RESUMEN
Epstein-Barr virus (EBV) is an oncogenic gamma-herpes virus associated with malignancies that develop in both lymphoid and epithelial cells including nasopharyngeal carcinoma (NPC). The EBV protein, latent membrane protein 2A (LMP2A), is expressed in NPC and can modulate epithelial proliferation, transformation and differentiation, and as such may promote malignancy. A key regulator of epithelial-cell differentiation is the transcription factor p63, a member of the p53 family. This study examines the potential contribution of p63 to LMP2A-mediated inhibition of epithelial-cell differentiation. Stable expression of LMP2A increased the protein level and stability of the DeltaNp63alpha isoform and in two epithelial cell lines, LMP2A interacted with DeltaNp63alpha under stable- and transient-expression systems. LMP2A and DeltaNp63alpha were localized to the cytoplasm and nuclear membrane and co-immunoprecipitated in the same fractions. Following induction of epithelial-cell differentiation by calcium, expression of differentiation markers was impaired in both DeltaNp63alpha- and LMP2A-expressing cells. Induction of p63alpha, association of p63alpha with LMP2A and impairment of differentiation required the PY and immunoreceptor tyrosine-based activation motif (ITAM) signaling motif of LMP2A. By associating with and being regulated by LMP2A, DeltaNp63alpha may function as a unique regulator of LMP2A effects on epithelial differentiation and contribute to EBV-associated epithelial cancers.
Asunto(s)
Células Epiteliales/patología , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Matriz Viral/fisiología , Calcio/fisiología , Diferenciación Celular , Línea Celular , Humanos , Transactivadores/análisis , Transactivadores/química , Factores de Transcripción , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/química , Proteínas de la Matriz Viral/análisisRESUMEN
Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.
Asunto(s)
Genes rel/fisiología , Linfoma , FN-kappa B/metabolismo , Transducción de Señal , Proteínas de la Matriz Viral/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Fosfatidilinositol 3-Quinasa Clase I , Proteínas del Citoesqueleto , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas con Dominio LIM , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Superficie Celular/metabolismo , Bazo/metabolismo , Factor 1 Asociado a Receptor de TNF/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIB/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de la Matriz Viral/genéticaRESUMEN
The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) functions to maintain latency in EBV-infected B lymphocytes. Although LMP2A is nonessential for the immortalization of B lymphocytes by EBV, its expression in B lymphocytes prevents viral reactivation by blocking B-cell receptor activation and signaling. LMP2A also provides an antiapoptotic signal in transgenic mice that express LMP2A in B lymphocytes. LMP2A activates phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase Akt in lymphocytes and epithelial cells. Here we show that EBV LMP2A activates the PI3K and beta-catenin signaling pathways in telomerase-immortalized human foreskin keratinocytes (HFK). LMP2A activated Akt in a PI3K-dependent manner, and the downstream Akt targets glycogen synthase kinase 3beta (GSK3beta) and the Forkhead transcription factor FKHR were phosphorylated and inactivated in LMP2A-expressing HFK cells. GSK3beta is a negative regulator of the Wnt signaling pathway, and inactivation of GSK3beta by LMP2A signaling led to stabilization of beta-catenin, the central oncoprotein of Wnt signaling. In LMP2A-expressing cells, beta-catenin accumulated in the cytoplasm and translocated into the nucleus via a two-step mechanism. The cytoplasmic accumulation of beta-catenin downstream of LMP2A was independent of PI3K signaling, whereas its nuclear translocation was dependent on PI3K signaling. In the nucleus, beta-catenin activated a reporter responsive to T-cell factor, and this activation was augmented by LMP2A coexpression. The Wnt pathway is inappropriately activated in 90% of colon cancers and is dysregulated in several other cancers, and these data suggest that activation of this pathway by LMP2A may contribute to the generation of EBV-associated cancers.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal/fisiología , Transactivadores/metabolismo , Proteínas de la Matriz Viral/fisiología , Transporte Activo de Núcleo Celular , Células Cultivadas , Proteínas de Unión al ADN/fisiología , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Herpesvirus Humano 4/patogenicidad , Humanos , Queratinocitos/metabolismo , Factor de Unión 1 al Potenciador Linfoide , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Telomerasa/genética , Factores de Transcripción/fisiología , beta CateninaRESUMEN
Opportunistic DNA viruses, particularly members of the herpesvirus family, are frequently the aetiological agents of HIV-associated oral lesions. Oral lesions common to the early phase of the AIDS epidemic, including Kaposi's sarcoma (KS), oral aphthous ulceration, AIDS-associated oral lymphoma, and oral hairy leukoplakia (OHL), have been tested for the prevalence of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). While EBV DNA is detected by PCR in all of these lesions, abundant viral replication can only be detected in OHL. In OHL, a novel state of EBV infection has been discovered with concurrent expression of replicative and transforming proteins, with all of these proteins contributing to the development of the lesion. Activation of signalling pathways and up-regulation of the viral receptor, proliferative and antiapoptotic genes by these proteins induce several of the histological features common to OHL, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected OHL tissue enables epithelial cell survival and may enhance viral replication. Detection of KSHV in these HIV-infected individuals has been localized only to their saliva. Replicative and latent KSHV gene products have been detected in association with the development of oral KS lesions. EBV, but not human cytomegalovirus (HCMV), has been detected by PCR in minor salivary gland biopsies of HIV-associated salivary gland disease. Human papillomaviruses (HPV) are associated with oral warts in HIV-positive individuals; a diagnosis that appears to be increasing in frequency in the era of highly active antiretroviral therapy. To date, there appears to be little increase in the incidence of HPV-associated oral cancer. The mechanisms of interaction between HIV and HPV are not fully understood. Expression of viral gene products is clearly important and necessary for the development of multiple AIDS-associated oral lesions.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/virología , Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/fisiología , Enfermedades de la Boca/virología , Papillomaviridae/fisiología , Apoptosis/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas Portadoras/análisis , División Celular/fisiología , Supervivencia Celular , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/complicaciones , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por VIH/complicaciones , Humanos , Leucoplasia Vellosa/virología , Linfoma Relacionado con SIDA/virología , Mucosa Bucal/virología , Neoplasias de la Boca/virología , Proteínas Oncogénicas Virales/análisis , Infecciones por Papillomavirus/complicaciones , Receptores Virales/fisiología , Proteínas Represoras , Saliva/virología , Enfermedades de las Glándulas Salivales/virología , Sarcoma de Kaposi/virología , Transducción de Señal/fisiología , Estomatitis Aftosa/virología , Regulación hacia Arriba/fisiología , Proteínas Virales/análisis , Replicación Viral , Verrugas/virologíaRESUMEN
Nasopharyngeal carcinoma (NPC) is universally associated with EBV infection. We have shown that the phosphonated nucleoside analog, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)-propyl]cytosine (HPMPC) strongly inhibits growth of NPC xenografts in nude mice by causing apoptosis (J. Neyts et al., Cancer Res., 58, 384-388, 1998). We, therefore, tested two additional members of this drug family that have different degrees of antiviral activity, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-2-(R)-(phosphonomethoxy)propyladenine (PMPA). Intratumoral injection of PMEA (75 microl of 2% solution) in C15 NPC xenografts, which are latently infected with EBV, slowed tumor growth moderately, whereas PMPA (75 microl of 2% solution) slowed tumor growth only marginally. Compared with the previous results showing complete regression of tumor, PMEA had less antitumoral effect than HPMPC, and PMPA had the least. After 4 weeks of preventive treatment, tumors formed in 12.5, 50, and 100% of mice treated with HPMPC, PMEA, and PMPA, respectively, in contrast to the development of tumors in all of the PBS-treated control mice. We also investigated the effect of each drug on the EBV-positive epithelial cell line NPC-KT in vitro. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed inhibition of growth of NPC-KT cells by HPMPC and PMEA, but not by PMPA, which correlates with the results observed in tumor xenografts. Growth inhibition was attributable to induction of apoptosis in NPC-KT cells as indicated by a DNA fragmentation assay. Cleavage of poly(ADP-ribose) polymerase after treatment of NPC-KT cells with HPMPC was observed, which suggested that the apoptosis may be mediated by caspase(s). The apoptotic effects of the drugs are independent of any effects on EBV DNA polymerase, which is not expressed in these latently infected NPCs. These results suggest that HPMPC as well as PMEA could provide an adjunctive treatment for NPC.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacología , Antineoplásicos/farmacología , Carcinoma/tratamiento farmacológico , Citosina/análogos & derivados , Neoplasias Nasofaríngeas/tratamiento farmacológico , Organofosfonatos , Compuestos Organofosforados/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma/patología , Carcinoma/prevención & control , División Celular/efectos de los fármacos , Cidofovir , Citosina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Inhibidores de Crecimiento/farmacología , Herpesvirus Humano 4 , Humanos , Ratones , Ratones Desnudos , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/prevención & control , Tenofovir , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Epstein-Barr virus (EBV) BamHI A mRNAs were originally identified in cDNA libraries from nasopharyngeal carcinoma, where they are expressed at high levels. The RNAs are differentially spliced to form several open reading frames and also contain the BARF0 open reading frame at the 3' end. One cDNA, RK-BARF0, included a potential endoplasmic reticulum-targeting signal peptide sequence. The RK-BARF0 protein is shown here to interact with the Notch4 ligand binding domain, using yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy. This interaction induces translocation of a portion of the full-length unprocessed Notch4 to the nucleus by using the Notch nuclear localization signal. These effects of RK-BARF0 on Notch intracellular location indicate that EBV possibly modulates Notch signaling. Unprocessed Notch4 was also detected in immunoprecipitated complexes from EBV-infected cells by using a rabbit antiserum raised against a BARF0-specific peptide. This finding provides additional evidence for expression of RK-BARF0 and its interaction with Notch during EBV infection. In EBV-infected, EBNA2-negative cells, RK-BARF0 induced the expression of EBV latent membrane protein 1 (LMP1), and this induction was dependent on the RK-BARF0/Notch interaction domain. The activation of LMP1 expression by RK-BARF0 may be responsible for expression of LMP1 in EBV latent infections in the absence of EBNA2.
Asunto(s)
Herpesvirus Humano 4/fisiología , Proteínas de la Membrana/fisiología , Proteínas Virales/fisiología , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Sistemas de Lectura Abierta , Conejos , Receptores Notch , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/biosíntesis , Proteínas Virales/químicaRESUMEN
LMP2A deletion and point mutants, with mutations in phosphotyrosine-containing protein-protein interaction motifs, were transiently expressed in 293 cells and their phosphorylation was examined in immune complex kinase assays as well as in vivo. In vitro LMP2A phosphorylation depended on tyrosine 112. In vivo, mutations of single tyrosines did not eliminate LMP2 phosphorylation, although mutation of the LMP2A ITAM decreased LMP2A phosphorylation. The relationship between LMP2A in vitro phosphorylation and that induced by cell-extracellular matrix (ECM) interactions was also investigated. While LMP2A was phosphorylated to higher levels in whole-cell extracts of stimulated cells, a difference in in vitro kinase assays with extracts from stimulated and unstimulated cells was not detected, indicating that the ECM-mediated regulation of LMP2A phosphorylation is lost in vitro. In the presence of LMP2A, several cellular proteins with molecular weights between 70 and 80 kDa were phosphorylated on tyrosine. This increase in cellular protein phosphorylation depended on the LMP2A ITAM motif and suggests that the ITAM may participate in signal-transduction events in epithelial cells.
Asunto(s)
Herpesvirus Humano 4/metabolismo , Proteínas de la Matriz Viral/metabolismo , Sitios de Unión , Línea Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Humanos , Mutagénesis , Fosforilación , Tirosina/genética , Tirosina/metabolismo , Proteínas de la Matriz Viral/genética , Familia-src Quinasas/metabolismoRESUMEN
The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase-Akt pathway.
Asunto(s)
Diferenciación Celular , Transformación Celular Neoplásica , Transformación Celular Viral , Herpesvirus Humano 4/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de la Matriz Viral/fisiología , Animales , División Celular , Línea Celular , Trasplante de Células , Colágeno , Células Epiteliales/virología , Fibroblastos , Humanos , Queratinocitos/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Técnicas de Cultivo de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Retroviridae/genética , Transducción de Señal , Proteínas de la Matriz Viral/metabolismoRESUMEN
Human herpesviruses are characterized by distinct states of infection. Typically in permissive herpesvirus infection, abundant virus production results in cell lysis. In latent transforming Epstein-Barr virus (EBV) infection, viral proteins that induce cell growth are expressed. The immunodeficiency-associated hairy leukoplakia (HLP) lesion is the only pathologic manifestation of permissive EBV infection; however, within HLP, viral proteins characteristic of latent infection have also been detected. In this study, we further analyzed expression of EBV latent genes and investigated their contribution to the unique histologic phenotype of HLP. Coexpression of lytic and transforming viral proteins was detected simultaneously within individual HLP keratinocytes. LMP1 has now been shown to be uniformly expressed in the affected tissue, and it is associated and colocalizes with tumor necrosis factor receptor-associated factor (TRAF) signaling molecules. Effects induced by activated TRAF signaling that were detected in HLP included activation of NF-kappaB and c-Jun terminal kinase 1 (JNK1) and upregulated expression of epidermal growth factor receptor (EGFR), CD40, A20, and TRAFs. This study identifies a novel state of EBV infection with concurrent expression of replicative and transforming proteins. It is probable that both replicative and latent proteins contribute to HLP development and induce many of the histologic features of HLP, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected HLP tissue enables epithelial cell survival and may enhance viral replication.
Asunto(s)
Transformación Celular Viral , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Leucoplasia Vellosa/patología , Leucoplasia Vellosa/virología , Infecciones Oportunistas Relacionadas con el SIDA/virología , Infecciones por Virus de Epstein-Barr/patología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Lengua/metabolismo , Lengua/virología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Activación Viral , Latencia del Virus/genética , Replicación ViralRESUMEN
Air pollution is a serious health problem in major cities in Mexico. The concentrations of monitored criteria pollutants have been above the US National Ambient Air Quality Standards for the last decade. To determine whether the number of primary malignant nasal and paranasal neoplasms has increased, we surveyed 256 such cases admitted to a major adult oncology hospital located in metropolitan Mexico City (MMC) for the period from 1976-1997 and to a tertiary hospital in Monterrey, an industrial city, for the period from 1993-1998. The clinical histories and histopathologic material were reviewed, and a brief clinical summary was written for each case. In the MMC hospital the number of newly diagnosed nasal and paranasal neoplasms per year for the period from 1976-1986 averaged 5.1, whereas for the next 11 years it increased to 12.5. The maximal increase was observed in 1995-1997, with an average of 20.3 new cases per year (P = 0.0006). The predominant neoplasms in these series were non-Hodgkin's lymphoma, squamous cell carcinoma, melanoma, adenocarcinoma, Schneiderian carcinoma, and nasopharyngeal carcinoma. In the Monterrey hospital a 2-fold increase in the numbers of newly diagnosed nasal and paranasal neoplasms was recorded between 1993 and 1998. The predominant MMC neoplasm in this series, namely nasal T-cell/natural killer cell non-Hodgkin's lymphoma, is potentially Epstein-Barr virus related. Nasal and paranasal malignant neoplasms are generally rare. Environmental causative factors include exposure in industries such as nickel refining, leather, and wood furniture manufacturing. Although epidemiologic studies have not addressed the relationship between outdoor air pollution and sinonasal malignant neoplasms, there is strong evidence for the nasal and paranasal carcinogenic effect of occupational aerosol complex chemical mixtures. General practitioners and ear, nose, and throat physicians working in highly polluted cities should be aware of the clinical presentations of these patients. Identification of this apparent increase in sinonasal malignant neoplasms in two urban Mexican polluted cities warrants further mechanistic and epidemiologic studies.
Asunto(s)
Contaminación del Aire/efectos adversos , Cavidad Nasal , Neoplasias Nasales/epidemiología , Neoplasias de los Senos Paranasales/epidemiología , Adenocarcinoma/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , California/epidemiología , Carcinoma/epidemiología , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Transicionales/epidemiología , Femenino , Humanos , Linfoma no Hodgkin/epidemiología , Masculino , Melanoma/epidemiología , México/epidemiología , Persona de Mediana Edad , Neoplasias Nasales/etiología , Exposición Profesional , Neoplasias de los Senos Paranasales/etiologíaRESUMEN
Nasopharyngeal carcinoma (NPC) is endemic in parts of southern China and is etiologically associated with Epstein-Barr virus (EBV) infection as well as other dietary and environmental factors. Loss of heterozygosity (LOH) on chromosome 3p has been described in NPC from the endemic region. In this study, tumors originating from both the NPC nonendemic area of northern China and the endemic area in southern China were analyzed for LOH at 8 microsatellite markers on chromosome 3. Allele loss was detected at D3S1300 in 3p14.2 in more than 50% of tumors from both the endemic and nonendemic areas, suggesting that LOH at this locus probably does not account for the endemicity of NPC in southern China. The 3p14.2 region encompasses FHIT, a candidate tumor suppressor gene previously shown to be rearranged in several NPC cell lines. In this study, analysis of FHIT gene structure and transcription in primary tumors did not support a role for FHIT in NPC. However, the high frequency of allele loss at 3p14.2 in NPC from endemic and nonendemic regions supports the possibility that an important tumor suppressor gene other than FHIT complements EBV transformation and resides in this region.
Asunto(s)
Ácido Anhídrido Hidrolasas , Cromosomas Humanos Par 3 , Enfermedades Endémicas , Neoplasias Nasofaríngeas/epidemiología , Neoplasias Nasofaríngeas/genética , Proteínas de Neoplasias , Alelos , China , Infecciones por Virus de Epstein-Barr , Eliminación de Gen , Genes Supresores de Tumor , Humanos , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Proteínas/genéticaRESUMEN
The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) interacts with the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) molecules, which are important for LMP1-mediated signaling. Two domains of LMP1 can independently activate NF-kB, carboxyl-terminal activating region 1 (CTAR1) and CTAR2. The activation of NF-kB by CTAR1 occurs through direct interaction of LMP1 with the TRAF molecules, whereas CTAR2 interacts with the TNFR-associated death domain protein (TRADD) to activate NF-kB and the c-Jun N-terminal kinase (JNK). A20, which is induced by LMP1 through NF-kB, can block NF-kB activation from both domains of LMP1 and inhibit JNK activation from CTAR2. A20 also has been shown to associate with TRAF1 and TRAF2. In this study, an interaction between LMP1 and A20 was detected that was increased by TRAF2 overexpression. A20 did not affect the association of TRAF1 with TRAF2 but did displace TRAF1 from the LMP1 complex. The interaction of LMP1 and TRADD was decreased in the presence of A20, and the LMP1-A20 association was decreased by TRADD, suggesting that A20 and TRADD both interact with LMP1 and may compete for binding. These data indicate that A20 alters the interactions between LMP1 and the TRAF molecules and TRADD, affecting the activation of NF-kB, JNK, and perhaps other TRAF-mediated signaling events.
Asunto(s)
Herpesvirus Humano 4/fisiología , Proteínas/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral , Proteínas de la Matriz Viral/metabolismo , Animales , Sitios de Unión , Carcinoma de Pulmón de Células no Pequeñas , Cisteína Endopeptidasas , Proteínas de Unión al ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares , Ratones , Modelos Químicos , FN-kappa B/metabolismo , Proteínas Nucleares , Proteínas/química , Proteínas/aislamiento & purificación , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Proteína de Dominio de Muerte Asociada a Receptor de TNF , Factor 1 Asociado a Receptor de TNF , Células Tumorales Cultivadas , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/aislamiento & purificaciónRESUMEN
The epidermal growth factor receptor (EGFR) is a potent stimulator of the mitogen-activated protein kinase (MAPK) signaling pathway. Chronic stimulation of the EGFR and of multiple steps in the MAPK signaling pathway is involved in the development of cancer. Several tumor viruses encode proteins that induce EGFR expression or stimulate EGFR-mediated signaling and are thus likely to play an important role in the transformation of virus-infected cells.
Asunto(s)
Transformación Celular Neoplásica , Receptores ErbB/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Virus Oncogénicos/metabolismo , Animales , Receptores ErbB/genética , Expresión Génica , Humanos , Proteínas Oncogénicas Virales/genética , Virus Oncogénicos/genéticaRESUMEN
The effect of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on the activation and differentiation of normal B cells was investigated. B cells of transgenic mice expressing LMP1 under the control of immunoglobulin promoter/enhancer displayed enhanced expression of activation antigens and spontaneously proliferated and produced antibody. Humoral immune responses of LMP1 transgenic mice in CD40-deficient or normal backgrounds revealed that LMP1 mimics CD40 signals to induce extrafollicular B cell differentiation but, unlike CD40, blocks germinal center formation. Thus, these specific properties of LMP1 may determine the site of primary B cell infection and the state of infection in the natural course of EBV infection, whereas subsequent loss of LMP1 expression may affect the site of persistent latent infection.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/metabolismo , Herpesvirus Humano 4/metabolismo , Activación de Linfocitos , Imitación Molecular , Proteínas de la Matriz Viral/metabolismo , Animales , Afinidad de Anticuerpos , Linfocitos B/metabolismo , Linfocitos B/virología , Antígenos CD40/genética , Diferenciación Celular , Femenino , Centro Germinal/inmunología , Centro Germinal/metabolismo , Herpesvirus Humano 4/fisiología , Inmunización , Cambio de Clase de Inmunoglobulina , Inmunoglobulinas/biosíntesis , Interleucina-4/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Transducción de Señal , Bazo/inmunología , Proteínas de la Matriz Viral/genéticaAsunto(s)
Artritis Infecciosa/virología , Herpesvirus Humano 4/aislamiento & purificación , Mononucleosis Infecciosa/complicaciones , Articulación de la Rodilla/virología , Adolescente , ADN Viral/aislamiento & purificación , Femenino , Herpesvirus Humano 4/genética , Humanos , Líquido Sinovial/virologíaRESUMEN
Sequence variations in the Epstein-Barr virus (EBV) latent membrane protein 1 gene have been described in numerous EBV-associated tumors with some of these variations, most notably a 30-base pair deletion in the cytoplasmic carboxyl-terminal domain, suggested as associated with an increase in tumorigenicity. In this study, EBV DNA sequence was determined from 92 tissue specimens or cell lines, including nasopharyngeal carcinoma, oral hairy leukoplakia, post-transplant lymphoma, post-transplant without pathology, mononucleosis, Burkitt's lymphoma, parotid tumor, and normal from distinct geographical regions. The amino- and carboxyl-terminal sequences and, in some cases, the full-length sequences of latent membrane protein 1 were determined. Characteristic sequence patterns distinguished strains, with the carboxyl-terminal sequence being the most informative in distinguishing among the strains. Phylogenetic relationships between strains were determined, as were signature amino acid changes that discriminate between them. A correlation between strain and disease or strain and geographic location was not detected. The sequence variation and signature sequences identified at least seven distinct strains, as well as hybrid strains that apparently result from recombination.
Asunto(s)
Infecciones por Herpesviridae/virología , Herpesvirus Humano 4/clasificación , Herpesvirus Humano 4/genética , Infecciones Tumorales por Virus/virología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Variación Genética , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Mononucleosis Infecciosa/virología , Neoplasias/virología , Filogenia , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Nasopharyngeal carcinoma (NPC), which is closely associated with the Epstein-Barr virus (EBV), is a highly metastatic malignant tumor. An important activity in tumor invasion and metastasis is that of the 92-kDa type IV collagenase or gelatinase, matrix metalloproteinase 9 (MMP-9), which mediates the degradation of the basement membrane and extracellular matrix. The expression of MMP-9 has been shown to be enhanced by the EBV oncoprotein, latent membrane protein 1 (LMP-1). LMP-1, which is expressed in NPC, has two essential signaling domains within the carboxy terminus, termed C-terminal activation regions 1 (CTAR-1) and CTAR-2. This study reveals that either signaling domain can activate the MMP-9 promoter and induce MMP-9 activity; however, LMP-1 deletion mutants lacking either CTAR-1 or CTAR-2 had a decreased ability to induce MMP-9 expression. The deletion of both activation regions completely abolished the induction of MMP-9 activity, while the cotransfection of both the CTAR-1 and CTAR-2 deletion mutants restored MMP-9 activity to levels produced by wild-type LMP-1. The NF-kappaB and activator protein 1 (AP-1) binding sites in the MMP-9 promoter were essential for the activation of MMP-9 gene expression by both CTAR-1 and CTAR-2. The induction of MMP-9 expression by LMP-1 and both CTAR-1 and CTAR-2 mutants was blocked by the overexpression of IkappaB. The tumor necrosis factor receptor-associated factor (TRAF) pathway also contributed to the activation of the MMP-9 promoter as shown by the use of TRAF-2 and TRAF-3 dominant-negative constructs. These data indicate that the activation of both the NF-kappaB and AP-1 pathways by LMP-1, CTAR-1, and CTAR-2 is necessary for the activation of MMP-9 expression. In NPC, LMP-1 may contribute to invasiveness and metastasis through the induction of MMP-9 transcription and enzymatic activity.
Asunto(s)
Colagenasas/genética , Regulación Enzimológica de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Proteínas de la Matriz Viral/metabolismo , Sitios de Unión , Colagenasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Proteínas I-kappa B , Metaloproteinasa 9 de la Matriz , Mutagénesis , FN-kappa B/metabolismo , Péptidos/genética , Regiones Promotoras Genéticas , Proteínas/metabolismo , Transducción de Señal , Factor 2 Asociado a Receptor de TNF , Factor 3 Asociado a Receptor de TNF , Factor de Transcripción AP-1/metabolismo , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/genéticaRESUMEN
The Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) is expressed in latently EBV-infected B cells, where it forms patches in the plasma membrane and interferes with B-cell receptor signal transduction through dominant-negative effects on protein kinases. LMP2 transcripts are detected in nasopharyngeal carcinoma, an epithelial-cell malignancy. In this study the function of LMP2A in epithelial cells was investigated. LMP2A was found to coprecipitate with protein kinase activities and to become phosphorylated in in vitro kinase assays. Analysis of LMP2A deletion mutants demonstrated that tyrosines implicated in interacting with Src family kinase SH2 domains and the SH2 domain of Csk, as well as the LMP2A immunoreceptor tyrosine-based activation motif, are important for its phosphorylation in epithelial cells. LMP2A tyrosine phosphorylation was triggered by cell adhesion to extracellular-matrix (ECM) proteins. Src family kinases, whose involvement in cell-ECM signaling and LMP2A phosphorylation in B lymphocytes has been well established, were found not to be responsible for LMP2A phosphorylation in epithelial cells. Instead, coexpression of Csk, a negative Src regulator, and LMP2A led to an increase in LMP2A phosphorylation both in nonadherent cells and upon cell adhesion. Csk also phosphorylated LMP2A in vitro. These results suggest that LMP2A has a different role in epithelial cells, where it interacts with cell adhesion-initiated signaling pathways. Although tyrosine phosphorylation of LMP2A occurs in both cell types, different protein kinases seem to be used: Src family kinases in B lymphocytes and Csk in epithelial cells.
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Proteínas de la Matriz Extracelular/fisiología , Proteínas Tirosina Quinasas/fisiología , Tirosina/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteína Tirosina Quinasa CSK , Adhesión Celular , Células Epiteliales/fisiología , Humanos , Fosforilación , Proteínas Quinasas/metabolismo , Células Tumorales Cultivadas , Dominios Homologos src , Familia-src QuinasasRESUMEN
The latent membrane protein 1 (LMP1) of the Epstein-Barr virus has transforming properties in rodent fibroblasts and is expressed in most of the cancers associated with Epstein-Barr virus (EBV) infection including posttransplant lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, and AIDS-related lymphomas. In this study, three lineages of LMP1 transgenic mice were established with LMP1 expressed under the control of the Ig heavy chain promoter and enhancer. Lymphoma developed in all three lineages, and the incidence of lymphoma increased significantly with age with lymphomas developing in 42% of transgenic mice over 18 months. The expression of LMP1 was detected at high levels in the lymphoma tissues but only at trace levels in normal lymphoid tissues. Gene rearrangement of the Ig heavy chain indicated monoclonality or oligoclonality in all lymphomas, some of the lymphoid hyperplastic spleens, and some histologically normal spleens. These data reveal that LMP1, without the expression of other EBV genes, is oncogenic in vivo and indicate that LMP1 is a major contributing factor to the development of EBV-associated lymphomas.
Asunto(s)
Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Linfoma de Células B/etiología , Linfoma de Células B/virología , Proteínas de la Matriz Viral/genética , Animales , Elementos de Facilitación Genéticos , Expresión Génica , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Genes Virales , Humanos , Linfoma de Células B/genética , Ratones , Ratones Transgénicos , Oncogenes , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The hairy leukoplakia lesion (HLP) is a unique example of a permissive infection with Epstein-Barr virus (EBV) in the tongue epithelium. HLP contains abundant replicating viral DNA and may be coinfected with multiple EBV strains. In this study, characterization of viral gene transcription within HLP biopsy specimens revealed that several genes, usually expressed in latently infected lymphocytes, are also transcribed in the HLP lesion. The BamHI W and C promoters, (Wp and Cp) are consistently active in the HLP lesion, resulting in transcription and processing of mRNAs that encode the Epstein-Barr nuclear antigens (EBNAs) EBNA-LP, EBNA1, EBNA2, EBNA3B, and EBNA3C. The EBNA2 protein has been shown to activate expression of the EBV receptor, CD21. In HLP, CD21 transcription is also detected, usually in samples that contain transcripts for EBNA2. Transcripts encoding the LMP1 gene, the LMP2 gene, and rightward transcripts from the BamHI A fragment of the EBV genome are also detected in HLP. These gene products are invariably expressed in latently infected lymphocytes. This pattern of transcription suggests that genes characteristic of latent infection are also expressed in HLP. The activation of Wp and expression of EBNA2 and CD21 may contribute to the unique ability of the HLP lesion to permit superinfection and viral replication of multiple EBV strains.