2-deoxyglucose induces Noxa-dependent apoptosis in alveolar rhabdomyosarcoma.

Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation-encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x(L) and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG-induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress-induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG-induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents.

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3.

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-ß expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-ß and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-ß and ISG15.

Actin cytoskeleton differentially modulates NF-kappaB-mediated IL-8 expression in myelomonocytic cells.

Many physiopathological events such as phagocytosis, pathogen invasion, cellular adhesion and chemotaxis governed by actin-based cytoskeleton are often accompanied by nuclear factor kappaB (NF-kappaB) activation and expression of pro-inflammatory genes. In the present study, we demonstrated that reorganization of actin cytoskeleton induced by Cytochalasin D (CytD), an actin-polymerization inhibitor, enhanced il-8 gene expression induced by TNFalpha and LPS in HL-60 monocyte-like cells. Both transcriptional and post-transcriptional mechanisms were involved. CytD potentiated NF-kappaB-mediated transcription induced by both TNFalpha and LPS but via different mechanisms. In the case of LPS, the perturbation of actin dynamics increased the TLR4 levels at the cell membrane and consequently enhanced the IKK complex activation and NF-kappaB nuclear translocation. However, the canonical pathway involving the IKK complex and leading to the NF-kappaB translocation into the nucleus was not affected by actin remodelling in the case of TNFalpha. Interestingly, actin disruption primed p65 phosphorylation induced by TNFalpha and LPS, on Ser(276) and Ser(536), respectively, which suggested actin cytoskeleton could also modulate p65 transactivating activity.

The varicella-zoster virus immediate-early 63 protein affects chromatin-controlled gene transcription in a cell-type dependent manner.

BACKGROUND: Varicella Zoster Virus Immediate Early 63 protein (IE63) has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. RESULTS: In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding on the promoters tested and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-kappaB dependent genes such as IL-8, ICAM-1, and IkappaBalpha, it modulates transcription of these genes upon TNFalpha induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. CONCLUSION: While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-kappaB dependent genes by the accelerated resynthesis of the inhibitor IkappaBalpha.

Varicella-zoster virus modulates NF-kappaB recruitment on selected cellular promoters.

Intercellular adhesion molecule 1 (ICAM-1) expression is down-regulated in the center of cutaneous varicella lesions despite the expression of proinflammatory cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha). To study the molecular basis of this down-regulation, the ICAM-1 induction of TNF-alpha was analyzed in varicella-zoster virus (VZV)-infected melanoma cells (MeWo), leading to the following observations: (i) VZV inhibits the stimulation of icam-1 mRNA synthesis; (ii) despite VZV-induced nuclear translocation of p65, p52, and c-Rel, p50 does not translocate in response to TNF-alpha; (iii) the nuclear p65 present in VZV-infected cells is no longer associated with p50 and is unable to bind the proximal NF-kappaB site of the icam-1 promoter, despite an increased acetylation and accessibility of the promoter in response to TNF-alpha; and (iv) VZV induces the nuclear accumulation of the NF-kappaB inhibitor p100. VZV also inhibits icam-1 stimulation of TNF-alpha by strongly reducing NF-kappaB nuclear translocation in MRC5 fibroblasts. Taken together, these data show that VZV interferes with several aspects of the immune response by inhibiting NF-kappaB binding and the expression of target genes. Targeting NF-kappaB activation, which plays a central role in innate and adaptive immune responses, leads to obvious advantages for the virus, particularly in melanocytes, which are a site of viral replication in the skin.

Promoter-dependent effect of IKKalpha on NF-kappaB/p65 DNA binding.

IKKalpha regulates many chromatin events in the nuclear phase of the NF-kappaB program, including phosphorylation of histone H3 and removal of co-repressors from NF-kappaB-dependent promoters. However, all of the nuclear functions of IKKalpha are not understood. In this study, using mouse embryonic fibroblasts IKKalpha knock-out and reexpressing IKKalpha after retroviral transduction, we demonstrate that IKKalpha contributes to NF-kappaB/p65 DNA binding activity on an exogenous kappaB element and on some, but not all, endogenous NF-kappaB-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKKalpha is crucial for p65 binding on kappaB sites of icam-1 and mcp-1 promoters but not on ikappabalpha promoter. The mutation of IKKalpha putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKKalpha activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the ikappabalpha promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKKalpha in a promoter-specific manner. Indeed, whereas the absence of IKKalpha induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the ikappabalpha promoter, where a better p65 binding occurs. We conclude that nuclear IKKalpha is required for p65 DNA binding in a gene-specific manner.

Histone deacetylase inhibitor trichostatin A sustains sodium pervanadate-induced NF-kappaB activation by delaying IkappaBalpha mRNA resynthesis: comparison with tumor necrosis factor alpha.

NF-kappaB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) alpha-elicited NF-kappaB activation and delays IkappaBalpha cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-kappaB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-kappaB signaling. This extension is similarly correlated with delayed IkappaBalpha cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFalpha, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of ikappabalpha mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-kappaB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFalpha, the presence of TSA provokes several impairments on the ikappabalpha promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys(14) and -Ser(10), respectively; (iii) decreased presence of phosphorylated p65-Ser(536); and (iv) reduction of IKKalpha binding. The recruitment of these proteins on the icam-1 promoter, another NF-kappaB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-kappaB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-kappaB activation by molecular mechanisms specific of the stimulus and the promoter.