RESUMO
TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.
Assuntos
Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cromatina/metabolismo , Fragmentação do DNA/fisiologia , DNA de Neoplasias/metabolismo , Embrião de Mamíferos/citologia , Células HeLa/citologia , Humanos , Fator de Crescimento Insulin-Like I/fisiologia , Camundongos , Modelos Biológicos , Mutação , Proteína Oncogênica v-akt/metabolismo , Proteínas Quinases/metabolismo , Ratos , Serina-Treonina Quinases TOR , Transfecção/métodos , Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
The major impediment to cure for many malignancies is the development of therapy resistance with resultant tumor progression. Genetic alterations leading to subversion of inherent apoptosis pathways are common themes in therapy resistance. Bcl-2 family proteins play a critical role in regulating mitochondrial apoptosis that governs chemotherapeutic effects, and defective engagement of these pathways contributes to treatment failure. We have studied the efficacy of BH3 peptidomimetics consisting of the minimal death, or BH3, domains of the proapoptotic BH3-only proteins Bid and Bad to induce apoptosis using neuroblastoma (NB) as a model system. We demonstrate that BH3 peptides, modified with an arginine homopolymer for membrane transduction (called r8-BidBH3 and r8-BadBH3, respectively), potently induce apoptosis in NB cells, including those with MYCN amplification. Cell death is caspase 9 dependent, consistent with a requirement for the intrinsic mitochondrial pathway. Substitutions at highly conserved residues within the r8-BidBH3 peptide abolish apoptotic efficacy supporting activity through specific BH domain interactions. Concomitant exposure to r8-BadBH3 and r8-BidBH3 at sublethal monotherapy doses revealed potent synergy consistent with a competitive displacement model, whereby BH3 peptides displace sequestered BH3 proteins to induce cell death. Further, BH3 peptides demonstrate antitumor efficacy in a xenograft model of NB in the absence of additional genotoxic or trophic stressors. These data provide proof of principle that targeted re-engagement of apoptosis pathways may be of therapeutic utility, and BH3-like compounds are attractive lead agents to re-establish therapy-induced apoptosis in refractory malignancies.
Assuntos
Apoptose , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Peptídeos/química , Fatores de Tempo , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
The expression of the proto-oncogene c-myc is frequently deregulated, via multiple mechanisms, in human breast cancers. Deregulated expression of c-myc contributes to mammary epithelial cell transformation and is causally involved in mammary tumorigenesis in MMTV-c-myc transgenic mice. c-Myc is known to promote cellular proliferation, apoptosis, genomic instability and tumorigenesis in several distinct tissues, both in vivo and in vitro. Expression of the proapoptotic regulatory gene bax is reduced or absent in human breast cancers, and c-Myc has been shown to regulate the expression of Bax, as well as cooperate with Bax in controlling apoptosis in a fibroblast model. Additionally, loss of bax reduces c-Myc-induced apoptosis in lymphoid cells and increases c-Myc-mediated lymphomagenesis in vivo. In order to assess whether loss of bax could influence c-Myc-induced apoptosis and tumorigenesis in the mammary gland in vivo, we generated MMTV-c-myc transgenic mice in which neither, one, or both wild-type alleles of bax were eliminated. Haploid loss of bax in MMTV-c-myc transgenic mice resulted in significantly reduced mammary tumour apoptosis. As anticipated for an apoptosis-regulatory gene, loss of the wild-type bax alleles did not significantly alter cellular proliferation in either mammary adenocarcinomas or dysplastic mammary tissues. However, in contrast to c-Myc-mediated lymphomagenesis, loss of one or both alleles of bax in MMTV-c-myc transgenic mice did not significantly enhance mammary tumorigenesis, despite evidence that haploid loss of bax might modestly increase mammary tumour multiplicity. Our results demonstrate that Bax contributes significantly to c-Myc-induced apoptosis in mammary tumours. In addition, they suggest that in contrast to c-Myc-induced lymphomagenesis, mammary tumorigenesis induced by deregulated c-myc expression requires some amount of Bax expression.
Assuntos
Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Divisão Celular/genética , Transformação Celular Neoplásica , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/fisiopatologia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/genética , Receptores Virais/genética , Animais , Western Blotting , Modelos Animais de Doenças , Regulação para Baixo , Genes myc , Humanos , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/farmacologia , Proteína X Associada a bcl-2RESUMO
Mcl1 is a Bcl2-related antiapoptotic protein originally isolated from human myeloid leukemia cells. Unlike Bcl2, expression has not been reported in CNS neurons. We isolated Mcl1 in a direct screen for candidate modifier genes of neuronal vulnerability by differential display of mRNAs upregulated following prolonged seizures in two mouse strains with contrasting levels of hippocampal cell death. Mcl1 is widely expressed in neurons, and transcription is rapidly induced in both strains. In resistant C57Bl/6J mice, Mcl1 protein levels remain persistently elevated in hippocampal pyramidal neurons after seizures, but fall rapidly in C3H/HeJ hippocampus, coinciding with extensive neuronal apoptosis. DNA damage and caspase-mediated cell death were strikingly increased in Mcl1-deficient mice when compared to +/+ littermates after similar seizures. We identify Mcl1 as a neuronal gene responsive to excitotoxic insult in the brain, and link relative levels of Mcl1 expression to inherited differences in neuronal thresholds for apoptosis.
Assuntos
Apoptose , Sistema Nervoso Central/patologia , Proteínas de Neoplasias/biossíntese , Neurônios/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Western Blotting , Morte Celular , Dano ao DNA , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Regulação para CimaRESUMO
Granzyme B (GzmB) is a serine protease that is used by activated cytotoxic T lymphocytes to induce target cell apoptosis. Although GzmB directly cleaves the Bcl2 family member BID on target cell entry, Bid-deficient (and Bax, Bak doubly deficient) cells are susceptible to GzmB-induced death, even though they fail to release cytochrome c from mitochondria. GzmB still induces mitochondrial depolarization in Bax, Bak double knockout cells without cytochrome c release or opening of the permeability transition pore. Because GzmB cannot directly cause depolarization of isolated mitochondria, novel intracellular factor(s) may be required for GzmB to depolarize mitochondria in situ. GzmB therefore utilizes two distinct mitochondrial pathways to amplify the proapoptotic signal that it delivers to target cells.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/fisiologia , Serina Endopeptidases/farmacologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Permeabilidade da Membrana Celular , Grupo dos Citocromos c/metabolismo , Imunofluorescência , Granzimas , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/fisiologia , Frações Subcelulares/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2 , 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
Critical issues in apoptosis include the importance of caspases versus organelle dysfunction, dominance of anti- versus proapoptotic BCL-2 members, and whether commitment occurs upstream or downstream of mitochondria. Here, we show cells deficient for the downstream effectors Apaf-1, Caspase-9, or Caspase-3 display only transient protection from "BH3 domain-only" molecules and die a caspase-independent death by mitochondrial dysfunction. Cells with an upstream defect, lacking "multidomain" BAX, BAK demonstrate long-term resistance to all BH3 domain-only members, including BAD, BIM, and NOXA. Comparison of wild-type versus mutant BCL-2, BCL-X(L) indicates these antiapoptotics sequester BH3 domain-only molecules in stable mitochondrial complexes, preventing the activation of BAX, BAK. Thus, in mammals, BH3 domain-only molecules activate multidomain proapoptotic members to trigger a mitochondrial pathway, which both releases cytochrome c to activate caspases and initiates caspase-independent mitochondrial dysfunction.
Assuntos
Apoptose/fisiologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Fator Apoptótico 1 Ativador de Proteases , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteína 11 Semelhante a Bcl-2 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular , Grupo dos Citocromos c/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Immunoblotting , Proteínas de Membrana/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl , Proteína bcl-XRESUMO
Chloroquine is a lysosomotropic agent that causes marked changes in intracellular protein processing and trafficking and extensive autophagic vacuole formation. Chloroquine may be cytotoxic and has been used as a model of lysosomal-dependent cell death. Recent studies indicate that autophagic cell death may involve Bcl-2 family members and share some features with caspase-dependent apoptotic death. To determine the molecular pathway of chloroquine-induced neuronal cell death, we examined the effects of chloroquine on primary telencephalic neuronal cultures derived from mice with targeted gene disruptions in p53, and various caspase and bcl-2 family members. In wild-type neurons, chloroquine produced concentration- and time-dependent accumulation of autophagosomes, caspase-3 activation, and cell death. Cell death was inhibited by 3-methyladenine, an inhibitor of autophagic vacuole formation, but not by Boc-Asp-FMK (BAF), a broad caspase inhibitor. Targeted gene disruptions of p53 and bax inhibited and bcl-x potentiated chloroquine-induced neuron death. Caspase-9- and caspase-3-deficient neurons were not protected from chloroquine cytotoxicity. These studies indicate that chloroquine activates a regulated cell death pathway that partially overlaps with the apoptotic cascade.
Assuntos
Amebicidas/farmacologia , Apoptose/genética , Caspases/metabolismo , Cloroquina/farmacologia , Genes bcl-2/fisiologia , Genes p53/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Camundongos , Camundongos Mutantes , Neurônios/metabolismo , Neurônios/ultraestrutura , Gravidez , Telencéfalo/efeitos dos fármacos , Telencéfalo/metabolismo , Telencéfalo/ultraestruturaRESUMO
The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in E mu-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in E mu-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type E mu-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions in ARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in E mu-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective of Bax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas from Bax-null E mu-myc transgenics lacked p53 alterations, whereas 27% of the tumors in Bax(+/-) E mu-myc transgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection of p53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.
Assuntos
Apoptose , Linfoma de Células B/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Linfócitos B/citologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2RESUMO
It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.
Assuntos
Apoptose/genética , Caspases/deficiência , Citocinas/deficiência , Meiose/genética , Oócitos/citologia , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Caspase 1/metabolismo , Caspase 10 , Caspase 2 , Caspases/genética , Caspases/metabolismo , Caspases Iniciadoras , Proteínas de Ciclo Celular , Citocinas/genética , Citocinas/farmacologia , Proteínas de Ligação a DNA , Feminino , Deleção de Genes , Interleucina-1/metabolismo , Interleucina-1/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Oócitos/enzimologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor , Proteína X Associada a bcl-2RESUMO
Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter-reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.
Assuntos
9,10-Dimetil-1,2-benzantraceno/análogos & derivados , Poluição Ambiental/efeitos adversos , Insuficiência Ovariana Primária/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Adulto , Animais , Apoptose , Feminino , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Microinjeções , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/transplante , Insuficiência Ovariana Primária/induzido quimicamente , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/deficiência , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Proteína X Associada a bcl-2RESUMO
Bid is a BH3 domain only pro-apoptotic member of the Bcl-2 family which interacts with Bax to regulate apoptosis. Bax-deficient embryos show decreased neuronal programmed cell death in vivo and resistance to cytosine arabinoside (AraC)-induced neuronal apoptosis in vitro. In this report, we demonstrate that Bid-deficient embryos show no neurodevelopmental abnormalities, and Bid-deficiency has no effect on the in vitro apoptotic response of either telencephalic neural precursor cells or neurons to AraC-induced death. We conclude that bid does not play an essential role in either naturally occurring or genotoxin-induced neuronal cell death.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Neurônios/citologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Encéfalo/citologia , Encéfalo/embriologia , Caspase 3 , Caspases/metabolismo , Citarabina/farmacologia , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Mutagênicos/farmacologia , Gravidez , Células-Tronco/citologiaRESUMO
Cell death is critical for the development and orderly maintenance of cellular homeostasis in metazoans. Developmental genetics in model systems, including Caenorhabditis elegans and Drosophila melanogaster, have helped to identify and order the components of cell-death pathways. An even more complex network of apoptotic pathways has evolved in higher organisms that possess homologs within each set of cell-death regulators. Whereas biochemical studies provide details of molecular mechanisms, genetic models reveal the essential physiologic roles. Transgenic and gene-ablated mice have helped to elucidate mammalian apoptotic pathways and identify the principal effect of each cell death regulator. Here, we review the details of the apoptotic machinery as revealed by mice deficient in critical components of cell-death pathways; we concentrate on cell-death regulators classified as members of the caspase and Bcl2 families or, broadly, as adaptors and mitochondrial released factors.
Assuntos
Caspases/metabolismo , Morte Celular/fisiologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases , Caspases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais , Proteína bcl-X , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.
Assuntos
Apoptose/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/metabolismo , Animais , Anticorpos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Biopolímeros , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Retículo Endoplasmático/metabolismo , Etoposídeo/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Camundongos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Estaurosporina/farmacologia , Transfecção , Raios Ultravioleta , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Receptor fas/imunologia , Receptor fas/fisiologiaRESUMO
A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB-CREB-binding protein (CBP) complex. When tested for binding to CREB or CBP individually, this MLL fragment interacted directly with CBP, but not with CREB. In vitro binding experiments refined the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain). The transactivation activity of MLL was dependent on CBP, as either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction proved effective at inhibiting MLL-mediated transactivation. Single amino acid substitutions within the MLL activation domain revealed that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, were critical for the interaction of MLL with CBP. Using purified components, we found that the MLL activation domain facilitated the binding of CBP to phosphorylated CREB. In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proto-Oncogenes , Transativadores/metabolismo , Fatores de Transcrição , Sítios de Ligação/genética , Proteína de Ligação a CREB , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plasmídeos , Ligação Proteica , Saccharomyces cerevisiae , Transativadores/química , Transativadores/genética , Ativação TranscricionalRESUMO
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Dopamina/metabolismo , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/genética , Animais , Sequência de Bases , Primers do DNA , Dimerização , Modelos Animais de Doenças , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2RESUMO
Multiple apoptotic pathways release cytochrome c from the mitochondrial intermembrane space, resulting in the activation of downstream caspases. In vivo activation of Fas (CD95) resulted in increased permeability of the mitochondrial outer membrane and depletion of cytochrome c stores. Serial measurements of oxygen consumption, NADH redox state and membrane potential revealed a loss of respiratory state transitions. This tBID-induced respiratory failure did not require any caspase activity. At early time points, re-addition of exogenous cytochrome c markedly restored respiratory functions. Over time, however, mitochondria showed increasing irreversible respiratory dysfunction as well as diminished calcium buffering. Electron microscopy and tomographic reconstruction revealed asymmetric mitochondria with blebs of herniated matrix, distended inner membrane and partial loss of cristae structure. Thus, apoptogenic redistribution of cytochrome c is responsible for a distinct program of mitochondrial respiratory dysfunction, in addition to the activation of downstream caspases.
Assuntos
Apoptose , Grupo dos Citocromos c/farmacologia , Mitocôndrias/efeitos dos fármacos , Animais , Cálcio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Bax is a Bcl-2 family member that promotes apoptosis and counters the protective effect of Bcl-2. Bax is a downstream effector of p53-induced apoptosis and is transcriptionally regulated by p53. Moreover, the introduction of Bax deficiency accelerates the onset of tumors in transgenic mice expressing truncated large T antigen. These results implicate Bax as a tumor suppressor. Consequently, we asked whether the levels of Bax expression would influence tumor development by comparing Bax-deficient and Bax transgenic mice in the presence or absence of p53. We found that Bax-deficient mice did not display an increased incidence of spontaneous cancers when followed for > 1.5 years. In addition, Bax-deficiency did not further accelerate oncogenesis in mice also deficient in p53. We generated Lck(pr)-Bax transgenic mice to examine the effects of overexpressed BAX on T-cell development and tumorigenesis. Lck(pr)-Bax mice show increased apoptosis consistent with the pro-apoptotic function of Bax. The introduction of p53-deficiency did not interfere with BAX-induced apoptosis; this is consistent with BAX operating downstream or independent of p53. However, we found that Lck(pr)-Bax/p53-deficient mice have an increased incidence of T-cell lymphomas when compared with p53-deficient mice. The Lck(pr)-Bax transgenic mice have an increased percentage of cells in cycle. These findings extend previous work suggesting that Bcl-2 family proteins regulate proliferation as well as cell death. We conclude that BAX-induced proliferation is synergistic with a defect in apoptosis contributed by p53-deficiency. Thus, the dual roles of BAX can either accelerate or inhibit tumorigenesis depending on the genetic context.
Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/deficiência , Animais , Apoptose/genética , Bromodesoxiuridina/metabolismo , Divisão Celular/genética , Sobrevivência Celular/genética , Intervalo Livre de Doença , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Gravidez , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Análise de Sobrevida , Timo/citologia , Timo/metabolismo , Timo/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2RESUMO
Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.
Assuntos
Regulação da Expressão Gênica , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T/citologia , Animais , Apoptose , Relação CD4-CD8 , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Sobrevivência Celular , Citometria de Fluxo , Imunofluorescência , Deleção de Genes , Marcação In Situ das Extremidades Cortadas , Janus Quinase 3 , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimento , Proteína X Associada a bcl-2RESUMO
BACKGROUND: Hypoxic-ischemic (H-I) injury to the neonatal brain has been shown to result in rapid cell death with features of acute excitotoxicity/necrosis as well as prominent delayed cell death with features of apoptosis such as marked caspase-3 activation. BAX, a pro-apoptotic molecule, has been shown to be required for apoptotic neuronal cell death during normal development but the contribution of endogenous BAX in cell death pathways following H-I injury to the developing or adult brain has not been studied. MATERIALS AND METHODS: Bax +/+, +/-, and -/- mice at post-natal day 7 were subjected to unilateral carotid ligation followed by exposure to 45 minutes of 8% oxygen. At different timepoints following H-I, brain tissue was studied by conventional histology, immunohistochemistry, immunofluorescence, Western blotting, and enzymatic assay to determine the extent and type of cell injury as well as the amount of caspase activation. RESULTS: We found that bax -/- mice had significantly less (38%) hippocampal tissue loss than mice expressing bax. Some of the remaining cell death in bax -/- mice, however, still had features of apoptosis including evidence of nuclear shrinkage and caspase-3 activation. Though bax -/- mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I, the density of cells with activated caspase-8 in the CA3 region of the hippocampus did not differ between bax +/- and bax -/- mice. CONCLUSIONS: These findings demonstrate that endogenous BAX plays a role in regulating cell death in the central nervous system (CNS) following neonatal H-I, a model of cerebral palsy. In addition, while BAX appears to modulate the caspase-3 activation following neonatal H-I, caspase-8 which is linked to death receptor activation, may contribute to apoptotic-like neuronal death in a BAX-independent manner.