Kenzo Takada

Epstein-Barr virus and gastric carcinoma

The Epstein-Barr virus (EBV) is detected in the tissue of about 10% of gastric carcinoma cases throughout the world. In each case, 100% of carcinoma cells are infected with EBV. Analysis of EBV in carcinoma biopsies indicates that carcinoma is formed by the proliferation of a single EBV infected cell. These findings suggest that EBV plays an important role in the development of EBV positive gastric carcinomas. The EBV genes expressed are EBV determined nuclear antigen 1 (EBNA1), two small non-polyadenylated RNAs known as EBER1 and EBER2, and the transcripts from the BamHI-A region (BARF0); in addition, some cases also express a small amount of latent membrane protein 2A (LMP2A). Epithelial cells are refractory to EBV infection in vitro. This has hampered the study of the role of EBV in epithelial malignancies. The use of recombinant EBV carrying a selectable marker has enabled this difficulty to be overcome. EBV infected cell clones can be obtained from most carcinoma cell lines examined, and it was found that cell to cell contact was an efficient mode of EBV infection. Furthermore, it was possible to immortalise primary gastric epithelial cells by EBV infection. The cells expressed identical EBV genes to those typically seen in EBV positive gastric carcinoma, and showed accelerated malignant properties, including growth in soft agarose and tumorigenicity in severe combined immunodeficient (SCID) mice. These results suggest that EBV contributes to the maintenance of the malignant phenotype of EBV positive gastric carcinoma.

Nationwide Survey of Hemophagocytic Lymphohistiocytosis in Japan

Hemophagocytic lymphohistiocytosis (HLH), a disorder of the mononuclear phagocyte system, can be classified into two distinct forms: primary HLH (FHL) and secondary HLH. To clarify the epidemiology and clinical outcome for each HLH subtype, we conducted a nationwide survey of HLH in Japan. Since 799 patients were diagnosed in 292 institutions of Japan between 2001 and 2005, the annual incidence of HLH was estimated as 1 in 800,000 per year. Among them, 567 cases were actually analyzed in this study. The most frequent subtype was Epstein-Barr virus (EBV)-associated HLH, followed by other infection- or lymphoma-associated HLH. Age distribution showed a peak of autoimmune disease- and infection-associated HLH in children, while FHL and lymphoma-associated HLH occurred almost exclusively in infants and the elderly, respectively. The 5-year overall survival rate exceeded 80% for patients with EBV- or other infection-associated HLH, was intermediate for those with FHL or B-cell lymphoma-associated HLH, and poor for those with T/NK cell lymphoma-associated HLH (<15%). Although this nationwide survey establishes the heterogeneous characteristics of HLH, the results should be useful in planning prospective studies to identify the most effective therapy for each HLH subtype.

Activation of DNA Methyltransferase 1 by EBV Latent Membrane Protein 2A Leads to Promoter Hypermethylation of PTEN Gene in Gastric Carcinoma

CpG island promoter methylation of tumor suppressor genes is one of the most characteristic abnormalities in EBV-associated gastric carcinoma (GC). Aberrant promoter methylation and expression loss of PTEN were evaluated in cancer tissues of GC by methylation-specific PCR and immunohistochemistry, respectively, showing that both abnormalities occurred concurrently in EBV-associated GC. PTEN abnormalities were reiterated in GC cell lines MKN-1 and MKN-7 infected with recombinant EBV, and DNA methyltransferase 1 (DNMT1) was commonly overexpressed in both cell lines. Stable and transient transfection systems in MKN-1 similarly showed that viral latent membrane protein 2A (LMP2A) up-regulated DNMT1, leading to an increase in methylation of the PTEN promoter. Importantly, the level of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) increased in the nuclei of LMP2A-expressing GC cells, and knockdown of STAT3 counteracted LMP2A-mediated DNMT1 overexpression. Immunohistochemistry for both pSTAT3 and DNMT1 showed diffuse labeling in the nuclei of the cancer cells in GC tissues, especially in EBV-associated GC. Taken together, LMP2A induces the phosphorylation of STAT3, which activates DNMT1 transcription and causes PTEN expression loss through CpG island methylation of the PTEN promoter in EBV-associated GC. LMP2A plays an essential role in the epigenetic abnormalities in host stomach cells and in the development and maintenance of EBV-associated cancer.

EB virus‐encoded RNAs are recognized by RIG‐I and activate signaling to induce type I IFN

Epstein–Barr virus (EBV)‐encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV‐infected cells, and are expected to show secondary structures with many short stem–loops. Retinoic acid‐inducible gene I (RIG‐I) is a cytosolic protein that detects viral double‐stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG‐I as dsRNA. Transfection of RIG‐I plasmid induced IFNs and IFN‐stimulated genes (ISGs) in EBV‐positive Burkitts lymphoma (BL) cells, but not in their EBV‐negative counterparts or EBER‐knockout EBV‐infected BL cells. Transfection of EBER plasmid or in vitro‐synthesized EBERs induced expression of type I IFNs and ISGs in RIG‐I‐expressing, EBV‐negative BL cells, but not in RIG‐I‐minus counterparts. EBERs activated RIG‐Is substrates, NF‐κB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co‐precipitated with RIG‐I. These results indicate that EBERs are recognized by RIG‐I and activate signaling to induce type I IFN in EBV‐infected cells.

Epstein–Barr virus RNA confers resistance to interferon-α-induced apoptosis in Burkitt’s lymphoma

We investigated whether Epstein–Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)‐α. EBV‐negative subclones isolated from EBV‐positive Burkitts lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN‐α treatment. On the other hand, EBV‐positive counterparts exhibited striking resistance against IFN‐α‐induced apoptosis. Transfection of an individual EBV latent gene into EBV‐negative BL cells revealed that EBV‐encoded poly(A)− RNAs (EBERs) were responsible for IFN resistance. EBERs bound double‐stranded (ds) RNA‐activated protein kinase (PKR), a key mediator of the antiviral effect of IFN‐α, and inhibited its phosphorylation. Transfection of dominant‐negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV‐negative BL cells resistant to IFN‐α‐induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA‐binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN‐α‐induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN‐induced apoptosis in virus‐associated tumors.

Epstein-Barr virus (EBV)–encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3

Epstein-Barr virus–encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by intermolecular base-pairing, giving rise to double-stranded RNA (dsRNA)–like molecules, and exists abundantly in EBV-infected cells. Here, we report that EBER induces signaling from the Toll-like receptor 3 (TLR3), which is a sensor of viral double-stranded RNA (dsRNA) and induces type I IFN and proinflammatory cytokines. A substantial amount of EBER, which was sufficient to induce signaling from TLR3, was released from EBV-infected cells, and the majority of the released EBER existed as a complex with a cellular EBER-binding protein La, suggesting that EBER was released from the cells by active secretion of La. Sera from patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), whose general symptoms are caused by proinflammatory cytokines contained EBER, and addition of RNA purified from the sera into culture medium induced signaling from TLR3 in EBV-transformed lymphocytes and peripheral mononuclear cells. Furthermore, DCs treated with EBER showed mature phenotype and antigen presentation capacity. These findings suggest that EBER, which is released from EBV-infected cells, is responsible for immune activation by EBV, inducing type I IFN and proinflammatory cytokines. EBER-induced activation of innate immunity would account for immunopathologic diseases caused by active EBV infection.

Epstein–Barr virus-encoded poly(A)– RNA supports Burkitt’s lymphoma growth through interleukin-10 induction

Akata and Mutu cell lines are derived from Burkitts lymphoma (BL) and retain the in vivo phenotype of Epstein–Barr virus (EBV) expression that is characterized by expression of EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNAs (EBERs) and transcripts from the BamHI A region (BARF0). We found that EBV‐positive Akata and Mutu cell clones expressed higher levels of interleukin (IL)‐10 than their EBV‐negative subclones at the transcriptional level. Transfection of an individual EBV latent gene into EBV‐negative Akata cells revealed that EBERs were responsible for IL‐10 induction. Recombinant IL‐10 enabled EBV‐negative Akata cells to grow in low (0.1%) serum conditions. On the other hand, growth of EBV‐positive Akata cells was blocked by treatment either with an anti‐IL‐10 antibody or antisense oligonucleotide against IL‐10. EBV‐positive BL biopsies consistently expressed IL‐10, but EBV‐negative BL biopsies did not. These results suggest that IL‐10 induced by EBERs acts as an autocrine growth factor for BL. EBERs, EBER1 and EBER2, are non‐polyadenylated RNAs and are 166 and 172 nucleotides long, respectively. The present findings indicate that RNA molecules could regulate cell growth.

Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.

Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3′-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.

Expression of Viral MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma

ABSTRACT Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.

Classification of Epstein–Barr Virus–Positive Gastric Cancers by Definition of DNA Methylation Epigenotypes

Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illuminas Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV(-)/low methylation, EBV(-)/high methylation, and EBV(+)/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV(+) tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV(+) and EBV(-)/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV(-)/high-methylation genes and commonly methylated gastric cancer genes (P = 2 × 10(-15) and 2 × 10(-34), respectively), but not among EBV(+) tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV(+)-associated de novo methylation. When recombinant EBV was introduced into the EBV(-)/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV(+) gastric cancers showed distinct methylation patterns likely attributable to EBV infection.