RESUMEN

BRE (Brain and Reproductive Organ-Expressed) is an anti-apoptotic protein and a core component of DNA-repair BRCA1-A complex. Microarray-detected high BRE gene expression has been found to be associated with better patient survival in AML (acute myeloid leukemia) with MLL-AF9 translocation, and radiotherapy-treated non-familial breast cancer. A recent finding suggests that the high BRE gene expression in MLL-AF9 AML could be attributed to the additional expression of a transcript variant encoding a novel C-terminal BRE isoform. Using THP-1 as the MLL-AF9 AML cell model, we found that ectopic expression of the C-terminal BRE, which could not form an intact BRCA1-A complex, indeed increased cellular sensitivity to chemotherapeutic drugs and inhibited cell proliferation, while the complete opposite was achieved by the ectopic expression of full-length BRE. Our findings suggest that the C-terminal BRE-encoding transcript could be responsible for better patient survival and may have therapeutic potential for cancer.

Asunto(s)

Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Antineoplásicos/farmacología , Apoptosis/genética , Línea Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/metabolismo , Transducción de Señal/efectos de los fármacos , Translocación Genética

RESUMEN

The BRE gene, alias BRCC45, produces a 44 kDa protein that is normally distributed in both cytoplasm and nucleus. In this study, we used adult fibroblasts isolated from wild-type (WT) and BRE knockout (BRE(-/-)) mice to investigate the functional role of BRE in DNA repair and cellular senescence. We compared WT with BRE(-/-) fibroblasts at different cell passages and observed that the mutant fibroblasts entered replicative senescence earlier than the WT fibroblasts. With the use of gamma irradiation to induce DNA damage in fibroblasts, the percentage of SA-ß-Gal(+) cells was significantly higher in BRE(-/-) fibroblasts compared with WT cells, suggesting that BRE is also associated with DNA damage-induced premature senescence. We also demonstrated that the gamma irradiation induced γ-H2AX foci, a DNA damage marker, persisted significantly longer in BRE(-/-) fibroblasts than in WT fibroblasts, confirming that the DNA repair process is impaired in the absence of BRE. In addition, the BRCA1-A complex recruitment and homologous recombination (HR)-dependent DNA repair process upon DNA damage were impaired in BRE(-/-) fibroblasts. Taken together, our results demonstrate a role for BRE in both replicative senescence and DNA damage-induced premature senescence. This can be attributed to BRE being required for BRCA1-A complex-driven HR DNA repair.

Asunto(s)

Daño del ADN , Replicación del ADN , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Animales , Células Cultivadas , Senescencia Celular , Genes BRCA1 , Ratones

RESUMEN

Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.

Asunto(s)

Apoptosis/fisiología , Carcinoma Pulmonar de Lewis/metabolismo , Inhibidores de Caspasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Proliferación Celular , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares , Proteína Inhibidora de la Apoptosis Ligada a X/genética

RESUMEN

BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-ß/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.

Asunto(s)

Diferenciación Celular , Condrogénesis , Silenciador del Gen , Proteínas del Tejido Nervioso/metabolismo , Osteogénesis , Células Madre/citología , Cordón Umbilical/citología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Quimiocinas/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Proteínas del Citoesqueleto/metabolismo , Epigénesis Genética , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/citología , Osteoblastos/metabolismo , Proteoma/metabolismo , Proteómica , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Cordón Umbilical/irrigación sanguínea

RESUMEN

Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) opens up the possibility of converting malignant cells into any cell type, including those best suited to be developed as cancer vaccines. Mouse models are needed to evaluate and optimize the therapeutic efficacy of such novel cancer vaccines. However, only human cancer cell lines have been reported as being reprogrammed into iPSCs. Here, we report a proof-of-principle study which shows that mouse cancer cells can be reprogrammed into iPSCs that are capable of subsequent differentiation. Four canonical reprogramming transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, were introduced by plasmid transfection into mouse Lewis lung carcinoma D122 harboring Nanog-GFP reporter. Green fluorescent cells were found clustered into embryonic stem cell (ESC)-like colonies expressing ESC markers, Oct4 and SSEA-1. Bisulfite genomic sequencing analyses of these cells revealed hypomethylation of the Nanog promoter. The expression of a host of pluripotency genes by these reprogrammed cells at levels similar to those of ESCs was confirmed by quantitative real-time PCR. Functional pluripotency of the reprogrammed cells was demonstrated by their ability to form embryoid bodies and differentiate into neuronal progenitors on retinoic acid treatment. This study indicates the feasibility of developing iPSC-based experimental cancer vaccines for immunotherapy in mouse models.

Asunto(s)

Carcinoma Pulmonar de Lewis/patología , Células Madre Pluripotentes Inducidas/citología , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Diferenciación Celular/fisiología , Reprogramación Celular , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Factor 4 Similar a Kruppel , Ratones , Transfección

RESUMEN

Co-deletion of chromosomes 1p and 19q is a common event in oligodendroglial tumors (OTs), suggesting the presence of OT-related genes. The aim of this study was to identify the target genes residing in the minimally deleted regions on chromosome 1p36.31-p36.32 that might be involved in OTs. A novel gene KIAA0495/p53-dependent apoptosis modulator (PDAM) was found frequently deregulated, with 37 of 58 (63.8%) OTs examined showing reduced expression compared with normal brain. Chromosome 1p loss and epigenetic modifications were the major mechanisms contributing to PDAM downregulation. The role of PDAM in chemosensitivity was also evaluated. PDAM knockdown had no effect on sensitivity to vincristine, lomustine, temozolomide and paclitaxel, but could induce cisplatin resistance in glioma cells harboring wild-type p53. B-cell CCL/lymphoma 2 (BCL2)-like 1 (BCL2L1) exhibited significant upregulation, while BCL2 showed partial derepression in PDAM-silenced cells after cisplatin treatment, suggesting that alteration of anti-apoptotic genes contributed in part to cisplatin resistance. Knockdown of BCL2L1 abrogated the induced cisplatin-resistant phenotype. Moreover, our data suggested that PDAM might function as a non-protein-coding RNA. Collectively, these findings suggest that PDAM deregulation may play a role in OT development and that PDAM may possess the capacity to modulate apoptosis via regulation of p53-dependent anti-apoptotic genes.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Oligodendroglioma/metabolismo , ARN Largo no Codificante/fisiología , ARN Interferente Pequeño/farmacología , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Antineoplásicos/farmacología , Línea Celular Tumoral , Niño , Cisplatino/farmacología , Regulación hacia Abajo/efectos de los fármacos , Interacciones Farmacológicas , Femenino , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Sensación Térmica/genética , Transfección , Proteínas Supresoras de Tumor/genética , Adulto Joven

RESUMEN

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.

Asunto(s)

Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Hígado/patología , Proteínas del Tejido Nervioso/biosíntesis , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Dietilnitrosamina/toxicidad , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Transfección , Regulación hacia Arriba

RESUMEN

Stress-responsive genes play critical roles in many biological functions that includes apoptosis, survival, differentiation and regeneration. We have identified a novel stress-responsive gene called BRE which interacts with TNF-receptor-1 and blocks the apoptotic effect of TNF-alpha. BRE enhances tumor growth in vivo and is up-regulated in hepatocellular and esophageal carcinomas. BRE also regulates the ubiquitination of the DNA repair complex BRCC, and the synthesis of steroid hormones. Here, we examined BRE-mRNA in cells after treatments with UV and ionizing radiation (IR). UV and IR treatment alone suppressed BRE-mRNA levels by more than 90% at 24 h, while hydroxyurea, fluorodeoxyuridine, aphidicolin, known inhibitors of S-phase DNA synthesis, had no significant effect. BRE protein expression was unaltered in cells treated with TNF-alpha, Interleukin-1 and Dexamethasone, while a threefold increase was observed following chorionic gonadotropin exposure. Although BRE plays a regulatory role in many different pathways, yet its expression is apparently under very stringent control.

Asunto(s)

Factores Biológicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Afidicolina/farmacología , Cadaverina/análogos & derivados , Cadaverina/farmacología , Línea Celular , Gonadotropina Coriónica/farmacología , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Dexametasona/farmacología , Dimetilsulfóxido/farmacología , Fluorodesoxiuridilato/farmacología , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Hidroxiurea/farmacología , Interleucina-1/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Estrés Fisiológico/efectos de la radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Rayos Ultravioleta

RESUMEN

OBJECTIVE: To evaluate the contraceptive ability of a synthetic Bin1b peptide in vivo in the rat. DESIGN: Basic research. SETTING: University laboratory animal service center. ANIMAL(S): A peptide-based immunization model was developed; rats were injected with the Bin1b specific peptide. INTERVENTION(S): A synthetic peptide segment, MCRSGERKGDICSDP-conjugated with KLH (Bin1b), was used to immunize male wistar rats. Freund's complete adjuvant was used as a control. MAIN OUTCOME MEASURE(S): Anti-Bin1b levels in sera were evaluated by enzyme-linked immunosorbent assay (ELISA). Anti-Bin1b and control antisera were used to evaluate sperm function inhibition in vitro. The fertility of immunized rats was determined by mating experiment. The testis and epididymides were analyzed by histology. RESULT(S): Histological studies showed no evidence of orchitis or epididymitis in Bin1b-immunized animals. ELISA results revealed that the titers of anti-Bin1b antibodies in serum increased with the immunization process in immunized rats. Sperm recovered from the corpus epididymidis of the Bin1b-immunized animals exhibited a significant decrease in motility. Immunization of Bin1b also caused a reduction (25%) in fertility after the mating experiment. CONCLUSION(S): The present study has demonstrated that immunization with Bin1b peptide specifically interferes with sperm motility, resulting in a compromised fertilizing capacity of sperm.

Asunto(s)

Anticoncepción Inmunológica/métodos , Anticonceptivos Masculinos/inmunología , Anticonceptivos Masculinos/farmacología , Motilidad Espermática/efectos de los fármacos , beta-Defensinas/inmunología , beta-Defensinas/farmacología , Reacción Acrosómica/efectos de los fármacos , Reacción Acrosómica/inmunología , Animales , Anticuerpos/sangre , Especificidad de Anticuerpos , Peso Corporal/efectos de los fármacos , Epidídimo/efectos de los fármacos , Femenino , Fertilidad/efectos de los fármacos , Masculino , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Wistar , Motilidad Espermática/inmunología

RESUMEN

Esophageal tumorigenesis is a complex and cascading process, involving the interaction of many genes and proteins. In this study, we have used the comparative proteomic approach to identify tumor-associated proteins and explore the carcinogenic mechanisms. Two-dimensional electrophoresis (2-DE) and MALDI-TOF MS analysis of esophageal carcinoma and control cells revealed 10 proteins that were upregulated. A further 10 proteins were downregulated. Among these 20 differentially expressed proteins, brain and reproductive organ-expressed (BRE) protein was identified as a potential tumor promoter. It was high expressed by the esophageal carcinoma cells, as confirmed by RT-PCR and immunoblotting. BRE has been reported to be a stress-responsive protein. To gain further insight into its function, BRE expression was silenced in esophageal carcinoma cells using BRE-specific small interference RNA. It was discovered that silencing BRE expression downregulated prohibitin expression, but upregulated tumor-suppressor p53 expression. Furthermore, cyclin A and CDK2 expressions were suppressed suggesting that BRE inhibited cell proliferation. These results implied that BRE plays a significant role in mediating antiapoptotic and proliferative responses in esophageal carcinoma cells.

Asunto(s)

Neoplasias Esofágicas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteómica , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Cartilla de ADN , Electroforesis en Gel Bidimensional , Neoplasias Esofágicas/patología , Humanos , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Epstein-Barr virus (EBV) latent infection is a critical event in nasopharyngeal carcinoma (NPC) tumorigenesis. EBV-encoded genes have been shown to be involved in immune evasion and in the regulation of various cellular signaling cascades. To elucidate the roles of EBV in NPC development, stable infection of EBV in nasopharyngeal epithelial cell lines was established. Similar to primary tumors of NPC, these infected cells exhibited a type II EBV latency expression pattern. In this study, multiple cellular signaling pathways in EBV-infected cells were investigated. We first demonstrated that in vitro EBV infection resulted in the activation of STAT3 and NFkappaB signal cascades in nasopharyngeal epithelial cells. Increased expression of their downstream targets (c-Myc, Bcl-xL, IL-6, LIF, SOCS-1, SOCS-3, VEGF, and COX-2) was also observed. Moreover, EBV latent infection induced the suppression of p38-MAPK activities, but did not activate PKR cascade. Our findings suggest that EBV latent infection is able to manipulate multiple cellular signal cascades to protect infected cells from immunologic attack and to facilitate cancer development.

Asunto(s)

Carcinoma/patología , Transformación Celular Viral , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/fisiopatología , Herpesvirus Humano 4/fisiología , Neoplasias Nasofaríngeas/patología , Nasofaringe/citología , Transducción de Señal , Carcinoma/inmunología , Carcinoma/virología , Línea Celular/fisiología , Línea Celular/virología , Línea Celular Tumoral , Citocinas/biosíntesis , Citocinas/genética , Células Epiteliales/fisiología , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Inflamación , Sistema de Señalización de MAP Quinasas , FN-kappa B/fisiología , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/virología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/fisiología , Latencia del Virus , eIF-2 Quinasa/fisiología

RESUMEN

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.

Asunto(s)

Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/fisiología , Proteómica/métodos , Proteínas Represoras/biosíntesis , Proteína p53 Supresora de Tumor/química , Animales , Anhidrasa Carbónica III/biosíntesis , Línea Celular , Proliferación Celular , Ratones , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/química , Prohibitinas , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , ARN Interferente Pequeño/metabolismo

RESUMEN

We previously demonstrated that the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) potently activates the cellular c-Jun N-terminal kinase (JNK) pathway by sequentially engaging an unknown adaptor, TRAF6, TAB1/TAK1, and JNKKs. We now show that BS69, a MYND domain-containing cellular protein, is the missing adaptor that bridges LMP1 and TRAF6, as the MYND domain and a separate region of BS69 bind to the carboxyl termini of LMP1 and TRAF6, respectively. While LMP1 promotes the interaction between BS69 and TRAF6, the complex formation between LMP1 and TRAF6 is BS69 dependent. A fraction of LMP1 and BS69 is constitutively colocalized in the membrane lipid rafts. Importantly, knockdown of BS69 by small interfering RNAs specifically inhibits JNK activation by LMP1 but not tumor necrosis factor alpha. Although overexpression of either BS69 or a mutant LMP1 without the cytoplasmic carboxyl tail is not sufficient to activate JNK, interestingly, when BS69 is covalently linked to the mutant LMP1, the chimeric protein restores the ability to activate JNK. This indicates that the recruitment and aggregation of BS69 is a prerequisite for JNK activation by LMP1.

Asunto(s)

Proteínas Portadoras/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , Proteínas Co-Represoras , Proteínas de Unión al ADN , Activación Enzimática , Humanos , Microdominios de Membrana/metabolismo , Ratones , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/fisiología , Técnicas del Sistema de Dos Híbridos

RESUMEN

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation.

Asunto(s)

Neoplasias/metabolismo , Neoplasias/patología , Proteínas del Tejido Nervioso/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Expresión Génica , Masculino , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias/genética , Proteínas del Tejido Nervioso/genética

RESUMEN

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.

Asunto(s)

Apoptosis/fisiología , Proteínas del Tejido Nervioso/fisiología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Núcleo Celular/metabolismo , Cicloheximida/farmacología , Citosol/metabolismo , Etopósido/farmacología , Expresión Génica , Células HeLa , Humanos , Células Jurkat , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/metabolismo

RESUMEN

OBJECTIVE: To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]). METHODS: The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF. RESULTS: The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp-2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5'-triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5'-monophosphate or cytidylyl (3'-5') guanosine, and, in turn, was better than to guanosine, while N7-methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp-2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils. CONCLUSION: A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto-unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single-strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.

Asunto(s)

Anticuerpos Antinucleares/sangre , ADN/inmunología , Ensayo de Inmunoadsorción Enzimática/normas , Guanosina Trifosfato/inmunología , Lupus Eritematoso Sistémico/inmunología , Especificidad de Anticuerpos , Niño , ADN/metabolismo , Reacciones Falso Positivas , Femenino , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Solubilidad

RESUMEN

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a common cancer in Hong Kong. The EBV-encoded LMP1 protein is believed to play an important role in cell transformation. We have previously identified a prevalent LMP1 variant (2117-LMP1) that is expressed in 86% of primary NPC in Hong Kong. In this study, the biologic phenotypes induced by 2117-LMP1 were compared with those of the prototypic B95.8-LMP1 in an immortalized nasopharyngeal epithelial cell line, NP69. The 2117-LMP1 could induce cell proliferation and resistance to apoptosis induced by growth factor deprivation. Expression of 2117-LMP1 also suppressed expression of p16, p21 and Bax but induced expression of CDK2 and A20. Compared with B95.8-LMP1, 2117-LMP1 could induce a higher migration ability in NP69 cells but was less efficient in inducing morphologic changes, anchorage-independent growth and cell invasion. Relatively weaker ability of 2117-LMP1 than B95.8-LMP1 in upregulation of vimentin, VEGF and MMP9 as well as in downregulation of E-cadherin was observed. 2117-LMP1 could activate higher level of NF-kappaB activity in HEK 293 cells than B95.8-LMP1. The present study supports a role of 2117-LMP1 in NPC development by enhancing cell proliferation, cell death inhibition and migration in premalignant nasopharyngeal epithelial cells. Furthermore, our study reveals significant functional differences between 2117-LMP1 and the prototypic B95.8-LMP1. Our results provide insights into the pathologic significance of this prevalent LMP1 variant, 2117-LMP1, in the development of NPC in the Hong Kong population.

Asunto(s)

Herpesvirus Humano 4/fisiología , Neoplasias Nasofaríngeas/virología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas de la Matriz Viral/fisiología , Quinasas CDC2-CDC28/metabolismo , Cadherinas/metabolismo , División Celular , Movimiento Celular , Supervivencia Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN , Hong Kong , Humanos , Péptidos y Proteínas de Señalización Intracelular , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Proteínas Nucleares , Fenotipo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Tumorales Cultivadas , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína X Asociada a bcl-2

RESUMEN

Epstein-Barr virus (EBV) is associated with several human diseases including infectious mononucleosis and nasopharyngeal carcinoma. EBV-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for cellular transformation caused by EBV. Expression of LMP1 in host cells constitutively activates both the c-Jun N-terminal kinase (JNK) and NF-kappaB pathways, which contributes to the oncogenic effect of LMP1. However, the underlying signaling mechanisms are not very well understood. Based mainly on overexpression studies with various dominant-negative constructs, LMP1 was generally thought to functionally mimic members of the tumor necrosis factor (TNF) receptor superfamily in signaling. In contrast to the prevailing paradigm, using embryonic fibroblasts from different knockout mice and the small interfering RNA technique, we find that the LMP1-mediated JNK pathway is distinct from those mediated by either TNF-alpha or interleukin-1. Moreover, we have further elucidated the LMP1-mediated JNK pathway by demonstrating that LMP1 selectively utilizes TNF receptor-associated factor 6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 to activate JNK.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Herpesvirus Humano 4/metabolismo , MAP Quinasa Quinasa 4 , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Portadoras/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 7 , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Estructura Secundaria de Proteína , Proteínas/metabolismo , Factor 6 Asociado a Receptor de TNF

RESUMEN

Mouse Bre, an evolutionarily conserved stress-modulating gene, like its human counterpart, is expressed in multiple alternative transcripts. The main transcript, which is ubiquitously expressed, encodes a protein that binds tumor necrosis factor receptor 1 (TNF-R1) and downregulates TNF-induced activation of NF-kappaB. Alternative splicing of mouse Bre occurs only at the 5' region of the gene, generating either nonfunctional transcripts or transcripts that can encode putative protein isoforms differ at the N-terminal sequence. In contrast, alternative splicing of human BRE occurs at either or both ends of the gene; only the 3' alternative splicing can generate functional transcripts that encode putative protein isoforms differ at the C-terminus, occurrence of the 5' alternative splicing only results in forming nonfunctional transcripts. Unlike the human BRE alternative transcripts which are coexpressed at considerable levels with the main transcript, the mouse counterparts are expressed in a restricted pattern and generally in low abundance except in the heart. Both species, however, share a type of Bre alternative transcripts generated by cryptic splicing at a nonstandard, noncanonical acceptor site. Thus, a highly conserved gene in two species can generate alternative transcripts different in both of the sequence structure and expression pattern, as well as a similar class of transcripts resulting from unconventional transcript processing.

Asunto(s)

Proteínas del Tejido Nervioso/genética , Empalme Alternativo , Animales , Northern Blotting , Secuencia Conservada , Expresión Génica , Humanos , Ratones , Miocardio , Proteínas Nucleares , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas