RESUMO
The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Biologia Computacional/métodos , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Nucleofosmina , Prognóstico , Retratamento , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversos , Sulfonamidas/uso terapêutico , Falha de Tratamento , Resultado do TratamentoRESUMO
1. The goal of the current study was to evaluate the genetic effects of the vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2) on feather maturity in the Qingyuan partridge chicken, Guangxi sanhuang chicken and Princess chicken. 2. Both SSCP-PCR and qPCR were employed to detect the polymorphism and gene expression of the VEGF and VEGFR-2 genes. 3. Four SNPs were identified in the VEGFR-2 gene. Exon10-A69G was associated with feather maturity (P < 0.01). Princess chickens with the genotype EF had higher feather maturity scores (P < 0.01). Higher expression levels of VEGF and VEGFR-2 were detected in the immature feather group of Qingyuan partridge chickens, especially in the skin. 4. The VEGF and VEGFR-2 genes play critical roles in feather maturity. In addition, exon10-A69G and genotype EF in the Princess chicken could potentially be utilised as genetic markers to improve efficiency in breeding.
Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Plumas/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Proteínas Aviárias/metabolismo , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , China , Plumas/metabolismo , Genótipo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Polo-like kinase 1 (Plk1) has an important role in mitosis. Volasertib (BI 6727), a potent and selective cell cycle kinase inhibitor, induces mitotic arrest and apoptosis by targeting Plk; this phase I study sought to determine its maximum tolerated dose (MTD) in Asian patients with advanced solid tumours. METHODS: Patients were enrolled simultaneously into two 3-week schedules of volasertib: a 2-h infusion on day 1 (schedule A) or days 1 and 8 (schedule B). Dose escalation followed a 3+3 design. The MTD was determined based on dose-limiting toxicities (DLT) in the first treatment course. RESULTS: Among 59 treated patients, the most common first course DLTs were reversible thrombocytopenia, neutropenia and febrile neutropenia; MTDs were 300 mg for schedule A and 150 mg for schedule B. Volasertib exhibited multi-exponential pharmacokinetics (PK), a long terminal half-life of â¼135 h, a large volume of distribution (>3000 l), and a moderate clearance. Partial responses were observed in two pre-treated patients (ureteral cancer; melanoma). Volasertib was generally well tolerated, with an adverse event profile consistent with its antimitotic mode of action and a favourable PK profile. CONCLUSIONS: These data support further development of volasertib and a harmonised dosing for Asian and Caucasian patients.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/uso terapêutico , Terapia de Salvação , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Terapia Combinada , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Meia-Vida , Doenças Hematológicas/induzido quimicamente , Humanos , Infusões Intravenosas , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias/terapia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Pteridinas/administração & dosagem , Pteridinas/efeitos adversos , Pteridinas/farmacocinética , Taiwan , Resultado do Tratamento , Quinase 1 Polo-LikeRESUMO
Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Morte Celular/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteína bcl-X/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Sobrevivência Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteína bcl-X/genéticaRESUMO
Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.
Assuntos
Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Cristalografia por Raios X , Dimerização , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas Virais/genéticaRESUMO
How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.
Assuntos
Citoplasma/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Caspases/metabolismo , Linhagem Celular , Grupo dos Citocromos c/farmacologia , Citoplasma/ultraestrutura , Ativação Enzimática , Imunofluorescência , Humanos , Microscopia Confocal , Microscopia Imunoeletrônica , Proteínas/imunologia , Proteína bcl-XRESUMO
Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.
Assuntos
Apoptose/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/citologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/genética , Inibidores de Caspase , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epistasia Genética , Expressão Gênica , Genes de Insetos/genética , Proteínas Inibidoras de Apoptose , Proteínas de Insetos/genética , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Apoptosis can be triggered by members of the Bcl-2 protein family, such as Bim, that share only the BH3 domain with this family. Gene targeting in mice revealed important physiological roles for Bim. Lymphoid and myeloid cells accumulated, T cell development was perturbed, and most older mice accumulated plasma cells and succumbed to autoimmune kidney disease. Lymphocytes were refractory to apoptotic stimuli such as cytokine deprivation, calcium ion flux, and microtubule perturbation but not to others. Thus, Bim is required for hematopoietic homeostasis and as a barrier to autoimmunity. Moreover, particular death stimuli appear to activate apoptosis through distinct BH3-only proteins.
Assuntos
Apoptose , Autoimunidade , Proteínas de Transporte/fisiologia , Leucócitos/fisiologia , Proteínas de Membrana , Proteínas Proto-Oncogênicas , Animais , Proteínas Reguladoras de Apoptose , Doenças Autoimunes/etiologia , Linfócitos B/fisiologia , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Cruzamentos Genéticos , Feminino , Marcação de Genes , Glomerulonefrite/etiologia , Células-Tronco Hematopoéticas/fisiologia , Homeostase , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Transdução de Sinais , Subpopulações de Linfócitos T/fisiologiaRESUMO
Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Anoikis , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Proteínas de Membrana , Proteínas Motores Moleculares/metabolismo , Miosina Tipo V , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Proteína 11 Semelhante a Bcl-2 , Proteínas de Transporte/genética , Linhagem Celular , Citoesqueleto/metabolismo , Dineínas , Perfilação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/química , Proteínas Proto-Oncogênicas/química , Proteína de Morte Celular Associada a bcl/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/isolamento & purificação , Proteína 11 Semelhante a Bcl-2 , Dicroísmo Circular , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/isolamento & purificação , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Idarubicina/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/isolamento & purificação , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/isolamento & purificação , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras/química , Alinhamento de SequênciaRESUMO
Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DeltaE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DeltaF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a RNA/fisiologia , Vaccinia virus/fisiologia , Vaccinia virus/patogenicidade , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Deleção de Genes , Genes Virais , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Vaccinia virus/genética , Proteínas Virais/genética , Virulência/genética , Virulência/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Compartimento Celular , Divisão Celular , Membrana Celular/enzimologia , Transformação Celular Neoplásica/patologia , Células Cultivadas , Chlorocebus aethiops , Análise Mutacional de DNA , Ativação Enzimática , Proteínas Ativadoras de GTPase , Inibidores do Crescimento , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurofibromina 1 , Oligodesoxirribonucleotídeos/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Transfecção , Proteínas Ativadoras de ras GTPaseAssuntos
Apoptose , Proteínas de Escherichia coli , Proteínas de Insetos , Proteínas , Animais , Proteínas de Bactérias/metabolismo , Ciclo Celular , Divisão Celular , Coenzima A Ligases/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Citocinas/metabolismo , Transdução de Sinais , Fator de Transcrição RelA , Fatores de Transcrição/metabolismoRESUMO
The BET (bromodomain and extraterminal domain) bromodomain-containing proteins, such as BRD4, are highly promising targets for treating lymphoid and myeloid malignancies. They act to modulate the expression of multiple genes that control diverse cellular processes including proliferation, survival and differentiation that are consequentially disrupted by small-molecule BET bromodomain inhibitors such as JQ1. By assessing the impact of these inhibitors on normal mouse hematopoietic cells or their transformed counterparts, we establish definitively that their cytotoxic action in vitro and in vivo relies predominantly on the activation of BAX/BAK-dependent mitochondrial (intrinsic) apoptosis. In large part, this is triggered by marked upregulation of the BH3-only protein BIM when the BET inhibitors suppress miR-17-92, a key post-transcriptional repressor of BIM expression. Thus, our study strongly suggests that mutations that permit the evasion of apoptosis (for example, BCL2 overexpression, BIM inactivation) are likely to blunt the activity of the BET bromodomain inhibitors and should be anticipated when therapy resistance develops. Strikingly, we also found that certain normal hematopoietic cells, especially those of lymphoid origin, are as prone to apoptosis induced by the BET inhibitors as their transformed counterparts, indicating that their susceptibility to BET inhibitors did not arise from oncogenic transformation.
Assuntos
Apoptose , Azepinas/farmacologia , Proteína 11 Semelhante a Bcl-2/fisiologia , Linfoma/patologia , MicroRNAs/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Transformada , Modelos Animais de Doenças , Sistema Hematopoético/citologia , História Antiga , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/antagonistas & inibidores , RNA Longo não CodificanteRESUMO
Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.
Assuntos
Proteínas de Choque Térmico HSP90/genética , Proteínas Quinases/genética , Animais , Apoptose , Morte Celular , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Necrose , Fosforilação , Proteínas Quinases/metabolismo , Translocação GenéticaRESUMO
Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, is an effective inhibitor of apoptotic cell death induced by many cytotoxic agents. Survival-promoting homologues of Bcl-2 include its close relative, Bcl-xL and the 19 kD protein encoded by the E1B gene of adenoviruses. Whether these proteins are functionally equivalent and whether they can antagonise all or only some pathways to apoptosis is unresolved. We have carried out a systematic comparison of Bcl-2, Bcl-xL and adenovirus E1B19kD activity, using several cell lines and a range of cytotoxic conditions. High levels of expression of each of these proteins inhibited apoptosis induced by growth factor deprivation or treatment with gamma-radiation, glucocorticoid and various cytotoxic drugs. In contrast, none of them could effectively counter apoptosis induced via the TNF receptor or Fas/APO-1 (CD95). Biochemical analysis revealed that all three proteins can associate with Bax and Bak, members of the Bcl-2 protein subfamily that can facilitate apoptosis. The results provide evidence that Bcl-2, Bcl-xL and adenovirus protein E1B19kD are indistinguishable in their ability to regulate the cell death effector machinery.
Assuntos
Proteínas E1B de Adenovirus/biossíntese , Apoptose , Ciclo Celular , Sobrevivência Celular , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Animais , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular , Cicloeximida/farmacologia , Ciclosporina/farmacologia , Dexametasona/farmacologia , Raios gama , Humanos , Interleucina-3/farmacologia , Células Jurkat , Cinética , Camundongos , Proteínas Recombinantes/farmacologia , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Proteína bcl-XRESUMO
The prototypic mammalian regulator of cell death is bcl-2, the oncogene implicated in the development of human follicular lymphoma. Several homologues of bcl-2 are now known. Using a PCR-based strategy we cloned a novel member of this gene family, denoted bcl-w. The gene, which is highly conserved between mouse and human, resides near the T-cell antigen receptor alpha gene within the central portion of mouse chromosome 14 and on human chromosome 14 at band q11. Enforced expression of bcl-w rendered lymphoid and myeloid cells refractory to several (but not all) cytotoxic conditions. Thus, like Bcl-2 and Bcl-x, the Bcl-w protein promotes cell survival, in contrast to other close homologues, Bax and Bak, which facilitate cell death. Comparison of the expected amino acid sequence of Bcl-w with that of these relatives helps to delineate residues likely to convey survival or anti-survival function. While expression of bcl-w was uncommon in B or T lymphoid cell lines, the mRNA was observed in almost all murine myeloid cell lines analysed and in a wide range of tissues. These findings suggest that bcl-w participates in the control of apoptosis in multiple cell types. Its functional similarity to bcl-2 also makes it an attractive candidate proto-oncogene.
Assuntos
Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Sobrevivência Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Clonagem Molecular , DNA Complementar , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Família Multigênica , Proteínas/fisiologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2 , Homologia de Sequência de AminoácidosRESUMO
Cell production within an organ is determined by the rate of immigration, proliferation, differentiation, emigration and death of cells. Abnormalities in any one of these processes will disturb normal control of cell production, thereby eliciting hyperplasia can be an early event in neoplasia. Cell death, apoptosis, is a physiological process responsible for removing unwanted cells. It is used in multi-cellular organisms for tissue remodelling during embryogenesis, regulation of cell turnover and as a defence strategy against invading pathogens. In this review article we describe the role of the bcl-2/ced-9 gene family in cancer and discuss the general implications of defects in the apoptosis program for tumourigenesis and resistance of cancer cells to chemotherapy in light of current knowledge of the molecular mechanisms of cell death.