Abstract
Ectopic expression of c-myc sensitises cells to a wide range of apoptotic stimuli by inducing the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. To elucidate the molecular mechanisms of mitochondrial permeabilisation in response to c-Myc activation, we carried out a biochemical fractionation analysis of Rat1 fibroblasts expressing an inducible c-Myc protein. We find that cytoplasmic extracts from cells in which c-Myc has been activated contain a soluble factor capable of inducing cytochrome c release from isolated mouse liver mitochondria. This factor is present only under growth factor deprivation conditions and its activity is inhibited by addition of Bcl-XL. The c-Myc-induced factor copurifies with full-length Bid, a “BH3-only” proapoptotic member of the Bcl-2 family, and antibodies raised against the BH3 domain of Bid inhibit c-Myc-induced cytochrome c releasing activity. These results are consistent with a model in which the activation of c-Myc regulates factors capable of enhancing the mitochondrial membrane destabilisation function of “BH3-only” proteins.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- 4-OHT:
-
4-hydroxytamoxifen
- OMM:
-
outer mitochondrial membrane
- FCS:
-
foetal calf serum
- DMEM:
-
Dulbecco's modified Eagle medium
- PMSF:
-
phenylmethylsulphonyl fluoride
- IGF-1:
-
insulin-like growth factor 1
References
Grandori C, Cowley SM, James LP and Eisenman RN (2000) The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell. Dev. Biol. 16: 653–699
Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ and Hancock DC (1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell. 69: 119–128
Harrington EA, Bennett MR, Fanidi A and Evan GI (1994) c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J 13: 3286–3295
Hueber AO and Evan GI (1998) Traps to catch unwary oncogenes. Trends Genet. 14: 364–367
Fanidi A, Harrington EA and Evan GI (1992) Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature 359: 554–556
Strasser A, Harris AW, Bath ML and Cory S (1990) Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 348: 331–333
Gross A, McDonnell JM and Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13: 1899–1911
Chen-Levy Z, Nourse J and Cleary ML (1989) The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation. Mol. Cell. Biol. 9: 701–710
Hockenbery D, Nunez G, Milliman C, Schreiber RD and Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348: 334–336
Vander Heiden MG, Chandel NS, Li XX, Schumacker PT, Colombini M and Thompson CB (2000) Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc. Natl. Acad. Sci. USA 97: 4666–4671
Vander Heiden MG, Li XX, Gottleib E, Hill RB, Thompson CB and Colombini M (2001) Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J. Biol. Chem. 276: 19414–19419
Minn AJ, Kettlun CS, Liang H, Kelekar A, Vander Heiden MG, Chang BS, Fesik SW, Fill M and Thompson CB (1999) Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms. EMBO J. 18: 632–643
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y and Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell- intrinsic death machinery. Cell 91: 231–241
Puthalakath H, Huang DC, O'Reilly LA, King SM and Strasser A (1999) The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Mol. Cell 3: 287–296
Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B and Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144: 891–901
Juin P, Hueber AO, Littlewood T and Evan G (1999) c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev. 13: 1367–1381
Littlewood TD, Hancock DC, Danielian PS, Parker MG and Evan GI (1995) A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 23: 1686–1690
Hovius R, Lambrechts H, Nicolay K and de Kruijff B (1990) Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim. Biophys. Acta 1021: 217–726
Luo X, Budihardjo I, Zou H, Slaughter C and Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481–490
Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D and Reed JC (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA. 95: 4997–5002
Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ, Matsuda H and Tsujimoto Y (1998) Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci USA. 95: 14681–14686
Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, Roth KA and Korsmeyer SJ (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400: 886–891
Downward J (1999) How BAD phosphorylation is good for survival. Nat Cell Biol. 1: E33–E35
Zha J, Weiler S, Oh KJ, Wei MC and Korsmeyer SJ (2000) Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science. 290: 1761–1765
Desagher S, Osen-Sand A, Montessuit S, Magnenat E, Vilbois F, Hochmann A, Journot L, Antonsson B and Martinou JC (2001) Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol. Cell 8: 601–611
Eskes R, Desagher S, Antonsson B and Martinou JC (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol. 20: 929–935
Hsu YT and Youle RJ (1998) Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J. Biol. Chem. 273: 10777–10783
Makin GW, Corfe BM, Griffiths GJ, Thistlethwaite A, Hickman JA and Dive C (2001) Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate. EMBO J. 20: 6306–6315
Evan G and Littlewood T (1998) A matter of life and cell death. Science 281: 1317–1322
Downward J (1998) Ras signalling and apoptosis. Curr. Opin. Genet. Dev. 8: 49–54.
Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J and Evan G (1997) Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385: 544–548
Brunet A, Datta SR and Greenberg ME (2001) Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol. 11: 297–305
Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB and Hay N (2001) Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev. 15: 1406–1418
Plas DR, Talapatra S, Edinger AL, Rathmell JC and Thompson CB (2001) Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology. J. Biol. Chem. 276: 12041–12048
Chang DW, Claassen GF, Hann SR and Cole MD (2000) The c-Myc transactivation domain is a direct modulator of apoptotic versus proliferative signals. Mol. Cell. Biol. 20: 4309–4319
Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, Ulrich E, Waymire KG, Mahar P, Frauwirth K, Chen Y, Wei M, Eng VM, Adelman DM, Simon MC, Ma A, Golden JA, Evan G, Korsmeyer SJ, MacGregor GR and Thompson CB (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell 6: 1389–1399
Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB and Korsmeyer SJ (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292: 727–730
Zong WX, Lindsten T, Ross AJ, MacGregor GR and Thompson CB (2001) BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev. 15: 1481–1486
Hueber AO, Zornig M, Lyon D, Suda T, Nagata S and Evan GI (1997) Requirement for the CD95 receptor-ligand pathway in c-Myc-induced apoptosis. Science 278: 1305–1309
Chou JJ, Li H, Salvesen GS, Yuan J and Wagner G (1999) Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell 96: 615–624
Esposti MD, Erler JT, Hickman JA and Dive C (2001) Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity. Mol. Cell. Biol. 21: 7268–7276
Lutter M, Fang M, Luo X, Nishijima M, Xie X and Wang X (2000) Cardiolipin provides specificity for targeting of tBid to mitochondria. Nat. Cell. Biol. 2: 754–761
Ott M, Robertson JD, Gogvadze V, Zhivotovsky B and Orrenius S (2002) Cytochrome c release from mitochondria proceeds by a two-step process. Proc. Natl. Acad. Sci. USA. 99: 1259–1263
Zamzami N and Kroemer G (2001) The mitochondrion in apoptosis: how Pandora's box opens. Nat. Rev. Mol. Cell. Biol. 2: 67–71
Guo QM, Malek RL, Kim S, Chiao C, He M, Ruffy M, Sanka K, Lee NH, Dang CV and Liu ET (2000) Identification of c-myc responsive genes using rat cDNA microarray. Cancer Res. 60: 5922–5928
Acknowledgements
We thank Patricia Warne for antibody purification, the Electron Microscopy unit of Cancer Research UK for assessing the purity of mitochondrial preparations, Victoria Cowling and Patrizia Stoppelli for their critical review of the manuscript, Philippe Juin for the stimulating critical support of this work, and all the members of the Signal Transduction Laboratory for helpful discussions. II was recipient of an EMBO fellowship.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by G Melino
Rights and permissions
About this article
Cite this article
Iaccarino, I., Hancock, D., Evan, G. et al. c-Myc induces cytochrome c release in Rat1 fibroblasts by increasing outer mitochondrial membrane permeability in a Bid-dependent manner. Cell Death Differ 10, 599–608 (2003). https://doi.org/10.1038/sj.cdd.4401211
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.cdd.4401211
Keywords
This article is cited by
-
Self-consumption: the interplay of autophagy and apoptosis
Nature Reviews Molecular Cell Biology (2014)
-
Apoptotic signaling by c-MYC
Oncogene (2008)
-
c-Myc primed mitochondria determine cellular sensitivity to TRAIL-induced apoptosis
The EMBO Journal (2007)
-
Hypertrophic growth in cardiac myocytes is mediated by Myc through a Cyclin D2-dependent pathway
The EMBO Journal (2006)
-
Role of mitochondrial membrane permeabilization in apoptosis and cancer
Oncogene (2004)
