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Oncolytic viruses (OVs) have shown potential in converting a "cold" tumor into a "hot" one and exhibit effectiveness in various cancer types. However, only a subset of patients respond to oncolytic virotherapy. It is important to understand the resistance mechanisms to OV treatment in pancreatic ductal adenocarcinoma (PDAC) to engineer oncolytic viruses. In this study, we used transcriptome RNA sequencing (RNA-seq) to identify Visfatin, which was highly expressed in the responsive tumors following OV treatment. To explore the antitumor efficacy, we modified OV-mVisfatin, which effectively inhibited tumor growth. For the first time, we revealed that Visfatin promoted the antitumor efficacy of OV by remodeling the tumor microenvironment, which involved enhancing CD8+ T cell and DC cell infiltration and activation, repolarizing macrophages towards the M1-like phenotype, and decreasing Treg cells using single-cell RNA sequencing (scRNA-seq) and flow cytometry. Furthermore, PD-1 blockade significantly enhanced OV-mVisfatin antitumor efficacy, offering a promising new therapeutic strategy for PDAC.
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Herpesvirus Humano 1 , Nicotinamida Fosforribosiltransferasa , Viroterapia Oncolítica , Virus Oncolíticos , Neoplasias Pancreáticas , Microambiente Tumoral , Animales , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Ratones , Viroterapia Oncolítica/métodos , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Herpesvirus Humano 1/genética , Línea Celular Tumoral , Virus Oncolíticos/genética , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Ratones Endogámicos C57BL , Humanos , Linfocitos T CD8-positivos/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , FemeninoRESUMEN
The low clinical response rate of checkpoint blockades, such as PD-1 and CTLA-4, highlighted the requirements of agonistic antibodies to boost optimal T cell responses. OX40, a co-stimulatory receptor on the T cells, plays a crucial role in promoting T cell survival and differentiation. However, the clinical efficacy of anti-OX40 agonistic antibodies was unimpressive. To explore the mechanism underlying the action of anti-OX40 agonists to improve the anti-tumor efficacy, we analyzed the dynamic changes of tumor-infiltrating immune cells at different days post-treatments using single-cell RNA-sequencing (scRNA-seq). In this study, we found that tumor-infiltrating regulatory T (Treg) cells were reduced after two rounds of anti-OX40 treatment, but the increase of infiltration and activation of CD8+ effector T cells, as well as M1 polarization in the tumor were only observed after three rounds of treatments. Moreover, our group first analyzed the antitumor effect of anti-OX40 treatments on regulating the macrophages and discovered the dynamic changes of vascular endothelial growth factor (VEGF) and CD40 signaling pathways on macrophages, indicating their possibility to being potential combination targets to improve the anti-OX40 agonists efficacy. The combination of VEGFR inhibitors or anti-CD40 agonist antibody with anti-OX40 agonists exhibited more remarkable inhibition of tumor growth. Therefore, the mechanism-driven combination of anti-OX40 agonists with VEGFR inhibitors or anti-CD40 agonists represented promising strategies.
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Linfocitos T Reguladores , Factor A de Crecimiento Endotelial Vascular , Anticuerpos , Inmunoterapia , MacrófagosRESUMEN
Oncolytic viruses are an emerging cancer treatment modality with promising results in clinical trials. The new generation of oncolytic viruses are genetically modified to enhance virus selectivity for tumor cells and allow local expression of therapeutic genes in tumors. The traditional technique for viral genome engineering based on homologous recombination using a bacterial artificial chromosome (BAC) system is laborious and time-consuming. With the advent of the CRISPR/Cas9 system, the efficiency of gene editing in human cells and other organisms has dramatically increased. In this report, we successfully applied the CRISPR/Cas9 technique to construct an HSV-based oncolytic virus, where the ICP34.5 coding region was replaced with the therapeutic genes murine interleukin 12 (IL12, p40-p35) and C-X-C motif chemokine ligand 11 (CXCL11), and ICP47 gene was deleted. The combination of IL12 and CXCL11 in oncolytic viruses showed considerable promise in colorectal cancer (CRC) treatment. Overall, our study describes genetic modification of the HSV-1 genome using the CRISPR/Cas9 system and provides evidence from principle studies for engineering of the HSV genome to express foreign genes.
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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Despite the promising outcome of immune checkpoint inhibitors and agonist antibody therapies in different malignancies, PDAC exhibits high resistance due to its immunosuppressive tumor microenvironment (TME). Ameliorating the TME is thus a rational strategy for PDAC therapy. The intratumoral application of oncolytic herpes simplex virus-1 (oHSV) upregulates pro-inflammatory macrophages and lymphocytes in TME, and enhances the responsiveness of PDAC to immunotherapy. However, the antitumor activity of oHSV remains to be maximized. The aim of this study is to investigate the effect of the CD40L armed oHSV on the tumor immune microenvironment, and ultimately prolong the survival of the PDAC mouse model. METHODS: The membrane-bound form of murine CD40L was engineered into oHSV by CRISPR/Cas9-based gene editing. oHSV-CD40L induced cytopathic effect and immunogenic cell death were determined by microscopy and flow cytometry. The expression and function of oHSV-CD40L was assessed by reporter cell assay. The oHSV-CD40L was administrated intratumorally to the immune competent syngeneic PDAC mouse model, and the leukocytes in TME and tumor-draining lymph node were analyzed by multicolor flow cytometry. Intratumoral cytokines were determined by ELISA. RESULTS: Intratumoral application of oHSV-CD40L efficiently restrained the tumor growth and prolonged the survival of the PDAC mouse model. In TME, oHSV-CD40L-treated tumor accommodated more maturated dendritic cells (DCs), which in turn activated T helper 1 and cytotoxic CD8+ T cells in an interferon-γ-dependent and interleukin-12-dependent manner. In contrast, the regulatory T cells were significantly reduced in TME by oHSV-CD40L treatment. Repeated dosing and combinational therapy extended the lifespan of PDAC mice. CONCLUSION: CD40L-armed oncolytic therapy endues TME with increased DCs maturation and DC-dependent activation of cytotoxic T cells, and significantly prolongs the survival of the model mice. This study may lead to the understanding and development of oHSV-CD40L as a therapy for PDAC in synergy with immune checkpoint blockade.
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Ligando de CD40/administración & dosificación , Carcinoma Ductal Pancreático/terapia , Viroterapia Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Simplexvirus , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral , Animales , Carcinoma Ductal Pancreático/inmunología , Línea Celular Tumoral , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia/prevención & control , Neoplasias Pancreáticas/inmunologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the major type of pancreatic malignancy with very poor prognosis. Despite the promising results of immune checkpoint inhibitors (ICIs) in some solid tumors, immunotherapy is less effective for PDAC due to its immunosuppressive tumor microenvironment (TME). In this report, we established an immunocompetent syngeneic PDAC model and investigated the effect of oncolytic herpes simplex virus-1 (oHSV) on the composition of TME immune cells. The oHSV treatment significantly reduced tumor burden and prolonged the survival of tumor-bearing mice. Further, by single cell RNA sequencing (scRNA-seq) and multicolor fluorescence-activated cell sorting (FACS) analysis, we demonstrated that oHSV administration downregulated tumor-associated macrophages (TAMs), especially the anti-inflammatory macrophages, and increased the percentage of tumor-infiltrating lymphocytes, including activated cytotoxic CD8+ T cells and T helper (Th)1 cells. Besides, the combination of oHSV and immune checkpoint modulators extended the lifespan of the tumor-bearing mice. Overall, our data suggested that oHSV reshapes the TME of PDAC by boosting the immune activity and leads to improved responsiveness of PDAC to immunotherapy.
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Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/terapia , Interacciones Microbiota-Huesped/inmunología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Simplexvirus/genética , Microambiente Tumoral/inmunología , Animales , Biomarcadores , Citocinas/metabolismo , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Viroterapia Oncolítica/métodosRESUMEN
Vav1 is a guanine nucleotide exchange factor (GEF) predominantly expressed in hematopoietic cells, and functions in the development and antigen-stimulated response of lymphocytes. Burkitt's lymphoma (BL) is characterized as transformed B cell lymphoma, and is highly associated with Epstein-Barr virus (EBV). EBV nuclear antigen 1 (EBNA1) is the only viral protein expressed across all three types of latency and essential for the persistence of EBV genome. It is not clear yet how EBNA1 contributes to the growth advantage of latently infected cells such as in EBV+ lymphoma B cells. Here, we reported that Vav1 interacts with EBNA1 via its C-terminal SH3 domain. This interaction suppresses the expression of a pro-apoptotic Bcl-2 family member, Bim, resulting in the resistance of the BL cells to apoptotic inductions. Our data uncovered Vav1 as a novel target for EBNA1, and suggested a pro-survival role of Vav1 in the pathogenesis of EBV associated BLs.
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Proteína 11 Similar a Bcl2/genética , Linfoma de Burkitt/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Humanos , Mapas de Interacción de ProteínasRESUMEN
Background: Current standard methods used to detect and monitor bladder cancer (BC) are invasive or have low sensitivity. We have previously reported in an international European study four non-invasive tests for BC diagnosis based on the gene expression patterns of urine. Objective: to validate the tests in an independent Asian cohort. Design, setting and participants: Prospective blinded study in which consecutive voided urine samples from BC patients and controls (n=520) were collected in the Fudan University Shanghai Cancer Center from 2014-2016. Gene expression values were quantified using TaqMan Arrays. The same cut-off as previously reported for discrimination between tumours and controls was used in this validation study. Results and limitations: Finally, a total of 257 tumour and 132 control urine samples were analysed. We found a high accuracy for the four gene classifiers in this independent Asian set, the classifiers composed of 5 and 10 genes achieved the best sensitivity (80.54% and 81.32%, respectively) maintaining a high specificity (91.67% and 85.61%, respectively). Sensitivity of 5-gene (GS_D5) and 10-gene (GS_D10) expression classifiers in recurrent BC cases (78 and 79%, respectively) is comparable to that of primary BC cases (82%). Cytology and NMP22 identified 67% and 40%, respectively, of tumours that have been diagnosed with our tests. In addition, influence of each studied gene was analyzed and showed similar gene rank between Chinese and Caucasian population. Conclusions: Our study proves that our non-invasive diagnostic BC tests can be reproduced in independent cohorts and in an external laboratory. All the four gene classifiers have shown equal or superior performance to the current gold standard in the present and previously reported validation studies. Consequently, they may be taken for consideration as molecular tests applicable to clinical practice in the management of BC. Patient summary: Our gene classifiers achieve sensitivities up to 90% in HR NMIBC and MIBC patients, while this achievement is comparatively lower in LR NMIBC ones.
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OBJECTIVE: To determine the performance of the prostate health index (PHI) in predicting pathologic outcomes of radical prostatectomy (RP) in Chinese patients with low-risk prostate cancer (PCa). METHODS: Of all consecutive patients who underwent RP in one tertiary center from September 2013 to January 2017, we prospectively examined the data of 140 patients with low-risk PCa based on the Prostate Cancer Research International: Active Surveillance (PRIAS) criteria. All patients were eligible for active surveillance, but underwent RP. Clinical and pathological data were collected. Logistic regression was used to evaluate the associations between the PHI and outcome of RP. The area under the receiver operating curve (AUC) was used to evaluate the accuracy of different models. Decision curve analysis was used to evaluate the potential clinical usefulness of making model-based decisions. RESULTS: Only 44 (31.4%) patients were finally confirmed to have organ-confined Gleason ≤6 PCa. A low PHI was significantly predictive of organ-confined Gleason ≤6 PCa (p = 0.001), while tPSA and f/tPSA were not associated with final pathology. In the multivariate analyses, addition of the PHI significantly increased the predictive accuracy (AUC = 0.767, 95% Cl 0.685-0.849, p < 0.001). CONCLUSION: The PRIAS criteria for active surveillance may not suitable for Chinese patients with PCa. Addition of the PHI to the PRIAS models improved the prognostic performance. If confirmed in future larger and multicenter studies, PHI may help us to identify patients eligible for AS in China.
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Pronóstico , Próstata/cirugía , Prostatectomía/efectos adversos , Neoplasias de la Próstata/cirugía , Anciano , Biopsia , Humanos , Masculino , Persona de Mediana Edad , Periodo Preoperatorio , Próstata/patología , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Factores de RiesgoRESUMEN
OBJECTIVE: To investigate the association between smoking and different prostate cancer (PCa) pathological subtypes incidence in Chinese men. PATIENTS AND METHODS: We prospectively included 1795 patients who underwent prostate biopsies in one tertiary center between March 2013 and April 2016. Clinical data and biopsy outcomes were collected. Logistic regression was used to evaluate the association between cigarette smoking and PCa incidence. RESULTS: A total of 737 men, 480 men and 58 men were diagnosed with PCa, high-grade PCa (HGPCa, grade group ≥ 4 as accepted by the 2014 ISUP) and intraductal carcinoma of the prostate (IDC-P), respectively. Current smokers had a significantly higher risk of HGPCa than never smokers (OR = 1.89, 95%CI: 1.44-2.48). No such association was observed for low-grade disease and cigarette smoking (OR = 0.84, 95%CI: 0.61-1.16). In a sub-analysis, men who had smoked longer than 30 years had a higher risk of HGPCa, compared with men who had smoked fewer than 30 years (OR = 1.50, 95%CI: 1.09-2.06). Current smokers were more likely to develop IDC-P than never smokers (OR = 2.29, 95%CI: 1.14-4.59). CONCLUSION: Among men in this Chinese biopsy cohort, current smoking was associated with highly malignant PCa incidence, such as HGPCa and IDC-P. The duration of smoking may be associated with HGPCa.
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Carcinoma Ductal , Próstata/patología , Neoplasias de la Próstata , Fumar/epidemiología , Anciano , Biopsia/métodos , Biopsia/estadística & datos numéricos , Carcinoma Ductal/epidemiología , Carcinoma Ductal/patología , China/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Neoplasias de la Próstata/epidemiología , Neoplasias de la Próstata/patología , Factores de Riesgo , Estadística como AsuntoRESUMEN
To investigate whether waist-hip ratio (WHR) is a better predictor of prostate cancer (PCa) incidence than body mass index (BMI) in Chinese men. Of consecutive patients who underwent prostate biopsies in one tertiary center between 2013 and 2015, we examined data on 1018 with PSA ≤20 ng/ml. Clinical data and biopsy outcomes were collected. Logistic regression was used to evaluate the associations between BMI, WHR and PCa incidence. Area under the ROC (AUC) was used to evaluate the accuracy of different prognostic models. A total of 255 men and 103 men were diagnosed with PCa and high grade PCa (HGPCa, Gleason score ≥8). WHR was an independent risk factor for both PCa (OR = 1.07 95%Cl 1.03-1.11) and HGPCa (OR = 1.14 95%Cl 1.09-1.19) detection, while BMI had no relationship with either PCa or HGPCa detection. Adding WHR to a multivariable model increased the AUC for detecting HGPCa from 0.66 (95%Cl 0.60-0.72) to 0.71 (95%Cl 0.65-0.76). In this Chinese cohort, WHR was significantly predictive of PCa and HGPCa. Adding WHR to a multivariable model increased the diagnostic accuracy for detecting HGPCa. If confirmed, including WHR measurement may improve PCa and HGPCa detection.
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Índice de Masa Corporal , Neoplasias de la Próstata/diagnóstico , Relación Cintura-Cadera , Anciano , Biomarcadores , Biomarcadores de Tumor , Biopsia , China , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Oportunidad Relativa , Pronóstico , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Curva ROC , Medición de Riesgo , Factores de RiesgoRESUMEN
Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the γ134.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32, and protein kinase C. Unlike wild-type virus, an HSV mutant devoid of γ134.5 or its amino terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild-type virus recruits protein kinase C to the nuclear membrane and triggers its activation, whereas the γ134.5 mutants fail to exert such an effect. Accordingly, the γ134.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells γ134.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino terminus from γ134.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by γ134.5 promotes HSV replication. IMPORTANCE: HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus-infected cells. We report that the γ134.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C, and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.
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Herpesvirus Humano 1/metabolismo , Lamina Tipo A/metabolismo , Fosforilación/fisiología , Proteínas Virales/metabolismo , Liberación del Virus/fisiología , Animales , Cápside/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virología , Chlorocebus aethiops , Citoplasma/metabolismo , Citoplasma/virología , Células HeLa , Humanos , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Lámina Nuclear/metabolismo , Lámina Nuclear/virología , Proteínas Nucleares/metabolismo , Nucleocápside/metabolismo , Proteína Quinasa C/metabolismo , Células Vero , Ensamble de Virus/fisiologíaRESUMEN
Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance.
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Proteínas Adaptadoras Transductoras de Señales/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Proteínas de la Membrana/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Proteínas Virales/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Herpes Simple/genética , Herpes Simple/patología , Herpesvirus Humano 1/genética , Humanos , Evasión Inmune/genética , Interleucina-2/genética , Interleucina-2/inmunología , Células Jurkat , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Antígenos de Linfocitos T , Transducción de Señal/genética , Linfocitos T/patología , Linfocitos T/virología , Factor 6 Asociado a Receptor de TNF/genética , Proteínas Virales/genéticaRESUMEN
Parthenolide (PTL) is a sesquiterpene lactone isolated from feverfew and exhibits potent antitumor activity against various cancers. Many studies indicate that PTL treatment leads to apoptosis, however, the mechanism has not been defined. Here, we observed that cells underwent autophagy shortly after PTL treatment. Inhibition of autophagy by knocking out autophagy associated gene atg5 blocked PTL-induced apoptosis. Surprisingly, PTL decreased the level of translation initiation factor eIF4E binding protein 1 (4E-BP1) in correlation with autophagy. Ectopic expression or shRNA knockdown of 4E-BP1 further verified the effect of 4E-BP1 on PTL-induced autophagy. Meanwhile, PTL elevated the cellular reactive oxygen species (ROS) which located upstream of the depletion of 4E-BP1, and contributed to the consequent autophagy. This study revealed 4E-BP1 as a trigger for PTL-induced autophagy and may lead to therapeutic strategy to enhance the efficacy of anticancer drugs.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Autofagia/efectos de los fármacos , Proteínas Portadoras/metabolismo , Fosfoproteínas/metabolismo , Sesquiterpenos/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Proteínas de Ciclo Celular , Factores Eucarióticos de Iniciación , Fibroblastos/metabolismo , Células HEK293 , Células HL-60 , Células HeLa , Humanos , Ratones , Fagosomas/metabolismo , Fosforilación/efectos de los fármacos , Plásmidos , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.
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Proteínas Portadoras/metabolismo , Núcleo Celular/virología , Herpesvirus Humano 1/fisiología , Proteínas Mitocondriales/metabolismo , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Vero , Liberación del Virus , Receptor de Lamina BRESUMEN
Vav proteins are guanine nucleotide exchange factors (GEFs) that activate a group of small G proteins (GTPases). Vav1 is predominantly expressed in hematopoietic cells, whereas Vav2 and Vav3 are ubiquitously distributed in almost all human tissues. All three Vav proteins contain conserved structural motifs and associate with a variety of cellular activities including proliferation, migration, and survival. Previous observation with Jurkat leukemia T cells showed that Vav1 possessed anti-apoptotic activity by enhancing Bcl-2 transcription. However the mechanism has not been unveiled. Here, we explored the effectors of Vav1 in promoting Bcl-2 expression in Jurkat cells and revealed that Rac2-Akt was specifically evoked by the expression of Vav1, but not Vav2 or Vav3. Although all three Vav isoforms existed in Jurkat cells, Rac2 was distinguishably activated by Vav1 and that led to enhanced Bcl-2 expression and cell survival. Akt was modulated downstream of Vav1-Rac2, and the activation of Akt was indispensable in the enhanced transcription of Bcl-2. Intriguingly, neither Vav2 nor Vav3 was able to activate Rac2-Akt pathway as determined by gene silencing approach. Our data illustrated a unique role of Vav1 in T leukemia survival by selectively triggering Rac2-Akt axis and elevating the expression of anti-apoptotic Bcl-2.
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Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Apoptosis , Células HEK293 , Humanos , Células Jurkat , Leucemia/metabolismo , Leucemia/patología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-vav/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-vav/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/genética , Proteína RCA2 de Unión a GTPRESUMEN
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17ß-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not ß. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-vav/genética , Activación Transcripcional , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1-20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.
Asunto(s)
Señalización del Calcio/fisiología , Activación de Linfocitos/fisiología , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Células HeLa , Humanos , Interleucina-2/biosíntesis , Interleucina-2/genética , Mutación , Unión Proteica/fisiología , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-vav/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citologíaRESUMEN
The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Gripe Humana/patología , Proteínas con Dominio LIM/metabolismo , Proteínas de Microfilamentos/metabolismo , Dominios PDZ/fisiología , Fragmentos de Péptidos/química , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the α subunit of translation initiation factor eIF2 (eIF2α), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2α. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2α by in vitro and in vivo assays. The binding site of eIF2α is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2α is independent of the phosphorylation status of eIF2α at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2α by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2α is crucial for the specific dephosphorylation of eIF2α by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2α via their binding motifs and thereby facilitates the protein synthesis and viral replication.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Herpesvirus Humano 1/fisiología , Biosíntesis de Proteínas , Proteína Fosfatasa 1/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Secuencias de Aminoácidos , Animales , Sitios de Unión , Chlorocebus aethiops , Factor 2 Eucariótico de Iniciación/genética , Células HEK293 , Células HeLa , Humanos , Fosforilación , Proteína Fosfatasa 1/genética , Eliminación de Secuencia , Células Vero , Proteínas Virales/genéticaRESUMEN
AIM: To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells. METHODS: Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨ(m)) was measured using DiOC(6)(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays. RESULTS: Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨ(m)) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression. CONCLUSION: Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.