Leukemia virus long terminal repeat activates NFκB pathway by a TLR3-dependent mechanism

AbstractThe long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulates specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFκB, and because the antiapoptotic and growth promoting activities of NFκB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFκB signaling. Here, we demonstrate that FeLV U3-LTR indeed upregulates the NFκB signaling pathway via activation of Ras-Raf-IκB kinase (IKK) and degradation of IκB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR−/− mouse embryo fibroblasts, we further demonstrate that although dsRNA-activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFκB by the LTR. Our study thus demonstrates involvement of a TLR3-dependent but PKR-independent dsRNA-mediated signaling pathway for NFκB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation

Ewing's sarcoma: Analysis of single nucleotide polymorphism in the EWS gene

AbstractWe aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p=0.02; Chi Square Test). About 300bp from the rs4820804 SNP lies a palindromic hexamer (5′-GCTAGC-3′) and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility

Immunohistochemical characterization of engrafted D283ev and D283hCD73 human MB tumors.

<p>Immunohistochemical labeling was performed as described in the Materials and Methods. Anti-synaptophysin and enolase immunostaining were used to evaluate whether the tumor samples were representative of human MB. The proliferation profile was determined using the anti-human Ki67 antibody. Tissue vascularization was visualized with an anti-human CD31 antibody. Labeling is indicated by arrows. Additional analyses are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140996#pone.0140996.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140996#pone.0140996.s003" target="_blank">S3 Fig</a>.</p

Ecto-5’-NT expression and activity following transfection of the D283 MB cell line.

<p>Ecto-5’-NT expression was determined by <b>(A)</b> RT-PCR analysis, <b>(B)</b> flow cytometry and <b>(C)</b> enzymatic activity as described in the Materials and Methods. <b>(D)</b> Cell proliferation indices for each cell line were obtained during five days of culture. (*) p < 0.05; (**) p < 0.01; and (***) p < 0.001 indicate significant differences compared to the Daoy cell line, and (#) p < 0.05; (##) p < 0.01; and (###) p < 0.001 indicate significant differences compared to the D283 cell line.</p

Immunohistochemical analysis of implanted MB.

<p>Immunohistochemistry slides were analyzed by a pathologist in a blinded manner. Analysis were performed using ten randomly chosen fields (x200) per tumor (Olympus BH-2 microscope). Ki67 positive cells were quantified by counting labeled cells in each field.</p><p>Immunohistochemical analysis of implanted MB.</p

Effect of APCP on MB cell proliferation.

<p>At 60% confluence, the cells were treated with 5 μM APCP for 24 and 48 h, and cell counting was performed as described in the Materials and Methods. Controls were considered 100%. The data were analyzed by a Student t-test, and (*) p < 0.05 and (***) p < 0.001 indicate significant differences compared to the control of each respective cell line.</p

Ecto-5’-NT expression in D283 decreases the growth of tumor cells.

<p>To determine human MB tumor growth in nude mice, equal amounts of Daoy, D283ev and D283hCD73 cells (1 × 10⁶ cells) were implanted by subcutaneous injection into the dorsal region of nude mice. During tumor growth, the following data were obtained: <b>(A)</b> Measurements of tumor mass, which determine tumor growth (mm³). <b>(B)</b> Prior to euthanasia, nude mice were injected with the IRDYE^® 800 CW PEG Contrast Agent, and images were captured in the Odyssey^®CLx Infrared Imaging System plus MousePOD <i>in vivo</i> Imaging Accessory. Thus, the location and size of the tumor could be qualitatively measured <i>in vivo</i>. The white circle highlights the tumor mass in each animal that was examined. <b>(C)</b> Detection of ecto-5’-NT immunoreactivity in D283ev and D283hCD73 tumors. The values represent the mean values ± SD (n = 10) for each analyzed group, where (*) p < 0.05.</p

P1 adenosine receptor expression in human MB lines.

<p>The relative expression levels of A₁, A_2A, A_2B and A₃ in Daoy, D283, D283ev and D283hCD73 MB cell lines were assessed by real-time PCR. Endogenous GAPDH expression was used to normalize the adenosine expression levels. * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate a significant difference between the analyzed samples and the Daoy cell line; # p < 0.05 corresponds to differences between the analyzed samples and the D283 cell line.</p