, Volume 11, Issue 9, pp 1473–1487 | Cite as

Caspase-activated DNase (CAD)-independent oligonucleosomal DNA fragmentation in chronic myeloid leukaemia cells; a requirement for serine protease and Mn2+-dependent acidic endonuclease activity

  • L. B. McGrath
  • V. Onnis
  • G. Campiani
  • D. C. Williams
  • D. M. Zisterer
  • M. M. Mc Gee


We have previously reported that the pro-apoptotic pyrrolobenzoxazepine, PBOX-6, induces apoptosis in chronic myelogenous leukaemia (CML) cells which is accompanied by oligonucleosomal DNA fragmentation. In this study we show that PBOX-6-induced oligonucleosomal DNA fragmentation occurs in the absence of caspase and CAD activation in CML cells. Dissection of the signalling pathway has revealed that induction of apoptosis requires the upstream activation of a trypsin-like serine protease that promotes the phosphorylation and inactivation of anti-apoptotic Bcl-2. In addition, in this system chymotrypsin-like serine proteases are dispensable for high molecular weight DNA fragmentation, however are required for the activation of a relatively small manganese-dependent acidic endonuclease that is responsible for oligonucleosomal fragmentation of DNA. Furthermore, we demonstrate mitochondrial involvement during PBOX-6-induced apoptosis and suggest the existence of unidentified mitochondrial effectors of apoptosis.


Caspase-independent Pyrrolobenzoxazepine Apoptosis Serine proteases Endonuclease 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239–257PubMedGoogle Scholar
  2. 2.
    Donovan M, Cotter TG (2004) Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death. BBA 1644(2–3):133–147PubMedGoogle Scholar
  3. 3.
    Vermeulen K, Van Bockstaele DR, Berneman ZN (2005) Apoptosis: mechanisms and relevance in cancer. Ann Haematol 84:627–639CrossRefGoogle Scholar
  4. 4.
    Reed RC, Pellecchia M Apoptosis-based therapies for hematologic malignancies. Blood 106(2):408–418Google Scholar
  5. 5.
    Saelens X, Festjens N, Vande Walle L, van Gurp M, van Loo G, Vandenabeele P (2004) Toxic proteins released from mitochondria in cell death. Oncogene 23(16):2861–2874. (Review)PubMedCrossRefGoogle Scholar
  6. 6.
    Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86(1):147–157PubMedCrossRefGoogle Scholar
  7. 7.
    Susin SA, Lorenzo HK, Zamzami N et al (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397(6718):441–446PubMedCrossRefGoogle Scholar
  8. 8.
    Verhagen AM, Ekert PG, Pakusch M et al (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102(1):43–53PubMedCrossRefGoogle Scholar
  9. 9.
    Li LY, Luo X, Wang X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412(6842):95–99PubMedCrossRefGoogle Scholar
  10. 10.
    Verhagen AM, Silke J, Ekert PG et al (2002) HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem 277(1):445–454 (Epub 2001 Oct 16)PubMedCrossRefGoogle Scholar
  11. 11.
    Choi WS, Lee EH, Chung CW et al (2001) Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2. J Neurochem 77(6):1531–1541PubMedCrossRefGoogle Scholar
  12. 12.
    Mathiasen IS, Jaattela M (2002) Triggering caspase-independent cell death to combat cancer. Trends Mol Med 8(5):212–220 (Review)PubMedCrossRefGoogle Scholar
  13. 13.
    Stenson-Cox C, FitzGerald U, Samali A (2003) In the cut and thrust of apoptosis, serine proteases come of age. Biochem Pharmacol 66(8):1469–1474 (Review)PubMedCrossRefGoogle Scholar
  14. 14.
    Hegde R, Srinivasula SM, Zhang Z et al (2002) Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem 277(1):432–438 (Epub 2001 Oct 17)PubMedCrossRefGoogle Scholar
  15. 15.
    Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284(5756):555–556PubMedCrossRefGoogle Scholar
  16. 16.
    Liu X, Zou H, Slaughter C, Wang X (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89(2):175–184PubMedCrossRefGoogle Scholar
  17. 17.
    Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391(6662):43–50PubMedCrossRefGoogle Scholar
  18. 18.
    Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391(6662):96–99PubMedCrossRefGoogle Scholar
  19. 19.
    Samejima K, Tone S, Earnshaw WC (2001) CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis. J Biol Chem 276(48):45427–45432 (Epub 2001 Sep 27)PubMedCrossRefGoogle Scholar
  20. 20.
    Donovan M, Cotter TG (2002) Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development. Cell Death Differ 9(11):1220–1231PubMedCrossRefGoogle Scholar
  21. 21.
    van Loo G, Schotte P, van Gurp M et al (2001) Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation. Cell Death Differ 8(12):1136–1142PubMedCrossRefGoogle Scholar
  22. 22.
    Yakovlev AG, Di X, Movsesyan V et al (2001) Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury. Mol Med 7(3):205–216PubMedGoogle Scholar
  23. 23.
    Kawabata H, Anzai N, Masutani H et al (1997) Mg2+- or Mn2+-dependent endonuclease activities of human myeloid leukemia cells capable of producing nucleosomal-size DNA fragmentation. Biochem Biophys Res Commun 233(1):133–138PubMedCrossRefGoogle Scholar
  24. 24.
    Cohen GM, Sun XM, Fearnhead H et al (1994) Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes. J Immunol 153(2):507–516PubMedGoogle Scholar
  25. 25.
    Khodarev NN, Ashwell JD (1996) An inducible lymphocyte nuclear Ca2+/Mg (2+)-dependent endonuclease associated with apoptosis. J Immunol 156(3):922–931PubMedGoogle Scholar
  26. 26.
    Assuncao Guimaraes C, Linden R (2004) Programmed cell deaths. Apoptosis and alternative deathstyles. Eur J Biochem 271(9):1638–1650PubMedCrossRefGoogle Scholar
  27. 27.
    Drucker BJ, Tamura S, Buchdunger E et al (1996) Effects of a selective inhibitor of the ABL tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566CrossRefGoogle Scholar
  28. 28.
    Zisterer DM, Campiani G, Nacci V, Williams DC (2000) Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents. J Pharmacol Exp Ther 293(1):48–59PubMedGoogle Scholar
  29. 29.
    Mc Gee MM, Campiani G, Ramunno A et al (2001) Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor Bcr-Abl. J Pharmacol Exp Ther 296(1):31–40PubMedGoogle Scholar
  30. 30.
    Mc Gee MM, Hyland E, Campiani G, Ramunno A, Nacci V, Zisterer DM (2002) Caspase-3 is not essential for DNA fragmentation in MCF-7 cells during apoptosis induced by the pyrrolo-1,5-benzoxazepine, PBOX-6. FEBS Lett 515(1–3):66–70PubMedCrossRefGoogle Scholar
  31. 31.
    Mc Gee MM, Campiani G, Ramunno A et al (2002) Activation of the c-Jun NH2 terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells. J Biol Chem 277(21):18383–18389PubMedCrossRefGoogle Scholar
  32. 32.
    Mc Gee MM, Greene LM, Ledwidge S et al (2004) Selective induction of apoptosis by PBOX-6 in Leukemia cells occurs via the JNK dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL. J Pharmacol Exp Ther 310(3):1084–1095PubMedCrossRefGoogle Scholar
  33. 33.
    Giannakis C, Forbes IJ, Zalewski PD (1991) Ca2+ /Mg2+ - dependent nuclease; tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+. Biochem Biophys. Res Commun 181:915Google Scholar
  34. 34.
    Murn J, Urleb U, Mlinaric-Rascan I (2004) Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like protease. Genes Cells 9(11):1103–1111PubMedCrossRefGoogle Scholar
  35. 35.
    Dong Z, Saikumar P, Patel Y, Weinberg JM, Venkatachalam MA (2000) Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochem J 347(Pt 3):669–677PubMedCrossRefGoogle Scholar
  36. 36.
    Fearnhead HO, Rivett AJ, Dinsdale D, Cohen GM (1995) A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli. FEBS Lett 357(3):242–246PubMedCrossRefGoogle Scholar
  37. 37.
    Rideout HJ, Zang E, Yeasmin M et al (2001) Inhibitors of trypsin-like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction. Neuroscience 107(2):339–352PubMedCrossRefGoogle Scholar
  38. 38.
    Gong B, Chen Q, Endlich B, Mazumder S, Almasan A (1999) Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth Differ 10(7):491–502PubMedGoogle Scholar
  39. 39.
    Huang Y, Sheikh MS, Fornace AJ Jr, Holbrook NJ (1999) Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells. Oncogene 18(23):3431–3439PubMedCrossRefGoogle Scholar
  40. 40.
    Yamada M, Hirasawa A, Shiojima S, Tsujimoto G (2003) Granzyme A mediates glucocorticoid-induced apoptosis in leukemia cells. FASEB J 17(12):1712–1714PubMedCrossRefGoogle Scholar
  41. 41.
    Blink E, Maianski NA, Alnemri ES, Zervos AS, Roos D, Kuijpers TW (2004) Intramitochondrial serine protease activity of Omi/HtrA2 is required for caspase-independent cell death of human neutrophils. Cell Death Differ 11(8):937–939PubMedCrossRefGoogle Scholar
  42. 42.
    Cilenti L, Lee Y, Hess S et al (2003) Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2. J Biol Chem 278(13):11489–11494 (Epub 2003 Jan 15)PubMedCrossRefGoogle Scholar
  43. 43.
    Widlak P, Garrard WT (2001) Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD. Mol Cell Biochem 218(1–2):125–130PubMedCrossRefGoogle Scholar
  44. 44.
    Nagata S (2005) DNA degradation in development and programmed cell death. Annu Rev Immunol 23:853–875 (Review)PubMedCrossRefGoogle Scholar
  45. 45.
    Kawane K, Fukuyama H, Yoshida H et al (2003) Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation. Nat Immunol 4(2):138–144PubMedCrossRefGoogle Scholar
  46. 46.
    Hughes FM Jr, Evans-Storms RB, Cidlowski JA (1998) Evidence that non-caspase proteases are required for chromatin degradation during apoptosis. Cell Death Differ 5(12):1017–1027PubMedCrossRefGoogle Scholar
  47. 47.
    Sane AT, Bertrand R (1998) Distinct steps in DNA fragmentation pathway during camptothecin-induced apoptosis involved caspase-, benzyloxycarbonyl- and N-tosyl-L-phenylalanylchloromethyl ketone-sensitive activities. Cancer Res 58(14):3066–3072PubMedGoogle Scholar
  48. 48.
    Eitel K, Wagenknecht B, Weller M (1999) Inhibition of drug-induced DNA fragmentation, but not cell death, of glioma cells by non-caspase protease inhibitors. Cancer Lett 142(1):11–16PubMedCrossRefGoogle Scholar
  49. 49.
    Widlak P, Li P, Wang X, Garrard WT (2000) Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates. J Biol Chem 275(11):8226–8232PubMedCrossRefGoogle Scholar
  50. 50.
    Cande C, Vahsen N, Kouranti I et al (2004) AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis. Oncogene 23(8):1514–1521PubMedCrossRefGoogle Scholar
  51. 51.
    Widlak P, Li LY, Wang X, Garrard WT (2001) Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I. J Biol Chem 276(51):48404–48409PubMedGoogle Scholar
  52. 52.
    Altairac S, Wright SC, Courtois Y, Torriglia A (2003) L-DNase II activation by the 24 kDa apoptotic protease (AP24) in TNFalpha-induced apoptosis. Cell Death Differ 10(9):1109–1111PubMedCrossRefGoogle Scholar
  53. 53.
    Nagata S, Nagase H, Kawane K, Mukae N, Fukuyama H (2003) Degradation of chromosomal DNA during apoptosis. Cell Death Differ 10(1):108–116 (Review)PubMedCrossRefGoogle Scholar
  54. 54.
    Arnoult D, Gaume B, Karbowski M, Sharpe JC, Cecconi F, Youle RJ (2003) Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J 22(17):4385–4399PubMedCrossRefGoogle Scholar
  55. 55.
    Liu X, Li P, Widlak P et al (1998) The 40-Kda subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc Natl Acad Sci USA 95:8461–8466PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  • L. B. McGrath
    • 1
  • V. Onnis
    • 2
  • G. Campiani
    • 3
  • D. C. Williams
    • 2
  • D. M. Zisterer
    • 2
  • M. M. Mc Gee
    • 1
  1. 1.UCD School of Biomolecular and Biomedical Science, Conway InstituteUniversity College DublinDublin 4Ireland
  2. 2.School of Biochemistry and ImmunologyTrinity CollegeDublin 2Ireland
  3. 3.Dipartimento Farmaco Chimico TecnologicoUniversita'delgi Studi di SienaSienaItaly

Personalised recommendations