RESUMEN

Lung cancer is the leading cause of cancer-related death in people and is mainly due to environmental factors such as smoking and radon. The National Toxicology Program (NTP) tests various chemicals and mixtures for their carcinogenic hazard potential. In the NTP chronic bioassay using B6C3F1 mice, the incidence of lung tumors in treated and control animals is second only to the liver tumors. In order to study the molecular mechanisms of chemically induced lung tumors, an understanding of the genetic changes that occur in spontaneous lung (SL) tumors from untreated control animals is needed. The authors have evaluated the differential transcriptomic changes within SL tumors compared to normal lungs from untreated age-matched animals. Within SL tumors, several canonical pathways associated with cancer (eukaryotic initiation factor 2 signaling, RhoA signaling, PTEN signaling, and mammalian target of rapamycin signaling), metabolism (Inositol phosphate metabolism, mitochondrial dysfunction, and purine and pyramidine metabolism), and immune responses (FcγR-mediated phagocytosis, clathrin-mediated endocytosis, interleukin 8 signaling, and CXCR4 signaling) were altered. Meta-analysis of murine SL tumors and human non-small cell lung cancer transcriptomic data sets revealed a high concordance. These data provide important information on the differential transcriptomic changes in murine SL tumors that will be critical to our understanding of chemically induced lung tumors and will aid in hazard analysis in the NTP 2-year carcinogenicity bioassays.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie

RESUMEN

Hepatocellular carcinoma (HCC) is an important cause of morbidity and mortality worldwide. Although the risk factors of human HCC are well known, the molecular pathogenesis of this disease is complex, and in general, treatment options remain poor. The use of rodent models to study human cancer has been extensively pursued, both through genetically engineered rodents and rodent models used in carcinogenicity and toxicology studies. In particular, the B6C3F1 mouse used in the National Toxicology Program (NTP) two-year bioassay has been used to evaluate the carcinogenic effects of environmental and occupational chemicals, and other compounds. The high incidence of spontaneous HCC in the B6C3F1 mouse has challenged its use as a model for chemically induced HCC in terms of relevance to the human disease. Using global gene expression profiling, we identify the dysregulation of several mediators similarly altered in human HCC, including re-expression of fetal oncogenes, upregulation of protooncogenes, downregulation of tumor suppressor genes, and abnormal expression of cell cycle mediators, growth factors, apoptosis regulators, and angiogenesis and extracellular matrix remodeling factors. Although major differences in etiology and pathogenesis remain between human and mouse HCC, there are important similarities in global gene expression and molecular pathways dysregulated in mouse and human HCC. These data provide further support for the use of this model in hazard identification of compounds with potential human carcinogenicity risk, and may help in better understanding the mechanisms of tumorigenesis resulting from chemical exposure in the NTP two-year carcinogenicity bioassay.

Asunto(s)

Carcinoma Hepatocelular/patología , Perfilación de la Expresión Génica/métodos , Estudios de Asociación Genética/métodos , Neoplasias Hepáticas/patología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Pruebas de Carcinogenicidad/métodos , Carcinógenos/toxicidad , Ciclo Celular , Biología Computacional , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos , Oncogenes , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba

RESUMEN

Tumor response in the B6C3F1 mouse, F344 rat, and other animal models following exposure to various compounds provides evidence that people exposed to these or similar compounds may be at risk for developing cancer. Although tumors in rodents and humans are often morphologically similar, underlying mechanisms of tumorigenesis are often unknown and may be different between the species. Therefore, the relevance of an animal tumor response to human health would be better determined if the molecular pathogenesis were understood. The underlying molecular mechanisms leading to carcinogenesis are complex and involve multiple genetic and epigenetic events and other factors. To address the molecular pathogenesis of environmental carcinogens, the authors examine rodent tumors (e.g., lung, colon, mammary gland, skin, brain, mesothelioma) for alterations in cancer genes and epigenetic events that are associated with human cancer. National Toxicology Program (NTP) studies have identified several genetic alterations in chemically induced rodent neoplasms that are important in human cancer. Identification of such alterations in rodent models of chemical carcinogenesis caused by exposure to environmental contaminants, occupational chemicals, and other compounds lends further support that they are of potential human health risk. These studies also emphasize the importance of molecular evaluation of chemically induced rodent tumors for providing greater public health significance for NTP evaluated compounds.

Asunto(s)

Carcinógenos Ambientales/toxicidad , Modelos Animales de Enfermedad , Neoplasias Experimentales/inducido químicamente , Animales , Bioensayo , Pruebas de Carcinogenicidad , Epigénesis Genética/efectos de los fármacos , Genes Relacionados con las Neoplasias/efectos de los fármacos , Humanos , Ratones , Programas Nacionales de Salud , Neoplasias/inducido químicamente , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Ratas , Ratas Endogámicas F344 , Medición de Riesgo

RESUMEN

A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg 3'-azido-3'-deoxythymidine (AZT)/kg body weight (BW) daily for the duration of gestation (18-19 days) [National Toxicology Program,2006]. The incidence of alveolar/bronchiolar adenomas and carcinomas in the 200 and 300 mg/kg groups was significantly higher (P = 0.027 and 0.007, respectively) in male offspring, but not in females (P = 0.338 and 0.315, respectively). The purpose of the present study was to evaluate K-ras mutation status in lung tumors from the female offspring in AZT exposed groups and to determine whether at the molecular level there were signature K-ras mutations in lung tumors that were different from spontaneous tumors. K-ras mutation was detected by cycle sequencing of polymerase chain reaction (PCR)-amplified DNA, isolated from formalin-fixed, paraffin-embedded lung tumors. K-ras mutations were detected in 17 of 28 (61%) lung tumors from the female offspring in AZT exposed groups. No K-ras mutations were detected in the 8 tumors examined from the female control group. The predominant mutations were Codon 12 G-->T transversions in the 50, 100, and 300 mg/kg groups, and Codon 12 G-->C transversions in the 200 and 300 mg/kg groups. K-ras Codon 12 G-->T transversions (TGT mutations) may be induced by oxidative DNA damage and 8-oxoguanine (8-oxoG), while K-ras Codon 12 G-->C transversions (CGT mutations) may be due to further oxidative lesions of guanine and 8-oxoG.

Asunto(s)

Genes ras/genética , Neoplasias Pulmonares/etiología , Mutación/efectos de los fármacos , Zidovudina/toxicidad , Animales , Femenino , Neoplasias Pulmonares/genética , Masculino , Exposición Materna/efectos adversos , Ratones , Embarazo , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/genética , Factores Sexuales

RESUMEN

The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.

Asunto(s)

Derivados del Benceno/toxicidad , Genes p53/genética , Genes ras , Neoplasias Pulmonares/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma Bronquioloalveolar/inducido químicamente , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/patología , Animales , Carcinoma/inducido químicamente , Carcinoma/genética , Carcinoma/patología , Codón , Exones , Inmunohistoquímica , Pérdida de Heterocigocidad , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos , Mutación Puntual

RESUMEN

National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Asunto(s)

Derivados del Benceno/toxicidad , Genes ras/genética , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/genética , Adenocarcinoma Bronquioloalveolar/inducido químicamente , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/patología , Animales , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos , Transducción de Señal/fisiología

RESUMEN

Cerebellar hypoplasia in fetal alcohol spectrum disorders (FASD) is associated with inhibition of insulin and insulin-like growth factor (IGF) signaling in the brain. Aspartyl (asparaginyl)-beta-hydroxylase (AAH) is a mediator of neuronal motility, and stimulated by insulin and IGF activation of PI3 kinase-Akt, or inhibition of GSK-3beta. Since ethanol inhibits PI3 Kinase-Akt and increases GSK-3beta activity in brain, we examined the effects of ethanol and GSK-3beta on AAH expression and directional motility in neuronal cells. Control and ethanol-exposed (100 mM x 48 h) human PNET2 cerebellar neuronal cells were stimulated with IGF-1 and used to measure AAH expression and directional motility. Molecular and biochemical approaches were used to characterize GSK-3beta regulation of AAH and neuronal motility. Ethanol reduced IGF-1 stimulated AAH protein expression and directional motility without inhibiting AAH's mRNA. Further analysis revealed that: (1) AAH protein could be phosphorylated by GSK-3beta; (2) high levels of GSK-3beta activity decreased AAH protein; (3) inhibition of GSK-3beta and/or global Caspases increased AAH protein; (4) AAH protein was relatively more phosphorylated in ethanol-treated compared with control cells; and (5) chemical inhibition of GSK-3beta and/or global Caspases partially rescued ethanol-impaired AAH protein expression and motility. Ethanol-impaired neuronal migration is associated with reduced IGF-I stimulated AAH protein expression. This effect may be mediated by increased GSK-3beta phosphorylation and Caspase degradation of AAH. Therapeutic strategies to rectify CNS developmental abnormalities in FASD should target factors underlying the ethanol-associated increases in GSK-3beta and Caspase activation, e.g. IGF resistance and increased oxidative stress.

Asunto(s)

Movimiento Celular/efectos de los fármacos , Etanol/farmacología , Oxigenasas de Función Mixta/metabolismo , Neuronas/fisiología , Actinas/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/farmacología , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/metabolismo , Fosforilación , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas

RESUMEN

BACKGROUND: Aspartyl (asparaginyl)-beta-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation. This study examines the expression, regulation and function of AAH, and its related transcripts, Humbug and Junctin, which lack catalytic domains, using SH-Sy5y neuroblastoma cells. RESULTS: Real time quantitative RT-PCR demonstrated 8- or 9-fold higher levels of Humbug than AAH and Junctin, and lower levels of all 3 transcripts in normal human brains compared with neuroblastic tumor cells. AAH and Humbug expression were significantly increased in response to insulin and IGF-I stimulation, and these effects were associated with increased directional motility. However, over-expression of AAH and not Humbug significantly increased motility. Treatment with chemical inhibitors of Akt, Erk MAPK, or cyclin-dependent kinase 5 (Cdk-5) significantly reduced IGF-I stimulated AAH and Humbug expression and motility relative to vehicle-treated control cells. In addition, significantly increased AAH and Humbug expression and directional motility were observed in cells co-transfected with Cdk-5 plus its p35 or p25 regulatory partner. Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase. CONCLUSION: AAH and Humbug are over-expressed in SH-Sy5y neuroblastoma cells, and their mRNAs are regulated by insulin/IGF-1 signaling through Erk MAPK, PI3 kinase-Akt, and Cdk-5, which are known mediators of cell migration. Although AAH and Humbug share regulatory signaling pathways, AAH and not Humbug mediates directional motility in SH-Sy5y neuroblastoma cells.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/farmacología , Oxigenasas de Función Mixta/genética , Neuroblastoma/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al Calcio , Línea Celular Tumoral , Movimiento Celular , Proteínas de Unión al ADN/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Hipoglucemiantes , Insulina/farmacología , Proteínas de la Membrana , Oxigenasas de Función Mixta/metabolismo , Proteínas Musculares , Proteínas del Tejido Nervioso , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Factores de Transcripción/farmacología , Transfección

RESUMEN

Phthalate chemical plasticizers can damage the fetal and postnatal mammalian testis, but several aspects of the injury mechanism remain unknown. Using a genome-wide microarray, the profile of testicular gene expression changes was examined following exposure of postnatal day 28 rats to a single, high dose (1000 mg/kg) of mono-(2-ethylhexyl) phthalate (MEHP). By microarray analysis, approximately 1675 nonredundant genes exhibited significant expression changes; the vast majority were observed at 12 h. Among the 36 genes significantly altered up to the 3-h time point, prominent functional categories were secreted, transcription, and signaling factors. Using quantitative PCR (qPCR), the dose-response of 24 genes was determined after a single MEHP exposure of 10, 100, or 1000 mg/kg. Increasing 114-fold by 12 h at 1000 mg/kg, Thbs1 (thrombospondin 1) showed the highest level of gene induction. The vast majority of genes analyzed by qPCR exhibited significant expression alterations at the lowest dose level. Interestingly, a unique, dose-dependent expression pattern was observed for the transcription factor Nr0b1, steroidogenic genes (Cyp17a1 and StAR), and a cholesterol metabolism gene (Dhcr7). For these genes, the direction of expression change at 10 or 100 mg/kg was opposite that observed at 1000 mg/kg. Gene profiling data at 1000 mg/kg MEHP were phenotypically anchored to increased germ cell apoptosis (6 and 12 h) and an interstitial neutrophil infiltrate (12 h). At 10 or 100 mg/kg MEHP, no testicular morphological changes were detected, but a significant increase in germ cell apoptosis was seen at 6 h. Finally, comparison of the prepubertal MEHP microarray data to similar data from fetal dibutyl phthalate (DBP) exposure showed conservation in both the identities of testicular genes altered and the direction of expression changes. For example, 60% of the genes altered within 3 h of prepubertal MEHP exposure also were changed following acute fetal DBP exposure, and the direction of expression change was highly preserved. These data demonstrate that similar genetic targets are altered following fetal and prepubertal phthalate exposure, suggesting that the initial mechanism of fetal and prepubertal phthalate-induced testicular injury is shared.

Asunto(s)

Dietilhexil Ftalato/análogos & derivados , Feto/efectos de los fármacos , Perfilación de la Expresión Génica , Testículo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Dietilhexil Ftalato/toxicidad , Relación Dosis-Respuesta a Droga , Feto/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , FN-kappa B/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Ratas , Maduración Sexual , Espermatozoides/efectos de los fármacos , Testículo/metabolismo , Trombospondina 1/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1

RESUMEN

BACKGROUND/AIMS: We measured aspartyl (asparaginyl)-beta-hydroxylase (AAH) gene expression in human hepatocelluar carcinoma and surrounding uninvolved liver at both the mRNA and protein level and examined the regulation and function of this enzyme. METHODS: Since growth of HCC is mediated by signaling through the insulin-receptor substrate, type 1 (IRS-1), we examined-if AAH is a downstream gene regulated by insulin and IGF-1 in HCC cells. In addition, IRS-1 regulation of AAH was examined in a transgenic (Tg) mouse model in which the human (h) IRS-1 gene was over-expressed in the liver, and an in vitro model in which a C-terminus truncated dominant-negative hIRS-1 cDNA (hIRS-DeltaC) was over-expressed in FOCUS HCC cells. The direct effects of AAH on motility and invasiveness were examined in AAH-transfected HepG2 cells. RESULTS: Insulin and IGF-1 stimulation increased AAH mRNA and protein expression and motility in FOCUS and Hep-G2 cells. These effects were mediated by signaling through the Erk MAPK and PI3 kinase-Akt pathways. Over-expression of hIRS-1 resulted in high levels of AAH in Tg mouse livers, while over-expression of hIRS-DeltaC reduced AAH expression, motility, and invasiveness in FOCUS cells. Finally, over-expression of AAH significantly increased motility and invasiveness in HepG2 cells, whereas siRNA inhibition of AAH expression significantly reduced directional motility in FOCUS cells. CONCLUSIONS: The results suggest that enhanced AAH gene activity is a common feature of human HCC and growth factor signaling through IRS-1 regulates AAH expression and increases motility and invasion of HCC cells. Therefore, AAH may represent an important target for regulating tumor growth in vivo.

Asunto(s)

Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundario , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Oxigenasas de Función Mixta/metabolismo , Animales , Biopsia , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Factor de Crecimiento Epidérmico/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Transgénicos , Oxigenasas de Función Mixta/genética , Invasividad Neoplásica , Fosfoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Some features of phthalate-induced testicular injury suggest that phthalates alter Sertoli-germ cell adhesion and G protein signaling. Celsr2 is a unique protein that has structural characteristics of both an adhesion molecule and a G protein coupled receptor (GPCR) and has been demonstrated to function in Sertoli-germ cell adhesion. Within 2 h of a 1-g/kg mono-(2-ethylhexyl) phthalate (MEHP) exposure, in vivo Sertoli cell celsr2 localization was altered; celsr2 immunostaining became concentrated in the basal aspect of Sertoli cells, and then a diffuse pattern emerged. Because GPCRs are regulated by phosphorylation, the hypothesis that phthalate exposure induces the phosphorylation of celsr2 was tested by examining phosphorylation in celsr2-transfected HeLa cells treated with MEHP. At concentrations of 1 microM or greater, MEHP transiently increased celsr2 phosphorylation on serine/threonine residues; celsr2 phosphorylation was increased by 15 min of exposure and returned to control levels after 60 min. Cells exposed to the inactive phthalate monoester mono-methyl phthalate showed no change in celsr2 phosphorylation. In addition, phosphorylation of the endogenous HeLa cell GPCR, Chemokine Receptor 4 (CXCR4), was not altered by exposure to MEHP. Inhibition of protein kinase C or casein kinase 1 prevented MEHP-induced celsr2 phosphorylation, while inhibition of protein kinase A or mitogen-activated protein kinase had no effect. These data show that MEHP exposure rapidly alters testicular celsr2 immunolocalization as well as celsr2 posttranslational modification in a model cell line.

Asunto(s)

Cadherinas/metabolismo , Quinasa de la Caseína I/metabolismo , Dietilhexil Ftalato/análogos & derivados , Proteína Quinasa C/metabolismo , Dietilhexil Ftalato/farmacología , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Fosforilación , Receptores CXCR4/metabolismo

RESUMEN

Familial Alzheimer's Disease (AD) has been linked to amyloid beta protein precursor (AbetaPP) and presenilin gene mutations. In sporadic AD, which accounts for the vast majority of cases, the pathogenesis of neurodegeneration is unknown; however, recent evidence suggests a role for oxidative stress. The present study demonstrates that transient hypoxic injury to cortical neurons causes several of the molecular and biochemical abnormalities that occur in AD including, mitochondrial dysfunction, impaired membrane integrity, increased levels of DNA damage, reactive oxygen species, phospho-tau, phospho-MAP-1B, and ubiquitin immunoreactivity, and AbetaPP cleavage with accumulation of Abeta-immunoreactive products. These abnormalities were associated with activation of kinases that phosphorylate tau, including glycogen synthase kinase 3beta (GSK-3beta), mitogen-activated protein kinase (MAPK), and cyclin-dependent kinase 5 (Cdk-5). Further studies showed that significant neuro-protection with sparing of mitochondrial function and membrane integrity could be achieved by pre-treating the cortical neurons with N-acetyl cysteine, glutathione, or inhibitors of GSK-3beta, MAP kinase, or AbetaPP gamma-secretase. Therefore, in the absence of underlying gene mutations, oxidative stress can cause AD-type abnormalities, including aberrant post-translational processing of neuronal cytoskeletal proteins and APP. Our results also suggest that pre-treatment with agents that block specific components of the AD neurodegeneration cascade may provide neuroprotection against oxidative stress-induced impairments in membrane integrity, mitochondrial function, and viability.

Asunto(s)

Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide/metabolismo , Corteza Cerebral/patología , Quinasas Ciclina-Dependientes/metabolismo , Hipoxia/complicaciones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/patología , Animales , Anticuerpos Monoclonales/inmunología , Corteza Cerebral/embriología , Estrés Oxidativo/fisiología , Fosfotransferasas/inmunología , Fosfotransferasas/metabolismo , Ensayo de Radioinmunoprecipitación , Ratas , Factores de Tiempo

RESUMEN

Dementia in Alzheimer's disease (AD) is correlated with cell loss that is mediated by apoptosis, mitochondrial (Mt) dysfunction, and possibly necrosis. Previous studies demonstrated increased expression of the nitric oxide synthase 3 (NOS3) gene in degenerating neurons of AD brains. For investigating the role of NOS3 overexpression as a mediator of neuronal loss, human PNET2 central nervous system-derived neuronal cells were infected with recombinant adenovirus vectors that expressed either human NOS3 or green fluorescent protein cDNA under the control of a CMV promoter. NOS3 overexpression resulted in apoptosis accompanied by increased levels of p53, p21/Waf1, Bax, and CD95. In addition, NOS3 overexpression impaired neuronal Mt function as demonstrated by the reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and nicotinamide adenine dinucleotide (reduced form)-tetrazolium reductase activities and MitoTracker Red fluorescence. These adverse effects of NOS3 were associated with increased cellular levels of reactive oxygen species and impaired membrane integrity and were not produced in cells that were transfected with a cDNA encoding catalytically inactive NOS3. Importantly, modest elevations in NOS3 expression, achieved by infection with low multiplicities of adenovirus-NOS3 infection, did not cause apoptosis but rendered the cells more sensitive to oxidative injury by H(2)O(2) or diethyldithiocarbamate. In contrast, treatment with NO donors did not enhance neuronal sensitivity to oxidative injury. These results suggest that NOS3-induced neuronal death is mediated by Mt dysfunction, oxidative injury, and impaired membrane integrity, rather than by NO production, and that neuroprotection from these adverse effects of NOS3 may be achieved by modulating intracellular levels of oxidative stress.

Asunto(s)

Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Apoptosis , Mitocondrias/enzimología , Neuronas/enzimología , Neuronas/patología , Óxido Nítrico Sintasa/metabolismo , Enfermedad de Alzheimer/etiología , Células Cultivadas , Humanos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an approximately 150-165 kD glycoprotein that is mutated in individuals with cystic fibrosis. Previous studies demonstrated expression of the CFTR gene in the hypothalamus, suggesting a potential role for this molecule in the regulation of systemic metabolic functions. Individuals with cystic fibrosis often exhibit wasting and marked reductions in body fat content. Since the hypothalamus is a late target of neurodegeneration in Alzheimer's disease (AD), we postulated that patients with end-stage AD and bodily wasting would have reduced levels of CFTR expression in the hypothalamus. METHODS: CFTR mRNA and protein were examined in postmortem hypothalamic tissue from 11 AD and 7 aged controls using in situ hybridization and immunohistochemical staining. Standardized sections that included the supra-optic, paraventricular, anterior, and ventromedial nuclei, and the lateral hypothalamus were studied. RESULTS: The density of CFTR+ neurons and the intensity of the CFTR hybridization signals were strikingly reduced in AD. Immunohistochemical staining studies demonstrated CFTR immunoreactivity most prominently distributed in small clusters of neurites (5 to 20 in number). Digital image quantification showed that the density of CFTR+ neurites was significantly reduced in AD relative to aged control samples (P=0.001). However, there was no evidence for selective involvement of particular hypothalamic nuclei. CONCLUSIONS: CFTR gene expression is down-regulated and its corresponding immunoreactivity reduced in AD relative to control hypothalamic tissue. Reduced CFTR expression in the hypothalamus may represent an important mechanism by which AD neurodegeneration contributes to body wasting in the late stages of disease.

Asunto(s)

Enfermedad de Alzheimer/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Hipotálamo/patología , Anciano , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Técnicas de Cultivo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Hibridación in Situ , Neuronas/metabolismo

RESUMEN

Aspartyl (asparaginyl) beta-hydroxylase (AAH) is overexpressed in various malignant neoplasms, and high levels of immunoreactivity mainly occur in infiltrating or metastasized tumors. In addition, AAH is abundantly expressed in normally invasive placental trophoblastic cells. These observations led to the hypothesis that AAH may have a role in motility and aggressive behavior of tumor cells. The present study demonstrates that AAH is overexpressed in primary human malignant neuroectodermal tumors, including medulloblastomas and neuroblastomas, and that AAH expression is at a low level or undetectable in the normal mature brain. In the Sy5y neuroblastoma cell line, endogenous expression of the approximately 86-kd AAH protein was demonstrated by Western blot analysis, and immunoreactivity predominantly localized to the cell surface by immunocytochemical staining and FACS analysis. Sy5y cells that were stably transfected with the human AAH cDNA had increased levels of proliferating cell nuclear antigen and Bcl-2, and reduced levels of p21/Waf1 and p16. In addition, increased AAH expression enhanced Sy5y cell motility, whereas antisense oligodeoxynucleotide inhibition of AAH significantly reduced Sy5y cell motility and increased the levels of p21/Waf1 and p16. The findings suggest that AAH overexpression contributes to the malignant phenotype of neuroectodermal tumor cells by increasing motility and enhancing proliferation, survival, and cell cycle progression. Because AAH expression is at a low level or undetectable in normal brain, the AAH gene may be a target for treating primitive neuroectodermal tumors.

Asunto(s)

Movimiento Celular/genética , Oxigenasas de Función Mixta/genética , Neuroblastoma/genética , Anticuerpos Monoclonales , Citometría de Flujo , Humanos , Neuroblastoma/enzimología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Células Tumorales Cultivadas

RESUMEN

Directional motility and invasion assays are largely based on the use of Boyden chambers or Transwell culture inserts in which porous membranes separate seeded cells from a chemotactic factor supplied in the medium outside the chamber. The major obstaclefor most currently available assays is that they lack a sensitive, easy, and reliable method of quantifying the nonmotile cell populations. Failure to accountfor all cells within the assay chamber prohibits the determination of percentages of migrated cells. Here we describe an ATP luminescence-based motility-invasion (ALMI) assay that circumvents this problem, enabling investigators to quantify directional cell migration or invasiveness easily. The ALMI assay is based on the detection of ATP in viable cells harvested from inert surfaces that do not generate background signals. We demonstrate how the ALMI assay can be used to assess the effects of various experimental conditions such as growth factor stimulation and ethanol exposure on cell migration. In addition, precoating the membranes with extracellular matrix molecules enabled the measurement of the cell invasion. In conclusion, the ALMI assay provides a reliable and flexible method to quantify cell motility and invasiveness using a luminescence microplate reader.

Asunto(s)

Adenosina Trifosfato/análisis , Movimiento Celular/efectos de los fármacos , Invasividad Neoplásica/fisiopatología , Neuroglía/efectos de los fármacos , Animales , Bovinos , Adhesión Celular , Recuento de Células , Línea Celular , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados , Relación Dosis-Respuesta a Droga , Etanol/farmacología , Matriz Extracelular , Sangre Fetal/metabolismo , Insulina/farmacología , Mediciones Luminiscentes , Membranas Artificiales , Neuroglía/clasificación , Neuroglía/fisiología , Ratas , Receptor IGF Tipo 1/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad