, Volume 10, Issue 5, pp 1031–1041 | Cite as

Role of phosphoinositide 3-kinase in the autophagic death of serum-deprived PC12 cells

  • A. Guillon-Munos
  • M. X. P. van Bemmelen
  • P. G. H. ClarkeEmail author


The death of serum-deprived undifferentiated PC12 cells shows both autophagic and apoptotic features. Since it is still controversial whether the autophagy is instrumental in the cell death or a mere epiphenomenon, we tested the effects of inhibiting the autophagy by a variety of phosphoinositide 3-kinase inhibitors, and provided evidence that the autophagy, or a related trafficking event, is indeed instrumental in the cell death. Furthermore, by comparing the effects of PI3-K inhibition and caspase-inhibition on autophagic and apoptotic cellular events, we showed that in this case the autophagic and apoptotic mechanisms mediate cell death by parallel pathways and do not act in series.


apoptosis autophagic cell death LY294002 PI3-kinases 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ogier-Denis E, Codogno P. Autophagy: A barrier or an adaptive response to cancer. Biochim Biophys Acta-Reviews on Cancer 2003; 1603 113–128.CrossRefGoogle Scholar
  2. 2.
    Blommaart EFC, Krause U, Schellens JPM, Vreeling-Sindelárová H, Meijer AJ. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 1997; 243 240–246.CrossRefPubMedGoogle Scholar
  3. 3.
    Petiot A, Ogier-Denis E, Blommaart EFC, Meijer AJ, Codogno P. Distinct classes of phosphatidylinositol 3’-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 2000; 275 992–998.CrossRefPubMedGoogle Scholar
  4. 4.
    Cuervo AM. Autophagy: In sickness and in health. Trends Cell Biol 2004; 14 70–77.CrossRefPubMedGoogle Scholar
  5. 5.
    Clarke PGH. Developmental cell death: Morphological diversity and multiple mechanisms. Anat Embryol 1990; 181 195–213.CrossRefPubMedGoogle Scholar
  6. 6.
    Knaapen MWM, Davies MJ, De Bie M, Haven AJ, Martinet W, Kockx MM. Apoptotic versus autophagic cell death in heart failure. Cardiovasc Res 2001; 51 304–312.CrossRefPubMedGoogle Scholar
  7. 7.
    Borsello T, Croquelois K, Hornung JP, Clarke PGH. N-methyl-D-aspartate-triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase pathway. Eur J Neurosci 2003; 18 473–485.CrossRefPubMedGoogle Scholar
  8. 8.
    Nixon RA, Cataldo AM, Mathews PM. The endosomal-lysosomal system of neurons in Alzheimer’s disease pathogenesis: A review. Neurochem Res 2000; 25 1161–1172.CrossRefPubMedGoogle Scholar
  9. 9.
    Kitanaka C, Kuchino Y. Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ 1999; 6 508–515.CrossRefPubMedGoogle Scholar
  10. 10.
    Bursch W, Hochegger K, Török L, Marian B, Ellinger A, Hermann RS. Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments. J Cell Sci 2000; 113 1189–1198.PubMedGoogle Scholar
  11. 11.
    Yanagisawa H, Miyashita T, Nakano Y, Yamamoto D. HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death. Cell Death Differ 2003; 10 798–807.CrossRefPubMedGoogle Scholar
  12. 12.
    Bursch W, Ellinger A, Kienzl H, et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: The role of autophagy. Carcinogenesis 1996; 17 1595–1607.PubMedGoogle Scholar
  13. 13.
    Isahara K, Ohsawa Y, Kanamori S, et al. Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases. Neuroscience 1999; 91 233–249.CrossRefPubMedGoogle Scholar
  14. 14.
    Ohsawa Y, Isahara K, Kanamori S, et al. An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins. Arch Histol Cytol 1998; 61 395–403.PubMedGoogle Scholar
  15. 15.
    Xue LZ, Fletcher GC, Tolkovsky AM. Autophagy is activated by apoptotic signalling in sympathetic neurons: An alternative mechanism of death execution. Molec Cell Neurosci 1999; 14 180–198.CrossRefPubMedGoogle Scholar
  16. 16.
    Martin DN, Baehrecke EH. Caspases function in autophagic programmed cell death in Drosophila. Development 2004; 131 275–284.CrossRefPubMedGoogle Scholar
  17. 17.
    Uchiyama Y. Autophagic cell death and its execution by lysosomal cathepsins. Arch Histol Cytol 2001; 64 233–246.PubMedGoogle Scholar
  18. 18.
    Clarke PGH. Identical populations of phagocytes and dying neurons revealed by intravascularly injected horseradish peroxidase, and by endogenous glutaraldehyde-resistant acid phosphatase, in the brains of chick embryos. Histochem J 1984; 16 955–969.CrossRefPubMedGoogle Scholar
  19. 19.
    Pearse AGE. Histochemistry, Theoretical and Applied, vol.1. Edinburgh, London, New York: Churchill Livingstone 1968.Google Scholar
  20. 20.
    Kobayashi T, Stang E, Fang KS, de M, Parton RG, Gruenberg J. A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature. 1998; 392 193–197.CrossRefPubMedGoogle Scholar
  21. 21.
    Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi networkBeclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Reports 2001; 2 330–335.CrossRefPubMedGoogle Scholar
  22. 22.
    Hornung JP, Koppel H, Clarke PGH. Endocytosis and autophagy in dying neurons: An ultrastructural study in chick embryos. J Comp Neurol 1989; 283 425–437.CrossRefPubMedGoogle Scholar
  23. 23.
    Clarke PGH, Oppenheim RW. Neuron death in vertebrate development: In vivo methods. Methods Cell Biol 1995; 46 277–321.PubMedGoogle Scholar
  24. 24.
    Tassa A, Roux MP, Attaix D, Bechet DM. Class III phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes. Biochem J 2003; 376 577–586.CrossRefPubMedGoogle Scholar
  25. 25.
    Foster FM, Traer CJ, Abraham SM, Fry MJ. The phosphoinositide (PI) 3-kinase family. J Cell Sci 2003; 116 3037–3040.CrossRefPubMedGoogle Scholar
  26. 26.
    Seglen PO, Gordon PB. 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Nat Acad Sci USA 1982; 79 1889–1892.PubMedGoogle Scholar
  27. 27.
    Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem 1998; 67 481–507.CrossRefPubMedGoogle Scholar
  28. 28.
    Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000; 351 95–105.CrossRefPubMedGoogle Scholar
  29. 29.
    Stein RC, Waterfield MD. PI3-kinase inhibition: A target for drug development? Mol Med Today 2000; 6 347–357.CrossRefPubMedGoogle Scholar
  30. 30.
    Takegawa K, DeWald DB, Emr SD. Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology. J Cell Sci 1995; 108 3745–3756.PubMedGoogle Scholar
  31. 31.
    Marte BM, Downward J. PKB/Akt: Connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem Sci 1997; 22 355–358.CrossRefPubMedGoogle Scholar
  32. 32.
    Luo HR, Hattori H, Hossain MA, et al. Akt as a mediator of cell death. Proc Natl Acad Sci USA 2003; 100 11712–11717.CrossRefPubMedGoogle Scholar
  33. 33.
    Spear N, Estevez AG, Barbeito L, Beckman JS, Johnson GV. Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase. J Neurochem 1997; 69 53–59.PubMedGoogle Scholar
  34. 34.
    Aki T, Mizukami Y, Oka Y, et al. Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia. Biochemical J 2001; 358 481–487.CrossRefGoogle Scholar
  35. 35.
    Aki T, Yamaguchi K, Fujimiya T, Mizukami Y. Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2. Oncogene 2003; 22 8529–8535.CrossRefPubMedGoogle Scholar
  36. 36.
    Sandvig K, van Deurs B. Toxin-induced cell lysis: Protection by 3-methyladenine and cycloheximide. Exp Cell Res. 1992; 200 253–262.CrossRefPubMedGoogle Scholar
  37. 37.
    Jia L, Dourmashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM. Inhibition of autophagy abrogates tumour necrosis factor α induced apoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol 1997; 98 673–685.Google Scholar
  38. 38.
    Jia L, Dourmashkin RR, Newland AC, Kelsey SM. Mitochondrial ultracondensation, but not swelling, is involved in TNF alpha-induced apoptosis in human T-lymphoblastic leukaemic cells. Leukemia Res 1997; 21 973–983.CrossRefGoogle Scholar
  39. 39.
    Brown WJ, DeWald DB, Emr SD, Plutner H, Balch WE. Role for Phosphatidylinositol 3-Kinase in the Sorting and Transport of Newly Synthesized Lysosomal-Enzymes in Mammalian-Cells. J Cell Biol 1995; 130 781–796.CrossRefPubMedGoogle Scholar
  40. 40.
    Shibata M, Kanamori S, Isahara K, et al. Participation of cathepsins B and D in apoptosis of PC12 cells following serum deprivation. Biochem Biophys Res Comm 1998; 251 199–203.CrossRefPubMedGoogle Scholar
  41. 41.
    Yu L, Alva A, Su H, et al. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 2004; 304 1500–1502.Google Scholar
  42. 42.
    Shimizu S, Kanaseki T, Mizushima N, et al. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biol 2004; 6 1221–1228.CrossRefPubMedGoogle Scholar
  43. 43.
    Lee CY, Baehrecke EH. Steroid regulation of autophagic programmed cell death during development. Development 2001; 128 1443–1455.PubMedGoogle Scholar
  44. 44.
    Piacentini M, Evangelisti C, Mastroberardino PG, Nardacci R, Kroemer G. Does prothymosin-α act as molecular switch between apoptosis and autophagy? Cell Death Differ 2003; 10 937–939.CrossRefPubMedGoogle Scholar
  45. 45.
    Chi SJ, Kitanaka C, Noguchi K, et al. Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells. Oncogene. 1999; 18 2281–2290.CrossRefPubMedGoogle Scholar
  46. 46.
    Zaidi AU, D’Sa-Eipper C, Brenner J, et al. Bcl-XL-Caspase-9 interactions in the developing nervous system: Evidence for multiple death pathways. J Neurosci 2001; 21 169–175.PubMedGoogle Scholar
  47. 47.
    Guimaraes CA, Benchimol M, Amarante-Mendes GP, Linden R. Alternative programs of cell death in developing retinal tissue. J Biol Chem 2003; 278 41938–41946.CrossRefPubMedGoogle Scholar
  48. 48.
    Shimoke K, Chiba H. Nerve growth factor prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced cell death via the Akt pathway by suppressing caspase-3-like activity using PC12 cells: Relevance to therapeutical application for Parkinson’s disease. J Neurosci Res 2001; 63 402–409.CrossRefPubMedGoogle Scholar
  49. 49.
    Kagedal K, Zhao M, Svensson I, Brunk UT. Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem J 2001; 359: 335–343.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • A. Guillon-Munos
    • 1
  • M. X. P. van Bemmelen
    • 2
  • P. G. H. Clarke
    • 1
    Email author
  1. 1.Département de Biologie Cellulaire et de MorphologieUniversity of LausanneLausanneSwitzerland
  2. 2.Département de Pharmacologie et de ToxicologieUniversity of LausanneSwitzerland

Personalised recommendations