A prognostic signature of G₂ checkpoint function in melanoma cell lines

As DNA damage checkpoints are barriers to carcinogenesis, G2 checkpoint function was quantified to test for override of this checkpoint during melanomagenesis. Primary melanocytes displayed an effective G2 checkpoint response to ionizing radiation (IR)-induced DNA damage. Thirty-seven percent of melanoma cell lines displayed a significant defect in G2 checkpoint function. Checkpoint function was melanoma subtype-specific with “epithelial-like” melanoma lines, with wild type NRAS and BRAF displaying an effective checkpoint, while lines with mutant NRAS and BRAF displayed defective checkpoint function. Expression of oncogenic B-Raf in a checkpoint-effective melanoma attenuated G2 checkpoint function significantly but modestly. Other alterations must be needed to produce the severe attenuation of G2 checkpoint function seen in some BRAF-mutant melanoma lines. Quantitative trait analysis tools identified mRNA species whose expression was correlated with G2 checkpoint function in the melanoma lines. A 165 gene signature was identified with a high correlation with checkpoint function (p < 0.004) and low false discovery rate (≤ 0.077). The G2 checkpoint gene signature predicted G2 checkpoint function with 77–94% accuracy. The signature was enriched in lysosomal genes and contained numerous genes that are associated with regulation of chromatin structure and cell cycle progression. The core machinery of the cell cycle was not altered in checkpoint-defective lines but rather numerous mediators of core machinery function were. When applied to an independent series of primary melanomas, the predictive G2 checkpoint signature was prognostic of distant metastasis-free survival. These results emphasize the value of expression profiling of primary melanomas for understanding melanoma biology and disease prognosis

Belgium: Transparent Entities: The Case of the French SCI under the Belgium-French Tax Treaty

peer reviewedThis book is a unique publication that gives a global overview of international tax disputes on double tax conventions, thereby filling a gap in the area of tax treaty case law. It covers the 41 most important tax treaty cases that were decided around the world in 2016. The systematic structure of each chapter allows for the easy and efficient study and comparison of the various methods adopted for applying and interpreting tax treaties in different cases. With the continuously increasing importance of tax treaties, Tax Treaty Case Law around the Globe 2017 is a valuable reference tool for anyone interested in tax treaty case law. This book is of interest to tax practitioners, multinational businesses, policymakers, tax administrators, judges and academics

Western immunoblot of IGFBP3 in maternal and fetal serum in non-diabetic (non-pregnant, first trimester, third trimester and cord samples) and type 1 diabetes (first trimester, third trimester and cord samples).

<p>Western immunoblot of IGFBP3 in maternal and fetal serum in non-diabetic (non-pregnant, first trimester, third trimester and cord samples) and type 1 diabetes (first trimester, third trimester and cord samples).</p

Maternal and Fetal placental growth hormone, IGF-I, and IGFBP3 in non diabetic and Type 1 diabetic pregnancy.

<p>Maternal and Fetal placental growth hormone, IGF-I, and IGFBP3 in non diabetic and Type 1 diabetic pregnancy.</p

Maternal and Fetal placental growth hormone, IGF-I, and IGFBP3 in non diabetic and Type 1 diabetic pregnancy.

<p>Data presented as median (range). PGH placental growth hormone, IGF-I insulin like growth factor 1, IGFBP3 insulin like growth factor binding protein 3.</p

A prognostic signature of G₂ checkpoint function in melanoma cell lines

As DNA damage checkpoints are barriers to carcinogenesis, G(2) checkpoint function was quantified to test for override of this checkpoint during melanomagenesis. Primary melanocytes displayed an effective G(2) checkpoint response to ionizing radiation (IR)-induced DNA damage. Thirty-seven percent of melanoma cell lines displayed a significant defect in G(2) checkpoint function. Checkpoint function was melanoma subtype-specific with “epithelial-like” melanoma lines, with wild type NRAS and BRAF displaying an effective checkpoint, while lines with mutant NRAS and BRAF displayed defective checkpoint function. Expression of oncogenic B-Raf in a checkpoint-effective melanoma attenuated G(2) checkpoint function significantly but modestly. Other alterations must be needed to produce the severe attenuation of G(2) checkpoint function seen in some BRAF-mutant melanoma lines. Quantitative trait analysis tools identified mRNA species whose expression was correlated with G(2) checkpoint function in the melanoma lines. A 165 gene signature was identified with a high correlation with checkpoint function (p < 0.004) and low false discovery rate (≤ 0.077). The G(2) checkpoint gene signature predicted G(2) checkpoint function with 77–94% accuracy. The signature was enriched in lysosomal genes and contained numerous genes that are associated with regulation of chromatin structure and cell cycle progression. The core machinery of the cell cycle was not altered in checkpoint-defective lines but rather numerous mediators of core machinery function were. When applied to an independent series of primary melanomas, the predictive G(2) checkpoint signature was prognostic of distant metastasis-free survival. These results emphasize the value of expression profiling of primary melanomas for understanding melanoma biology and disease prognosis

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells.

AbstractThe Jagged/Notch pathway has been implicated in TGFβ1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFβ1-stimulated gene responses in human kidney epithelial cells in vitro. TGFβ1 treatment of HK-2 and RPTEC cells for 24h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of γ-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFβ1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by γ-secretase inhibition, but other TGFβ1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFβ1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFβ1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFβ1, but did not affect α-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFβ1-responsive gene subset. Increased Jagged1 expression upon TGFβ1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFβ1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFβ1 and Notch cascades in kidney epithelia

AMP-activated Protein Kinase Mediates Apoptosis in Response to Bioenergetic Stress through Activation of the Pro-apoptotic Bcl-2 Homology Domain-3-only Protein BMF*

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation