Apoptosis

, Volume 11, Issue 4, pp 497–507 | Cite as

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

Reports
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Abstract

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.

Keywords

apoptosis ARK DCP-1 DRICE DRONC drosophila survival 

Abbreviations

APAF-1

apoptotic protease-activating factor 1

BH domain

Bcl-2 homology domain

dsRNA

double stranded RNA

IAP

inhibitor of apoptosis protein

RNAi

RNA interference

S2

Schneider cell line 2

FITC

fluoresceine isothiocyanate

zVAD-fmk

benzoyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

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References

  1. 1.
    Danial NN, Korsmeyer SJ. Cell death: Critical control points. Cell 2004; 116: 205–219.PubMedCrossRefGoogle Scholar
  2. 2.
    Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis. Curr Opin Cell Biol 2003; 15: 691–699.PubMedCrossRefGoogle Scholar
  3. 3.
    Zamzami N, Kroemer G. Apoptosis: mitochondrial membrane permeabilization—the (w)hole story? Curr Biol 2003; 13: 71–73.CrossRefGoogle Scholar
  4. 4.
    Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479–489.PubMedCrossRefGoogle Scholar
  5. 5.
    Green DR, Evan GI. A matter of life and death. Cancer Cell 2002; 1: 19–30.PubMedCrossRefGoogle Scholar
  6. 6.
    Kumar S, Doumanis J. The fly caspases. Cell Death Differ 2000; 7: 1039–1044.PubMedCrossRefGoogle Scholar
  7. 7.
    Salvesen GS, Abrams JM. Caspase activation—stepping on the gas or releasing the brakes? Lessons from humans and flies. Oncogene 2004; 23: 2774–2784.PubMedCrossRefGoogle Scholar
  8. 8.
    Dorstyn L, Mills K, Lazebnik Y, Kumar S. The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells. J Cell Biol 2004; 167: 405–410.PubMedCrossRefGoogle Scholar
  9. 9.
    Dorstyn L, Read S, Cakouros D, Huh JR, Hay BA, Kumar S. The role of cytochrome c in caspase activation in Drosophila melanogaster cells. J Cell Biol 2002; 156: 1089–1098.PubMedCrossRefGoogle Scholar
  10. 10.
    Varkey J, Chen P, Jemmerson R, Abrams JM. Altered cytochrome c display precedes apoptotic cell death in Drosophila. J Cell Biol 1999; 144: 701–710.PubMedCrossRefGoogle Scholar
  11. 11.
    Zimmermann KC, Ricci JE, Droin NM, Green DR. The role of ARK in stress-induced apoptosis in Drosophila cells. J Cell Biol 2002; 156: 1077–1087.PubMedCrossRefGoogle Scholar
  12. 12.
    Bergmann A, Yang AY, Srivastava M. Regulators of IAP function: Coming to grips with the grim reaper. Curr Opin Cell Biol 2003; 15: 717–724.PubMedCrossRefGoogle Scholar
  13. 13.
    Igaki T, Yamamoto-Goto Y, Tokushige N, Kanda H, Miura M. Down-regulation of DIAP1 triggers a novel Drosophila cell death pathway mediated by Dark and DRONC. J Biol Chem 2002; 277: 23103–23106.PubMedCrossRefGoogle Scholar
  14. 14.
    Kaiser WJ, Vucic D, Miller LK. The Drosophila inhibitor of apoptosis D-IAP1 suppresses cell death induced by the caspase drICE. FEBS Lett 1998; 440: 243–248.PubMedCrossRefGoogle Scholar
  15. 15.
    Wilson R, Goyal L, Ditzel M, et al. The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat Cell Biol 2002; 4: 445–450.PubMedCrossRefGoogle Scholar
  16. 16.
    Martin SJ. Destabilizing influences in apoptosis: Sowing the seeds of IAP destruction. Cell 2002; 109: 793–796.PubMedCrossRefGoogle Scholar
  17. 17.
    Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004; 305: 626–629.PubMedCrossRefGoogle Scholar
  18. 18.
    Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA, Gruss P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 1998; 94: 727–737.PubMedCrossRefGoogle Scholar
  19. 19.
    Yoshida H, Kong YY, Yoshida R, et al. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 1998; 94: 739–750.PubMedCrossRefGoogle Scholar
  20. 20.
    Brunet CL, Gunby RH, Benson RS, Hickman JA, Watson AJ, Brady G. Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers. Cell Death Differ 1998; 5: 107–115.PubMedCrossRefGoogle Scholar
  21. 21.
    Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol 2000; 10: 369–377.PubMedCrossRefGoogle Scholar
  22. 22.
    Newmeyer DD, Ferguson-Miller S. Mitochondria: Releasing power for life and unleashing the machineries of death. Cell 2003; 112: 481–490.PubMedCrossRefGoogle Scholar
  23. 23.
    Ricci JE, Waterhouse N, Green DR. Mitochondrial functions during cell death, a complex (I-V) dilemma. Cell Death Differ 2003; 10: 488–492.PubMedCrossRefGoogle Scholar
  24. 24.
    Vernooy SY, Copeland J, Ghaboosi N, Griffin EE, Yoo SJ, Hay BA. Cell death regulation in Drosophila: conservation of mechanism and unique insights. J Cell Biol 2000; 150: 69–76.CrossRefGoogle Scholar
  25. 25.
    Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today 1997; 18: 44–51.PubMedCrossRefGoogle Scholar
  26. 26.
    Harosh I, Mezzina M, Harris PV, Boyd JB. Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos. Eur J Biochem 1992; 210: 455–460.PubMedCrossRefGoogle Scholar
  27. 27.
    Clemens JC, Worby CA, Simonson-Leff N, et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA 2000; 97: 6499–6503.PubMedCrossRefGoogle Scholar
  28. 28.
    Muro I, Hay BA, Clem RJ. The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated, processed form of the apical caspase DRONC. J Biol Chem 2002; 277: 49644–49650.PubMedCrossRefGoogle Scholar
  29. 29.
    Hawkins CJ, Yoo SJ, Peterson EP, Wang SL, Vernooy SY, Hay BA. The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM. J Biol Chem 2000; 275: 27084–27093.PubMedGoogle Scholar
  30. 30.
    Meier P, Silke J, Leevers SJ, Evan GI. The Drosophila caspase DRONC is regulated by DIAP1. EMBO J 2000; 19: 598–611.PubMedCrossRefGoogle Scholar
  31. 31.
    Marsden VS, O’Connor L, O’Reilly LA, et al. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 2002; 419: 634–637.PubMedCrossRefGoogle Scholar
  32. 32.
    Arama E, Agapite J, Steller H. Caspase activity and a specific cytochrome c are required for sperm differentiation in Drosophila. Dev Cell 2003; 4: 687–697.PubMedCrossRefGoogle Scholar
  33. 33.
    Muro I, Monser K, Clem RJ. Mechanism of Dronc activation in Drosophila cells. J Cell Sci 2004; 117: 5035–5041.PubMedCrossRefGoogle Scholar
  34. 34.
    Rodriguez A, Chen P, Oliver H, Abrams JM. Unrestrained caspase-dependent cell death caused by loss of Diap1 function requires the Drosophila Apaf-1 homolog, Dark. EMBO J 2002; 21: 2189–2197.PubMedCrossRefGoogle Scholar
  35. 35.
    Chautan M, Chazal G, Cecconi F, Gruss P, Golstein P. Interdigital cell death can occur through a necrotic and caspase-independent pathway. Curr Biol 1999; 9: 967–970.PubMedCrossRefGoogle Scholar
  36. 36.
    Deshmukh M, Kuida K, Johnson EM. Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization. J Cell Biol 2000; 150: 131–143.PubMedCrossRefGoogle Scholar
  37. 37.
    Haraguchi M, Torii S, Matsuzawa S, et al. Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2. J Exp Med 2000; 191: 1709–1720.PubMedCrossRefGoogle Scholar
  38. 38.
    Rodriguez A, Oliver H, Zou H, Chen P, Wang X, Abrams JM. Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway. Nat Cell Biol 1999; 1: 272–279.PubMedCrossRefGoogle Scholar
  39. 39.
    Kuida K, Haydar TF, Kuan C, et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998; 94: 325–337.PubMedCrossRefGoogle Scholar
  40. 40.
    Kuida K, Zheng TS, Na S, et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 1996; 384: 368–372.PubMedCrossRefGoogle Scholar
  41. 41.
    Scott CL, Schuler M, Marsden VS, et al. Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation. J Cell Biol 2004; 164: 89–96.PubMedCrossRefGoogle Scholar
  42. 42.
    Chipuk JE, Green DR. Opinion: Do inducers of apoptosis trigger caspase-independent cell death? Nat Rev Mol Cell Biol 2005; 6: 268–275.CrossRefGoogle Scholar
  43. 43.
    Yokokura T, Dresnek D, Huseinovic N, et al. Dissection o DIAP1 functional domains via a mutant replacement strategy. J Biol Chem 2004; 279: 52603–52612.PubMedCrossRefGoogle Scholar
  44. 44.
    Muro I, Means JC, Clem RJ. Cleavage of the apoptosis inhibitor DIAP1 by the apical caspase DRONC in both normal and apoptotic Drosophila cells. J Biol Chem 2005; 280: 18683–18688.PubMedCrossRefGoogle Scholar
  45. 45.
    Vernooy SY, Chow V, Su J, et al. Drosophila Bruce can potently suppress Rpr- and Grim-dependent but not Hid-dependent cell death. Curr Biol 2002; 12: 1164–1168.PubMedCrossRefGoogle Scholar
  46. 46.
    Ditzel M, Wilson R, Tenev T, et al. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nat Cell Biol 2003; 5: 467–473.PubMedCrossRefGoogle Scholar
  47. 47.
    Tenev T, Zachariou A, Wilson R, Ditzel M, Meier P. IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Nat Cell Biol 2005; 7: 70–77.PubMedCrossRefGoogle Scholar
  48. 48.
    Claveria C, Caminero E, Martinez A, Campuzano S, Torres M. GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway. EMBO J 2002; 21: 3327–3336.PubMedCrossRefGoogle Scholar
  49. 49.
    Claveria C, Torres M. Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators. Biochem Biophys Res Commu 2003; 304: 531–537.CrossRefGoogle Scholar
  50. 50.
    Claveria C, Martinez A; Torres M. A Bax/Bak-independent mitochondrial death pathway triggered by Drosophila Grim GH3 domain in mammalian cells. J Biol Chem 2004; 279: 1368–1375.PubMedCrossRefGoogle Scholar
  51. 51.
    McCarthy JV, Dixit VM. Apoptosis induced by Drosophila reaper and grim in a human system. Attenuation by inhibitor of apoptosis proteins (cIAPs). J Biol Chem 1998; 273: 24009–24015.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  1. 1.Division of Cellular ImmunologyLa Jolla Institute for Allergy and ImmunologySan Diego
  2. 2.Department of ImmunologySt. Jude Children’s Research HospitalMemphisUSA

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