RESUMO
Imatinib, the anti-Abl tyrosine kinase inhibitor used as first-line therapy in chronic myeloid leukemia (CML), eliminates CML cells mainly by apoptosis and induces autophagy. Analysis of imatinib-treated K562 cells reveals a cell population with cell cycle arrest, p27 increase and senescence-associated beta galactosidase (SA-ß-Gal) staining. Preventing apoptosis by caspase inhibition decreases annexin V-positive cells, caspase-3 cleavage and increases the SA-ß-Gal-positive cell population. In addition, a concomitant increase of the cell cycle inhibitors p21 and p27 is detected emphasizing the senescent phenotype. Inhibition of apoptosis by targeting Bim expression or overexpression of Bcl2 potentiates senescence. The inhibition of autophagy by silencing the expression of the proteins ATG7 or Beclin-1 prevents the increase of SA-ß-Gal staining in response to imatinib plus Z-Vad. In contrast, in apoptotic-deficient cells (Bim expression or overexpression of Bcl2), the inhibition of autophagy did not significantly modify the SA-ß-Gal-positive cell population. Surprisingly, targeting autophagy by inhibiting ATG5 is accompanied by a strong SA-ß-Gal staining, suggesting a specific inhibitory role on senescence. These results demonstrate that in addition to apoptosis and autophagy, imatinib induced senescence in K562 CML cells. Moreover, apoptosis is limiting the senescent response to imatinib, whereas autophagy seems to have an opposite role.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Senescência Celular , Piperazinas/toxicidade , Pirimidinas/toxicidade , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 7 Relacionada à Autofagia , Proteína 11 Semelhante a Bcl-2 , Proteína Beclina-1 , Benzamidas , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.
Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/fisiologia , Autofagia/fisiologia , Morte Celular/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Expression of the pro-apoptotic protein Bax in yeast Saccharomyces cerevisiae induces a release of cytochrome c accompanied by a decrease of the amount of cytochrome c oxidase. Here we show that the decrease of cytochrome c oxidase is due to the activation of mitochondrial protease Yme1p, of which cytochrome c oxidase subunit 2 (Cox2p) is a substrate. The absence of Yme1p slightly delays Bax-induced cell death, suggesting a role of this protease in yeast cell death and thus of its mammalian homologue in apoptosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteases Dependentes de ATP , Adenosina Trifosfatases/genética , Apoptose/fisiologia , Respiração Celular , Genes Fúngicos , Genes myc , Humanos , Técnicas In Vitro , Mitocôndrias/enzimologia , Fenótipo , Proteínas Proto-Oncogênicas/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína X Associada a bcl-2RESUMO
During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.
Assuntos
Apoptose/fisiologia , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Humanos , Membranas Intracelulares/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/metabolismo , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Canais de Ânion Dependentes de Voltagem , Leveduras/fisiologia , Proteína X Associada a bcl-2RESUMO
The interaction of the anti-apoptotic members of the Bcl-2 family with mitochondria, through their hydrophobic C-terminus, has been proposed to play a crucial role in the execution phase of apoptosis. We report here that a substitution of the C-terminal end of pro-apoptotic bax by that of anti-apoptotic bcl-xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae. In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells. These results suggest that the C-terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Membrana Celular/fisiologia , Citosol/fisiologia , Glioma , Humanos , Células K562 , L-Lactato Desidrogenase/análise , Mitocôndrias/fisiologia , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Deleção de Sequência , Transfecção , Células Tumorais Cultivadas , Raios Ultravioleta , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
A new system for bax-expression in yeast has been devised to investigate bax's effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (non-repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-xL.
Assuntos
Grupo dos Citocromos c/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas Proto-Oncogênicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Apoptose , Fermentação , Expressão Gênica , Humanos , Microscopia Eletrônica , Consumo de Oxigênio , Proteínas Proto-Oncogênicas c-bcl-2/genética , Saccharomyces cerevisiae/citologia , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Recent studies that attempt to explore the action of pro- and anti-apoptotic proteins of the bcl2 family demonstrate the crucial role of relocalization of cytochrome c from the mitochondrial intermembrane space to the cytosol. This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bax. In mammalian mitochondria, the mechanism of relocalization is thought to involve the opening of the so-called permeability transition pore. We show in this paper: (a) that bax-induced release of cytochrome c in yeast does not involve any permeability transition of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. This suggests that a permeability transition of the inner mitochondrial membrane is not an event required for the relocalization of cytochrome c in yeast. (b) The outer-membrane voltage-dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax-induced release of cytochrome c or in the prevention of this release by bcl-xL. (c) Bax devoid of its C-terminal putative hydrophobic alpha-helix is as efficient as full-length bax to allow the relocalization of cytochrome c, demonstrating this segment of the protein is not required for membrane-targeting. (d) We finally observe that the action of bax on the outer mitochondrial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bax inserted to mitochondria.
Assuntos
Trifosfato de Adenosina/metabolismo , Grupo dos Citocromos c/metabolismo , Mitocôndrias/metabolismo , Porinas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Canais de Ânion Dependentes de Voltagem , Proteína X Associada a bcl-2RESUMO
It has been suggested that the C-terminal domain of Bcl-2 family members may contain a signal anchor sequence that targets these proteins to the mitochondrial outer membrane. We have investigated the consequence of deleting this domain upon cytochrome c release in yeast strains that coexpress truncated forms of Bax (i.e. BaxA) and Bcl-X(L) (i.e. Bcl-X(L)delta). We find that (i) Bax(delta) is as efficient as full-length Bax in promoting cytochrome c release, but Bcl-x(L)delta has remarkably reduced rescuing ability compared to full-length Bcl-x(L); (ii) full-length Bcl-X(L) protein acts by relocalizing Bax from the mitochondrial fraction to the soluble cytosolic fraction; (iii) Bax undergoes N-terminal cleavage when expressed in yeast, which is prevented by coexpression of Bcl-X(L), suggesting that Bcl-x(L) may mask the cleavage site of Bax through a direct physical interaction of the two proteins.