. 19.
    Gold R., Schmied M., Giegerich G., Breitschopf H., Hartung H. P., Toyka K. V. and Lassmann H. (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab. Invest. 71, 219-22Google Scholar
  20. 20.
    Lopes S., Jurisicova A., Sun J. G. and Casper R. F. (1998) Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod. 13, 896–900.PubMedCrossRefGoogle Scholar
  21. 21.
    Coates P. J., Save V., Ansari B. and Hall P. A. (1995) Demonstration of DNA damage/repair in individual cells using in situ end labeling: association of p53 with sites of DNA damage. J. Pathol. 176, 19–26.PubMedCrossRefGoogle Scholar
  22. 22.
    Assad M., Lemieux N. and Rivard C. H. (1997) Immunogold electron microscopy in situ end-labeling (EM-ISEL): assay for biomaterial DNA damage detection. Biomed. Mater. Eng. 7, 391–400.PubMedGoogle Scholar
  23. 23.
    Brooks P. J. (2000) Brain atrophy and neuronal loss in alcoholism: a role for DNA damage? Neurochem. Int. 37, 403–412.PubMedCrossRefGoogle Scholar
  24. 24.
    Kisby G. E., Kabel H., Hugon J. and Spencer P. (1999) Damage and repair of nerve cell DNA in toxic stress. Drug Metab. Rev. 31, 589–618.PubMedCrossRefGoogle Scholar
  25. 25.
    Tateyama H., Tada T., Hattori H., Murase T., Li W. X. and Eimoto T. (1998) Effects of prefixation and fixation times on apoptosis detection by in situ endlabeling of fragmented DNA. Arch. Pathol. Lab. Med. 122, 252–255.PubMedGoogle Scholar
  26. 26.
    Schallock K., Schulz-Schaeffer W. J., Giese A. and Kretzschmar H. A. (1997) Postmortem delay and temperature conditions affect the in situ end-labeling (ISEL) assay in brain tissue of mice. Clin. Neuropathol. 16, 133–136.PubMedGoogle Scholar
  27. 27.
    Labat-Moleur F., Guillermet C., Lorimier P., Robert C., Lantuejoul S., Brambilla E. and Negoescu A. (1998) TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement. J. Histochem. Cytochem. 46, 327–334.PubMedGoogle Scholar
  28. 28.
    Clarke P. G. H. (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat. Embryol. 181, 195–213.PubMedCrossRefGoogle Scholar
  29. 29.
    Migheli A., Piva R., Wei J., Attanasio A., Casolino S., Dlouhy S. R., Bayer S. A. and Ghetti B. (1997) Diverse cell death pathways result from a single missense mutation in weaver mouse. Am. J. Pathol. 151, 1629–1638.PubMedGoogle Scholar
  30. 30.
    Migheli A., Attanasio A. and Schiffer D. (1995) Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labeling techniques. J. Pathol. 176, 27–35.PubMedCrossRefGoogle Scholar
  31. 31.
    Kanoh M., Takemura G., Misao J., Hayakawa Y., Aoyama T., Nishigaki K., Noda T., Fujiwara T., Fukuda K., Minatoguchi S. and Fujiwara H. (1999) Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy-Not apoptosis but DNA repair. Circulation 99, 2757–2764.PubMedGoogle Scholar
  32. 32.
    Thiry M. (1991) In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell. J. Histochem. Cytochem. 39, 871–874.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Antonio Migheli
    • 1
  1. 1.Laboratory of Neuropathology, Department of NeuroscienceUniversity of TurinTurinItaly

Personalised recommendations