Urska Cvek

Translation Initiation Factor eIF4G-1 Binds to eIF3 through the eIF3e Subunit

eIF3 in mammals is the largest translation initiation factor (∼800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2α from complexes sedimenting at ∼40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.

Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes

ABSTRACT The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells. IMPORTANCE Here, we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but also resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells, indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a “hit-and-run” fashion, with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally induced epigenetic changes can be reversed pharmacologically, providing therapeutic interventions to EBV-associated malignancies.

Epstein-Barr virus-induced epigenetic alterations following transient infection.

Epstein–Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBVs contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV‐negative, transiently infected clones to uninfected controls identified expression changes in more than 1,000 genes. Among downregulated genes, several genes known to be deoxyribonucleic acid (DNA) methylated in cancer were identified including E‐cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV‐positive cells were retained after viral loss, indicating an epigenetic effect. Repression of PYCARD expression was a result of increased promoter CpG methylation, whereas loss of E‐cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E‐cadherin promoter. Rather, repression of E‐cadherin was consistent with the formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5′ end of the E‐cadherin gene was observed in an EBV‐negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, whereas its own contribution to the oncogenic process is masked.

Retinoids and skin: microarrays shed new light on chemopreventive action of all-trans retinoic acid.

Despite the use of retinoids in the clinic for many years, their mode of action in the prevention of skin cancer is still unclear. Recent microarray analyses of the chemopreventive effect of all‐trans retinoic acid (ATRA), one of the primary naturally occurring biologically active retinoids, in the two‐stage mouse skin chemical carcinogenesis model have provided novel insight into their action. Comparison of the gene expression profiles of control skin to skin subjected to the two‐stage protocol for 3 wk, with or without ATRA, has shown that approximately half of the genes regulated by 12‐o‐tetradecanoylphorbol‐13‐acetate (TPA) are oppositely regulated when ATRA is coadministered with TPA. It was further shown the Raf/Mek/Erk branch of mitogen‐activated protein (MAP) kinase pathway contains a disproportionate number of oppositely regulated genes, thereby implicating it as one of the key pathways involved in tumor promotion by TPA, that is blocked by ATRA. This result has pointed the way toward the detailed study of Raf/Mek/Erk pathway signaling in skin cancer development and its potential as a target pathway for chemoprevention by ATRA and other chemopreventive drugs.

Identification of Genes Correlated with Early-Stage Bladder Cancer Progression

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed ∼1,900 unique genes differentially expressed (≥3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers. Cancer Prev Res; 3(6); 776–86. ©2010 AACR.

Bioinformatics Multivariate Analysis Determined a Set of Phase-Specific Biomarker Candidates in a Novel Mouse Model for Viral Myocarditis

Background—Myocarditis is an inflammatory disease of the cardiac muscle and is mainly caused by viral infections. Viral myocarditis has been proposed to be divided into 3 phases: the acute viral phase, the subacute immune phase, and the chronic cardiac remodeling phase. Although individualized therapy should be applied depending on the phase, no clinical or experimental studies have found biomarkers that distinguish between the 3 phases. Theiler’s murine encephalomyelitis virus belongs to the genus Cardiovirus and can cause myocarditis in susceptible mouse strains. Methods and Results—Using this novel model for viral myocarditis induced with Theiler’s murine encephalomyelitis virus, we conducted multivariate analysis including echocardiography, serum troponin and viral RNA titration, and microarray to identify the biomarker candidates that can discriminate the 3 phases. Using C3H mice infected with Theiler’s murine encephalomyelitis virus on 4, 7, and 60 days post infection, we conducted bioinformatics analyses, including principal component analysis and k-means clustering of microarray data, because our traditional cardiac and serum assays, including 2-way comparison of microarray data, did not lead to the identification of a single biomarker. Principal component analysis separated heart samples clearly between the groups of 4, 7, and 60 days post infection. Representative genes contributing to the separation were as follows: 4 and 7 days post infection, innate immunity–related genes, such as Irf7 and Cxcl9; 7 and 60 days post infection, acquired immunity–related genes, such as Cd3g and H2-Aa; and cardiac remodeling–related genes, such as Mmp12 and Gpnmb. Conclusions—Sets of molecules, not single molecules, identified by unsupervised principal component analysis, were found to be useful as phase-specific biomarkers.

Multidimensional Visualization Techniques for Microarray Data

Analysis of high-dimensional micro array expression data is based mostly on the statistical approaches that are indispensable for the study of biological systems. To aid the analysis and exploration of such data, the process of analyzing such data is often enhanced with visual, data mining and other computational techniques. We utilize a set of tools for the visual analysis of data aimed at generating the hypotheses. We show the usability of classic and novel multi-dimensional visualization tools in life sciences. Additionally, we survey and show several multidimensional visualization tools applied to the process of data exploration using a urothelial cell carcinoma of the bladder time course. These tools have the potential of uncovering non-trivial relationships and structures in the data.

2D and 3D Neural-Network Based Visualization of High-Dimensional Biomedical Data

In this paper we integrate self-organizing map algorithm (SOM) with scatter plot and Radviz, extending these visualizations into the third dimension and reducing overlap. Classic visualizations are used as the two- dimensional base, combined with a self-organizing map that extends them into the third dimension, with an adjusted neighborhood function. This approach solves the problem of overlap where more than one point plots to the same space and uncovers additional information about relationships inherent in high-dimensional data sets, including distribution of points, outliers and associations. Case studies are presented on a microarray and miRNA data sets.

Blood circulating microparticle species in relapsing-remitting and secondary progressive multiple sclerosis. A case-control, cross sectional study with conventional MRI and advanced iron content imaging outcomes.

BACKGROUNDnAlthough multiple sclerosis (MS) is thought to represent an excessive and inappropriate immune response to several central nervous system (CNS) autoantigens, increasing evidence also suggests that MS may also be a neurovascular inflammatory disease, characterized by endothelial activation and shedding of cell membrane microdomains known as microparticles into the circulation.nnnOBJECTIVEnTo investigate the relationships between these endothelial biomarkers and MS.nnnMETHODSnWe examined the relative abundance of CD31(+)/PECAM-1, CD51(+)CD61(+) (αV-β3) and CD54(+) (ICAM-1) bearing microparticles in sera of healthy individuals, patients with relapsing-remitting MS, and secondary-progressive MS. We also investigated the correlation among circulating levels of different microparticle species in MS with conventional MRI (T2- and T1-lesion volumes and brain atrophy), as well as novel MR modalities [assessment of iron content on susceptibility-weighted imaging (SWI)-filtered phase].nnnRESULTSnDifferences in circulating microparticle levels were found among MS groups, and several microparticle species (CD31(+)/CD51(+)/CD61(+)/CD54(+)) were found to correlate with conventional MRI and SWI features of MS.nnnCONCLUSIONnThese results indicate that circulating microparticles profiles in MS may support mechanistic roles for microvascular stress and injury which is an underlying contributor not only to MS initiation and progression, but also to pro-inflammatory responses.

Both MC1 and MC3 Receptors Provide Protection From Cerebral Ischemia-Reperfusion–Induced Neutrophil Recruitment

Objective—Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC1 and MC3. Approach and Results—Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC3/4 antagonist, suggesting the importance of early MC1 signaling. However, by 2-hour reperfusion, MC1-mediated effects were reduced, and MC3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC1 mutant and MC3−/− mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells. Conclusions—These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics.