Death Signal-induced Localization of p53 Protein to Mitochondria

The mechanism of p53-mediated apoptosis after cellular stress remains poorly understood. Evidence suggests that p53 induces cell death by a multitude of molecular pathways involving activation of target genes and transcriptionally independent direct signaling. Mitochondria play a key role in apoptosis. We show here that a fraction of p53 protein localizes to mitochondria at the onset of p53-dependent apoptosis but not during p53-independent apoptosis or p53-mediated cell cycle arrest. The accumulation of p53 to mitochondria is rapid (within 1 h after p53 activation) and precedes changes in mitochondrial membrane potential, cytochromec release, and procaspase-3 activation. Immunoelectron microscopy and immuno-fluorescence-activated cell sorter analysis of isolated mitochondria show that the majority of mitochondrial p53 localizes to the membranous compartment, whereas a fraction is found in a complex with the mitochondrial import motor mt hsp70. After induction of ectopic p53 without additional DNA damage in p53-deficient cells, p53 again partially localizes to mitochondria, preceding the onset of apoptosis. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL abrogates stress signal-mediated mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback signaling loop between p53 and mitochondrial apoptotic regulators. Importantly, bypassing the nucleus by targeting p53 to mitochondria using import leader fusions is sufficient to induce apoptosis in p53-deficient cells. We propose a model where p53 can contribute to apoptosis by direct signaling at the mitochondria, thereby amplifying the transcription-dependent apoptosis of p53. The mechanism of p53-mediated apoptosis after cellular stress remains poorly understood. Evidence suggests that p53 induces cell death by a multitude of molecular pathways involving activation of target genes and transcriptionally independent direct signaling. Mitochondria play a key role in apoptosis. We show here that a fraction of p53 protein localizes to mitochondria at the onset of p53-dependent apoptosis but not during p53-independent apoptosis or p53-mediated cell cycle arrest. The accumulation of p53 to mitochondria is rapid (within 1 h after p53 activation) and precedes changes in mitochondrial membrane potential, cytochromec release, and procaspase-3 activation. Immunoelectron microscopy and immuno-fluorescence-activated cell sorter analysis of isolated mitochondria show that the majority of mitochondrial p53 localizes to the membranous compartment, whereas a fraction is found in a complex with the mitochondrial import motor mt hsp70. After induction of ectopic p53 without additional DNA damage in p53-deficient cells, p53 again partially localizes to mitochondria, preceding the onset of apoptosis. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL abrogates stress signal-mediated mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback signaling loop between p53 and mitochondrial apoptotic regulators. Importantly, bypassing the nucleus by targeting p53 to mitochondria using import leader fusions is sufficient to induce apoptosis in p53-deficient cells. We propose a model where p53 can contribute to apoptosis by direct signaling at the mitochondria, thereby amplifying the transcription-dependent apoptosis of p53. tumor necrosis factor-α mitochondrial actinomycin D Tdt-mediated dUTP-X nick end labeling interleukin-3 proliferating cell nuclear antigen camptothecin wild type retinoblastoma The mechanism of p53-mediated apoptosis after cellular stress remains unclear. Current evidence suggests that p53 induces cell death by a multitude of molecular pathways involving transactivation of target genes and direct signaling events (for review see Refs. 1.Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6716) Google Scholar, 2.Harris C.C. J. Natl. Cancer Inst. 1996; 88: 1442-1455Crossref PubMed Scopus (649) Google Scholar, 3.Gottlieb T.M. Oren M. Semin. Cancer Biol. 1998; 8: 359-368Crossref PubMed Scopus (217) Google Scholar). A growing and diverse list of p53-inducible genes are candidates for participating in the apoptotic action of p53. These include genes with proven roles in apoptotic pathways such as Bax (4.Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (303) Google Scholar),Fas/APO-1 (5.Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. Zhang W.-W. Kruzel E. Radinsky R. Mol. Cell. 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Science. 1995; 270: 96-99Crossref PubMed Scopus (1301) Google Scholar). Likewise, APO-1 is fully dispensable for p53-dependent apoptosis as shown by Fas-deficient mice (so called lpr mice) (13.Reinke V. Lozano G. Oncogene. 1997; 15: 1527-1534Crossref PubMed Scopus (61) Google Scholar). An interesting series of p53-induced redox-modulating genes named PIGs (p53-inducedgenes) were found in an apoptosis screen of colorectal carcinoma cells (17.Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2234) Google Scholar). The p53 target PIG3 is related to the plant NADPH oxidoreductase TED2 and is a potent generator of reactive oxygen species. The isolation of PIG genes strongly suggests that oxidative damage to mitochondria is of central importance in p53-controlled cell death. However, overexpressed PIG3 alone does not induce apoptosis, and most PIGs are induced by p53 regardless of whether cells undergo apoptosis or growth arrest (17.Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2234) Google Scholar). Evidence for transcription-independent pathways for p53-mediated apoptosis is accumulating in 3.Gottlieb T.M. Oren M. Semin. Cancer Biol. 1998; 8: 359-368Crossref PubMed Scopus (217) Google Scholar). cell p53-induced apoptosis in the of gene or protein A. M. Nature. PubMed Scopus Google Scholar, A.J. N. 8: PubMed Scopus Google Scholar, N. J. Cancer 1999; PubMed Scopus Google Scholar). Furthermore, of protein induce p53-dependent apoptosis in the of transactivation Y. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the transcriptionally p53 and p53 to to DNA or as and other target as potent of apoptosis in cell Y. S. E. Oren M. 1995; PubMed Scopus Google Scholar, Y. S. E. A. K. 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Mitochondria undergo critical changes that a These changes include of the of and reactive oxygen and mitochondrial with membrane in critical of mitochondrial is the rapid of such as the the cytochromec is the for activation the pathway (for review see Refs. Reed J.C. Science. 1998; Google and J.C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Bcl-2 and Bcl-xL in the mitochondrial cells death by mitochondrial of the of apoptosis. thereby the mitochondrial and mitochondrial and the of Reed J.C. Science. 1998; Google Scholar, J.C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google N. I. G. Oncogene. 1998; PubMed Scopus Google Scholar). the transcription-independent of p53 and the importance of mitochondria in p53-dependent apoptosis (17.Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2234) Google Scholar), the in to transactivation role in the p53 a role at regulatory found that a but fraction of p53 protein to mitochondria at the onset of p53-dependent apoptosis after DNA damage or This does not during p53-independent apoptosis or during p53-mediated cell cycle arrest. The of p53 to mitochondria is preceding the of and procaspase-3 activation and is by of anti-apoptotic Importantly, p53 the nucleus and targeting to mitochondria by using mitochondrial import leader is sufficient to induce apoptosis in p53-deficient cells. These a role of the p53 protein in direct apoptotic signaling at mitochondria, are a in the cell death The action of p53 at the mitochondrial to a to transactivation in the thereby amplifying apoptotic and cells were in with and cells were in with actinomycin and tumor necrosis factor-α were to Mitochondria were by as D. J. Biol. Full Text PDF PubMed Google Scholar). cells were in and to for in were with and to mitochondria of 3 of After nuclear for the a and at for at Mitochondria were at the by the of in a of of and for The and and nuclear and protein protein of mitochondrial and protein or cell were to analysis and with and for The were and for and of D. for mt and of B. for with mt or mt and and and and or for For the of mitochondrial p53 to induced of were with to and and by cells for were with 1 of and for by the fraction of cells a events For microscopy the in or For p53-independent apoptosis, cells were with and D PubMed Scopus Google Scholar). 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Full Text PDF PubMed Google Scholar), in mitochondria and by with other cellular apoptosis by cell cycle analysis or and by or microscopy (e.g. A and not cell wild type the 1 and 1 and and the colorectal carcinoma 1 were and after apoptosis induction by DNA damage using camptothecin A or and after apoptosis induction by stress using 1 cell a of p53 to the mitochondrial fraction of cells. for were with the cell type not were found in cells wild type and and in cells with wild type not undergo p53-dependent apoptosis after DNA damage J. J. D. J.L. 1998; PubMed Scopus Google Scholar). mitochondrial p53 only a fraction of induced cellular a of cells, of induced p53 after camptothecin as by of p53 not accumulation for other nuclear such as and the not were for by using for the nucleus and and were and or and the of and the in the mitochondrial to in the mitochondrial mitochondrial were by microscopy and found to with with the of and membrane mitochondrial p53 accumulation is a that in of stress and in and cells. the is of to p53-dependent apoptosis. cells to p53-independent apoptosis by the Mitochondria are a of the (for review see M.E. Krammer P.H. 1998; PubMed Scopus Google Scholar). contrast to in p53-dependent cell p53 not to mitochondria cells were induced to undergo p53-independent death by the D that mitochondrial cells with actinomycin D not of induction of cellular p53 and a of apoptosis that to the with for with 1 These that mitochondrial p53 does not a of cellular p53 or is a of cells cell death. the strongly that mitochondrial p53 is of a regulatory that a p53-dependent apoptotic are for cell to a of DNA damage is the activation of arrest apoptosis, to induce the accumulation of mitochondrial p53. contrast to cells, the and wild type to p53 in mitochondria after camptothecin 1 or not with a of the induction of cellular p53 1 and a with to and but apoptosis. These and to mitochondrial p53 accumulation during apoptosis but not during cell cycle arrest, is by the transactivation of p53. in that mitochondrial cells to a death to p53 after only 1 h and to to the of nuclear p53 induction between 3 and the to procaspase-3 as by the of after that of cells after in of the changes in the mitochondrial membrane in are to well the cells undergo apoptotic death Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of and Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), as by a and and a of that at h with cells This fully after h as by mitochondrial show that the of mitochondrial p53 precedes mitochondrial membrane release, and procaspase-3 activation. We that mitochondrial p53 accumulation is an again of an and not a of apoptosis or a of to in cells. p53 to mitochondria of cells, independent with cells that of cells nuclear but not the of mitochondrial p53 to by This mitochondrial p53 We techniques that mitochondrial p53 in apoptosis. analysis of mitochondria cells and after DNA isolated by and for p53 or as Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). that mitochondria cells in the p53 and to the as the to protein of the mitochondrial membrane and not in and an mitochondria cells with for h in and with p53 as by a in but not with the These p53 the membranous The of the p53 by the for after to to mitochondrial and of the membrane Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) These were the p53 isolated mitochondria were after as that the p53 and is the as and the for the of in a p53 by microscopy of mitochondria for and cells were isolated as by in and were with p53 or at the by at and only at and mitochondria cells labeling with the mitochondrial membrane compartment, were the only were with mitochondria in the of DNA damage or with mitochondria of DNA damage alone only not mitochondria were to with to the or membrane or not of p53 isolated mitochondria cells p53 p53 of with For at were in a of with For at were a mechanism for mitochondrial p53 for a between the mitochondrial import protein mt and called is the motor that mitochondria and the or the R. W. Biol. 1996; Full Text PDF PubMed Scopus Google Scholar, N. M. Biol. 1997; Full Text Full Text PDF PubMed Google Scholar). a in complex between p53 and mt p53 with and to mt isolated mitochondria of cells. 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This suggests a regulatory feedback signaling loop between p53 and at the of the strongly that p53 to mitochondria to the apoptotic being a However, strongly not an role show to mitochondria, is competent in apoptosis by and without additional DNA other can at an apoptotic the mitochondrial The to is the nucleus and overexpressed in mitochondria, of apoptosis in p53-deficient cells that in to nuclear a to a transcriptionally p53 protein at at mitochondria at as as nuclear The for the transactivation function of p53 apoptosis mitochondrial p53 as leads to mitochondrial that can the and to cell death. These a transcription-independent function of p53. However, mitochondrial function is an to the the transcription-dependent action of in apoptosis is to in that the of p53 that were after targeting that are induced during stress or are in cells. This is by cells with the nuclear p53 The mitochondrial of were not were to the apoptotic The to tumor cells that are the by G. S. A. J. 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