RESUMO
PURPOSE: Diagnosing heparin-induced thrombocytopenia, a potentially catastrophic immune-mediated disorder, continues to pose significant challenges for clinicians, as both clinical and laboratory tools lack specificity. There is mounting evidence supporting a positive correlation between definitive heparin-induced thrombocytopenia and optical density (OD) positivity from the widely available anti-platelet factor 4 enzyme-linked immunosorbent assays (PF4 ELISAs). However, the clinical features distinguishing these patients remain poorly understood. PATIENTS AND METHODS: To better characterize this group, we conducted a case-controlled, retrospective chart review of patients from two large, urban academic institutions who underwent a PF4 ELISA at a central laboratory. Associations between OD and 18 clinical characteristics were calculated using the Fisher's exact test for categorical variables and Wilcoxon rank-sum test for continuous variables. RESULTS: In total, 184 negative patients (OD <0.7), and 121 positive patients (OD >0.7), including 74 low-positive patients (0.7< OD <1.4) and 47 high-positive patients (OD >1.4) were identified. Several clinical variables were significantly different in the negative group compared with the positive group, including hospital day (P<0.001), previous admission within the past 3 months (P<0.001), and the presence of a new thrombus (P=0.003). However, many of these variables were not different between the negative and low-positive group, and were only distinct between the negative and high-positive group. When the low-positive and high-positive groups were compared, only the 4T score was significantly different (P=0.003). CONCLUSION: These data indicate that those with OD >1.4 form a distinct clinical group and support the clinical utility of the 4T score.
RESUMO
The hostile tumor microenvironment results in the generation of intracellular stresses including hypoxia and nutrient deprivation. In order to adapt to such conditions, the cell utilizes several stress-response mechanisms, including the attenuation of protein synthesis, the inhibition of cellular proliferation, and induction of autophagy. Autophagy leads to the degradation of cellular contents, including damaged organelles and mutant proteins, which the cell can then use as an alternate energy source. Two integral changes to the signaling milieu to promote such a response include inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of eIF2α. This review will describe how conditions found in the tumor microenvironment regulate mTORC1 as well as eIF2α, the downstream impact of these modifications, and the implications in tumorigenesis. We will then discuss the remarkable similarities and overlapping function of these 2 signaling pathways, focusing on the response to amino acid deprivation, and present a new model involving crosstalk between them based on our recent work.
Assuntos
Fator de Iniciação 2 em Eucariotos/fisiologia , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adaptação Fisiológica , Autofagia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosforilação , Processamento de Proteína Pós-Traducional , Receptor Cross-Talk , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
Amino acid deprivation promotes the inhibition of the kinase complex mTORC1 (mammalian target of rapamycin complex 1) and activation of the kinase GCN2 (general control nonrepressed 2). Signaling pathways downstream of both kinases have been thought to independently induce autophagy. We showed that these two amino acid-sensing systems are linked. We showed that pharmacological inhibition of mTORC1 led to activation of GCN2 and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) in a mechanism dependent on the catalytic subunit of protein phosphatase 6 (PP6C). Autophagy induced by pharmacological inhibition of mTORC1 required PP6C, GCN2, and eIF2α phosphorylation. Although some of the PP6C mutants found in melanoma did not form a strong complex with PP6 regulatory subunits and were rapidly degraded, these mutants paradoxically stabilized PP6C encoded by the wild-type allele and increased eIF2α phosphorylation. Furthermore, these PP6C mutations were associated with increased autophagy in vitro and in human melanoma samples. Thus, these data showed that GCN2 activation and phosphorylation of eIF2α in response to mTORC1 inhibition are necessary for autophagy. Additionally, we described a role for PP6C in this process and provided a mechanism for PP6C mutations associated with melanoma.
Assuntos
Autofagia/fisiologia , Ativação Enzimática/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Melanoma/genética , Melanoma/fisiopatologia , Complexos Multiproteicos/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Aminoácidos/deficiência , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ativação Enzimática/efeitos dos fármacos , Técnicas de Introdução de Genes , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Espectrometria de Massas , Alvo Mecanístico do Complexo 1 de Rapamicina , Microscopia de Fluorescência , Mutação/genética , Fosfoproteínas Fosfatases/genética , Fosforilação/efeitos dos fármacos , Sirolimo/farmacologia , Tunicamicina/farmacologiaRESUMO
Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTC) and the rapid degradation of their mRNAs by nonsense-mediated RNA decay (NMD). We used virtual library screening, targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD-targeted mRNAs at nanomolar concentrations, with minimal toxicity in cell-based assays. As expected, pharmacologic NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC-mutated p53, pharmacologic NMD inhibition combined with a PTC "read-through" drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacologic NMD inhibitors can restore mRNA integrity in the presence of PTC and can be used as part of a strategy to restore full-length protein in a variety of genetic diseases.
Assuntos
Códon sem Sentido , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/genética , Proteínas de Transporte/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Helicases , RNA Mensageiro/genética , Transativadores/genética , Regulação para Cima/efeitos dos fármacosRESUMO
UNLABELLED: Recent whole genome melanoma sequencing studies have identified recurrent mutations in the gene encoding the catalytic subunit of serine/threonine phosphatase 6 (PPP6C/PP6C). However, the biochemical, functional, and clinical ramifications of these mutations are unknown. Sequencing PP6C from patients with melanoma (233 primary and 77 metastatic specimens) with extended prospective clinical outcome revealed a large number of hotspot mutations in patients with both primary and metastatic melanoma. Despite minimal association between stage and presence of PP6C mutations in patients with primary melanoma, a subpopulation of cells within each tumor did contain PP6C mutations, suggesting PP6C mutation is an early, but non-tumor-initiating event in melanoma. Among patients with primary melanoma with PP6C mutations, patients with stop mutations had significantly shorter recurrence-free survival compared with patients without stop mutations. In addition, PP6C mutations were independent of commonly observed BRAF and NRAS mutations. Biochemically, PP6C mutations could be classified as those that interact with PP6C regulatory subunits and those that do not. Mutations that did not bind to PP6C regulatory subunits were associated with increased phosphorylation of Aurora kinase, a PP6C substrate, and mitotic defects. However, both classes of PP6C mutations led to increased sensitivity to Aurora kinase inhibition. Together, these data support for the first time that PP6C mutations are molecularly, biochemically, and clinically heterogeneous. IMPLICATIONS: PP6C mutations have distinct functional and clinical consequences in melanoma, and confer sensitivity to Aurora A kinase inhibitors.
Assuntos
Aurora Quinases/antagonistas & inibidores , Melanoma/enzimologia , Melanoma/genética , Mutação , Fosfoproteínas Fosfatases/genética , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Aurora Quinases/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/patologia , Estadiamento de Neoplasias , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Prognóstico , Inibidores de Proteínas Quinases , Subunidades Proteicas , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologiaRESUMO
Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that destabilizes aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes. This raises the possibility that NMD itself is under regulatory control. Indeed, several recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including microRNA circuits and NMD itself. Cellular stress also modulates the magnitude of NMD by mechanisms that are beginning to be understood. Here, we review the evidence that NMD is regulated and discuss the physiological role for this regulation. We propose that the efficiency of NMD is altered in some cellular contexts to regulate normal biological events. In disease states-such as in cancer-NMD is disturbed by intrinsic and extrinsic factors, resulting in altered levels of crucial NMD-targeted mRNAs that lead to downstream pathological consequences. This article is part of a Special Issue entitled: RNA Decay mechanisms.
Assuntos
MicroRNAs/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Estresse Fisiológico/genética , Códon sem Sentido , Humanos , Neoplasias/genética , Neoplasias/patologia , Splicing de RNA/genéticaRESUMO
Nonsense-mediated RNA decay (NMD) is an mRNA surveillance mechanism which rapidly degrades select cytoplasmic mRNAs. We and others have shown that NMD is a dynamically regulated process inhibited by amino acid deprivation, hypoxia, and other cellular stresses commonly generated by the tumor microenvironment. This inhibition of NMD can result in the accumulation of misfolded, mutated, and aggregated proteins, but how cells adapt to these aberrant proteins is unknown. Here we demonstrate that the inhibition of NMD activates autophagy, an established protein surveillance mechanism, both in vitro and in vivo. Conversely, the hyperactivation of NMD blunts the induction of autophagy in response to a variety of cellular stresses. The regulation of autophagy by NMD is due, in part, to stabilization of the documented NMD target ATF4. NMD inhibition increases intracellular amino acids, a hallmark of autophagy, and the concomitant inhibition of autophagy and NMD, either molecularly or pharmacologically, leads to synergistic cell death. Together these studies indicate that autophagy is an adaptive response to NMD inhibition and uncover a novel relationship between an mRNA surveillance system and a protein surveillance system, with important implications for the treatment of cancer.
Assuntos
Autofagia/genética , Degradação do RNAm Mediada por Códon sem Sentido , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Morte Celular/genética , Cloroquina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa/efeitos dos fármacos , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/imunologia , RNA Helicases , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Preclinical and clinical studies demonstrate the feasibility of treating ß-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human ß-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human ß-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human ß-globin through a novel mechanism that links the rate of transcription of the transgenic ß-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+) cells isolated from patients affected by ß-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the ß-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from ß-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.
Assuntos
Anemia Falciforme/terapia , Terapia Genética/métodos , Hemoglobinas/metabolismo , Talassemia beta/terapia , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/genética , Animais , Anquirinas/genética , Antígenos CD34/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Células Precursoras Eritroides/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hemoglobinas/genética , Humanos , Elementos Isolantes/genética , Lentivirus/genética , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Globinas beta/genética , Talassemia beta/sangue , Talassemia beta/genéticaRESUMO
The Myc transcription factor plays a vital role in both normal cellular physiology and in many human cancers. We have recently demonstrated that nonsense-mediated RNA decay (NMD), a mechanism that rapidly degrades select mRNAs, is inhibited by the stress-induced phosphorylation of translation initiation factor eIF2α, and this inhibition stabilizes many transcripts necessary for tumorigenesis. Here, we demonstrate that NMD is inhibited by high Myc expression. We show that the phosphorylation of eIF2α, likely due to the ability of Myc to generate reactive oxygen species and augment endoplasmic reticulum stress, is necessary for the inhibition of NMD by Myc. The inhibition of NMD both stabilizes and up-regulates multiple Myc targets, suggesting that the inhibition of NMD may play an important role in the dynamic regulation of genes by Myc.
Assuntos
Linfócitos B/metabolismo , Regulação da Expressão Gênica/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Linfócitos B/citologia , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/fisiologiaRESUMO
RATIONALE: Human atherosclerotic plaques contain large numbers of cells deprived of O(2). In murine atherosclerosis, because the plaques are small, it is controversial whether hypoxia can occur. OBJECTIVE: To examine if murine plaques contain hypoxic cells, and whether hypoxia regulates changes in cellular lipid metabolism and gene expression in macrophages. METHODS AND RESULTS: Aortic plaques from apolipoprotein-E-deficient mice were immunopositive for hypoxia-inducible transcription factor (HIF-1α) and some of its downstream targets. Murine J774 macrophages rendered hypoxic demonstrated significant increases in cellular sterol and triglycerides. The increase in sterol content in hypoxic macrophages correlated with elevated 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity and mRNA levels. In addition, when macrophages were incubated with cholesterol complexes, hypoxic cells accumulated 120% more cholesterol, predominately in the free form. Cholesterol-efflux assays showed that hypoxia significantly decreased efflux mediated by ATP-binding cassette subfamily A member 1 (ABCA1), whose sub cellular localization was altered in both J774 and primary macrophages. Furthermore, in vivo expression patterns of selected genes from cells in hypoxic regions of murine plaques were similar to those from J774 and primary macrophages incubated in hypoxia. The hypoxia-induced accumulation of sterol and decreased cholesterol efflux was substantially reversed in vitro by reducing the expression of the hypoxia-inducible transcription factor, HIF-1α. CONCLUSION: Hypoxic regions are present in murine plaques. Hypoxic macrophages have increased sterol content due to the induction of sterol synthesis and the suppression of cholesterol efflux, effects that are in part mediated by HIF-1α.
Assuntos
Aterosclerose/metabolismo , Hipóxia/metabolismo , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Transporte Biológico , Linhagem Celular , Colesterol/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genética , Interferência de RNA , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2α phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumor-promoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis.
Assuntos
Códon sem Sentido/genética , Neoplasias/genética , Estabilidade de RNA , Microambiente Tumoral/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Helicases , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Transplante HeterólogoRESUMO
Two related ER oxidation 1 (ERO1) proteins, ERO1α and ERO1ß, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1α and ERO1ß. The peak amplitude of calcium transients in homozygous Ero1α mutant adult cardiomyocytes was reduced to 42.0 ± 2.2% (n=10, P ≤ 0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of ß adrenergic blockade (P ≤ 0.01). In addition, mice lacking ERO1α were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31 ± 0.02 in the mutant (n=20) vs. 0.23 ± 0.03 in the wild type (n=18); P ≤ 0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.
Assuntos
Glicoproteínas/metabolismo , Miócitos Cardíacos/fisiologia , Retículo Sarcoplasmático/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Sinalização do Cálcio , Hipóxia Celular , Retículo Endoplasmático/metabolismo , Acoplamento Excitação-Contração/efeitos dos fármacos , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Hemodinâmica , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mutagênese Insercional , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Oxirredutases , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Propanolaminas/farmacologiaRESUMO
Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2α. eIF2α phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2α phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2Bδ variant 1 (V1). Replacement of total eIF2Bδ with V1 renders cells insensitive to eIF2α phosphorylation; specifically, they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2Bδ in ER stress response-deficient cells restores the stress response. Our data suggest that V1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2α phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases that result in ER stress.
Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Isoformas de Proteínas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Regulação da Expressão Gênica , Humanos , Neuroblastoma , Isoformas de Proteínas/genética , Estresse FisiológicoRESUMO
Nonsense-mediated RNA decay (NMD) has long been viewed as an important constitutive mechanism to rapidly eliminate mutated mRNAs. More recently, it has been appreciated that NMD also degrades multiple nonmutated transcripts and that NMD can be regulated by wide variety of cellular stresses. Many of the stresses that inhibit NMD, including cellular hypoxia and amino acid deprivation, are experienced in cells exposed to hostile microenvironments, and several NMD-targeted transcripts promote cellular adaptation in response to these environmental stresses. Because adaptation to the microenvironment is crucial in tumorigenesis, and because NMD targets many mutated tumor suppressor gene transcripts, the regulation of NMD may have particularly important implications in cancer. This review briefly outlines the mechanisms by which transcripts are identified and targeted by NMD and reviews the evidence showing that NMD is a regulated process that can dynamically alter gene expression. Although much of the focus in NMD research has been in identifying the proteins that play a role in NMD and identifying NMD-targeted transcripts, recent data about the potential functional significance of NMD regulation, including the stabilization of alternatively spliced mRNA isoforms, the validation of mRNAs as bona fide NMD targets, and the role of NMD in tumorigenesis, are explored.
Assuntos
Transformação Celular Neoplásica/genética , Códon sem Sentido/genética , Regulação Neoplásica da Expressão Gênica/genética , Estabilidade de RNA/genética , Estresse Fisiológico/genética , Processamento Alternativo/genética , Animais , Humanos , Isoformas de Proteínas/genética , RNA Mensageiro/genéticaAssuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/administração & dosagem , Antifúngicos/administração & dosagem , Desoxicitidina/análogos & derivados , Fluconazol/administração & dosagem , Infecções por HIV/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Organofosfonatos/administração & dosagem , Oxazinas/administração & dosagem , Combinação Trimetoprima e Sulfametoxazol/administração & dosagem , Adenina/administração & dosagem , Terapia Antirretroviral de Alta Atividade/métodos , Desoxicitidina/administração & dosagem , Combinação de Medicamentos , Combinação Efavirenz, Emtricitabina, Fumarato de Tenofovir Desoproxila , Infecções por HIV/sangue , Infecções por HIV/complicações , Infecções por HIV/diagnóstico por imagem , Humanos , Linfoma Difuso de Grandes Células B/sangue , Linfoma Difuso de Grandes Células B/complicações , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons/métodos , RadiografiaRESUMO
The clinical relevance of heparin-induced antibodies (HIA) in the absence of thrombocytopenia remains to be defined. The aims of this study were (i) to determine the prevalence of HIA in patients treated by dialysis, (ii) to determine the prevalence of thrombocytopenia and heparin-induced thrombocytopenia (HIT), and (iii) to test whether HIA are associated with adverse outcomes. Sera from 740 patients treated by hemodialysis (HD, n=596) and peritoneal dialysis (PD, n=144) were tested for HIA (IgG, IgA or IgM) by masked investigators at approximately six months after enrolment in the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE) study. We assessed, with time-to-event Cox proportional hazards models, whether the presence of HIA predicted any of four clinical outcomes: arterial cardiovascular events, venous thromboembolism, vascular access occlusion and mortality. HIA prevalence was 10.3% overall. HIA positivity did not predict development of thrombocytopenia or any of the four clinical outcomes over a mean follow-up of 3.6 years, with hazard ratios for arterial cardiovascular events of 0.98 (95% confidence interval 0.70-1.37), venous thromboembolism 1.39 (0.17-11.5), vascular access occlusion 0.82 (0.40-1.71), and mortality 1.18 (0.85-1.64). Chronic intermittent heparin exposure was associated with a high seroprevalence of HIA. In dialysis patients these antibodies were not an independent risk factor for cardiovascular events and mortality. Our data do not suggest that dialysis patients should be monitored for HIA antibodies in the absence of thrombocytopenia.
Assuntos
Anticorpos/química , Doenças Cardiovasculares/metabolismo , Heparina/química , Trombocitopenia/sangue , Adulto , Idoso , Doenças Cardiovasculares/etiologia , Feminino , Heparina/metabolismo , Humanos , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal , Contagem de Plaquetas , Diálise Renal , Risco , Trombocitopenia/induzido quimicamenteRESUMO
Many tumors are hypoxic, and cells that are experimentally rendered hypoxic display a variety of phenotypes which allow them to adapt to the micro-environment. These phenotypes include a shift from aerobic to anaerobic metabolism, a diminution of reactive oxygen species, an arrest of proliferation, apoptosis, and a secretion of pro-angiogenic growth factors. Some of these hypoxic phenotypes are re-capitulated in normoxic tumor cells (e.g., an increase in anaerobic metabolism), and some tumors have undergone mutations that allow them to bypass the cell cycle arrest and apoptosis typically seen in hypoxic cells. Hypoxic regulation of gene expression is responsible for many hypoxia-induced phenotypes, and here we review a variety of mechanisms by which gene expression is altered in hypoxic cells. These include transcription by HIF-1, the hypoxia inducible transcription factor, and other hypoxia-inducible transcription factors, including ones generated by hypoxic activation of the integrated stress response. Recent data from our laboratory demonstrate that nonsense mediated RNA decay is also regulated in hypoxic cells and thus may play an important role in hypoxic gene regulation and hypoxic phenotypes.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Hipóxia Celular/fisiologia , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Ciclo Celular/fisiologia , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Metástase Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nonsense-mediated RNA decay (NMD) rapidly degrades both mutated mRNAs and nonmutated cellular mRNAs in what is thought to be a constitutive fashion. Here we demonstrate that NMD is inhibited in hypoxic cells and that this inhibition is dependent on phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). eIF2alpha phosphorylation is known to promote translational and transcriptional up-regulation of genes important for the cellular response to stress. We show that the mRNAs of several of these stress-induced genes are NMD targets and that the repression of NMD stabilizes these mRNAs, thus demonstrating that the inhibition of NMD augments the cellular stress response. Furthermore, hypoxia-induced formation of cytoplasmic stress granules is also dependent on eIF2alpha phosphorylation, and components of the NMD pathway are relocalized to these granules in hypoxic cells, providing a potential mechanism for the hypoxic inhibition of NMD. Our demonstration that NMD is inhibited in hypoxic cells reveals that the regulation of NMD can dynamically alter gene expression and also establishes a novel mechanism for hypoxic gene regulation.
Assuntos
Códon sem Sentido , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fator 4 Ativador da Transcrição/genética , Anaerobiose , Animais , Hipóxia Celular/genética , Camundongos , Fases de Leitura Aberta , Fosforilação , RNA Mensageiro/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
The Id proteins play an important role in proliferation, differentiation and tumorigenesis. Many tumors are hypoxic, but it is unknown if expression of Id proteins is regulated in hypoxic cells. Here we show that Id-1 is down-regulated in multiple primary, immortalized, and neoplastic hypoxic cell lines, and the transcriptional repressor ATF-3 is both necessary and sufficient for this hypoxia-induced repression of Id-1. Hypoxic up-regulation of ATF-3 is due in part to activation of the unfolded protein response, a cellular stress response. Remarkably, we observe that the unfolded protein response is de-regulated in all neuroblastoma cell lines tested. Indeed, in the absence of ATF-3 the hypoxia-induced transcription factor HIF-1 up-regulates Id-1 in hypoxic neuroblastoma cells. Hypoxic neuroblastoma cells diminish expression of some neuronal differentiation markers, and forced expression of ATF-3 in hypoxic neuroblastoma cells represses Id-1 and prevents the loss of these markers. The divergent regulation of Id proteins in distinct hypoxic cells may explain some of the varied effects hypoxia has on cellular differentiation and proliferation.
Assuntos
Hipóxia , Proteína 1 Inibidora de Diferenciação/metabolismo , Neuroblastoma/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Humanos , Camundongos , Modelos Biológicos , Desnaturação Proteica , Dobramento de Proteína , Transcrição Gênica , Regulação para CimaRESUMO
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1alpha is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1alpha can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1alpha is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1alpha in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1alpha by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1alpha significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1alpha expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1alpha promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1alpha within tumor compartments, we generated cells with combined disruptions of both HIF-1alpha and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1alpha did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1alpha is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1alpha is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo.