DNA Fragmentation in Mammalian Apoptosis and Tissue Homeostasis

  • Ming Xu
  • Jianhua Zhang


Apoptosis is a highly regulated physiological process critical in development and tissue homeostasis. Abnormal apoptosis can lead to disease conditions including neurodegeneration, autoimmunity and cancer. DNA fragmentation is an integral part of apoptosis and has long been suspected to be of critical importance in cleaning up potentially antigenic DNA and genetic materials capable of inducing neoplasmic transformation in neighboring cells. Direct evidence for this role of DNA fragmentation in apoptosis however, is still lacking. The identification of a heterodimeric DNA fragmentation factor composed of a 45 and 40 kDa subunit (termed DFF45 and DFF40, or ICAD for Inhibitor of Caspase Activated DNase and CAD for Caspase Activated DNase, respectively) as well as endonuclease G (EndoG) provides a timely opportunity for addressing the physiological significance of DNA fragmentation in apoptosis and tissue homeostasis. We previously generated a DFF45 mutant mouse in which the DFF activity is abolished. We found that DFF45-deficient thymocytes are resistant to DNA fragmentation both in vivo and in cultured primary cells exposed to various apoptotic stimuli. Interestingly, DFF45-deficient thymocytes and mouse embryonic fibroblasts (MEFs) are partially resistant to apoptosis in response to several apoptotic-inducing agents. There are more granule cells in the dentate gyrus of the hippocampal formation in DFF45 mutant mice than in normal control mice. This increased neuronal cell number correlates with enhanced spatial and non-spatial learning and memory retention in DFF45 mutant mice compared with control mice. These results suggest that DFF45 is critical for DNA fragmentation and a deficiency in DFF45 can affect timely completion of apoptosis and consequently tissue homeostasis and proper cellular function. Likely due to the unaffected EndoG activity however, residual DNA fragmentation can be found in DFF45-deficient splenocytes and MEFs. In a collaborative effort, we are generating EndoG mutant mice and mice with combined deficiencies of DFF45 and EndoG to investigate how DFF and EndoG jointly function to insure proper apoptosis and tissue homeostasis.


Mutant Mouse Tissue Homeostasis Mammalian Apoptosis Systemic Lupus Erythematosus Development Neuronal Cell Number 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abrams, J.M. (1999). An emerging blueprint for apoptosis in Drosophila. Trends Cell Biol. 9, 435–440.PubMedCrossRefGoogle Scholar
  2. Adachi, M., Suematsu, S., Kondo, T., Ogasawara, J., Tanaka, T., Yoshida, N., and Nagata, S. (1995). Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver. Nat. Genet. 11, 294–300.PubMedCrossRefGoogle Scholar
  3. Adams, J.M., and Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326.PubMedCrossRefGoogle Scholar
  4. Boulares, A.H., Zoltoski, A.J., Yakovlev, A., Xu, M., and Smulson, M.E. (2001). Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of TNF-induced apoptosis. J. Biol. Chem. 276, 38185–38192.PubMedGoogle Scholar
  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. Caron, H., Peter, M., van Sluis, P., Speleman, F., de Kraker, J. et al. (1995). Evidence for two tumour suppressor loci on chromosomal bands 1p35–36 involved in neuroblastoma: one probably imprinted, another associated with N-myc amplification. Hum. Mol. Genet. 4, 535–539.PubMedCrossRefGoogle Scholar
  7. Chen, Y.Z., Soeda, E., Yang, H.W., Takita, J., Chai, L., Horii, A., Inazawa, J., Ohki, M., and Hayashi, Y. (2001). Homozygous deletion in a neuroblastoma cell line defined by a high-density STS map spanning human chromosome band 1p36. Genes Chromosomes Cancer 31, 326–332.PubMedCrossRefGoogle Scholar
  8. Cote, J., and Ruiz-Carrilo, A. (1993). Primers for mitochondrial DNA replication generated by endonuclease G. Science 261, 765–769.PubMedCrossRefGoogle Scholar
  9. Counis, M.F., and Torriglia, A. (2000). Dnases and apoptosis. Biochem. Cell Biol. 78, 405–414.PubMedCrossRefGoogle Scholar
  10. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50.PubMedCrossRefGoogle Scholar
  11. Fadok, V.A., and Henson, P.M. (1998). Apoptosis: getting rid of the bodies. Curr. Biol. 8, R693–695.PubMedCrossRefGoogle Scholar
  12. Gross, A., McDonnell, J.M., and Korsmeyer, S.J. (1999). Bcl-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899–1911.PubMedCrossRefGoogle Scholar
  13. Halenbeck, R., MacDonald, H., Roulston, A., Chen, T.T., Conroy, L., and Williams, L.T. (1998). CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. Curr. Biol. 8, 537–540.PubMedCrossRefGoogle Scholar
  14. Hengartner, M.O. (2001). Apoptosis: corralling the corpses. Cell 104, 325–328.Google Scholar
  15. Hobbs, J.A., Cho, S.Y., Roberts, T.J., Sriram, V., Zhang, J., Xu, M., and Brutkiewicz, R.R. (2001). Selective loss of NKT cells by apoptosis following infection with lymphocytic choriomeningitis virus. J. Virol. 75, 10746–10754.PubMedCrossRefGoogle Scholar
  16. Horvitz, H.R. (1999). Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res. 59, 1701s - 1706s.PubMedGoogle Scholar
  17. Ikeda, S., and Ozaki, K. (1997). Action of mitochondrial endonuclease G on DNA damaged by L-ascorbic acid, peplomycin, and cis-diamminedichloroplatinum (II). Biochem. Biophys. Res. Comm. 235, 291–294.PubMedCrossRefGoogle Scholar
  18. Imyanitov, E.N., Birrell, G.W., Filippovich, I., Sorokina, N., Arnold, J., Mould, M.A., Wright, K., Walsh, M., Mok, S.C., Lavin, M.F. et al. (1999). Frequent loss of heterozygosity at 1p36 in ovarian adenocarcinomas but the gene encoding p73 is unlikely to be the target. Oncogene 18, 4640–4642.PubMedCrossRefGoogle Scholar
  19. Jacobson, M.D., Weil, M., and Raff, M.C. (1997). Programmed cell death in animal development. Cell 88, 347–354.PubMedCrossRefGoogle Scholar
  20. Judson, H., van Roy, N., Strain, L., Vandesompele, J., Van Gele, M., Speleman, F., and Bonthron, D.T. (2000). Structure and mutation analysis of the gene encoding DNA fragmentation factor 40 (caspase-activated nuclease), a candidate neuroblastoma tumour suppressor gene. Hum. Genet. 106, 406–413.PubMedCrossRefGoogle Scholar
  21. Kawane, K., Fukuyama, H., Adachi, M., Sakahira, H., Copeland, N.G., Gilbert, D.J., Jenkin, N.A., and Nagata, S. (1999). Structure and promoter analysis of murine CAD and ICAD genes. Cell Death Differ. 6, 745–752.PubMedCrossRefGoogle Scholar
  22. Kawane, K., Fukuyama, H., Kondoh, G., Takeda, J., Ohsawa, Y., Uchiyama, Y., and Nagata, S. (2001). Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science 292, 1546–1549.PubMedCrossRefGoogle Scholar
  23. Kempermann, G., Kuhn, H.G., and Gage, F.H. (1997). More hippocampal neurons in adult mice living in an enriched environment. Nature 386, 493–495.PubMedCrossRefGoogle Scholar
  24. Leek, J.P., Can, I.M., Bell, S.M., Markham, A.F., and Lench, N.J. (1997). Assignment of the DNA fragmentation factor gene (DFFA) to human chromosome bands 1p36.3->p36.2 by in situ hybridization. Cytogenet. Cell Genet. 79, 212–213.PubMedCrossRefGoogle Scholar
  25. Li, L.Y., Luo, X., and Wang, X. (2001). Endonuclease G (EndoG) is an apoptotic DNase when released from Mitochondria. Nature 412, 95–99.PubMedCrossRefGoogle Scholar
  26. Lindsten, T., Ross, A:J., King, A., Zong, W.X., Rathmell, J.C., Shiels, H.A., Ulrich, E., Waymire, K.G., Mahar, P., Frauwirth, K., et al. (2000). The combined functions of proapoptotic Bc1–2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell 6, 1389–1399.Google Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. Liu, X., Li, P., Widlak, P., Zou, H., Luo, X., Garrard, W.T., and Wang, X. (1998). DFF40 induces DNA fragmentation and chromatin condensation during apoptosis. Proc. Natl. Acad. Sci. USA 95, 8461–8466.PubMedCrossRefGoogle Scholar
  29. Mcllroy, D., Tanaka, M., Sakahira, H., Fukuyama, H., Suzuki, M., Yamamura, K., Ohsawa, Y., Uchiyama, Y., and Nagata, S. (2000). An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes. Genes Dev. 14, 549–558.Google Scholar
  30. McQuade, J.S., Vorhees, C., Xu, M., and Zhang, J. (2002). Enhanced non-spatial learning and memory in DFF45 knockout mice compared to wild-type mice. Physiol. Beh. 76, 315–320.CrossRefGoogle Scholar
  31. Milner, B., Squire, L.R., and Kandel, E.R. (1998). Cognitive neuroscience and the study of memory. Neuron 20, 445–468.PubMedCrossRefGoogle Scholar
  32. Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355–365.PubMedCrossRefGoogle Scholar
  33. Nagata, S. (2000). Apoptotic DNA fragmentation. Exp. Cell Res. 256, 12–18.PubMedCrossRefGoogle Scholar
  34. Napirei, M., Karsunky, H., Zevnik, B, Stephan, H., Mannherz, H.G., and Moroy, T. (2000). Features of systemic lupus erythematosus in Dnasel-deficient mice. Nat. Genet. 25, 177–181.PubMedCrossRefGoogle Scholar
  35. Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R., and Sikorska, M. (1993). Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679–3684.PubMedGoogle Scholar
  36. Ohira, M., Kageyama, H., Mihara, M., Furuta, S., Machida, T., Shishikura, T., Takayasu, H., Islam, A., Nakamura, Y., Takahashi, M. et al. (2000). Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line. Oncogene 19, 4302–4307.PubMedCrossRefGoogle Scholar
  37. Oliveri, M., Dap, A., Cantoni, C., Lunardi, C., Millo, R., and Puccetti, A. (2001). DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis. Eur. J. Immunol. 31, 743–751.PubMedCrossRefGoogle Scholar
  38. Parrish, J., Li, L., Klotz, K., Ledwich, D., Wang, X., and Xue, D. (2001). C. elegans mitochondrial endonuclease G is important for DNA fragmentation and progression of apoptosis. Nature 412, 90–94.PubMedCrossRefGoogle Scholar
  39. Peitsch, M.C., Mannherz, H.G., and Tschopp, J. (1994). The apoptosis endonucleases: cleaning up after cell death? Trends Cell Biol. 4, 37–41.PubMedCrossRefGoogle Scholar
  40. Ranger, A.M„ Malynn, B.A., and Korsmeyer, S.J. (2001). Mouse models of cell death. Nat. Genet. 28, 113–118.Google Scholar
  41. Ruiz-Carrillo, A., and Renaud, J. (1987). Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes. EMBO J. 6, 401–407.PubMedGoogle Scholar
  42. Sakahira, H., Enari, M., and Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96–99.PubMedCrossRefGoogle Scholar
  43. Sakahira, H., Enari, M., Ohsawa, Y., Uchiyama, Y., and Nagata, S. (1999). Apoptotic nuclear morphological change without DNA fragmentation, Curr. Biol. 9, 543–546.PubMedCrossRefGoogle Scholar
  44. Samejima, K., Tone, S., and Earnshaw, W.C. (2001). CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis. J. Biol. Chem. 276, 45427–45432.PubMedCrossRefGoogle Scholar
  45. Slane, J., Lee, H., Vorhees, C., Zhang, J., and Xu, M. (2000). DNA fragmentation factor 45 deficient mice exhibit enhanced spatial learning and memory compared to wild-type control mice. Brain Res, 867, 70–79.PubMedCrossRefGoogle Scholar
  46. Strasser, A., Whittingham, S., Vaux, D.L., Bath, M.L., Adams, J.M., Cory, S., and Harris, A.W. (1991). Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88, 8661–8665.PubMedCrossRefGoogle Scholar
  47. Strasser, A., O’Connor, L., and Dixit, V.M. (2000). Apoptosis signaling. Annu. Rev. Biochem. 69, 217–245.PubMedCrossRefGoogle Scholar
  48. Susin, S.A., Lorenzo, H.K., Zamzami, N, Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., et al. (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441–446.PubMedCrossRefGoogle Scholar
  49. Thomas, D., Du, C., Xu, M., Wang, X., and Ley, T.J. (2000). DFF45/ICAD can be directly process by granzyme B during the induction of apoptosis. Immunity 12, 621–632.PubMedCrossRefGoogle Scholar
  50. Wakeland, E.K., Liu, K., Graham, R.R., and Behrens, T.W. (2001). Delineating the genetic basis of systemic lupus erythematosis. Immunity 15, 397–408.PubMedCrossRefGoogle Scholar
  51. Wang, X. (2001). The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922–2933.PubMedGoogle Scholar
  52. Widlak, P., Li, P., Wang, X., and Garrard, W.T. (2000). Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates. J. Biol. Chem. 275, 8226–8232.PubMedCrossRefGoogle Scholar
  53. Widlak, P., Li, L.Y., Wang, X., and Garrard, W.T. (2001). Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I. J. Biol. Chem. 276, 48404–48409.PubMedGoogle Scholar
  54. Wu, Y-C., Stanfield, G.M., and Horvitz, H.R. (2000). NUC-1, a Caenorhabditis elegans Dnase II homolog, functions in an intermediate step of DNA degradation during apoptosis. Genes Dev. 14, 536–548.PubMedGoogle Scholar
  55. Yakovlev, A.G., Di, X., Movsesyan, V., Mullins, P.G.M., Wang, G., Boulares, H., Zhang, J., Xu, M., and Faden, A.I. (2001). Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury. Mol. Medicine 7, 205–216.Google Scholar
  56. Zhang, J., Liu, X., Scherer, D.C., Van Kaer, L., Wang, X., and Xu, M. (1998). Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45. Proc. Natl. Acad. Sci. USA 95, 12480–12485.PubMedCrossRefGoogle Scholar
  57. Zhang, J., Wang, X., Bove, K.E., and Xu, M. (1999). DNA fragmentation factor 45-deficient cells are more resistant to apoptosis and exhibit different dying morphology than wild-type control cells. J. Biol. Chem. 274, 37450–37454.PubMedCrossRefGoogle Scholar
  58. Zhang, J., Lee, H., Lou, D.W., Boivin, G., and Xu, M. (2000). Lack of obvious 50 kilobase pair DNA fragments in DNA fragmentation factor 45-deficient thymocytes upon activation of apoptosis. Biochem. Biophys. Res. Comm. 274, 225–229.PubMedCrossRefGoogle Scholar
  59. Zhang, J., and Xu, M. (2000). DNA fragmentation in apoptosis. Cell Res. 10, 205–211.PubMedCrossRefGoogle Scholar
  60. Zhang, J., Lee, H., Agarwala, A., Lou, D.W., and Xu, M. (2001). DNA fragmentation factor 45 mutant mice exhibit resistance to kainic acid-induced neuronal cell death. Biochem. Biophy. Res. Comm. 285, 1143–1149.CrossRefGoogle Scholar
  61. Zhang, J., and Xu, M. (2002). Apoptotic DNA degradation and tissue homeostasis. Trends Cell Biol. 12, 84–89.PubMedCrossRefGoogle Scholar
  62. Zhang, J., Xu, M., and Aronow, B. (2002). Expression profiles of 109 apoptosis pathway-related genes in 82 mouse tissues and experimental conditions. Biochem. Biophy. Res. Comm. 297, 537–544.CrossRefGoogle Scholar
  63. Zheng, T.S., Hunot, S., Kuida, K., Momoi, T., Srinivasan, A., Nicholson, D.W., Lazebnik, Y., and Flavell, R.A. (2000). Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat. Med. 6, 1241–1247.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2003

Authors and Affiliations

  1. 1.Department of Cell Biology, Neurobiology and AnatomyUniversity of Cincinnati Medical CenterCincinnatiUSA

Personalised recommendations