RESUMEN
Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.
Asunto(s)
Linfocitos B , Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Proteínas Proto-Oncogénicas c-myc , Latencia del Virus , Animales , Linfoma de Burkitt/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/genética , Humanos , Ratones , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Linfocitos B/virología , Linfocitos B/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Apoptosis , Proteínas Virales/metabolismo , Proteínas Virales/genéticaRESUMEN
Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinomas (NPCs) in humans, but the mechanism(s) for this effect has been difficult to study because EBV cannot transform normal epithelial cells in vitro and the EBV genome is often lost when NPC cells are grown in culture. Here we show that the latent EBV protein, LMP1 (Latent membrane protein 1), induces cellular proliferation and inhibits spontaneous differentiation of telomerase-immortalized normal oral keratinocytes (NOKs) in growth factor-deficient conditions by increasing the activity of the Hippo pathway effectors, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). We demonstrate that LMP1 enhances YAP and TAZ activity in NOKs both by decreasing Hippo pathway-mediated serine phosphorylation of YAP and TAZ and increasing Src kinase-mediated Y357 phosphorylation of YAP. Furthermore, knockdown of YAP and TAZ is sufficient to reduce proliferation and promote differentiation in EBV-infected NOKs. We find that YAP and TAZ are also required for LMP1-induced epithelial-to-mesenchymal transition. Importantly, we demonstrate that ibrutinib (an FDA-approved BTK inhibitor that blocks YAP and TAZ activity through an off-target effect) restores spontaneous differentiation and inhibits proliferation of EBV-infected NOKs at clinically relevant doses. These results suggest that LMP1-induced YAP and TAZ activity contributes to the development of NPC.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Neoplasias Nasofaríngeas , Humanos , Diferenciación Celular , Proliferación Celular , Células Epiteliales/metabolismo , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.
Asunto(s)
Linfocitos B , Linfoma de Burkitt , Herpesvirus Humano 4 , Factores Reguladores del Interferón , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Herpesvirus Humano 4/metabolismo , Humanos , Factores Reguladores del Interferón/metabolismo , Fenotipo , Proteínas Virales/metabolismoRESUMEN
Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Neoplasias Nasofaríngeas , Telomerasa , Antibacterianos/metabolismo , Proliferación Celular , Herpesvirus Humano 4/metabolismo , Humanos , Queratinocitos , FN-kappa B/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Telomerasa/genética , Activación ViralRESUMEN
Epidermal growth factor receptor (EGFR) is a causal factor in carcinoma, yet many carcinoma patients are resistant to EGFR inhibitors. Potential insight into this resistance stems from prior work that showed EGFR in normal epithelial cells docks to the extracellular domain of the plasma membrane proteoglycan syndecan-4 (Sdc4) engaged with α3ß1 and α6ß4 integrins. We now report that this receptor complex is modified by the recruitment of syndecan-2 (Sdc2), the Recepteur d'Origine Nantais (RON) tyrosine kinase, and the cellular signaling mediator Abelson murine leukemia viral oncogene homolog 1 (ABL1) in triple-negative breast carcinoma and head and neck squamous cell carcinoma, where it contributes to EGFR kinase-independent proliferation. Treatment with a peptide mimetic of the EGFR docking site in the extracellular domain of Sdc4 (called SSTNEGFR) disrupts the entire complex and causes a rapid, global arrest of the cell cycle. Normal epithelial cells do not recruit these additional receptors to the adhesion mechanism and are not arrested by SSTNEGFR. Although EGFR docking with Sdc4 in the tumor cells is required, cell cycle progression does not depend on EGFR kinase. Instead, progression depends on RON kinase, activated by its incorporation into the complex. RON activates ABL1, which suppresses p38 mitogen-activated protein kinase and prevents a p38-mediated signal that would otherwise arrest the cell cycle. These findings add to the growing list of receptor tyrosine kinases that support tumorigenesis when activated by their association with syndecans at sites of matrix adhesion and identify new potential targets for cancer therapy.
Asunto(s)
Carcinoma , Ciclo Celular , Receptores ErbB , Proteínas Tirosina Quinasas Receptoras , Sindecano-2 , Sindecano-4 , Carcinoma/patología , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Sindecano-2/metabolismo , Sindecano-4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Activación Viral , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/patología , Vía de Señalización Hippo , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/virología , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Latencia del VirusRESUMEN
EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Antígenos Nucleares del Virus de Epstein-Barr/genética , Eliminación de Gen , Herpesvirus Humano 4/fisiología , Enfermedad de Hodgkin , Linfoma de Células B Grandes Difuso , Proteínas Virales/genética , Latencia del Virus , Animales , Línea Celular , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/patogenicidad , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/virología , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/virología , Ratones , Proteínas Virales/metabolismoRESUMEN
Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.
Asunto(s)
Linfocitos B/metabolismo , Factores de Transcripción NFATC/genética , Animales , Linfocitos B/virología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr , Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidad , Humanos , Ratones , Regiones Promotoras Genéticas/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Activación Viral , Latencia del VirusRESUMEN
Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.
Asunto(s)
Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Sangre Fetal/metabolismo , Herpesvirus Humano 4/genética , Linfoma/virología , Animales , Linfocitos B/virología , Transformación Celular Viral , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Herpesvirus Humano 4/fisiología , Humanos , Células Asesinas Naturales/inmunología , Linfoma/genética , Linfoma/patología , Linfoma de Células B , Ratones , Mutagénesis Sitio-Dirigida , Análisis de Secuencia de ARN , Eliminación de Secuencia , Linfocitos T/inmunología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Latencia del Virus/genéticaRESUMEN
The human DNA mismatch repair (MMR) protein MLH1 has essential roles in the correction of replication errors and the activation of cell cycle checkpoints and cytotoxic responses to DNA damage that contribute to suppression of cancer risk. MLH1 functions as a heterodimer with the PMS2 protein, and steady state levels of PMS2 are very low in MLH1-deficient cells. Unique to MLH1 among MutL-homolog proteins, and conserved in identified eukaryotic MLH1 proteins, is the so-called C-terminal homology domain (CTH). The function of these C-terminal 20-30 amino acids is not known. We investigated the effect of a C-terminal truncation of human MLH1 (MLH1-L749X) on mammalian MMR by testing its activity in MLH1-deficient cells. We found the CTH to be essential for suppression of spontaneous mutation, activation of a cytotoxic response to 6-thioguanine, and maintenance of normal steady state levels of PMS2. Co-expression in doubly mutant Mlh1-/-; Pms2-/- fibroblasts showed that MLH1-L749X was unable to stabilize PMS2. Over-expression of MLH1-L749X did not reduce stabilization of PMS2 mediated by wild-type MLH1, indicating that truncation of the CTH reduces the ability to compete with wild-type MLH1 for interaction with PMS2. Lack of PMS2 stabilization also was observed with a previously reported pathogenic truncation (MLH1-Y750X), but not with two different point mutations in the CTH. Biochemical assays demonstrated that truncation of the CTH reduced the stability of heterodimers, although MLH1-L749X retained significant capacity for interaction with PMS2. Thus, the CTH of human MLH1 is necessary for error correction, checkpoint signaling, and for promoting interaction with, and the stability of, PMS2. Analysis of the CTH role in stabilizing PMS2 was facilitated by a novel intracellular assay for MLH1-PMS2 interaction. This assay should prove useful for identifying additional amino acids in MLH1 and PMS2 necessary for interaction in cells, and for determining the functional consequences of MLH1 mutations identified in human cancers.
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Adenosina Trifosfatasas/metabolismo , Disparidad de Par Base/genética , Proteínas Portadoras/farmacología , Proteínas Portadoras/fisiología , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/farmacología , Proteínas Nucleares/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Ratones , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Mutación , Proteínas Nucleares/genética , Homología de Secuencia de Aminoácido , Supresión Genética , Tioguanina/farmacologíaRESUMEN
BACKGROUND: A majority of individuals with type 2 diabetes will eventually require exogenous insulin therapy to achieve or maintain glycemic control. This review provides practical recommendations for adding insulin therapy for patients with type 2 diabetes whose glucose levels are inadequately controlled with oral medications. METHODS: We used a systematic review of MEDLINE to retrieve relevant articles from 1990 to 2004 using the search terms insulin therapy, combination oral therapy, glycemic control, insulin analogs, insulin glargine, and basal insulin, which we supplemented with a review of clinical practice guidelines from the American Diabetes Association and the American Association of Clinical Endocrinologists. RESULTS: Type 2 diabetes mellitus is becoming more common in the United States and is likely to increase in prevalence as obesity, a risk factor for type 2 diabetes, likewise increases. Treatment often begins with oral monotherapy, but after 3 years of treatment, more than half of patients will require more than one pharmacological agent, and eventually most patients will require insulin. Adding insulin to oral therapy at an earlier stage in treatment provides improved glycemic control without promoting increased hypoglycemia or weight gain, lowers the risk of microvascular complications by 25%, and reduces the amount of insulin patients require. Various insulin preparations, including the newer analog insulins, with different onsets and durations of action are available to help meet individual patients' dosing needs. CONCLUSIONS: The addition of insulin to oral antidiabetic therapy can improve glycemic control. Newer insulin analogs can emulate normal physiologic insulin secretion and potentially limit diabetes-related comorbidity.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Insulina/uso terapéutico , Administración Oral , Glucemia/análisis , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/epidemiología , Quimioterapia Combinada , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/uso terapéutico , Insulina/efectos adversos , Insulina/análogos & derivados , Persona de Mediana Edad , Obesidad/complicaciones , Guías de Práctica Clínica como Asunto , Prevalencia , Estados Unidos/epidemiologíaRESUMEN
Humans, non-human primates and rodents show declines in spatial memory abilities with increased age. Some of these declines in mice are related to changes in the expression of the epsilon2 (epsilon2) (NR2B) subunit of the N-methyl-D-aspartate receptor. The purpose of this study was to determine whether primates show changes during aging in the mRNA expression of the NR2B subunit. In situ hybridization was performed on tissue sections from three different ages of Rhesus monkeys (Macaca mulatta; 6-8, 10-12, and 24-26 years). There was a significant decrease in the mRNA expression of the NR2B subunit overall in the prefrontal cortex and in the caudate nucleus between young and old monkeys. There were no significant changes in NR2B mRNA expression in the hippocampus or the parahippocampal gyrus. The results in the prefrontal cortex, caudate and hippocampus were similar to those seen previously in C57BL/6 mice during aging, which suggests that mice may be useful as a model for primates to further examine the age-related changes in the expression of the NR2B subunit of the NMDA receptor in several important regions of the brain.
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Envejecimiento/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Expresión Génica/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Hibridación in Situ/métodos , Macaca mulatta , Masculino , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
Activin responsiveness of the murine GnRH receptor gene promoter is mediated at a regulatory element we termed the GnRH receptor activating sequence (GRAS). Here, we have sought to define the complex of transcription factors that interact at this element. Consistent with activin regulation at GRAS, gel shift analyses and yeast one-hybrid assays reveal Smad4 interaction at the 5' end of GRAS. While overexpression of Smad3 activates a GRAS reporter, Smad3 binding at GRAS was not detectable. A functional interaction of Smad3 at GRAS was, however, detectable in yeast expressing Smad4. Thus, Smad3 interaction at GRAS appears to be dependent on the presence of Smad4. Mutations located at the 3' end of GRAS do not affect Smad binding but eliminate functional activity. Thus, Smad binding alone cannot account for the functional attributes of GRAS. Consistent with this notion, we find that AP-1 binding is immediately juxtaposed to and, in fact, partially overlaps the Smad binding site. Finally, a recently identified member of the forkhead family of transcription factors, FoxL2, is also capable of interacting at GRAS. Furthermore, FoxL2 activation at GRAS is lost with mutation of either the 5' Smad binding site or a putative forkhead binding site located at the 3' end of the element. We suggest that GRAS is a composite regulatory element whose functional activity is dependent on the organization of a multi-protein complex consisting of Smads, AP-1 and a member of the forkhead family of DNA binding proteins.
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Receptores LHRH/genética , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Activinas , Animales , Sitios de Unión , Proteínas de Unión al ADN/fisiología , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead , Ratones , Mutación , Proteínas Nucleares/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Smad , Proteína smad3 , Proteína Smad4 , Transactivadores/fisiología , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Homer protein expression is dependent in part on neuronal activity and sleep. Homer1a differs in function from Homer1bc although both are involved in synaptic scaffolding. The effects of sleep loss, time of day and afferent activity on these molecules were investigated. Rats were sacrificed at the midpoint of either their activity or sleep phases and RNA was prepared from somatosensory cortex samples. Homer1a and 1bc mRNA levels were determined by real time PCR. There were greater amounts of both Homer1a and 1bc in night time samples. In controls, there was more Homer1bc than 1a both day and night. Sleep loss upregulated Homer1a in the morning but not at night. Homer1bc was much less affected by manipulations of sleep. Thus, Homer1a may be a state- and activity-dependent synaptic scaling factor.
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Proteínas Portadoras/metabolismo , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/fisiología , Privación de Sueño/metabolismo , Corteza Somatosensorial/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Andamiaje Homer , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Vibrisas/inervación , Vibrisas/fisiologíaRESUMEN
BACKGROUND: The number of treatment options in the diabetes arena has grown dramatically in a short period of time, with a corresponding increase in the breadth and depth of literature from which physicians and diabetes organizations make evidence-based decisions. Thus, the purpose of this article is to provide an up-to-date review of the literature describing current treatment options and guidelines available for the management of type 2 diabetes and prevention of its complications. METHODS: Pubmed searches were conducted for recent literature pertaining to the prevention of complications in type 2 diabetes. Comprehensive search terms were devised to identify articles describing micro- and macrovascular complications including nephropathy, neuropathy, retinopathy, and cardiovascular disease associated with type 2 diabetes. CONCLUSIONS: The current body of literature demonstrates that a significant reduction in the incidence of diabetic complications is achievable with early initiation and long-term maintenance of controlled blood glucose and cardiovascular risk factors. Screening for diabetic complications should be initiated early and continued at regular intervals to ensure early pharmacological intervention.
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Complicaciones de la Diabetes/terapia , Diabetes Mellitus Tipo 2/terapia , Complicaciones de la Diabetes/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Humanos , Incidencia , Guías de Práctica Clínica como Asunto , PubMedRESUMEN
BACKGROUND: Insulin detemir, a long-acting basal insulin analog, is labeled for once-daily or twice-daily dosing in patients with type 1 (T1DM) or type 2 (T2DM) diabetes mellitus. Protocols for some earlier clinical studies of detemir evaluated twice-daily dosing, which may have generated the misperception that detemir should be prescribed twice daily for most patients. This review examines pharmacokinetic and pharmacodynamic (PK/PD), observational, and controlled studies that have evaluated once-daily and twice-daily detemir in patients with T2DM to determine the efficacy and safety of once-daily dosing. METHODS: PubMed was searched using the keywords "detemir," "once daily," "twice daily," and "type 2 diabetes" with the limits of clinical trial, human, and English. RESULTS: Detemir has a relatively flat time-action profile and duration of action of up to 24 hours for patients with T2DM. Once-daily dosing is the most commonly used detemir regimen reported in observational studies, and controlled clinical studies indicate that once-daily dosing controls glycosylated hemoglobin when detemir is administered alone or in combination with a prandial insulin or oral antidiabetes drugs. In comparative clinical trials, detemir had a similar time-action profile and duration of action to another long-acting insulin analog, glargine, with less within-subject variability. Once-daily detemir was associated with no weight gain or less weight gain than comparator regimens. For patients who had not achieved glycemic control with a basal dose of once-daily detemir, adding a prandial insulin provided better glycemic control, less postprandial hypoglycemia, and a lower total daily dose of detemir than twice-daily detemir. Involvement of a multidisciplinary team and the use of a holistic approach for the treatment of T2DM patients are recommended to achieve and maintain the best patient outcomes. CONCLUSION: Results from PK/PD, observational, and controlled clinical studies support a once-daily detemir regimen alone or in combination with a prandial insulin or oral antidiabetes drugs.