Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32

Response gene to complement-32 (RGC-32) activates cyclin-dependent kinase 1, regulates the cell cycle and is deregulated in many human tumours. We previously showed that RGC-32 expression is upregulated by the cancer-associated Epstein-Barr virus (EBV) in latently infected B cells through the relief of translational repression. We now show that EBV infection of naïve primary B cells also induces RGC-32 protein translation. In EBV-immortalised cell lines, we found that RGC-32 depletion resulted in cell death, indicating a key role in B cell survival. Studying RGC-32 translational control in EBV-infected cells, we found that the RGC-32 3′untranslated region (3′UTR) mediates translational repression. Repression was dependent on a single Pumilio binding element (PBE) adjacent to the polyadenylation signal. Mutation of this PBE did not affect mRNA cleavage, but resulted in increased polyA tail length. Consistent with Pumilio-dependent recruitment of deadenylases, we found that depletion of Pumilio in EBV-infected cells increased RGC-32 protein expression and polyA tail length. The extent of Pumilio binding to the endogenous RGC-32 mRNA in EBV-infected cell lines also correlated with RGC-32 protein expression. Our data demonstrate the importance of RGC-32 for the survival of EBV-immortalised B cells and identify Pumilio as a key regulator of RGC-32 translation

Complement membrane attack and tumorigenesis: a systems biology approach

Tumor development driven by inflammation is now an established phenomenon, but the role that complement plays remains uncertain. Recent evidence has suggested that various components of the complement (C) cascade may influence tumor development in disparate ways; however, little attention has been paid to that of the membrane attack complex (MAC). This is despite abundant evidence documenting the effects of this complex on cell behavior, including cell activation, protection from/induction of apoptosis, release of inflammatory cytokines, growth factors, and ECM components and regulators, and the triggering of the NLRP3 inflammasome. Here we present a novel approach to this issue by using global gene expression studies in conjunction with a systems biology analysis. Using network analysis of MAC-responsive expression changes, we demonstrate a cluster of co-regulated genes known to have impact in the extracellular space and on the supporting stroma and with well characterized tumor-promoting roles. Network analysis highlighted the central role for EGF receptor activation in mediating the observed responses to MAC exposure. Overall, the study sheds light on the mechanisms by which sublytic MAC causes tumor cell responses and exposes a gene expression signature that implicates MAC as a driver of tumor progression. These findings have implications for understanding of the roles of complement and the MAC in tumor development and progression, which in turn will inform future therapeutic strategies in cancer

Myasthenia gravis thymus: complement vulnerability of epithelial and myoid cells, complement attack on them, and correlations with autoantibody status.

Am J Pathol. 2007 Sep;171(3):893-905. Epub 2007 Aug 3. Myasthenia gravis thymus: complement vulnerability of epithelial and myoid cells, complement attack on them, and correlations with autoantibody status. Leite MI, Jones M, Ströbel P, Marx A, Gold R, Niks E, Verschuuren JJ, Berrih-Aknin S, Scaravilli F, Canelhas A, Morgan BP, Vincent A, Willcox N. Department of Clinical Neurology, University of Oxford, Oxford, United Kingdom. Abstract In early-onset myasthenia gravis, the thymus contains lymph node-type infiltrates with frequent acetylcholine receptor (AChR)-specific germinal centers. Our recent evidence/two-step hypothesis implicates hyperplastic medullary thymic epithelial cells (expressing isolated AChR subunits) in provoking infiltration and thymic myoid cells (with intact AChR) in germinal center formation. To test this, we screened for complement attack in a wide range of typical generalized myasthenia patients. Regardless of the exact serology, thymi with sizeable infiltrates unexpectedly showed patchy up-regulation of both C5a receptor and terminal complement regulator CD59 on hyperplastic epithelial cells. These latter also showed deposits of activated C3b complement component, which appeared even heavier on infiltrating B cells, macrophages, and especially follicular dendritic cells. Myoid cells appeared particularly vulnerable to complement; few expressed the early complement regulators CD55, CD46, or CR1, and none were detectably CD59(+). Indeed, when exposed to infiltrates, and especially to germinal centers, myoid cells frequently labeled for C1q, C3b (25 to 48%), or even the terminal C9, with some showing obvious damage. This early/persistent complement attack on both epithelial and myoid cells strongly supports our hypothesis, especially implicating exposed myoid cells in germinal center formation/autoantibody diversification. Remarkably, the similar changes place many apparent AChR-seronegative patients in the same spectrum as the AChR-seropositive patients. PMID: 17675582 [PubMed - indexed for MEDLINE]PMCID: PMC1959483Free PMC Article Images from this publication.See all images (6) Free text Figure 1 Distribution of complement receptors C3aR, C5aR, and CR1 (receptor for C3b and C4b) (all in red) in epithelial areas and/or infiltrates in thymi from non-MG controls (A and B), AChRAb+ (C–E), or SNMG (F) MG patients. A and B: In control thymi, occasional mTECs are weakly C5aR+, as in some areas in MG thymi, bu... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 2 Distribution of complement regulators CD46, CD55, and CD59 (all in red) in epithelial areas and infiltrates in control (A and D) and MG thymi (B, C, and E–I). Cytokeratin (CK, green). A: In controls, CD46 (A) and CD55 (not shown) expression is minimal; in MG, both are much stronger in the MEBs than in the nMe... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 3 Labeling for C1q and C3b complement fragments (both in red) in epithelial areas and infiltrates in MG and control thymi. Cytokeratin (CK, green). A and B: In MG, there is extensive patchy labeling for C1q in mTECs and other cells in MEBs and in infiltrates and GC in AChRAb+ (A) or SNMG (B) samples. C: In co... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 4 Rarity of complement regulators on myoid cells. In both control (not shown) and MG thymi (A), myoid cells (MC) are uniformly CD59− (red), even when exposed to infiltrates, but ∼5% of the latter express detectable CD55 (red) (B, inset). (Donors both female: A, 20 years of age; B, 16 years of age). Desmin (De, ... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 5 Labeling for C1q, C3b, or C9 (all in red) on exposed myoid cells (MC) in MG thymi. Desmin (De, green). A and B: Some exposed myoid cells label for C1q in AChRAb+ (A) or SNMG (B) MG samples, in which many of them label for C3b (C and D; enlarged in insets) and some for C9 in AChRAb+ (E) or SNMG (F) samples. Note aggr... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905.Figure 6 Percentages of myoid cells exposed to the infiltrates in non-MG controls and MG patient subgroups. Their rarity in the control and MuSKAb+ samples reflects the paucity of infiltrates. There were significantly fewer myoid cells/mm2 in the AChRAb+ group than in the controls (see mini-table below; *P < 0.0... Myasthenia Gravis Thymus Am J Pathol. 2007 September;171(3):893-905

The role of the complement system in traumatic brain injury: a review

Traumatic brain injury (TBI) is an important cause of disability and mortality in the western world. While the initial injury sustained results in damage, it is the subsequent secondary cascade that is thought to be the significant determinant of subsequent outcomes. The changes associated with the secondary injury do not become irreversible until some time after the start of the cascade. This may present a window of opportunity for therapeutic interventions aiming to improve outcomes subsequent to TBI. A prominent contributor to the secondary injury is a multifaceted inflammatory reaction. The complement system plays a notable role in this inflammatory reaction; however, it has often been overlooked in the context of TBI secondary injury. The complement system has homeostatic functions in the uninjured central nervous system (CNS), playing a part in neurodevelopment as well as having protective functions in the fully developed CNS, including protection from infection and inflammation. In the context of CNS injury, it can have a number of deleterious effects, evidence for which primarily comes not only from animal models but also, to a lesser extent, from human post-mortem studies. In stark contrast to this, complement may also promote neurogenesis and plasticity subsequent to CNS injury. This review aims to explore the role of the complement system in TBI secondary injury, by examining evidence from both clinical and animal studies. We examine whether specific complement activation pathways play more prominent roles in TBI than others. We also explore the potential role of complement in post-TBI neuroprotection and CNS repair/regeneration. Finally, we highlight the therapeutic potential of targeting the complement system in the context of TBI and point out certain areas on which future research is needed

Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe

Abstract 1157: Identification of miRNAs that target the Epithelial-Mesenchymal Transition regulator, Zeb2, using transcriptome PCR arrays

Abstract Epithelial-mesenchymal transition (EMT) is the process of molecular reprogramming from polarized immotile epithelial cells to motile mesenchymal cells and is activated during cancer cell invasion of tissues and metastasis. Zeb2 is a transcription factor that has been implicated in EMT, in part, through decreasing levels of E-cadherin. Loss of E-cadherin leads to cell motility which is required for tumor invasion and metastasis. The miRNA family of miR-200 has been shown to inhibit EMT through direct targeting of Zeb2. Currently, only members of the miR-200 family (miR-200bc/429, miR-200a/141) have been identified to target and decrease the expression of Zeb2. Therefore, to identify additional potential miRNAs that target Zeb2, a reverse genetics approach was used. The SureFind Transcriptome PCR Array is an array of 90 cDNA preparations obtained from HeLa cells that were transfected with various miRNA mimics to simulate overexpression of the mature miRNAs. Using a cancer-focused array, qPCR was performed to quantify the expression of Zeb2 wherein changes in mRNA expression identified specific miRNAs that alter Zeb2 expression. In addition to confirming miR-200c (miR-200ab/141/429 were not on the array) as a regulator of Zeb2, five new miRNAs, not previously predicted to target Zeb2 using bioinformatics or reported in the literature, were identified as targeting Zeb2 mRNA. Four of the miRNAs, Let-7g, miR-122, miR-142-5p, and miR-29b, caused at least a two-fold decrease in Zeb2 expression compared to the negative control, which was from cells transfected with a negative mimic control. In addition one miRNA, miR-34c-5p caused a 2.4 fold increase in Zeb2 expression, identifying it as a potential positive regulator of Zeb2 expression. Next to determine if the identified miRNAs inhibit translation of Zeb2, a reporter consisting of firefly luciferase coupled to the 3’UTR of Zeb2 was used to measure the activity of these miRNAs toward the 3’UTR of Zeb2. Let-7g, miR-142-5p, miR-200c, and miR-29b inhibited luciferase activity by at least 1.5 fold compared to negative mimic control. Interestingly, miR-34c-5p and miR-122 exhibited no statistical difference in luciferase activity compared to the negative control. An explanation for this is that miR-34c-5p and miR-122 do not target the 3’UTR of Zeb2 or that they require contextual cues from full-length RNA for their activity. In conclusion, five novel miRNA/Zeb2 interactions were identified in addition to the previously reported miR-200 family. To date only miR-200 has been implicated in EMT though it is possible that let-7g, miR-122, miR-142-5p and miR-29b maybe involved in inhibition of EMT through downregulation of Zeb2 expression. Additionally, these experiments demonstrated that the SureFIND Transcriptome PCR Arrays identify novel miRNA/gene interactions. This application is for research use only, not diagnostic/clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1157. doi:10.1158/1538-7445.AM2011-1157</jats:p

Abstract 1845: Development of a quantitative targeted RNA-Seq methodology for use in differential gene expression analysis

Abstract RNA Sequencing (RNA-Seq) uses the capabilities of Next Generation high-throughput sequencing (NGS) methods to provide insight into the transcriptome of a cell as it generates millions of reads. Whole transcriptome sequencing can be used to quantify gene expression on a transcriptome-wide scale, identify splice variants, quantify allele specific expression, and characterize fusion transcripts. Development of a highly reproducible and sensitive targeted quantitative sequencing method would aid in facilitating a deeper understanding and characterization of the roles of a specific set of genes, while enabling much higher sample throughput and significant cost savings relative to whole-transcriptome sequencing. In this study, we report a targeted RNA-Seq technology, QIAseq RNA, which makes use of several methodologies to deliver an extremely flexible, highly precise tool for characterizing gene expression. QIASeq RNA incorporates 12-base random molecular barcodes into each unique target strand which benefits quantifying gene expression in a given multiplexed sample. Counting the number of molecular tags allows one to determine the sequence coverage per target and adjust experimental conditions to use the read budget of any sequencing platform most efficiently. Using either the Illumina or Ion Torrent platforms, users can choose to multiplex up to 96 RNA samples from 12 to 1000-plex expression panels. No mRNA selection or rRNA removal or blocking is required. The entire protocol, from cDNA synthesis to finished library, which is ready for sequencing, can be accomplished in under one day. Custom assays for a specific target site can add the ability to distinguish between isoforms or identify allele specific expression. We explore the capabilities of this system by profiling large numbers of genes in a cell model system's response to small molecule treatment. Changes in gene expression by these treatments were measured by targeted RNA NGS, and fold-changes in gene expression due to chemical perturbation were characterized. Complex gene relationships in perturbed pathways were mapped using QIAGEN's Ingenuity Pathway Analysis (IPA) tool. The IPA tool also facilitated the elucidation of the impact of gene expression changes in the context of biological processes, molecular interactions, cellular phenotypes and disease. This article will provide application data for GRRC including a discussion of technical challenges faced when profiling large numbers of genes in a large cohort. Citation Format: Eric Lader, Melanie Hussong, Matthew Fosbrink. Development of a quantitative targeted RNA-Seq methodology for use in differential gene expression analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1845.</jats:p