Skip to main content
SpringerLink
Log in

Determination of Three-Dimensional Distribution of Apoptotic DNA Damage by Combination of TUNEL and Quick-Freezing and Deep-Etching Techniques

  • Protocol
In Situ Detection of DNA Damage

Part of the book series: Methods in Molecular Biology ((MIMB,volume 203))

  • 905 Accesses

Abstract

Apoptosis is a widespread phenomenon, which plays an important role in many physiological events as well as pathological processes (1). It was originally defined by its unique ultrastructural features, which were detected by electron microscopy (2,3). They are cytoplasmic shrinkage, nuclear chromatin condensation along nuclear margin, cell fragmentation into apoptotic bodies, and phagocytosis by adjacent epithelial cells or macrophages. It is also known that normal epithelial cell proliferation and death in mammalian prostatic glands depend upon an appropriate level of circulating androgen in blood (4,5). Therefore, the prostatic epithelial cells routinely undergo apoptosis by androgen withdrawal following castration in experimental male animals (6,7,8).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Ansari B., Coates P. J., Greenstein B. D. and Hall P. A. (1993) In situ end-labeling detects DNA strand breaks in apoptosis and other physiological and pathological states. J. Pathol. 170, 1–8.

    Article  PubMed  CAS  Google Scholar 

  2. Dini L. Coppola S. Ruzittu M. T. and Ghibelli L. 1996 Multiple pathways for apoptotic nuclear fragmentation. Exp. Cell Res. 223 340–347

    Google Scholar 

  3. Falcieri E., Zamai L., Santi S., Cinti C., Gobbi P., Bosco D., Cataldi A., Betts C. and Vitale M. (1994) The behaviour of nuclear domains in the course of apoptosis. Histochemistry 102, 221–231.

    Article  PubMed  CAS  Google Scholar 

  4. Evans G. S. and Chandler J. A. (1987) Cell proliferation studies in the rat prostate: II. the effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation. The Prostate 11, 339–351.

    Article  PubMed  CAS  Google Scholar 

  5. Furuya T., Kubo M., Ueno A., Fujii Y., Baba T. and Ohno S. (2000) His-tochemical study of apoptotic epithelial cells depending on testosterone in primary cultured rat prostatic tissues. Histol. Histopathol. 15, 385–394.

    PubMed  CAS  Google Scholar 

  6. Banerjee P. P., Banerjee S., Tilly K. I., Tilly J. L., Brown T. R. and Zirkin B. R. (1995) Lobe-specific apoptotic cell death in rat prostate after androgen ablation by castration. Endocrinology 136, 4368–4376.

    Article  PubMed  CAS  Google Scholar 

  7. English H. F., Kyprianou N. and Isaacs J. T. (1989) Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration. The Prostate 15, 233–250.

    Article  PubMed  CAS  Google Scholar 

  8. Kubo M., Uchiyama H., Ueno A., Terada N., Fujii Y., Baba T. and Ohno S. (1998) Three-dimensional ultrastructure of apoptotic nuclei in rat prostatic epithelial cells revealed by a quick-freezing and deep-etching method. The Prostate 35, 193–202.

    Article  PubMed  CAS  Google Scholar 

  9. Kyprianou N., English H. F. and Isaacs J. T. (1988) Activation of a Ca2+-Mg2+-dependent endonuclease as an early event in castration-induced prostatic cell death. The Prostate 13, 103–117.

    Article  PubMed  CAS  Google Scholar 

  10. Montague J. W. and Cidlowski J. A. (1996) Cellular catabolism in apoptosis: DNA degradation and endonuclease activation. Experientia 52, 957–962.

    Article  PubMed  CAS  Google Scholar 

  11. Allen T. D. (1989) The organization and substructure of chromatin fibres in theinterphase nucleus as studied by scanning electron microscopy. Scanning Microsc. [Suppl.] 3, 77–86.

    CAS  Google Scholar 

  12. Haggis G. H. and Bond E. F. (1978) Three-dimensional view of the chromatin in freeze-fractured chicken erythrocyte nuclei. J. Microsc. 115, 225–234.

    Google Scholar 

  13. Tanaka K. and Iino A. (1973) Demonstration of fibrous components in hepatic interphase nuclei by high resolution scanning electron microscopy. Exp. Cell Res. 81, 40–46.

    Article  Google Scholar 

  14. Gaffney E. F., O’Neill A. J. and Staunton M. J. (1995) In situ end-labeling, light microscopic assessment and ultrastructure of apoptosis in lung carcinoma. J. Clin. Pathol. 48, 1017–1021.

    Article  PubMed  CAS  Google Scholar 

  15. Gavrieli Y., Sherman Y. and Ben-Sasson S. A. (1992) Identification of programmed cell death in situ via specific lebeling of nuclear DNA fragmentation. J. CellBiol. 119, 493–501.

    Article  CAS  Google Scholar 

  16. Kasagi N., Adachi H., Ueno E., Hayashi H. and Ito H. (1996) Initial phase of apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling in UV-irradiated HL-60 cells: morphological and biochemical findings of DNA fragmentation. Biomed. Res. 17, 385–393.

    CAS  Google Scholar 

  17. Wijsman J. H., Jonker R. R., Keijzer R., van de Velde C. J. H., Cornelisse C. J. and van Dierendonk 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.

    PubMed  CAS  Google Scholar 

  18. 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.

    PubMed  CAS  Google Scholar 

  19. 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.

    Article  PubMed  CAS  Google Scholar 

  20. Saitoh T. (2000) Detection of VP-16-treated HL-60 cell apoptosis by TUNEL electron microscopy. Ultrastruc. Pathol. 24, 99–103.

    Article  CAS  Google Scholar 

  21. Sanders E. J. and Wride M. A. (1996) Ultrastructural identification of apoptotic nuclei using the TUNEL technique. Histochem. J. 28, 275–281.

    Article  PubMed  CAS  Google Scholar 

  22. Allera C., Lazzarini G., Patrone E., Alberti I., Barboro P., Sanna P., Melchiori A., Parodi S. and Balbi C. (1997) The condensation of chromatin in apoptotic thymocytes shows a specific structural change. J. Biol. Chem. 272, 10,817–10,822.

    Article  PubMed  CAS  Google Scholar 

  23. Weaver V. M., Carson C. E., Walker P. R., Chaly N., Lach B., Raymond Y., Brown D. L. and Sikorska M. (1996) Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes. J. Cell Sci. 109, 45–56.

    PubMed  CAS  Google Scholar 

  24. Ohno S. and Fujii Y. (1990) Three-dimensional and histochemical studies of peroxisomes in cultured hepatocytes by quick-freezing and deep-etching method. Histochem. J. 22, 143–154.

    Article  PubMed  CAS  Google Scholar 

  25. Ohno S. and Fujii Y. (1991) Three-dimensional studies of the cytoskeleton of cultured hepatocytes: a quick-freezing and deep-etching study. Virchows Arch. [A] Pathol. Anat. 418, 61–70.

    Article  CAS  Google Scholar 

  26. Anzai N., Kawabata H., Hirama T., Masutani H., Ueda Y., Yoshida Y. and Okuma M. (1995) Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different beween human CD34+ cells and their granulocytic descendants. Blood 86, 917–923.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy, Yamanashi Medical University, Yamanashi, Japan

    Shinichi Ohno, Takeshi Baba, Nobuo Terada & Yasuhisa Fuji

Authors
  1. Shinichi Ohno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takeshi Baba
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nobuo Terada
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yasuhisa Fuji
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Baylor College of Medicine, Houston, TX

    Vladimir V. Didenko

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Ohno, S., Baba, T., Terada, N., Fuji, Y. (2002). Determination of Three-Dimensional Distribution of Apoptotic DNA Damage by Combination of TUNEL and Quick-Freezing and Deep-Etching Techniques. In: Didenko, V.V. (eds) In Situ Detection of DNA Damage. Methods in Molecular Biology, vol 203. Humana Press. https://doi.org/10.1385/1-59259-179-5:55

Download citation

  • DOI: https://doi.org/10.1385/1-59259-179-5:55

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-952-0

  • Online ISBN: 978-1-59259-179-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation