Molecular Mechanisms Regulating the Expression of Non-Classical Major Histocompatibility Class I Molecules

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Phosphatidylinositol 3-kinase signaling in thymocytes : the need for stringent control

The thymus serves as the primary site for the lifelong formation of new T lymphocytes; hence, it is essential for the maintenance of an effective immune system. Although thymocyte development has been widely studied, the mechanisms involved are incompletely defined. A comprehensive understanding of the molecular events that control regular thymocyte development will not only shed light on the physiological control of T cell differentiation but also probably provide insight into the pathophysiology of T cell immunodeficiencies, the molecular basis that underpins autoimmunity, and the mechanisms that instigate the formation of T cell lymphomas. Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in thymocyte development, although not all of their downstream mediators have yet been identified. Here, we discuss experimental evidence that argues for a critical role of the PI3K-phosphoinositide-dependent protein kinase (PDK1)-protein kinase B (PKB) signaling pathway in the development of both normal and malignant thymocytes, and we highlight molecules that can potentially be targeted therapeutically

MICA Expression Is Regulated by Cell Adhesion and Contact in a FAK/Src-Dependent Manner

MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell–cell contact or loss of cell–matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis

MAP kinase-interacting kinase 1 regulates SMAD2-dependent TGF-β signaling pathway in human glioblastoma

Glioblastoma multiforme (GBM) is the most common aggressive brain cancer with a median survival of approximately 1 year. In a search for novel molecular targets that could be therapeutically developed, our kinome-focused microarray analysis identified the MAP (mitogen-activated protein) kinase-interacting kinase 1 (MNK1) as an attractive theranostic candidate. MNK1 overexpression was confirmed in both primary GBMs and glioma cell lines. Inhibition of MNK1 activity in GBM cells by the small molecule CGP57380 suppressed eIF4E phosphorylation, proliferation, and colony formation whereas concomitant treatment with CGP57380 and the mTOR inhibitor rapamycin accentuated growth inhibition and cell-cycle arrest. siRNA-mediated knockdown of MNK1 expression reduced proliferation of cells incubated with rapamycin. Conversely, overexpression of full-length MNK1 reduced rapamycin-induced growth inhibition. Analysis of polysomal profiles revealed inhibition of translation in CGP57380 and rapamycin-treated cells. Microarray analysis of total and polysomal RNA from MNK1-depleted GBM cells identified mRNAs involved in regulation of TGF-? pathway. Translation of SMAD2 mRNA as well as TGF-?-induced cell motility and vimentin expression was regulated by MNK1 signaling. Tissue microarray analysis revealed a positive correlation between the immunohistochemical staining of MNK1 and SMAD2. Taken together, our findings offer insights into how MNK1 pathways control translation of cancer-related mRNAs including SMAD2, a key component of the TGF-? signaling pathway. Furthermore, they suggest MNK1-controlled translational pathways in targeted strategies to more effectively treat GBM

Deltex-1 Activates Mitotic Signaling and Proliferation and Increases the Clonogenic and Invasive Potential of U373 and LN18 <em>Glioblastoma</em> Cells and Correlates with Patient Survival

<div><p><i>Glioblastoma</i> (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including <i>microRNA-21</i>- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low <i>DTX1</i> levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets. </p> </div

A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.

The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis

Proliferation and mitotic signaling pathways are modified by DTX1 in established glioma cell lines.

<p>(A) Western blot analysis of signaling cascade proteins. Blots for total EGFR (t-EGFR), phosphorylated Akt/PKB (p-Akt/PKB), total Akt/PKB (t-Akt/PKB), phosphorylated Erk (p-Erk), total Erk (t-Erk), Mcl-1 and β-actin (actin) are shown. (B) Proliferation analysis by BrdU incorporation assay. Relative average values of 5 individual experiments are shown per genotype. (C) Proliferation analysis by cell counting. Equal numbers of cells were seeded, grown for 3d under standard conditions and counted afterwards. (D) Apoptosis in U373 cells after treatment with 100 µM temozolomide (TMZ) or DMSO control. Relative values of sub-G₁ cells measured by PI staining are shown normalized to control cells treated with vector control. Averages of at least three independent experiments are shown. Values are normalized to controls. Error bars: ±SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Migration and invasion potential of glioma cells is regulated by DTX1.

<p>(A) Boyden chamber trans-well migration and invasion assay with U373 and LN18 glioma cells on collagen coated membranes with 8 µm porosity. Counts were performed after 24h. (B) Scratch test wound healing assay. A scratch wound was inflicted and immediately imaged (time 0h). Follow up images were taken after 12, 24 and 48 hours. Wound closing was assessed using standard imaging software. (C) Western blot analysis of known pro-migratory factors in glioblastoma probing for Snail-1, Akt2/PKBβ, and beta-actin. (D) Boyden chamber trans-well migration assay with U373-DTX1-myc cells not treated (white), treated with a miR-21 inhibitor (α-miR-21, green) or an inhibitor control (α-CTRL , yellow). Average values are shown from at least three individual experiments. Error bars: ±SEM. * p<0.05, *** p<0.001.</p

DTX1 controls miR-21 expression in a p300 dependent manner.

<p>(A) Real time quantitative PCR analysis of miR-21 expression in U373 cells with modified DTX1 levels. (B) p300 and (C) miR-21 expression levels in U373-DTX1-myc cells transfected with siRNA targeting p300 (sip300, red) or control siRNA (siCTRL, blue) analyzed by qPCR normalized to U373-DTX1-myc. (D) miR-21 expression in U373-DTX1-myc cells treated with miR-21 inhibitor (α-miR-21, green) or an inhibitor control (α-CTRL, yellow). Average relative expression values of at least four independent experiments are shown. Error bars: ±SEM. * p<0.05, ** p<0.01, *** p<0.001. (E) Western blot analysis of signaling cascades in U373-DTX1-myc cells transfected with sip300 or siCTRL. Blots were probed for phosphorylated and total Akt/PKB, phosphorylated and total Erk, Mcl-1 and actin.</p