36 research outputs found

    Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits

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    The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40 S ribosomal subunit. in the present study, we have identified amino acid residues in eIF2beta required for binding to eIF2gamma in yeast. Alteration of six residues in the central region of eIF2beta abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of (131)Tyr and (132)Ser by alanine residues ((YS)-Y-131), although abolishing the binding to eIF2gamma in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2gamma in vivo was shown by co-immunoprecipitation. the (YS)-Y-131 mutation in eIF2beta allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. the combination of this mutation with the (254)Ser --> Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2beta and eIF2gamma defines the correct usage of the AUG codon for translation initiation.Universidade Federal de SĂŁo Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 SĂŁo Paulo, BrazilUniversidade Federal de SĂŁo Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 SĂŁo Paulo, BrazilWeb of Scienc

    Quantifying cell cycle-dependent drug sensitivities in cancer using a high throughput synchronisation and screening approach.

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    BACKGROUND: Chemotherapy and targeted agent anti-cancer efficacy is largely dependent on the proliferative state of tumours, as exemplified by agents that target DNA synthesis/replication or mitosis. As a result, cell cycle specificities of a number of cancer drugs are well known. However, they are yet to be described in a quantifiable manner. METHODS: A scalable cell synchronisation protocol used to screen a library of 235 anti-cancer compounds exposed over six hours in G1 or S/G2 accumulated AsPC-1 cells to generate a cell cycle specificity (CCS) score. FINDINGS: The synchronisation method was associated with reduced method-related cytotoxicity compared to nocodazole, delivering sufficient cell cycle purity and cell numbers to run high-throughput drug library screens. Compounds were identified with G1 and S/G2-associated specificities that, overall, functionally matched with a compound's target/mechanism of action. This annotation was used to describe a synergistic schedule using the CDK4/6 inhibitor, palbociclib, prior to gemcitabine/AZD6738 as well as describe the correlation between the CCS score and published synergistic/antagonistic drug schedules. INTERPRETATION: This is the first highly quantitative description of cell cycle-dependent drug sensitivities that utilised a tractable and tolerated method with potential uses outside the present study. Drug treatments such as those shown to be G1 or S/G2 associated may benefit from scheduling considerations such as after CDK4/6 inhibitors and being first in drug sequences respectively. FUNDING: Cancer Research UK (CRUK) Institute core grants C14303/A17197 and C9545/A29580. The Li Ka Shing Centre where this work was performed was generously funded by CK Hutchison Holdings Limited, the University of Cambridge, CRUK, The Atlantic Philanthropies and others

    Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B.

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    The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-ÎşB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress

    Metabolic Control by S6 Kinases Depends on Dietary Lipids

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    Targeted deletion of S6 kinase (S6K) 1 in mice leads to higher energy expenditure and improved glucose metabolism. However, the molecular mechanisms controlling these effects remain to be fully elucidated. Here, we analyze the potential role of dietary lipids in regulating the mTORC1/S6K system. Analysis of S6K phosphorylation in vivo and in vitro showed that dietary lipids activate S6K, and this effect is not dependent upon amino acids. Comparison of male mice lacking S6K1 and 2 (S6K-dko) with wt controls showed that S6K-dko mice are protected against obesity and glucose intolerance induced by a high-fat diet. S6K-dko mice fed a high-fat diet had increased energy expenditure, improved glucose tolerance, lower fat mass gain, and changes in markers of lipid metabolism. Importantly, however, these metabolic phenotypes were dependent upon dietary lipids, with no such effects observed in S6K-dko mice fed a fat-free diet. These changes appear to be mediated via modulation of cellular metabolism in skeletal muscle, as shown by the expression of genes involved in energy metabolism. Taken together, our results suggest that the metabolic functions of S6K in vivo play a key role as a molecular interface connecting dietary lipids to the endogenous control of energy metabolism

    mTOR Inhibitors Synergize on Regression, Reversal of Gene Expression, and Autophagy in Hepatocellular Carcinoma

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    Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advance

    elF2alfa phosphorylation and the integrated stress response in the pilocarpine induce status epilepticus

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    A fosforilacao da subunidade a do fator de inicio de traducao eIF2, como resposta a inumeros estresses, leva 'a inibicao da sintese proteica geral das celulas, cuja extensao e de extrema relevancia para a sobrevivencia ou morte das celulas de acordo com o padrao de mensagens especificas traduzidas como parte da resposta integrada de estresse. Neste trabalho, foram investigados os efeitos da inducao da epilepsia do lobo temporal por pilocarpina sobre a sintese proteica no encefalo de camundongos. Durante os primeiros 30 minutos de status epilepticus ocorre uma drastica dissociacao dos polissomos associada a extensa fosforilacao de eIF2a. A fosforilacao de eIF2 e extensa e persistente no cortex (CTX) e no hipocampo (HPC), e transiente e moderada em areas nao relacionadas, como o tronco encefalico. eIF2a(P) tem papel central na resposta integrada ao stress (ISR). Varios elementos desta via foram investigados. Foi detectada ativacao das quinases de e1F2, PERK, GCN2 e PKR, em amostras totais de encefalo de animais em SE, porem nenhuma destas quinases de eIF2 e responsavel pela fosforilacao de eIF2 durante SE 30 min no CTX ou HPC. Outros elementos da resposta ISR foram analisados. Os mRNA's codificando BiP e CHOP, assim como a proteina BiP, tem seus niveis aumentados em amostras de cerebro total, porem nao foi detectado aumento no CTX ou HPC. O fator transcricional XBP-1 encontra-se parcialmente ativado em tempos tardios apos o inicio do SE, no CTX e HPC, sugerindo ativacao da UPR em uma populacao restrita de neuronios ou em niveis muito baixos. O estudo das bases moleculares que levam a fosforilacao de eIF2 na epilepsia e a resposta das celulas neuronais a inibicao da sintese proteica como parte da ISR podem ajudar na elucidacao dos eventos que resultam na morte neuronal causada pelas crises epilepticasBV UNIFESP: Teses e dissertaçõe

    Longitudinal tracking of T cell lymphomas in mice using flow cytometry

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    Summary: T cell hematological cancer has a complex interplay with host immune cells, but the ability to experimentally discriminate transferred cancer cells from host cells by flow cytometry is technically challenging. Here, we present a flow cytometry protocol to evaluate cancer cell and host immune phenotypes following transplant of a T cell lymphoma bearing a congenic marker (CD45.2) into a syngeneic host (CD45.1). We describe steps for isolation of primary immune cells from mice, staining preparation with flow cytometry antibody cocktails, and analysis by flow cytometry.For complete details on the use and execution of this protocol, please refer to Kuczynski et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics
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