RESUMO
The inhibitors of apoptosis (IAPs) suppress apoptosis through the inhibition of the caspase cascade and thus are key proteins in the control of cell death. Here we have isolated the protein XIAP-associated factor 1 (XAF1) on the basis of its ability to bind XIAP, a member of the IAP family. XIAP suppresses caspase activation and cell death in vitro, and XAF1 antagonizes these XIAP activities. Expression of XAF1 triggers a redistribution of XIAP from the cytosol to the nucleus. XAF1 is ubiquitously expressed in normal tissues, but is present at low or undetectable levels in many different cancer cell lines. Loss of control over apoptotic signalling is now recognized as a critical event in the development of cancer. Our results indicate that XAF1 may be important in mediating the apoptosis resistance of cancer cells.
Assuntos
Caspases/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Northern Blotting , Western Blotting , Inibidores de Caspase , Sobrevivência Celular , Meios de Cultura Livres de Soro , Etoposídeo/farmacologia , Genes Reporter , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Dedos de ZincoRESUMO
X-linked inhibitor of apoptosis protein (XIAP) is a potent modulator of programmed cell death. XIAP specifically binds and inhibits the function of caspase-3, -7, and -9, key effector proteases of apoptosis. We recently isolated, by yeast two-hybrid screening, a novel 34-kDa zinc finger protein, XIAP-associated factor 1 (XAF1). Both the caspase inhibiting and the anti-apoptotic abilities of XIAP were found to be blocked by overexpressed XAF1. Here, we report the isolation and characterization of the human XAF1 gene. The xaf1 gene consists of seven exons spanning 18 kb. Fluorescence in situ hybridization analysis localized the xaf1 locus at 17p13.2, telomeric to the p53 gene. The xaf1 locus was further refined to YAC 746C10, approximately 3 cM distal to TP53. Microsatellite analysis of the xaf1 locus using the NCI 60 cell line panel revealed significantly decreased heterozygosity at all three polymorphic markers tested, suggesting that allelic loss of the xaf1 gene is prevalent in cancer cell lines. Examination of the same NCI cell line panel for xaf1 RNA expression demonstrated that cancer cell lines exhibited very low levels of mRNA relative to normal human liver. In contrast, XIAP mRNA levels were relatively high in the majority of cancer cell lines tested. We propose that a high level of XIAP to XAF1 expression in cancer cells may provide a survival advantage through the relative increase of XIAP anti-apoptotic function.
Assuntos
Proteínas de Neoplasias/genética , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Apoptose , Proteínas Reguladoras de Apoptose , Biomarcadores Tumorais , Mapeamento Cromossômico , Feminino , Deleção de Genes , Duplicação Gênica , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular , Perda de Heterozigosidade , Masculino , Proteínas de Neoplasias/metabolismo , Placenta , Gravidez , Ligação Proteica , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Transient forebrain ischemia produced by four-vessel occlusion (4-VO) triggers the delayed death of CA1 neurons in the hippocampus, resulting in behavioral deficits of spatial learning performance. We demonstrate that CA1 neuronal loss induced by 4-VO (12 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. Virally mediated overexpression of the anti-apoptotic gene X chromosome-linked inhibitor of apoptosis protein (XIAP) prevented both the production of catalytically active caspase-3 and degeneration of CA1 neurons after transient forebrain ischemia. CA1 neurons protected in this manner appeared to function normally, as assessed by immunohistochemical detection of the neuronal activity marker nerve growth factor inducible-A and by spatial learning performance in the Morris water maze. These findings indicate that caspase-3 activation is a key event in ischemic neuronal death and that blockade of this event by XIAP overexpression permits CA1 neurons to survive and operate properly after an ischemic insult.
Assuntos
Apoptose/genética , Hipocampo/citologia , Ataque Isquêmico Transitório/fisiopatologia , Proteínas do Tecido Nervoso/genética , Cromossomo X , Animais , Comportamento Animal/fisiologia , Química Encefálica/genética , Caspase 3 , Caspases/metabolismo , Fragmentação do DNA , Regulação Enzimológica da Expressão Gênica/fisiologia , Hipocampo/irrigação sanguínea , Hipocampo/química , Masculino , Aprendizagem em Labirinto/fisiologia , Proteína Inibidora de Apoptose Neuronal , Neurônios/química , Neurônios/citologia , Neurônios/enzimologia , Proteínas/genética , Ratos , Ratos Wistar , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
IAPs (inhibitor of apoptosis proteins) are a recently identified family of proteins that function in the cell death pathway to inhibit programmed cell death. We have earlier reported cloning of four human IAPs: NAIP, hiap-1, hiap-2, and xiap. To facilitate studies of these genes, we have undertaken the cloning of their murine homologues. We report here the cloning, mapping, and preliminary characterization (including cellular and tissue distribution profile) of the murine homologue of the human X-linked IAP (xiap). The mouse gene called miap-3 (for murine IAP-3; GenBank Accession No. nuc 1 U88990) has a coding region of 1.5 kb that encodes a protein of 55 kDa and has 87 and 94% homology with its human homologue at DNA and amino acid levels, respectively. Northern blot analysis reveals an 8-kb miap-3 transcript in all tissues examined to date. miap-3 is composed of six exons and five introns spanning approximately 20 kb. miap-3 has been assigned to the A3-A5 region of mouse chromosome X by FISH analysis.
Assuntos
Apoptose , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Mapeamento Cromossômico , Éxons , Genoma , Humanos , Proteínas Inibidoras de Apoptose , Íntrons , Camundongos , Dados de Sequência Molecular , Proteínas/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Bone marrow is the major site of hematopoiesis in the adult mammal. Bone marrow contains a highly organized microenvironment for the support of hematopoietic stem and progenitor cells, including the production of growth factors. Bone marrow cells also produce negative regulatory factors which may regulate hematopoiesis and inflammatory responses. In this paper we describe Reptimed, a unique bone marrow-derived factor with inhibitory activity for myelopoiesis and in vitro growth of myeloid cell lines. Reptimed was partially purified from bone marrow supernatants using a combination of solid-phase extraction and size exclusion chromatography. Reptimed is < 1000 Da MW and is water soluble. Reptimed inhibited growth of granulocyte-macrophage and macrophage colonies as well as proliferation of several myeloid leukemia cell lines. Reptimed may be part of a hemoregulatory circuit.
Assuntos
Fatores Biológicos/fisiologia , Medula Óssea/fisiologia , Hematopoese/fisiologia , Animais , Fatores Biológicos/análise , Medula Óssea/química , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
Genomic and cDNA clones encoding mouse inhibitor of apoptosis protein 1 and 2 (Miap1 and Miap2) were isolated and characterized. The genes encoding the 602-amino-acid MIAP1 protein and the 612-amino-acid MIAP2 protein are contained within a 57-kb locus in a tandem head-to-tail arrangement. The Miap1 gene consists of nine exons spanning 24 kb, and the Miap2 gene consists of seven exons spanning 21 kb. Fluorescence in situ hybridization analysis mapped the locus to chromosome 9A2, which is syntenic with portions of the human 11q22-q23 region containing the human homologues HIAP1 and HIAP2. Sequencing of the complete Miap1 and Miap2 cDNAs revealed an unusually long 5' untranslated region in the Miap2 transcript, which may indicate nonscanning ribosomal initiation of translation.
Assuntos
Apoptose , Mapeamento Cromossômico , Proteínas/genética , Sequência de Aminoácidos , Animais , Proteína 3 com Repetições IAP de Baculovírus , Sequência de Bases , DNA Complementar , Humanos , Hibridização in Situ Fluorescente , Proteínas Inibidoras de Apoptose , Camundongos , Dados de Sequência Molecular , Distribuição Tecidual , Ubiquitina-Proteína LigasesRESUMO
A reverse phase high pressure liquid chromatographic method is presented for the separation and determination of residues of the carbamates oxamyl and methomyl on vegetables. A liquid-liquid extraction and cleanup procedure is applied to the vegetable extract. Samples are eluted from a muBondapak C18 column and quantitated by ultraviolet absorbance at 240 nm. Recovery data for vegetable samples spiked at 2 ppm are presented.