RESUMO
VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca(2+) across the OMM and because Ca(2+) has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca(2+) levels ([Ca(2)(+)]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca(2+) and induce VDAC1 over-expression. Direct elevation of [Ca(2+)]i by the Ca(2+)-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca(2+)]i using the cell-permeable Ca(2+)-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca(2+)]i levels and apoptosis induction, as induced by H2O2 or As2O3, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca(2+)-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca(2+)]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca(2+) promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Quelantes/farmacologia , Citocromos c/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Ionomicina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Membranas Mitocondriais/efeitos dos fármacos , Multimerização Proteica , Tapsigargina/farmacologia , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
The voltage-dependent anion channel (VDAC), located at the outer mitochondria membrane (OMM), mediates interactions between mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca(2+), and metabolites. Substantial evidence points to VDAC1 as being a key player in apoptosis, regulating the release of apoptogenic proteins from mitochondria, such as cytochrome c, and interacting with anti-apoptotic proteins. Recently, we demonstrated that VDAC1 oligomerization is a general mechanism common to numerous apoptogens acting via different initiating cascades and proposed that a protein-conducting channel formed within a VDAC1 homo/hetero oligomer mediates cytochrome c release. However, the molecular mechanism responsible for VDAC1 oligomerization remains unclear. Several studies have shown that mitochondrial Ca(2+) is involved in apoptosis induction and that VDAC1 possesses Ca(2+)-binding sites and mediates Ca(2+) transport across the OMM. Here, the relationship between the cellular Ca(2+) level, [Ca(2+)]i, VDAC1 oligomerization and apoptosis was studied. Decreasing [Ca(2+)]i using the cell-permeable Ca(2+) chelating reagent BAPTA-AM was found to inhibit VDAC1 oligomerization and apoptosis, while increasing [Ca(2+)]i using Ca(2+) ionophore resulted in VDAC1 oligomerization and apoptosis induction in the absence of apoptotic stimuli. Moreover, induction of apoptosis elevated [Ca(2+)]i, concomitantly with VDAC1 oligomerization. AzRu-mediated inhibition of mitochondrial Ca(2+) transport decreased VDAC1 oligomerization, suggesting that mitochondrial Ca(2+) is required for VDAC1 oligomerization. In addition, increased [Ca(2+)]i levels up-regulate VDAC1 expression. These results suggest that Ca(2+) promotes VDAC1 oligomerization via activation of a yet unknown signaling pathway or by increasing VDAC1 expression, leading to apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Assuntos
Apoptose , Cálcio/metabolismo , Mitocôndrias/patologia , Multimerização Proteica/efeitos dos fármacos , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Immunoblotting , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tapsigargina/farmacologiaRESUMO
Acute Myeloid Leukemia (AML) is a severe disease with a very high relapse rate. AML relapse may be attributable to leukemic stem cells (LSC). Notably, the "cancer stem cell" theory, which relates to LSCs, is controversial and criticized due to the technical peculiarities of the xenotransplant of human cells into mice. In this study, we searched for possible LSCs in an immunocompetent synergetic mice model. First, we found phenotypic heterogeneity in the ML23 leukemia line. We prospectively isolated a sub-population using the surface markers cKit+CD9-CD48+Mac1-/low, which have the potency to relapse the disease. Importantly, this sub-population can pass in syngeneic hosts and retrieve the heterogeneity of the parental ML23 leukemia line. The LSC sub-population resides in various organs. We present a unique gene expression signature of the LSC in the ML23 model compared to the other sub-populations. Interestingly, the ML23 LSC sub-population expresses therapeutic targeted genes such as CD47 and CD93. Taken together, we present the identification and molecular characterization of LSCs in a syngeneic murine model.
RESUMO
Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2 or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.
Assuntos
Apoptose/fisiologia , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Fator de Indução de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Citocromos c/metabolismo , Primers do DNA/genética , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Animal models are necessary to study cancer and develop treatments. After decades of intensive research, effective treatments are available for only a few types of leukemia, while others are currently incurable. Our goal was to generate novel leukemia models in immunocompetent mice. We had achieved abilities for overexpression of multiple driving oncogenes simultaneously in normal primary cells, which can be transplanted and followed in vivo. Our experiments demonstrated the induction of primary malignant growth. Leukemia lines that model various types of leukemia, such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), were passaged robustly in congenic wild-type immunocompetent mice. These novel leukemia lines, which may complement previous models, offer the flexibility to generate tailored models of defined oncogenes of interest. The characterization of our leukemia models in immunocompetent animals can uncover the mechanisms of malignancy progression and offer a unique opportunity to stringently test anti-cancer chemotherapies.
Assuntos
Transformação Celular Viral , Células-Tronco Hematopoéticas/virologia , Lentivirus/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Mieloide Aguda/genética , Oncogenes , Animais , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Imunocompetência , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/virologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/virologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Transplante Isogênico , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
The voltage-dependent anion channel (VDAC1), lying in the mitochondrial outer membrane (OMM), mediates the transport of ions and metabolites, thus controlling the cross talk between mitochondria and the rest of the cell. VDAC1 has also been recognized as a key protein in mitochondria-mediated apoptosis, is the proposed target for the pro- and antiapoptotic Bcl-2-family of proteins and is involved in apoptotic protein release from the mitochondria. Questions, however, remain as to if and how VDAC1 mediates the transfer of apoptotic proteins across the OMM. Our recent studies suggest that upon apoptosis induction, VDAC1 oligomerizes to form a new large pore allowing the passage of a folded protein, like cytochrome c. This review provides insight into the central role of VDAC1 in mammalian cell life and death and emphasizes VDAC1 function in apoptosis, focusing on VDAC1 oligomerization as a key step in mitochondria-mediated apoptosis and key structural features of VDAC1 that mediate its apoptotic function.
Assuntos
Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Multimerização Proteica , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Apoptose , Humanos , Modelos BiológicosRESUMO
Accumulating evidence implicates that the voltage-dependent anion channel (VDAC) functions in mitochondrion-mediated apoptosis and as a critical player in the release of apoptogenic proteins, such as cytochrome c, triggering caspase activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c-conducting channel remain unknown. Here the relationship between VDAC oligomerization and the induction of apoptosis was examined. We demonstrated that apoptosis induction by various stimuli was accompanied by highly increased VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using bioluminescence resonance energy transfer technology. VDAC oligomerization was induced in all cell types and with all apoptosis inducers used, including staurosporine, curcumin, As(2)O(3), etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-α), H(2)O(2), and UV irradiation, all acting through different mechanisms yet all involving mitochondria. Moreover, correlation between the levels of VDAC oligomerization and apoptosis was observed. Furthermore, the apoptosis inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited VDAC oligomerization. Finally, a caspase inhibitor had no effect on VDAC oligomerization and cytochrome c release. We propose that VDAC oligomerization is involved in mitochondrion-mediated apoptosis and may represent a general mechanism common to numerous apoptogens acting via different initiating cascades. Thus, targeting the oligomeric status of VDAC, and hence apoptosis, offers a therapeutic strategy for combating cancers and neurodegenerative diseases.
Assuntos
Apoptose/fisiologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/metabolismo , Animais , Inibidores de Caspase , Caspases/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Estrutura Quaternária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Research over the past decade has extended the prevailing view of the mitochondrion to include functions well beyond the generation of cellular energy. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organellar communication, aging, cell proliferation, diseases and cell death. Thus, mitochondria play a central role in the regulation of apoptosis (programmed cell death) and serve as the venue for cellular decisions leading to cell life or death. One of the mitochondrial proteins controlling cell life and death is the voltage-dependent anion channel (VDAC), also known as mitochondrial porin. VDAC, located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, thereby controlling cross-talk between mitochondria and the rest of the cell. VDAC is also a key player in mitochondria-mediated apoptosis. Thus, in addition to regulating the metabolic and energetic functions of mitochondria, VDAC appears to be a convergence point for a variety of cell survival and cell death signals mediated by its association with various ligands and proteins. In this article, we review what is known about the VDAC channel in terms of its structure, relevance to ATP rationing, Ca(2+) homeostasis, protection against oxidative stress, regulation of apoptosis, involvement in several diseases and its role in the action of different drugs. In light of our recent findings and the recently solved NMR- and crystallography-based 3D structures of VDAC1, the focus of this review will be on the central role of VDAC in cell life and death, addressing VDAC function in the regulation of mitochondria-mediated apoptosis with an emphasis on structure-function relations. Understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of functions, all important for cell life and death. This review also provides insight into the potential of VDAC1 as a rational target for new therapeutics.
Assuntos
Mitocôndrias/fisiologia , Canais de Ânion Dependentes de Voltagem/fisiologia , Animais , Apoptose/fisiologia , Humanos , Camundongos , Transdução de SinaisRESUMO
The release of mitochondrial-intermembrane-space pro-apoptotic proteins, such as cytochrome c, is a key step in initiating apoptosis. Our study addresses two major questions in apoptosis: how are mitochondrial pro-apoptotic proteins released and how is this process regulated? Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) plays a central role in mitochondria-mediated apoptosis. Here, we demonstrate that the N-terminal domain of VDAC1 controls the release of cytochrome c, apoptosis and the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to apoptosis, induced by various stimuli. Employing a variety of experimental approaches, we show that hexokinase and Bcl2 confer protection against apoptosis through interaction with the VDAC1 N-terminal region. We also demonstrate that apoptosis induction is associated with VDAC oligomerization. These results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the mechanism of cytochrome c release and how anti-apoptotic proteins regulate apoptosis and promote tumor cell survival.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Linhagem Celular , Citocromos c/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Proper cell activity requires an efficient exchange of molecules between mitochondria and cytoplasm. Lying in the outer mitochondrial membrane, VDAC assumes a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. As such, it has been recognized that VDAC plays a crucial role in regulating the metabolic and energetic functions of mitochondria. Indeed, down-regulation of VDAC1 expression by shRNA leads to a decrease in energy production and cell growth. VDAC has also been recognized as a key protein in mitochondria-mediated apoptosis through its involvement in the release of apoptotic proteins located in the inter-membranal space and as the proposed target of pro- and anti-apoptotic members of the Bcl2-family and of hexokinase. Questions, however, remain as to if and how VDAC mediates the transfer of apoptotic proteins from the inter-membranal space to the cytosol. The diameter of the VDAC pore is only about 2.5-3 nm, insufficient for the passage of a folded protein like cytochrome c. New work, however, suggests that pore formation involves the assembly of homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins, such as Bax. Thus, VDAC appears to represent a convergence point for a variety of cell survival and cell death signals. This review provides insight into the central role of VDAC in mammalian cell life and death, emphasizing VDAC function in the regulation of mitochondria-mediated apoptosis and, as such, its potential as a rational target for new therapeutics.