Epigenetic mechanisms in virus-induced tumorigenesis

About 15–20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis

Transfusion of fresh frozen plasma in non-bleeding ICU patients -TOPIC TRIAL: study protocol for a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Fresh frozen plasma (FFP) is an effective therapy to correct for a deficiency of multiple coagulation factors during bleeding. In past years, use of FFP has increased, in particular in patients on the Intensive Care Unit (ICU), and has expanded to include prophylactic use in patients with a coagulopathy prior to undergoing an invasive procedure. Retrospective studies suggest that prophylactic use of FFP does not prevent bleeding, but carries the risk of transfusion-related morbidity. However, up to 50% of FFP is administered to non-bleeding ICU patients. With the aim to investigate whether prophylactic FFP transfusions to critically ill patients can be safely omitted, a multi-center randomized clinical trial is conducted in ICU patients with a coagulopathy undergoing an invasive procedure.</p> <p>Methods</p> <p>A non-inferiority, prospective, multicenter randomized open-label, blinded end point evaluation (PROBE) trial. In the intervention group, a prophylactic transfusion of FFP prior to an invasive procedure is omitted compared to transfusion of a fixed dose of 12 ml/kg in the control group. Primary outcome measure is relevant bleeding. Secondary outcome measures are minor bleeding, correction of International Normalized Ratio, onset of acute lung injury, length of ventilation days and length of Intensive Care Unit stay.</p> <p>Discussion</p> <p>The Transfusion of Fresh Frozen Plasma in non-bleeding ICU patients (TOPIC) trial is the first multi-center randomized controlled trial powered to investigate whether it is safe to withhold FFP transfusion to coagulopathic critically ill patients undergoing an invasive procedure.</p> <p>Trial Registration</p> <p>Trial registration: Dutch Trial Register NTR2262 and ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01143909">NCT01143909</a></p

Gamma-Secretase-Dependent and -Independent Effects of Presenilin1 on β-Catenin·Tcf-4 Transcriptional Activity

Presenilin1 (PS1) is a component of the γ-secretase complex mutated in cases of Familial Alzheimer's disease (FAD). PS1 is synthesized as a 50 kDa peptide subsequently processed to two 29 and 20 kDa subunits that remain associated. Processing of PS1 is inhibited by several mutations detected in FAD patients. PS1 acts as negative modulator of β-catenin·Tcf-4 transcriptional activity. In this article we show that in murine embryonic fibroblasts (MEFs) the mechanisms of action of the processed and non-processed forms of PS1 on β-catenin·Tcf-4 transcription are different. Whereas non-processed PS1 inhibits β-catenin·Tcf-4 activity through a mechanism independent of γ-secretase and associated with the interaction of this protein with plakoglobin and Tcf-4, the effect of processed PS1 is prevented by γ-secretase inhibitors, and requires its interaction with E- or N-cadherin and the generation of cytosolic terminal fragments of these two cadherins, which in turn destabilize the β-catenin transcriptional cofactor CBP. Accordingly, the two forms of PS1 interact differently with E-cadherin or β-catenin and plakoglobin: whereas processed PS1 binds E-cadherin with high affinity and β-catenin or plakoglobin weakly, the non-processed form behaves inversely. Moreover, contrarily to processed PS1, that decreases the levels of c-fos RNA, non-processed PS1 inhibits the expression c-myc, a known target of β-catenin·Tcf-4, and does not block the activity of other transcriptional factors requiring CBP. These results indicate that prevention of PS1 processing in FAD affects the mechanism of repression of the transcriptional activity dependent on β-catenin

Transposable Elements Are a Major Cause of Somatic Polymorphism in Vitis vinifera L.

Through multiple vegetative propagation cycles, clones accumulate mutations in somatic cells that are at the origin of clonal phenotypic diversity in grape. Clonal diversity provided clones such as Cabernet-Sauvignon N°470, Chardonnay N° 548 and Pinot noir N° 777 which all produce wines of superior quality. The economic impact of clonal selection is therefore very high: since approx. 95% of the grapevines produced in French nurseries originate from the French clonal selection. In this study we provide the first broad description of polymorphism in different clones of a single grapevine cultivar, Pinot noir, in the context of vegetative propagation. Genome sequencing was performed using 454 GS-FLX methodology without a priori, in order to identify and quantify for the first time molecular polymorphisms responsible for clonal variability in grapevine. New generation sequencing (NGS) was used to compare a large portion of the genome of three Pinot noir clones selected for their phenotypic differences. Reads obtained with NGS and the sequence of Pinot noir ENTAV-INRA® 115 sequenced by Velasco et al., were aligned on the PN40024 reference sequence. We then searched for molecular polymorphism between clones. Three types of polymorphism (SNPs, Indels, mobile elements) were found but insertion polymorphism generated by mobile elements of many families displayed the highest mutational event with respect to clonal variation. Mobile elements inducing insertion polymorphism in the genome of Pinot noir were identified and classified and a list is presented in this study as potential markers for the study of clonal variation. Among these, the dynamic of four mobile elements with a high polymorphism level were analyzed and insertion polymorphism was confirmed in all the Pinot clones registered in France

The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes

Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL). By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP) or modified histones (chromatin IP, ChIP), our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced

Discovery of DNA methylation markers in cervical cancer using relaxation ranking

<p>Abstract</p> <p>Background</p> <p>To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in <it>in vitro </it>(cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.</p> <p>Aim</p> <p>To apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.</p> <p>Results</p> <p>The application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed <it>in silico</it>. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is <it>CCNA1</it>) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.</p> <p>Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (<it>SST</it>, <it>HTRA3 </it>and <it>NPTX1</it>) are not methylated in normal cervix tissue.</p> <p>Conclusion</p> <p>The application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown <it>in silico </it>and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.</p

B-cell depletion is protective against anti-AAV capsid immune response: a human subject case study

Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors due to aspects of the clinical application, such as: (i) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii) a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT) for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors