Characterization of a new CCCTC-binding factor binding site as a dual regulator of Epstein-Barr virus latent infection.

Distinct viral gene expression characterizes Epstein-Barr virus (EBV) infection in EBV-producing marmoset B-cell (B95-8) and EBV-associated gastric carcinoma (SNU719) cell lines. CCCTC-binding factor (CTCF) is a structural chromatin factor that coordinates chromatin interactions in the EBV genome. Chromatin immunoprecipitation followed by sequencing against CTCF revealed 16 CTCF binding sites in the B95-8 and SNU719 EBV genomes. The biological function of one CTCF binding site (S13 locus) located on the BamHI A right transcript (BART) miRNA promoter was elucidated experimentally. Microscale thermophoresis assay showed that CTCF binds more readily to the stable form than the mutant form of the S13 locus. EBV BART miRNA clusters encode 22 miRNAs, whose roles are implicated in EBV-related cancer pathogenesis. The B95-8 EBV genome lacks a 11.8-kb EcoRI C fragment, whereas the SNU719 EBV genome is full-length. ChIP-PCR assay revealed that CTCF, RNA polymerase II, H3K4me3 histone, and H3K9me3 histone were more enriched at S13 and S16 (167-kb) loci in B95-8 than in the SNU719 EBV genome. 4C-Seq and 3C-PCR assays using B95-8 and SNU719 cells showed that the S13 locus was associated with overall EBV genomic loci including 3-kb and 167-kb region in both EBV genomes. We generated mutations in the S13 locus in bacmids with or without the 11.8-kb BART transcript unit (BART(+/-)). The S13 mutation upregulated BART miRNA expression, weakened EBV latency, and reduced EBV infectivity in the presence of EcoRI C fragment. Another 3C-PCR assay using four types of BART(+/-)·S13(wild-type(Wt)/mutant(Mt)) HEK293-EBV cells revealed that the S13 mutation decreased DNA associations between the 167-kb region and 3-kb in the EBV genome. Based on these results, CTCF bound to the S13 locus along with the 11.8-kb EcoRI C fragment is suggested to form an EBV 3-dimensional DNA loop for coordinated EBV BART miRNA expression and infectivity.

Quercetin Synergistically Inhibit EBV-Associated Gastric Carcinoma with Ganoderma lucidum Extracts.

Mycotherapy has been shown to improve the overall response rate during cancer treatment and reduce some chemotherapy-related adverse events. Ganoderma lucidum is a traditional mushroom used for pharmaceutical purposes. G. lucidum extracts (GLE) showed potential antitumor activities against several cancers. These tumor inhibitory effects of GLE were attributed to the suppression of the proliferation and metastasis of cancer cells. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is defined as the monoclonal proliferation of carcinoma cells with latent EBV infection. The inhibitory effects of GLE against EBVaGC are questionable. The aim of this study was to investigate GLE as potential antitumor agents and a counterpart of quercetin (QCT) for the cotreatment in suppressing EBVaGC development. Therefore, this study conducted antitumor assays using a EBVaGC xenograft mice model and found that GLE could suppress tumor development. These inhibitory effects were significantly augmented by the low concentration of the quercetin (QCT) cotreatment in the xenograft mice. The addition of GLE in low concentrations synergistically reinforced QCT-induced apoptosis and EBV lytic reactivation. GLE contains various polysaccharides and triterpenes, such as ganoderic acid. Interestingly, the addition of ganoderic acid A (GAA) could produce similar bioactive effects like GLE in QCT-mediated antitumor activity. The GAA addition in low concentrations synergistically reinforced QCT-induced apoptosis and EBV lytic reactivation. GAA was sufficiently effective as much as GLE. Therefore, our results suggested that QCT-supplemented GLE could be a potential food adjunct for the prevention of EBVaGC development.

Adenosine Induces EBV Lytic Reactivation through ADORA1 in EBV-Associated Gastric Carcinoma.

Cordyceps species are known to contain numerous bioactive compounds, including cordycepin. Extracts of Cordyceps militaris (CME) are used in diverse medicinal purposes because of their bioactive components. Cordycepin, one of the active components of CME, exhibits anti-proliferative, pro-apoptotic, and anti-inflammatory effects. Cordycepin structurally differs from adenosine in that its ribose lacks an oxygen atom at the 3' position. We previously reported that cordycepin suppresses Epstein⁻Barr virus (EBV) gene expression and lytic replication in EBV-associated gastric carcinoma (EBVaGC). However, other studies reported that cordycepin induces EBV gene expression and lytic reactivation. Thus, it was reasonable to clarify the bioactive effects of CME bioactive compounds on the EBV life cycle. We first confirmed that CME preferentially induces EBV gene expression and lytic reactivation; second, we determined that adenosine in CME induces EBV gene expression and lytic reactivation; third, we discovered that the adenosine A1 receptor (ADORA1) is required for adenosine to initiate signaling for upregulating BZLF1, which encodes for a key EBV regulator (Zta) of the EBV lytic cycle; finally, we showed that BZLF1 upregulation by adenosine leads to delayed tumor development in the EBVaGC xenograft mouse model. Taken together, these results suggest that adenosine is an EBV lytic cycle inducer that inhibits EBVaGC development.

Phylogenetic comparison of Epstein-Barr virus genomes.

Technologies used for genome analysis and whole genome sequencing are useful for us to understand genomic characterization and divergence. The Epstein-Barr virus (EBV) is an oncogenic virus that causes diverse diseases such as Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma (HL), and gastric carcinoma (GC). EBV genomes found in these diseases can be classified either by phases of EBV latency (type-I, -II, and -III latency) or types of EBNA2 sequence difference (type-I EBV, type-II EBV or EBV-1, EBV-2). EBV from EBV-transformed lymphoblastoid cell line (LCL) establishes type-III latency, EBV from NPC establishes type-II latency, and EBV from GC establishes type-I latency. However, other important factors play key roles in classifying numerous EBV strains because EBV genomes are highly diverse and not phylogenetically related to types of EBV-associated diseases. Herein, we first reviewed previous studies to describe molecular characteristics of EBV genomes. Then, using comparative and phylogenetic analyses, we phylogenetically analyzed molecular variations of EBV genomes and proteins. The review of previous studies and our phylogenetic analysis showed that EBV genomes and proteins were highly diverse regardless of types of EBV-associated diseases. Other factors should be considered in determining EBV taxonomy. This review will be helpful to understand complicated phylogenetic relationships of EBV genomes.

Alteration of DNA Methylation in Gastric Cancer with Chemotherapy.

Epigenetic alterations such as DNA methylation, histone acetylation, and chromatin remodeling can control gene expression by regulating gene transcription. DNA methylation is one of the frequent epigenetic events that play important roles in cancer development. Cancer cells can gain significant resistance to anticancer drugs and escape programmed cell death through major epigenetic changes, including DNA methylation. To date, several research groups have identified instances of both (i) hypermethylation of tumor suppressor genes, and (ii) global hypomethylation of oncogenes. These changes in DNA methylation status could be used as biomarkers for the diagnosis and prognosis of cancer patients undergoing chemotherapies or other clinical therapies. Herein, we describe genes for which methylation is dependent upon anticancer drug resistance in patients with gastric cancer; we then suggest a significant epigenetic target to focus on for overcoming anticancer drug resistance.