A global view of the oncogenic landscape in nasopharyngeal carcinoma : an integrated analysis at the genetic and expression levels

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors

Background Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. Results Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. Conclusions Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties

Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-κB pathway in carcinoma cells by inhibiting IKK phosphorylation

Background The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-κB. Results In this report we demonstrate that EBNA1 inhibits the canonical NF-κB pathway in carcinoma lines by inhibiting the phosphorylation of IKKα/β. In agreement with this observation we find a reduction in the phosphorylation of IκBα and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-κB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-κB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-κB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied. Conclusions Inhibition of p65 NF-κB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-κB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-κB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC

Investigation of Prader-Willi-like Phenotype using a Whole Genome Array

IntroductionPrader-Willi syndrome (PWS) is characterised byobesity, short stature, small hands and feet, neonatalhypotonia with difficulty in feeding at birth,hypogonadism and eye problems. At about two years ofage the feeding difficulties with poor suck are graduallyreplaced by hyperphagia and obsession with food,leading to the obesity. In addition to developmentaldelay which is manifested by short stature, small handsand feet, growth hormone deficiency andhypogenitalism/hypogonadism, there are alsobehavioural characteristics including learningdisabilities, temper tantrums, aggression, repetitivespeech, obsessive compulsive behaviour, sleep disorderand skin picking (Cassidy and Driscoll, 2009). Thisdisparate collection of symptoms led Holm et al (1993)to define the major and minor characteristics whichallowed a clinical diagnosis of this the most commongenetic form of obesity. Consensus diagnostic criteriawere defined and weighted scores in which the majorcriteria were awarded one point and the minor criteriahalf a point calculated. A score of 8 or more is clinicallydiagnostic for PWS.The majority of people with PWS have a paternallyderived deletion of approximately 5-7Mb in 15q11-q13,others have maternal disomy of chromosome 15(UPD15mat) and a minority have a defect of theimprinting centre located in exon 1 of the SNRPN genewhich leads to a maternal imprint on the paternallyderived chromosome. Any of these abnormalities willresult in loss of the paternal contribution to the Prader-Willi syndrome critical region (PWSCR), demonstratedby loss of a paternally derived unmethylated band at theimprinting centre and a lack of expression of the SNRPNgene. Although these do not differentiate between thedifferent genetic types of PWS they are diagnostic forthe syndrome (Cassidy and Driscoll, 2009; Ramsden etal, 2010; Zeschnigk et al, 1997).Within 15q11-q13 the complex imprintedSNURF/SNRPN gene hosts several untranslated snoRNAgenes located within intronic sequences. The finding ofa microdeletion involving SNORD116 in a boy with PWSled to the identification of this snoRNA as the candidategene for the syndrome (Sahoo et al, 2008).In the course of a large study of PWS in the UK(Whittington et al, 2001; Soni et al, 2007) three peoplewere identified who fulfilled the criteria for a clinicaldiagnosis of the syndrome but not the geneticlaboratory diagnostic criteria.The Affymetrix Cytogenetics Whole-Genome 2.7M arraywhile providing high resolution whole genome coveragereliably detects changes in copy number. Deletionsand/or duplications present in all three participants ifinvolved in annotated genes could potentiallycontribute to the Prader-Willi-like phenotype.Candidate genes can subsequently be evaluated toestimate their transcription levels and compared withthose shown by people with PWS and with unaffectedindividuals

Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection

pstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE: Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases

The Length of the Terminal Repetition in Adenovirus-2 DNA

Adenovirus-2 DNA was end-labeled by partial digestion with Escherichia coli exonuclease III and resynthesis with the DNA polymerase from avian myeloblastosis virus and α-(32)P-labeled deoxyribonucleoside triphosphates. This end-labeled DNA was cleaved with several specific endonucleases and the terminal fragments were characterized by gel electrophoresis and pyrimidine tract analysis. Two endonucleases gave identical fragments from both ends, presumably from cleavage within the inverted terminal repetition, while all other endonucleases gave dissimilar fragments from the two ends. From the sizes of these fragments it is estimated that the inverted terminal repetition is between 100 and 140 nucleotide pairs long