Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis

Article metrics

  • A Corrigendum to this article was published on 23 September 2015


Various molecules such as cytokines and anticancer drugs, as well as factor deprivation, rapidly induce apoptosis (programmed cell death)1,2, which is morphologically characterized by cell shrinkage and the blebbing of plasma membranes and by nuclear condensation3,4. Caspases, particularly caspase 3, are proteases that are activated during apoptosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin5,6. Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA7,8,9. In the accompanying Article10, wehave identified and molecularly cloned a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD). Here we show that caspase 3 cleaves ICAD and inactivates its CAD-inhibitory effect. We identified two caspase-3 cleavage sites in ICAD by site-directed mutagenesis. When human Jurkat cells were transformed with ICAD-expressing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation. The ICAD transformants were also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase. Our results indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, and that ICAD works as an inhibitor of this process.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Inactivation of ICAD by caspase-3 cleavage.
Figure 2: Identification of caspase 3 cleavage sites in ICAD.
Figure 3: Killing of Jurkat cells without DNA fragmentation.


  1. 1

    Nagata, S. Apoptosis by death factor. Cell 88, 355–365 (1997).

  2. 2

    Raff, M. C. Social controls on cell survival and cell death. Nature 356, 397–400 (1992).

  3. 3

    Wyllie, A. H., Kerr, J. F. R. & Currie, A. R. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–306 (1980).

  4. 4

    Earnshaw, W. C. Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7, 337–343 (1995).

  5. 5

    Henkart, P. A. ICE family protease: mediators of all apoptotic cell death? Immunity 4, 195–201 (1996).

  6. 6

    Martin, S. & Green, D. Protease activation during apoptosis: death by a thousand cuts. Cell 82, 349–352 (1995).

  7. 7

    Compton, M. M. Abiochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metast. Rev. 11, 105–119 (1992).

  8. 8

    Wyllie, A. H., Morris, R. G., Smith, A. L. & Dunlop, D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 66–77 (1984).

  9. 9

    Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556 (1980).

  10. 10

    Enari, M. et al. Acaspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50 (1998).

  11. 11

    Enari, M., Talanian, R. V., Wong, W. W. & Nagata, S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 380, 723–726 (1996).

  12. 12

    Longthorne, V. & Williams, G. Caspase activity is required for commitment to Fas-mediated apoptosis. EMBO J. 16, 3805–3812 (1997).

  13. 13

    Armstrong, R. C. et al. Fas-induced activation of the cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors. J. Biol. Chem. 271, 16850–16855 (1996).

  14. 14

    Thornberry, N. A. et al. Acombinatorial approach defines specificities of members of the caspase family and granzyme B. J. Biol. Chem. 272, 17907–17911 (1997).

  15. 15

    Talanian, R. V. et al. Substrate specificities of caspase family proteases. J. Biol. Chem. 272, 9677–9682 (1997).

  16. 16

    Chinnaiyan, A. M. et al. Molecular ordering of the cell death pathway. J. Biol. Chem. 271, 4573–4576 (1996).

  17. 17

    Koopman, G. et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415–1420 (1994).

  18. 18

    Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Method. 65, 55–63 (1983).

  19. 19

    Liu, X., Zou, H., Slaughter, C. & Wang, X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184 (1997).

  20. 20

    Ellis, R. E., Yuan, J. & Horvitz, H. R. Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7, 663–698 (1991).

  21. 21

    Ucker, D. S. et al. Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes. Mol. Cell. Biol. 12, 3060–3069 (1992).

  22. 22

    Peitsch, M., Mannherz, H. & Tschopp, J. The apoptosis endonucleases: cleaning up after cell death? Trends Cell Biol. 4, 37–41 (1994).

  23. 23

    Cohen, J. J., Duke, R. C., Fadok, V. A. & Sellins, K. S. Apoptosis and programmed cell death in immunity. Annu. Rev. Immunol. 10, 267–293 (1992).

  24. 24

    Higuchi, R. in PCR Protocols: A guide to Methods and Applications 177–188 (Academic, San Diego, (1990)).

  25. 25

    Blanar, M. A. & Rutter, W. J. Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos. Science 256, 1014–1018 (1992).

  26. 26

    Mizushima, S. & Nagata, S. pEF-BOS: a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322 (1990).

  27. 27

    Itoh, N. et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233–243 (1991).

  28. 28

    Lamarre, D. et al. Structural and functional analysis of poly(ADP-ribose) polymerase: an immunological study. Biochim. Biophys. Acta 950, 147–160 (1988).

Download references


We thank R. V. Talanian for the caspase 3 expression plasmid, G. G. Poirier for anti-human poly(ADP-ribose) polymerase, M. A. Blanar for pGEX-2T[128/129], and S. Kumagai for secretarial assistance. This work was supported in parts by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture in Japan.

Author information

Correspondence to Shigekazu Nagata.

Rights and permissions

Reprints and Permissions

About this article

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.