RESUMEN
AIMS/HYPOTHESIS: There is convincing evidence that endoplasmic reticulum (ER) stress is implicated in the pathogenesis of diabetes and its complications; however, the mechanisms are not fully understood. This study aimed to dissect the role and signalling pathways of activating transcription factor 4 (ATF4) in ER-stress-associated endothelial inflammation and diabetic retinopathy. METHODS: ER stress and ATF4 activity were manipulated by complementary pharmacological and genetic approaches in cultured retinal endothelial (TR-iBRB) cells. Diabetes was induced by streptozotocin in heterozygous Atf4 knockout and wild-type mice. ER stress markers, inflammatory cytokines and adhesion molecules, activation of the signal transducer and activator of transcription 3 (STAT3) pathway, and retinal vascular permeability were measured. RESULTS: High-glucose treatment resulted in rapid induction of ER stress, activation of ATF4, and increased production of inflammatory factors in TR-iBRB cells. Suppressing ER stress or inhibiting ATF4 activity markedly attenuated high-glucose-induced production of intercellular adhesion molecule 1, TNF-α and vascular endothelial growth factor. Conversely, enhancing ER stress or overexpressing Atf4 was sufficient to induce endothelial inflammation, which was, at least in part, through activation of the STAT3 pathway. Furthermore, knockdown of the Stat3 gene or inhibiting STAT3 activity restored ER homeostasis in cells exposed to high glucose and prevented ATF4 activation, suggesting that STAT3 is required for high-glucose-induced ER stress. Finally, we showed that downregulation of Atf4 significantly ameliorated retinal inflammation, STAT3 activation and vascular leakage in a mouse model of type 1 diabetes. CONCLUSIONS/INTERPRETATION: Taken together, our data reveal a pivotal role of ER stress and the ATF4/STAT3 pathway in retinal endothelial inflammation in diabetic retinopathy.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Retinopatía Diabética/metabolismo , Hiperglucemia/metabolismo , Inflamación/metabolismo , Vasos Retinianos/patología , Factor de Transcripción STAT3/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Tipo 1/fisiopatología , Retinopatía Diabética/fisiopatología , Retículo Endoplásmico/patología , Endotelio Vascular/patología , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Vasos Retinianos/fisiopatología , Transducción de SeñalRESUMEN
This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely ß2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Catálisis , Diseño de Fármacos , Factor 1 Inducible por Hipoxia/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Isoformas de Proteínas , Especificidad por SustratoRESUMEN
Traumatic spinal cord injury (SCI) produces a complex syndrome that is expressed across multiple endpoints ranging from molecular and cellular changes to functional behavioral deficits. Effective therapeutic strategies for CNS injury are therefore likely to manifest multi-factorial effects across a broad range of biological and functional outcome measures. Thus, multivariate analytic approaches are needed to capture the linkage between biological and neurobehavioral outcomes. Injury-induced neuroinflammation (NI) presents a particularly challenging therapeutic target, since NI is involved in both degeneration and repair. Here, we used big-data integration and large-scale analytics to examine a large dataset of preclinical efficacy tests combining five different blinded, fully counter-balanced treatment trials for different acute anti-inflammatory treatments for cervical spinal cord injury in rats. Multi-dimensional discovery, using topological data analysis (TDA) and principal components analysis (PCA) revealed that only one showed consistent multidimensional syndromic benefit: intrathecal application of recombinant soluble TNFα receptor 1 (sTNFR1), which showed an inverse-U dose response efficacy. Using the optimal acute dose, we showed that clinically-relevant 90 min delayed treatment profoundly affected multiple biological indices of NI in the first 48 h after injury, including reduction in pro-inflammatory cytokines and gene expression of a coherent complex of acute inflammatory mediators and receptors. Further, a 90 min delayed bolus dose of sTNFR1 reduced the expression of NI markers in the chronic perilesional spinal cord, and consistently improved neurological function over 6 weeks post SCI. These results provide validation of a novel strategy for precision preclinical drug discovery that is likely to improve translation in the difficult landscape of CNS trauma, and confirm the importance of TNFα signaling as a therapeutic target.
Asunto(s)
Inteligencia Artificial , Modelos Neurológicos , Traumatismos de la Médula Espinal/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Inyecciones Espinales , Ratas Long-Evans , Receptores Tipo I de Factores de Necrosis Tumoral/farmacología , Proteínas Recombinantes/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patologíaRESUMEN
Importin alpha's mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin alpha's, alpha1's, alpha2's, and alpha3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster we describe the isolation and phenotypic characterization of the first animal importin alpha1 mutant. Animal alpha1's are more similar to ancestral plant and fungal alpha1-like genes than to animal alpha2 and alpha3 genes. Male and female importin alpha1 (Dalpha1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dalpha1 mutant phenotypes were rescued by Dalpha1 transgenes, but not by Dalpha2 or Dalpha3 transgenes. Genetic interactions between the ectopic expression of Dalpha1 and the karyopherins CAS and importin beta1 suggest that high nuclear levels of Dalpha1 are deleterious. We conclude that Dalpha1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dalpha1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin alpha-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin alpha-gene family.
Asunto(s)
Drosophila melanogaster/genética , Gametogénesis/genética , alfa Carioferinas/genética , Animales , Cruzamientos Genéticos , Cartilla de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Femenino , Fertilidad/genética , Eliminación de Gen , Masculino , Meiosis , Recombinación GenéticaRESUMEN
A more complete understanding of calcium's role in cell division requires knowledge of the timing, magnitude, and duration of changes in cytoplasmic-free calcium, [Ca2+]i, associated with specific mitotic events. To define the temporal relationship of changes in [Ca2+]i to cellular and chromosomal movements, we have measured [Ca2+]i every 6-7 s in single-dividing Pt K2 cells using fura-2 and microspectrophotometry, coupling each calcium measurement with a bright-field observation. In the 12 min before discernable chromosome some separation, 90% of metaphase cells show at least one transient of increased [Ca2+]i, 72% show their last transient within 5 min, and a peak of activity is seen at 3 min before chromosome separation. The mean [Ca2+]i of the metaphase transients is 148 +/- 31 nM (61 transients in 35 cells) with an average duration of 21 +/- 14 s. The timing of these increases makes it unlikely that these transient increases in [Ca2+]i are acting directly to trigger the start of anaphase. However, it is possible that a transient rise in calcium during late metaphase is part of a more complex progression to anaphase. In addition to these transient changes, a gradual increase in [Ca2+]i was observed starting in late anaphase. Within the 2 min surrounding cytokinesis onset, 82% of cells show a transient increase in [Ca2+]i to 171 +/- 48 nM (53 transients in 32 cells). The close temporal correlation of these changes with cleavage is consistent with a more direct role for calcium in this event, possibly by activating the contractile system. To assess the specificity of these changes to the mitotic cycle, we examined calcium changes in interphase cells. Two-thirds of interphase cells show no transient increases in calcium with a mean [Ca2+]i of 100 +/- 18 nM (n = 12). However, one-third demonstrate dramatic and repeated transient increases in [Ca2+]i. The mean peak [Ca2+]i of these transients is 389 +/- 70 nM with an average duration of 77 s. The necessity of any of these transient changes in calcium for the completion of mitotic or interphase activities remains under investigation.
Asunto(s)
Calcio/metabolismo , Mitosis , Anafase , Animales , División Celular , Línea Celular , Citofotometría , Interfase , Metafase , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , FN-kappa B/metabolismo , Virus Sindbis/fisiología , Células 3T3 , Acetilcisteína/farmacología , Animales , Antivirales/farmacología , Proteínas de Unión al ADN/genética , Vectores Genéticos , Peróxido de Hidrógeno/toxicidad , Ratones , Inhibidor NF-kappaB alfa , Subunidad p50 de NF-kappa B , Ratas , Estaurosporina/toxicidad , Factores de Tiempo , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SV-induced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 microM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Over-expression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SV-induced NF-kappa B activation. To determine if NF-kappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.
Asunto(s)
Apoptosis , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Virus Sindbis/fisiología , Reactivos de Sulfhidrilo/farmacología , Acetilcisteína/farmacología , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Quelantes/farmacología , ADN , Ditiotreitol/farmacología , Hierro , Peroxidación de Lípido/efectos de los fármacos , Masculino , Mercaptoetanol/farmacología , Ratones , Datos de Secuencia Molecular , FN-kappa B/antagonistas & inhibidores , Neuroblastoma/patología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Pirrolidinas/farmacología , Ratas , Tiocarbamatos/farmacología , Células Tumorales CultivadasRESUMEN
Resistance of human renal cell carcinoma (RCC) and melanoma to the apoptosis-inducing effects of IFNs was postulated to result from epigenetic silencing of genes by DNA methylation, a common feature of human cancers. To reverse silencing, 5-AZA-deoxycytidine (5-AZA-dC) or selective depletion of DNA methyltransferase 1 (DNMT1) by phosphorothioate oligonucleotide antisense (DNMT1 AS) were employed in cells resistant (<5% terminal deoxynucleotidyl transferase-mediated nick-end labeling positive) to apoptosis induction by IFN-alpha2 and IFN-beta (ACHN, SK-RC-45, and A375). 5-AZA-dC and DNMT1 AS similarly depleted available DNMT1 protein and, at doses that did not cause apoptosis alone, resulted in apoptotic response to IFNs. The proapoptotic tumor suppressor RASSF1A was reactivated by DNMT1 inhibitors in all three cell lines. This was associated with demethylation of its promoter region. IFNs augmented RASSF1A protein expression after reactivation by DNMT1 inhibition. In IFN-sensitive WM9 melanoma cells, expression of RASSF1A was constitutive but also augmented by IFNs. RASSF1A small interfering RNA reduced IFN-induced apoptosis in WM9 cells and in DNMT1-depleted ACHN cells. Conversely, lentiviral expression of RASSF1A but not transduction with empty virus enabled IFN-induced apoptosis. IFN induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL-neutralizing antibody inhibited apoptotic response to IFN in RASSF1A-expressing ACHN cells. Accordingly, RASSF1A markedly sensitized to recombinant TRAIL. Normal kidney epithelial cells, although expressing RASSF1A, did not undergo apoptosis in response to IFN or TRAIL but had >400-fold higher TRAIL decoy receptor 1 expression than transduced ACHN cells (real-time reverse transcription-PCR). Results identified RASSF1A as regulated by IFNs and participating in IFN-induced apoptosis at least in part by sensitization to TRAIL.
Asunto(s)
Apoptosis/efectos de los fármacos , Interferón-alfa/farmacología , Interferón beta/farmacología , Proteínas Supresoras de Tumor/biosíntesis , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/deficiencia , Resistencia a Antineoplásicos , Epigénesis Genética , Silenciador del Gen , Células HeLa , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Lentivirus/genética , Lentivirus/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Glicoproteínas de Membrana/farmacología , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Ligando Inductor de Apoptosis Relacionado con TNF , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
OBJECTIVE(S): To determine whether operator-dependent obstetric complications occur at higher rates in July at teaching hospitals using a large, nationwide sample of deliveries. STUDY DESIGN: Data for this study were obtained from an administrative dataset, the Nationwide Inpatient Sample, for the years 1998 to 2002. Singleton deliveries and singleton livebirth admissions among Medicaid patients at teaching hospitals with OB/GYN residents working on the Labor and Delivery ward were identified. Outcomes for various complications for these patients in the month of July were compared to those occurring in the months from August to June. RESULTS: The 26,546 women in our cohort who delivered in July were compared to the 272,584 women delivering during August to June. There were no statistically significant differences in the rates of cesarean delivery, urethral/bladder injury, third or fourth degree lacerations, wound complications, postpartum hemorrhage, transfusion, shoulder dystocia, chorioamnionitis or anesthesia-related complications. The 26,175 singleton livebirth admissions in July were compared to 266,158 such admissions in August to June. There were no statistically significant differences in the rates of brachial plexus injury (0.2 vs 0.2%, P=0.824) or birth asphyxia (0.1 vs 0.1%, P=0.643). CONCLUSION(S): This study shows no increased rate of operator-dependent complications of delivery at teaching hospitals nationwide in the month of July.
Asunto(s)
Parto Obstétrico/estadística & datos numéricos , Internado y Residencia/estadística & datos numéricos , Obstetricia/educación , Complicaciones del Embarazo/epidemiología , Estaciones del Año , Adulto , Cesárea/estadística & datos numéricos , Parto Obstétrico/efectos adversos , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Embarazo , SeguridadRESUMEN
Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.
Asunto(s)
Hidroxiquinolinas/farmacología , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Procolágeno-Prolina Dioxigenasa/genética , Inhibidores de Prolil-Hidroxilasa/farmacología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Adenosina Trifosfato/agonistas , Adenosina Trifosfato/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas , Línea Celular , Regulación de la Expresión Génica , Ácido Glutámico/toxicidad , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/genética , Ratones , Neuronas/citología , Neuronas/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Estrés Oxidativo , Procolágeno-Prolina Dioxigenasa/deficiencia , Procolágeno-Prolina Dioxigenasa/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway in non-neuronal cells that culminates in the stabilization of the transcriptional activator hypoxia-inducible factor-1 (HIF-1) and increased expression of gene products that mediate hypoxic adaptation. We examined the hypothesis that iron chelators prevent oxidative stress-induced death in cortical neuronal cultures by inducing expression of HIF-1 and its target genes. We report that the structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic cortical neuronal cultures. The protective effects of iron chelators are correlated with their ability to enhance DNA binding of HIF-1 and activating transcription factor 1(ATF-1)/cAMP response element-binding protein (CREB) to the hypoxia response element in cortical cultures and the H19-7 hippocampal neuronal cell line. We show that mRNA, protein, and/or activity levels for genes whose expression is known to be regulated by HIF-1, including glycolytic enzymes, p21(waf1/cip1), and erythropoietin, are increased in cortical neuronal cultures in response to iron chelator treatment. Finally, we demonstrate that cobalt chloride, which also activates HIF-1 and ATF-1/CREB in cortical cultures, also prevents oxidative stress-induced death in these cells. Altogether, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress.
Asunto(s)
Apoptosis/fisiología , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/farmacología , Ciclinas/genética , Proteínas de Unión al ADN/metabolismo , Deferoxamina/farmacología , Eritropoyetina/genética , Regulación de la Expresión Génica , Neuronas/citología , Neuronas/fisiología , Proteínas Nucleares/metabolismo , Estrés Oxidativo/fisiología , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 1 , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Cobalto/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inducción Enzimática/efectos de los fármacos , Feto , Fructosa-Bifosfato Aldolasa/biosíntesis , Fructosa-Bifosfato Aldolasa/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Glucólisis , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , L-Lactato Deshidrogenasa/biosíntesis , L-Lactato Deshidrogenasa/genética , Mimosina/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Ratas , Ratas Sprague-DawleyRESUMEN
We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.
Asunto(s)
Apoptosis , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Leupeptinas/farmacología , Complejos Multienzimáticos/metabolismo , Relación Dosis-Respuesta a Droga , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal , Virus Sindbis/fisiologíaRESUMEN
Epidemiological evidence has suggested an association between diets rich in antioxidants and diminished risks of various types of cancer. Proposed mechanisms for protective effects of antioxidants have involved inhibition of free radical-mediated DNA damage. Recent data suggest that antioxidants may prevent or eliminate cancerous cells through their ability to inhibit proliferation or to induce programmed cell death (PCD). To begin to identify cell cycle and cell death regulatory factors involved in antioxidant-induced growth arrest and PCD, we have studied colorectal carcinoma cells (CRCs) that differ in expression of the tumor suppressor protein p53, and of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). The antioxidants, N-acetylcysteine (NAC) and vitamin E either inhibited proliferation in a p53-independent manner without affecting cell viability or induced cell death. Growth arrest was not associated with upregulation of the CDK inhibitors p21(Waf1/Cip1), p18(ink4c) or p16(ink4a), but was associated with a decrease in reactive oxygen species (ROS). In contrast to previous observations, the absence of p21(Waf1/Cip1) increased susceptibility of CRCs to antioxidant-induced PCD. NAC decreased levels of retinoblastoma protein (Rb) phosphorylation in all cells tested, but Rb was cleaved only in cells which underwent NAC-induced death. Although NAC decreased ROS in all cells studied, cell lines in which PCD occurred had higher baseline levels of ROS than cell lines in which proliferation was blocked. These observations suggest that expression of p21(Waf1/Cip1) and basal levels of ROS are important determinants of outcome after antioxidant treatment.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Ciclinas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Vitamina E/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Humanos , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
Previous studies demonstrate that resting intracellular calcium in cultured skin fibroblasts declines due to in vivo aging and is further depressed by Alzheimer's disease. These data suggest that altered calcium homeostasis may underlie the deficits in cell function (e.g., cell spreading) that also occur in these cells. Depressed cytosolic free calcium in fibroblasts from aged and Alzheimer donors can be elevated by various drug treatments. 3,4-Diaminopyridine, serum, N-formyl-methionyl-leucyl-phenylalanine and bradykinin increased cytosolic free calcium transiently although the rate of the increase was slower and the magnitude of the rise was less in cells from aged and Alzheimer donors when compared to young donors. Four minutes after N-formyl-methionyl-leucyl-phenylalanine or bradykinin treatment cytosolic free calcium returned to resting levels in all six cell lines. Six minutes after either serum or 3,4-diaminopyridine treatments, however, cytosolic free calcium in cells from aged and Alzheimer donors remained elevated at concentrations similar to the resting calcium level in young cells. Bradykinin and serum were effective in the absence of extracellular calcium but 3,4-diaminopyridine and N-formyl-methionyl-leucyl-phenylalanine were not. These demonstrate that dynamic, as well as resting calcium homeostasis, is altered in cultured skin fibroblasts from aged and Alzheimer donors.
Asunto(s)
4-Aminopiridina/análogos & derivados , Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Amifampridina , Aminopiridinas/farmacología , Bradiquinina/farmacología , Citosol/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Piel/metabolismoRESUMEN
Previous studies have shown that certain tumor cell lines which naturally express high levels of the epidermal growth factor receptor (EGFR) undergo apoptosis when exposed to epidermal growth factor. Whether this phenomenon is a direct result of receptor overexpression or some other genetic alteration renders these cells sensitive to apoptosis is yet to be established. We show that experimentally increasing the level of EGFR expression predictably leads to apoptosis in a variety of cell types which requires an active tyrosine kinase but not EGFR autophosphorylation sites. Expression of a dominant negative Ras mutant in EGFR overexpressing cells results in a significant potentiation of EGFR induced apoptosis suggesting that Ras activation is a key survival signal generated by the EGFR. We propose that potentiation of EGFR induced apoptosis by dominant negative Ras results, at least in part, by a block of Akt activation.
Asunto(s)
Apoptosis , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas p21(ras)/genética , Western Blotting , Receptores ErbB/metabolismo , Genes Dominantes , Humanos , Mutación , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Iron is the most abundant transition metal in the brain, where it functions as an important cofactor in a host of vital metabolic processes and plays an absolutely essential role in cell viability. Free iron is also very toxic when present in high concentrations, thus placing this essential metal at the core of neurotoxic injury in a number of neurological disorders. The pivotal role of iron in cellular homeostasis, including its latent toxicity, necessitates a tight regulation of iron metabolism. Oxygen and iron appear to play an important role in iron homeostasis. They appear to exert their homeostatic role by modulating the proteins involved in a complex interplay between iron sensing, transport, and storage. These key regulatory proteins include ferritin (intracellular storage), transferrin (extracellular transport), transferrin receptor, and iron regulatory protein (sensor of intracellular iron concentration). The interplay of iron and oxygen is most intriguing in the setting of stroke, where hypoxia and free iron appear to interact in causing the subsequent neuronal death.
Asunto(s)
Hipoxia-Isquemia Encefálica , Quelantes del Hierro/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Electrones , Humanos , Hierro/metabolismo , Modelos Biológicos , Modelos Químicos , Neuronas/fisiología , Oxidación-Reducción , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/prevención & controlRESUMEN
We have previously shown that glutamate-induced cystine deprivation of embryonic cortical neurons leads to intracellular depletion of the antioxidant glutathione, consequent oxidative stress and apoptotic cell death. To test the hypothesis that glutathione depletion and oxidative stress represent a common pathway of neuronal apoptosis, we examined the effect of a variety of antioxidants on serum deprivation-induced death in embryonic cortical neurons. A host of antioxidant agents, capable of abrogating glutathione depletion-induced apoptosis in cortical neurons, were unable to inhibit serum deprivation-induced death in these cells. To test whether serum deprivation and glutathione depletion involve different or antagonistic pathways, we serum-deprived cortical neurons at the time of induction of glutathione depletion. Surprisingly, we found that serum deprivation diminished glutathione depletion-induced death as compared to cultures treated with growth factors or serum. These observations suggest that serum deprivation antagonizes the cell death signaling pathway activated by glutathione depletion and that serum and growth factors can enhance susceptibility to oxidative stress. Consistent with these conclusions, we show that growth factors or serum added in combination with antioxidants possess superior survival promoting effects as compared to either agent alone.
Asunto(s)
Apoptosis , Corteza Cerebral/citología , Corteza Cerebral/embriología , Medio de Cultivo Libre de Suero , Glutatión/administración & dosificación , Estrés Oxidativo , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Hidroxianisol Butilado/farmacología , Células Cultivadas , Cistina/farmacología , Deferoxamina/farmacología , Insulina/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Several observations indirectly suggest that intracellular calcium regulation may be altered by aging and Alzheimer's disease. Thus, calcium homeostasis was examined directly in skin fibroblasts from Alzheimer's patients and compared to cells from normal young and elderly controls. Alterations in both bound and free calcium were noted; cells from Alzheimer's donors have higher levels of bound calcium but lower concentrations of free intracellular calcium when compared to cells from young and normal aged donors. These changes in calcium homeostasis may be physiologically significant, since processes that require transient elevations of intracellular free calcium, such as cell spreading, decline in the Alzheimer's cells. In summary, cultured skin fibroblasts from normal aged and Alzheimer's patients demonstrate deficits in calcium homeostasis and other metabolic processes when compared to cells from young donors.
Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Homeostasis , HumanosRESUMEN
OBJECTIVE: L-buthionine sulfoximine (BSO) inhibits glutathione synthesis and may modulate tumor resistance to some alkylating agents, but it has not been proven effective in the treatment of intracranial neoplasms. To evaluate this drug for the treatment of brain tumors, we studied the use of BSO for potentiating the antineoplastic effect of 4-hydroxyperoxycyclophosphamide (4-HC) in the rat 9L glioma model. METHODS: The survival of male Fischer 344 rats with intracranial 9L gliomas was measured after implantation of controlled-release polymers containing one of the following: no drug, BSO, 4-HC, or both BSO and 4-HC. The efficacy of intracranial 4-HC treatment was assessed with and without serial systemic intraperitoneal BSO injections. Tissue glutathione levels were measured in the brains, tumors, and livers of animals treated with intraperitoneal injections or local delivery of BSO. RESULTS: The median survival of animals treated with intracranial polymers containing 4-HC was 2.3 times greater than that of controls. This survival benefit was doubled by local delivery of BSO. In contrast, systemic BSO therapy did not improve survival time. In animals that were treated systemically, both liver and tumor glutathione levels were significantly lower than they were in control animals. In the locally treated animals, glutathione levels were reduced in the brain tumor but not in the liver. CONCLUSION: These results demonstrate that local but not systemic delivery of BSO enhances the antineoplastic effect of 4-HC in this rat 9L glioma model. In addition, because local delivery of BSO within the brain did not deplete glutathione levels systemically, this method of treatment may be safer than systemic administration of BSO.
Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Butionina Sulfoximina/administración & dosificación , Ciclofosfamida/administración & dosificación , Glioma/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Encéfalo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Butionina Sulfoximina/uso terapéutico , Ciclofosfamida/análogos & derivados , Ciclofosfamida/uso terapéutico , Combinación de Medicamentos , Implantes de Medicamentos , Sinergismo Farmacológico , Glioma/metabolismo , Glioma/patología , Glutatión/antagonistas & inhibidores , Inyecciones Intraperitoneales , Hígado/metabolismo , Masculino , Ratas , Ratas Endogámicas F344 , Valores de Referencia , Análisis de SupervivenciaRESUMEN
In previous experiments rats pretreated with slow-release d-amphetamine (d-Amp) pellets for 4 1/2 days, given a 12-hr drug-free period, and then injected with d-Amp have been found to show a behavioral syndrome which has similarities to that induced by acute injections of the hallucinogens LSD and mescaline. The present results indicate that rats administered this same drug regimen have large decreases in Dopamine (DA), dihydroxyphenyl acetic acid (Dopac), and homovanillic acid (HVA) in caudate nucleus, smaller decreases in DA with no changes in Dopac and HVA levels in nucleus accumbens, but no alterations in 5-hydroxytryptamine (5HT) and 5-hydroxyindole acetic acid (5HIAA) levels in caudate, accumbens, brainstem and hippocampus. Increased 5HIAA levels are found in rats sacrificed with pellets intact following 3 days of continuous d-Amp administration, while sleep deprived and in motor stereotypies. The late and hallucinatory stage following continuous d-amp is correlated more closely with alterations in dopamine than of 5HT.