21 research outputs found
Identifying and Characterizing Interplay between Hepatitis B Virus X Protein and Smc5/6
Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression. In this review, we discuss in detail how the interplay between HBx and Smc5/6 was identified and characterized. We also discuss what is known regarding the repression of cccDNA transcription by Smc5/6, the timing of HBx expression, and the potential role of HBx in promoting hepatocellular carcinoma (HCC)
The NF-κB RelB protein is an oncogenic driver of mesenchymal glioma.
High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-κB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-κB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-κB (RelB-mediated) pathway to treat these deadly tumors
Identification and characterization of type III polyketide synthase genes from culturable endophytes of ethnomedicinal plants
Molecular characterization of two alkylresorcylic acid synthases from <em>Sordariomycetes</em> fungi
Smc5/6 silences episomal transcription by a three-step function
In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process. The first step is entrapment of the episomal DNA by a mechanism dependent on Smc5/6 ATPase activity and a function of its Nse4a subunit for which the Nse4b paralog cannot substitute. The second step results in Smc5/6 recruitment to promyelocytic leukemia nuclear bodies by SLF2 (the human ortholog of Nse6). The third step promotes silencing through a mechanism requiring Nse2 but not its SUMO ligase activity. By contrast, the related cohesin and condensin complexes fail to bind to or silence episomal DNA, indicating a property unique to Smc5/6
RelB controls glioma cell motility and invasion.
<p><i>In vitro</i> scratch assays were performed to compare the motility of <i>(A)</i> U87 cells expressing shRNA control, shRelB-1 cells, shRelB-1+vector and shRelB+mRelB; <i>(B)</i> U87 cells expressing pLenti6 vector, pLenti6-mRelB, or pLenti6-hRelB. Photographs were taken of cells pre-scratch, 0 hours and 20–24 hours post-scratch. <i>(C)</i> Western blot was performed on U87 wild type or shRelB-3 cells using antibodies to RelB and actin. (D<i>)</i> Representative photographs of a side view of U87 cells invading three-dimensional collagen matrices. Arrow indicates the surface of the collagen matrix. <i>(E)</i> Average numbers of invading cells per field from 3 independent fields (+/− SD). <i>(F)</i> Average invasion distances (n = 100 cells) +/− SEM. <i>(G)</i> Representative photographs of a side view of U87-shRelB cell invasion +/− rescue with mRelB. <i>(H)</i> Quantification of number of invading cells from <i>G</i>. <i>(I)</i> invasion distance from G. Data shown are average numbers of invading cells per field from 3 independent fields (+/− SD). <i>(J)</i> Representative photographs of a side view of U87 cells overexpressing hRelB invading collagen matrices. <i>(K)</i> Quantification of invasion from J. Data shown are average numbers of invading cells per field from 3 independent fields (+/− SD). <i>(L)</i> Invasion distance from panel J.</p
Kaplan-Meier curves from TCGA (The Cancer Genome Atlas) data analysis show the effect of RelB overexpression on time for tumor progression <i>(A)</i> and patient survival <i>(B)</i>.
<p>Kaplan-Meier curves from TCGA (The Cancer Genome Atlas) data analysis show the effect of RelB overexpression on time for tumor progression <i>(A)</i> and patient survival <i>(B)</i>.</p
RelB controls tumorigenesis glioma tumorigenesis <i>in vivo</i> and is a prognostic indicator of glioma patient survival.
<p><i>(A)</i> Subcutaneous xenografts of DiD-labeled U87 shControl and shRelB-1 cells were allowed to grow for 4 weeks (n = 4). Average volume of tumors was determined based on caliper measurement of tumor diameter. Inset shows representative <i>in vivo</i> tumor images taken with an <i>In Vivo</i> Kodak FX Imager. <i>(B)</i> Orthotopic intracranial injection of DiD-labeled U87 shControl, shRelB-1 and shRelB-3 cells were allowed to grow for 4 weeks. Representative <i>in vivo</i> tumor images from one experiment are shown (n = 3). <i>(C)</i> Western blot analysis was performed on patient-derived glioma cells. <i>(D)</i> Comparison of RelB protein and mRNA levels among the indicated cells. To compare RelB protein expression in U87 and BT cells, western blot data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057489#pone-0057489-g001" target="_blank">Figs. 1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057489#pone-0057489-g005" target="_blank">5B</a> were quantified and normalized to Actin. RelB mRNA levels were quantified by real-time PCR. <i>(E)</i> Intracranial tumor growth of DiD-labeled BT25 glioma cells expressing shRNA control or shRNA-RelB-3 was evaluated by <i>in vivo</i> fluorescence imaging 4 weeks after intracranial innoculation. Representative tumor images from one experiment are shown (n = 3). Similar results were seen with shRelB-1 cells (data not shown). (F) H&E and KI67 staining of frozen brain sections after 4 weeks of tumor growth. Yellow arrows indicate tumor borders. <i>(G)</i> Western blot analysis of RelB levels in BT25 shControl and shRelB-3 cells.</p
RelB promotes glioma cell proliferation and survival.
<p><i>(A)</i> Western blot analysis of glioma cells using indicated antibodies. <i>(B)</i> Western blot analysis was used to assess RelB expression in U87 cells stably expressing a scrambled shRNA control or RelB targeting shRNAs. <i>(C)</i> MTS assays performed on U87 shRNA control, shRelB-1 and shRelB-3 cell lines. Error bars indicate standard deviation (SD), n = 4. <i>(D)</i> A Bioluminescent assay to measure Caspase 3/7 activity was performed on U87 cells expressing the indicated shRNA constructs. Error bars indicate SD. <i>(E)</i> Quantitative real-time PCR examining levelsof Bcl-2 and c-FLIP mRNA in RelB knockdown cells. Error bars indicate standard error (n = 3).</p