Faculty Bibliography

BACKGROUND:Aberration in the pattern of DNA methylation is one of the hallmarks of cancer. We present data suggesting that dysregulation of MBD2, a recently characterized member of a novel family of methylated DNA binding proteins, is involved in tumorigenesis. Two functions were ascribed to MBD2, DNA demethylase activity and repression of methylated genes. METHODS:Multiple antisense expression and delivery systems, transfection, electrotransfer and adenoviral were employed to demonstrate that MBD2 is essential in tumorigenesis, both ex vivo and in vivo. RESULTS:Inhibition of MBD2 by antisense expression resulted in inhibition of anchorage-independent growth of antisense transfected cancer cells or cells infected with an adenoviral vector expressing MBD2 antisense. Xenograft tumors treated with an adenoviral vector expressing MBD2 antisense or xenografts treated with electrotransferred plasmids expressing MBD2 antisense showed reduced growth. CONCLUSIONS:These results support the hypothesis that one or both of the functions described for MBD2 are critical in tumorigenesis and that MBD2 is a potential anticancer target.

One of the initial responses of an organism to infection by pathogenic viruses is the synthesis of antiviral cytokines such as the type I interferons (interferon-alpha/beta), interleukins, and other proinflammatory cytokines and chemokines. Interferons provide a first line of defence against virus infections by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response. Interferons stimulate cells in the local environment to activate a network of interferon-stimulated genes, which encode proteins that have antiviral, antiproliferative and immunomodulatory activities. The present review focuses on recent reports that describe the activation of multiple signalling pathways following virus infection, new candidate genes that are implicated in the establishment of the antiviral state, and the strategies used by viruses and their specific viral products to antagonize and evade the host antiviral response.

Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-beta enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

The mechanisms of cellular recognition for virus infection remain poorly understood despite the wealth of information regarding the signaling events and transcriptional responses that ensue. Host cells respond to viral infection through the activation of multiple signaling cascades, including the activation of NF-kappaB, c-Jun/ATF-2 (AP-1), and the interferon regulatory factors (IRFs). Although viral products such as double-stranded RNA (dsRNA) and the processes of viral binding and fusion have been implicated in the activation of NF-kappaB and AP-1, the mechanism(s) of IRF-1, IRF-3, and IRF-7 activation has yet to be fully elucidated. Using recombinant measles virus (MeV) constructs, we now demonstrate that phosphorylation-dependent IRF-3 activation represents a novel cellular detection system that recognizes the MeV nucleocapsid structure. At low multiplicities of infection, IRF-3 activation is dependent on viral transcription, since UV cross-linking and a deficient MeV containing a truncated polymerase L gene failed to induce IRF-3 phosphorylation. Expression of the MeV nucleocapsid (N) protein, without the requirement for any additional viral proteins or the generation of dsRNA, was sufficient for IRF-3 activation. In addition, the nucleocapsid protein was found to associate with both IRF-3 and the IRF-3 virus-activated kinase, suggesting that it may aid in the colocalization of the kinase and the substrate. Altogether, this study suggests that IRF-3 recognizes nucleocapsid structures during the course of an MeV infection and triggers the induction of interferon production.

Recent molecular, biochemical, and gene disruption studies have demonstrated the essential role of interferon (IFN) regulatory factor-3, (IRF-3) and IRF-7 in the activation of type I IFN gene expression and the induction of the antiviral state. Both transcription factors share structural and functional properties, as well as a common mechanism of activation through C-terminal phosphorylation. The purpose of this review is to summarize recent investigations indicating that similar signalling pathways are likely involved in the activation of IRF-3 and IRF-7. Moreover, unique biochemical events, such as coactivator association and differential recognition of cis-acting elements, also illustrate the capacity of IRF-3 and IRF-7 to selectively regulate type I IFN and IFN-stimulated gene (ISG) expression.