RESUMEN
To retarget oncolytic herpes simplex virus (oHSV) to cancer-specific antigens, we designed a novel, double-retargeted oHSV platform that uses single-chain antibodies (scFvs) incorporated into both glycoprotein H and a bispecific adapter expressed from the viral genome to mediate infection predominantly via tumor-associated antigens. Successful retargeting was achieved using a nectin-1-detargeted HSV that remains capable of interacting with herpesvirus entry mediator (HVEM), the second canonical HSV entry receptor, and is, therefore, recognized by the adapter consisting of the virus-binding N-terminal 82 residues of HVEM fused to the target-specific scFv. We tested both an epithelial cell adhesion molecule (EpCAM)- and a human epidermal growth factor receptor 2-specific scFv separately and together to target cells expressing one, the other, or both receptors. Our results show not only dose-dependent, target receptor-specific infection in vitro, but also enhanced virus spread compared with single-retargeted virus. In addition, we observed effective infection and spreading of the EpCAM double-retargeted virus in vivo. Remarkably, a single intravenous dose of the EpCAM-specific virus eliminated all detectable tumors in a subcutaneous xenograft model, and the same intravenous dose seemed to be harmless in immunocompetent FVB/N mice. Our findings suggest that our double-retargeted oHSV platform can provide a potent, versatile, and systemically deliverable class of anti-cancer therapeutics that specifically target cancer cells while ensuring safety.
RESUMEN
We aimed to identify novel biomarkers in amniotic fluid (AF) that predict the outcome of emergency cerclage in women with cervical insufficiency. This retrospective cohort study included 40 singleton pregnant women who received emergency cerclage for cervical insufficiency (17-25 weeks) and underwent amniocentesis. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify AF proteins in pooled samples (n = 16) using a nested case-control approach. The six candidate biomarkers of interest were validated by enzyme-linked immunosorbent assays (ELISA) in the final cohort (n = 40). The differentially expressed proteins (DEPs) were analyzed by pathway analysis software. The primary outcome measure was failure of emergency cerclage [defined as spontaneous preterm delivery (SPTD) at < 34 weeks of gestation after cerclage placement]. Sixty-eight proteins were differentially expressed (P < 0.001) in AF from SPTD cases and near-term controls, of which 44 (64.7%) were upregulated and 24 (35.3%) were downregulated. Validation by ELISA confirmed that AF from women with cerclage failure contained significantly higher levels of myeloperoxidase, lactoferrin, glucose-6-phosphate isomerase, lipocalin-2, and lymphocyte cytosolic protein 1, the first four of which were independent of cervical dilatation at presentation. The five pathways with the most differentially regulated proteins were actin cytoskeleton signaling, acute phase response signaling, ILK signaling, glycolysis, and gluconeogenesis. Proteomic analyses of AF in this study identified DEPs and specific protein pathways related to poor prognosis after emergency cerclage for cervical insufficiency. Four novel independent biomarkers in AF for cerclage failure have been identified using proteomics.
Asunto(s)
Líquido Amniótico/metabolismo , Cerclaje Cervical/métodos , Nacimiento Prematuro/prevención & control , Incompetencia del Cuello del Útero/cirugía , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Cromatografía Liquida , Femenino , Humanos , Embarazo , Nacimiento Prematuro/metabolismo , Pronóstico , Proteómica , Estudios Retrospectivos , Espectrometría de Masas en Tándem , Incompetencia del Cuello del Útero/metabolismo , Adulto JovenRESUMEN
AIM OF THE STUDY: To identify proteins of which depletion are associated with the poor 6-month neurological outcome of out-of-hospital cardiac arrest survivors. METHODS: Seven healthy volunteers and 34 out-of-hospital cardiac arrest survivors admitted to the intensive care unit (ICU) and underwent targeted-temperature management were enrolled. According to the 6-month cerebral performance category (CPC) scale, patients were divided into the good (CPC 1-2) and poor (CPC 3-5) outcome groups. Blood samples were obtained at 0, 24, and 72â¯h after admission to the ICU. RESULTS: With proteomic approaches, we found 23 proteins that showed group-differences between the sera pooled from 7 study groups: healthy volunteers, the good outcome groups (0, 24, and 72â¯h), and the poor outcome groups (0, 24, and 72â¯h). We selected 7 candidate proteins of which intensities were different between the good and poor outcome groups (>2-fold change) and excluded 5 proteins related to haemolysis or remaining high abundant proteins. To confirm the 2 identified proteins: retinal dehydrogenase 1 and Kallistatin, we performed enzyme-linked immunosorbent assay with individual serum. Finally, old age (odds ratioâ¯=â¯1.055; 95% confidence interval, 1.002-1.112; pâ¯=â¯0.043) and low serum kallistatin level at 0â¯h (odds ratioâ¯=â¯0.784; 95% confidence interval, 0.618-0.995; pâ¯=â¯0.046) were independently associated with the poor 6-month neurological outcome. CONCLUSION: The depletion of serum kallistatin at admission to the ICU was associated with the poor neurological outcome of out-of-hospital cardiac arrest survivors.
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Paro Cardíaco Extrahospitalario/sangre , Serpinas/sangre , Adulto , Factores de Edad , Análisis de Varianza , Biomarcadores/sangre , Reanimación Cardiopulmonar , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Humanos , Hipotermia Inducida/métodos , Proteómica , Recuperación de la Función , Retinal-Deshidrogenasa/sangreRESUMEN
Cripto-1 and OCT4, expressed in stem cells and cancers, play important roles in tumorigenesis. Here, we demonstrate that Cripto-1 expression is regulated by OCT4 in human embryonic carcinoma NCCIT cells. The endogenous expression of Cripto-1 and OCT4 is significantly reduced during differentiation. Cripto-1 expression is increased by OCT4 overexpression, but decreased by shRNA-mediated OCT4 knockdown. OCT4 overexpression significantly activates Cripto-1 transcriptional activity. A 5'-upstream minimal promoter sequence in the gene-encoding Cripto-1 is significantly activated by OCT4 overexpression. Mutation of the putative OCT4-binding site abolishes OCT4-mediated activation of the Cripto-1 promoter. The OCT4 transactivation domains mediate transcriptional activity of the Cripto-1 minimal promoter through direct interaction. Taken together, OCT4 plays an important role as a transcriptional activator of Cripto-1 expression in NCCIT cells.
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Carcinoma Embrionario/genética , Proteínas Ligadas a GPI/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Neoplasias/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Sitios de Unión/genética , Carcinoma Embrionario/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Proteínas Ligadas a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mutación , Proteínas de Neoplasias/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Activación TranscripcionalRESUMEN
Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.
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Carcinoma Embrionario/genética , Factor 3 de Diferenciación de Crecimiento/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Neoplasias Testiculares/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Transcripción GenéticaRESUMEN
We examined the molecular mechanism of OCT4 gene regulation by polyomavirus enhancer activator 3 (PEA3) in NCCIT cells. Endogenous PEA3 and OCT4 were significantly elevated in undifferentiated cells and reduced upon differentiation. PEA3 knockdown led to a reduction in OCT4 levels. OCT4 promoter activity was significantly up-regulated by dose-dependent PEA3 overexpression. Deletion and site-directed mutagenesis of the OCT4 promoter revealed a putative binding site within the conserved region 2 (CR2). PEA3 interacted with the binding element within CR2 in NCCIT cells. This study reveals the molecular details of the mechanism by which the oncogenic factor PEA3 regulates OCT4 gene expression as a transcriptional activator.
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Desarrollo Embrionario , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Bases , Carcinogénesis , Línea Celular Tumoral , Humanos , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas/genética , Seminoma/patología , Eliminación de SecuenciaRESUMEN
The epithelium-specific ETS transcription factor-1 (ESE-1) is physiologically important in the pathogenesis of various diseases. Recently, OCT4, a transcription factor involved in stem cell pluripotency, has been implicated in tumorigenesis. In this study, we invested the molecular mechanism by which ESE-1 regulates transcription of OCT4 in NCCIT human embryonic carcinoma cells. Real-time PCR analysis revealed that OCT4 levels were high in undifferentiated NCCIT cells but significantly decreased upon retinoic acid-mediated differentiation, concomitant with up-regulation of ESE-1 expression. OCT4 mRNA level rose following shRNA-mediated knockdown of ESE-1, but declined when ESE-1 was overexpressed, suggesting that the expression levels of OCT4 and ESE-1 may be coordinated in an opposite manner. Promoter-reporter assays revealed that induced OCT4 promoter activity in NCCIT cells was significantly down-regulated by ESE-1 overexpression in a dose-dependent manner. The inhibitory effect of ESE-1 on OCT4 promoter activity was relieved by co-expression of an ESE-1 mutant lacking the transactivation domain, but not by mutants lacking other domains. Serial deletion and site-directed mutagenesis of the OCT4 promoter revealed that a potential ETS binding site (EBS) is present in the conserved region 2 (CR2). ESE-1 interacted with the EBS element in CR2 and enrichment of CR2 significantly increased upon RA-mediated differentiation of NCCIT cells, suggesting that this binding is likely to be involved in ESE-1-mediated repression of OCT4 promoter activity upon differentiation. Taken together, the results of this study reveal the molecular details of the mechanism by which the oncogenic factor ESE-1 regulates expression of the stem cell transcription factor OCT4 in pluripotent NCCIT cells.
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Proteínas de Unión al ADN/metabolismo , Células Madre de Carcinoma Embrionario/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Proteínas de Unión al ADN/genética , Células Madre de Carcinoma Embrionario/citología , Técnicas de Silenciamiento del Gen , Humanos , Mutación , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-ets/genética , Factores de Transcripción/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.