RESUMO
Japanese encephalitis virus (JEV), a mosquitoborne flavivirus, causes acute encephalitis and nervous damage. Previous studies have demonstrated that JEV induces apoptosis in infected cells. However, to date the mechanisms of JEVinduced apoptosis are unclear. In order to identify the viral proteins associated with JEVinduced apoptosis, pEGFPnonstructural protein 3 (NS3) 1619 (expressing the JEV NS3 intact protein, including the protease and helicase domains), pEGFPNS3 1180 (expressing the protease domain) and pEGFPNS3 163619 (expressing the helicase domain) were transfected into target cells to study cell death. Results demonstrate that the JEV NS3 intact protein and protease and helicase domains induce cell death. In addition, cell death was identified to be significantly higher in cells transfected with the NS3 protease domain compared with the intact protein and helicase domain. Caspase activation was also analyzed in the current study. NS3 intact protein and NS3 protease and helicase domains activated caspase9/3dependent and independent pathways. However, caspase8 activity was not found to be significantly different in NS3transfected cells compared with control. In summary, the present study demonstrates that the NS3 helicase and protease domains of JEV activate caspase9/3dependent and independent cascades and trigger cell death.
Assuntos
Apoptose , Caspases/metabolismo , DNA Helicases/metabolismo , Vírus da Encefalite Japonesa (Espécie)/enzimologia , Proteínas não Estruturais Virais/metabolismo , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Chlorocebus aethiops , DNA Helicases/química , DNA Helicases/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transfecção , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/ß-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.
Assuntos
Carcinoma Hepatocelular/genética , Variações do Número de Cópias de DNA , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Animais , Carcinoma Hepatocelular/patologia , Biologia Computacional , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/patologia , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
Phagocytic clearance of dying cells is found in many phagocytes. It has been shown that dying cells can be phagocytosed by other phagocytic cells through autophagic or apoptotic cellular death. To date, whether cancer cells have such phagocytic activity has not been studied. In this study, our data shows that RC-RNase can trigger cell death in human breast cancer MCF-7 cells through the apoptotic pathway. Interestingly, when treated with cytotoxic protein, the remaining MCF-7 cells can phagocytose the dying MCF-7 cells via autophagocytic activity, demonstrated directly by real-time image observation and electron microscopy analysis. To sum up, this study demonstrates for the first time that RC-RNase can trigger apoptosis and autophagocytosis in MCF-7 cancer cells.